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Reduced Expression of Interleukin-11 in Bone Marrow Stromal Cells of Senescence-Accelerated Mice (SAMP6): Relationship to Osteopenia with Enhanced Adipogenesis
Abstract
Aging is associated with an increase in bone marrow adipose tissue and a reduction in bone turnover. The P6 strain of senescence-accelerated mice (SAM) exhibit an early decrease in bone mass with a reduction in bone remodeling. In the bone marrow, suppressed osteoblastogenesis and osteoclastogenesis with enhanced adipogenesis are observed. The present study was undertaken to clarify the mechanism of age-related changes in bone turnover using bone marrow cells from SAMP6 mice. Because interleukin (IL)-11 has been shown to potently inhibit adipogenesis and to stimulate osteoclast formation, the effect of IL-11 on the differentiation of bone marrow cells was examined. The impaired formation of both osteoblasts and osteoclasts was restored and the enhanced formation of adipocytes was suppressed by the addition of 10 pM recombinant human IL-11. Other cytokines that activate gp130 as a common signal transducer, IL-6 and leukemia inhibitory factor, did not have such effects. Sequence analysis of the entire coding region of IL-11 cDNA obtained from SAMP6 stromal cells revealed no mutations. Constitutively secreted IL-11 protein into culture media, and its mRNA expression stimulated by transforming growth factor beta were reduced in stromal cells from SAMP6 compared with those in control mice. These results demonstrate that the expression of IL-11 is reduced in bone marrow cells of SAMP6 and suggest that the reduction in IL-11 actions is involved in the impairment of both osteoblastogenesis and osteoclastogenesis in these mice. There is a possibility that alterations in IL-11 actions may be associated with the age-related impairment in bone metabolism.
... IL-11 was also found to be upregulated by mechanical loading and to enhance osteoblastogenesis by decreasing the expression of the canonical Wnt pathway inhibitor dickkopf2 (Dkk2) [123]. Expression of IL-11 is decreased in the stromal cells of normal aging mice [124] and in the SAM/P6 mouse model of accelerated aging [125]. SAM/P6 mice exhibit decreased bone mass, suppressed osteoblastogenesis and increased bone marrow adiposity [125], similar to the reduced bone formation and increased marrow fat content observed in humans [10]. ...
... Expression of IL-11 is decreased in the stromal cells of normal aging mice [124] and in the SAM/P6 mouse model of accelerated aging [125]. SAM/P6 mice exhibit decreased bone mass, suppressed osteoblastogenesis and increased bone marrow adiposity [125], similar to the reduced bone formation and increased marrow fat content observed in humans [10]. Exogenous administration of IL-11 normalised the impaired osteoblastogenesis and reduced adipogenesis in cultured bone marrow stromal cells from SAM/P6 mice [125]. ...
... SAM/P6 mice exhibit decreased bone mass, suppressed osteoblastogenesis and increased bone marrow adiposity [125], similar to the reduced bone formation and increased marrow fat content observed in humans [10]. Exogenous administration of IL-11 normalised the impaired osteoblastogenesis and reduced adipogenesis in cultured bone marrow stromal cells from SAM/P6 mice [125]. Consistent with a possible role for IL-11 in protecting against senile osteoporosis, transgenic mice overexpressing IL-11 displayed increased bone formation, BMD and bone mass, even at an advanced age, compared to wild-type litter-mates [122]. ...
The number of mature osteoblasts and marrow adipocytes in bone is influenced by the differentiation of the common mesenchymal progenitor cell towards one phenotype and away from the other. Consequently, factors which promote adipogenesis not only lead to fatty marrow but also inhibit osteoblastogenesis, resulting in decreased osteoblast numbers, diminished bone formation and, potentially, inadequate bone mass and osteoporosis. In addition to osteoblast and bone adipocyte numbers being influenced by this skewing of progenitor cell differentiation towards one phenotype, mature osteoblasts and adipocytes secrete factors which may evoke changes in the cell fate and function of each other. This review examines the endogenous factors, such as PPAR-γ2, Wnt, IGF-1, GH, FGF-2, oestrogen, the GP130 signalling cytokines, vitamin D and glucocorticoids, which regulate the selection between osteoblastogenesis and adipogenesis and the interrelationship between fat and bone. The role of adipokines on bone, such as adiponectin and leptin, as well as adipose-derived oestrogen, is reviewed and the role of bone as an energy regulating endocrine organ is discussed.
... 18 In addition, bone marrow-derived MSC (BMSC) isolated from the long bones of SAMP6 mice have a reduced capacity for osteoblastogenesis and an increased tendency to differentiate toward the adipocyte lineage. [19][20][21] Such alterations in cell differentiation have been observed in BMSC from osteoporotic patients and have led to speculation that bone deterioration associated with age-related osteoporosis may be directly related to an inability to produce a sufficient number of functionally active osteoblasts. 22,23 This, in turn, may have significant ramifications for fracture repair due to a limited availability of bone-forming cells and a subsequent reduction in bone mineralization. ...
... [17][18][19]38,39 Furthermore, alterations in the osteogenic and adipogenic differentiation profile of mesenchymal stem cells within the bone marrow of SAMP6 mice have also been reported during this period. [19][20][21] In the present study, we confirm that BMSC isolated from SAMP6 mice have a reduced capacity for osteoblast differentiation, and adipogenic differentiation was significantly increased. It was therefore envisaged that such deficiencies in the bone-forming potential of SAMP6 marrow may have significant implications for bone repair in this osteoporotic model. ...
... In addition, comparable levels of IL-11 mRNA expression were also observed in these cells (data not shown), a cytokine previously implicated in the impairment of osteoblastogenesis in bone marrow cells from SAMP6 mice. 20 Therefore, this may offer some explanation as to why no delays in fracture repair were observed in SAMP6 mice, despite there being a clear deficit in marrow osteogenesis. However, the potential involvement of osteoprogenitors from other sources, both local and remote, should not be overlooked, and further studies are required to assess their contribution to the repair process. ...
Bone marrow osteogenesis in senile osteoporotic bone is impaired and, as such, may have significant implications on the successful outcome of fracture repair. Here we utilize a well-established murine model of senile osteoporosis, the P6 strain of senescence-accelerated mice (SAMP6), to investigate fracture healing in aged osteoporotic bone. A femoral osteotomy was created in SAMP6 and in non-osteoporotic age-matched control R1 senescence-resistant mice (SAMR1). The course of fracture healing was evaluated over a period of 42 days using quantitative microCT and histological analysis. The differentiation capabilities of bone mesenchymal progenitor cells derived from SAMP6 and SAMR1 mice was examined, and their osteogenic potential determined. Although preliminary in vitro analysis confirmed that bone marrow-derived stem cells (BMSC) isolated from SAMP6 mice had a reduced osteogenic capacity, no significant deficit in fracture repair as determined by quantitative microCT could be detected. This was supported by histology assessment, where complete bridging of the fracture gap was evident by day 28 and was fully healed day 42 in both SAMP6 and SAMR1 mice. Further in vitro studies revealed that periosteal-derived progenitor cells (PDPC) isolated from SAMP6 mice had an osteogenic potential comparable to that observed in SAMR1 mice. In conclusion, fracture healing in SAMP6 mice is not detrimentally affected by impairment of BMSC osteogenesis, suggesting that bone marrow-mediated repair processes are dispensable for normal bone healing in this senile osteoporotic fracture model. Furthermore, the influence of PDPC in the repair process may partly explain the absence of any detectable deficits in fracture repair in SAMP6 mice.
... Interpreting the results of these studies is further complicated by variations in the cell source site, isolation procedures, culture conditions, assay conditions, metrics, and developmental time-points being evaluated. The clearest trends are observed in murine osteoporosis/osteopenia models, including SAMP6 [6,7] and aged C57BL/6 [8] mice, which exhibit low bone mass and/or bone material and mechanical defects accompanied by altered mesenchymal progenitor properties (i.e., reduced numbers, proliferative capacity, or osteogenic differentiation capacity). ...
... A total of 2 × 106 cells were injected into sham-operated or ovariectomized female ddY mice (8-week-old, Central lab animal, Korea) via tail vein on postoperative day 4 and sacrificed at day 24 after injection (n = 6 per group). Microcomputed tomography (μCT) and histological analysis were performed, as reported previously [7,21]. Trabecular morphometry within the proximal tibia was quantified using high resolution microcomputed tomography (μCT, Skyscan 1076 μCT, Aartselaar, Belgium). ...
Osteoblast and adipocyte are derived from common mesenchymal progenitor cells. The bone loss of osteoporosis is associated with altered progenitor differentiation from an osteoblastic to an adipocytic lineage. In this study, a comparative analysis of gene expression profiling using cDNA microarray and realtime-PCR indicated that Zinc finger protein 467 (Zfp467) involved in adipocyte and osteoblast differentiation of cultured adipose derived stem cells (ADSCs). Our results showed that RNA interference for Zfp467 in ADSCs inhibited adipocyte formation and stimulated osteoblast commitment. The mRNA levels of osteogenic and adipogenic markers in ADSCs were regulated by si-Zfp467. Zfp467 RNAi in ADSCs could restore bone function and structure in an ovariectomized (OVX)-induced osteoporotic mouse model. Thus Zfp467 play an important role in ADSCs differentiation to adipocyte and osteoblast. This has relevance to therapeutic interventions in osteoporosis, including si-Zfp467-based therapies currently available, and may be of relevance for the use of adipose-derived stem cells for tissue engineering.
... In this review, we summarize current understanding of the regulation of osteoblast differentiation by various stimuli and inhibitors, with special focus on the role of interleukin (IL)-11 in mechanical stress and PTH-induced stimulation, along with aging and glucocorticoid-induced suppression of osteoblast differentiation. genesis was suppressed by the addition of IL-11 [2]. Other cytokines that activate gp130 as a common signal transducer, IL-6 and leukemia inhibitory factor, did not have such effects. ...
... Using senescence-accelerated mice (SAMP6), we demonstrated that these mice exhibit early decrease in bone mass and a reduction in bone turnover, that both osteoblastogenesis and osteoclastogenesis are suppressed and adipogenesis is enhanced from bone marrow stromal cells of SAMP6 mice, and that the expression of IL-11 is decreased in SAMP6 bone marrow stromal cells [2]. IL-11 is expressed in bone marrow stromal cells and is involved in the regulation of multiple biological processes such as enhancement of myeloid cell growth [3,4], inhibition of adipogenesis [3,5], and stimulation of osteoclast formation [6]. ...
Mechanical stress and parathyroid hormone (PTH) are major stimulators, and aging and glucocorticoids excess are important suppressors of osteoblast differentiation. Mechanical stress and PTH stimulate interleukin (IL)-11 expression in cells of osteoblast lineage by enhancing transcription of IL-11 gene via an increase in intracellular Ca²⁺. The elevated Ca²⁺ activates extracellular signal-regulated kinase (ERK) to enhance phosphorylation of cyclic AMP response element-binding protein (CREB), which binds to the fosB gene promoter and enhances ΔFosB expression. ΔFosB dimerizes with JunD on the IL-11 gene promoter to enhance its transcription. Both mechanical stress and PTH also stimulate phosphorylation of Smad1 via an activation of protein kinase Cδ (PKCδ). Phosphorylated Smad1 binds to the IL-11 gene promoter and forms complex with ΔFosB/JunD to further enhance IL-11 gene transcription. The increased IL-11 then suppresses expression of Wnt inhibitors, including Dickkopf 1 (Dkk1) and 2, and enhances Wnt signaling to stimulate osteoblast differentiation and inhibit adipocyte differentiation. The suppression of osteoblast differentiation by aging involves a decrease in IL-11 gene transcription by a reduction in JunD binding to the activator protein (AP)-1 site of the IL-11 gene promoter. Glucocorticoids inhibit transcriptional activation of IL-11 gene by an interaction of glucocorticoid-glucocorticoid receptor (GR) complex with ΔFosB/JunD heterodimer. Thus, factors that enhance osteoblast differentiation stimulate, and those which suppress osteoblast differentiation inhibit IL-11 gene transcription, and IL-11 enhances Wnt signaling by suppressing expression of its inhibitors. These observations are consistent with the notion that IL-11 mediates stimulatory and inhibitory signals of osteoblast differentiation by affecting Wnt signaling.
... For example, a decrease in bone volume in age-related and steroid-induced osteoporosis is accompanied by an increase in marrow adipose tissue (see for example, [4,5]). A variety of experimental manipulations in primary bone marrow stromal cells and cell lines have contributed molecular and cellular insight into the mechanisms underlying the apparent reciprocal relationship between the two lineages (see for example, [1,6,7,8,9,10,11]). These studies have led to the suggestion that regulated lineage allocation of stem or multipotential progenitor cells or a fate switch from osteoblast lineage (ObL) to adipocyte lineage (AdL) occurs under certain conditions, including aging. ...
... We also suggest that ObL cells are heterogeneous with respect to expression of non-osteoblastic phenotypic traits, and capacity for alternative fate choices with at least some maintaining capacity for fate switches even at relatively late osteoblast differentiation stages. As described [1,6,7,8,9,11], bone marrow cell models in vivo and in vitro including ours [18], all support a model in which adipocytes form at the expense of osteoblasts, which may be processed at least in part by the PPARc-mediated downregulation of Runx2 [16,28]. Only a few studies have addressed adipogenic potential in the calvaria cell model. ...
Background:
Understanding fate choice and fate switching between the osteoblast lineage (ObL) and adipocyte lineage (AdL) is important to understand both the developmental inter-relationships between osteoblasts and adipocytes and the impact of changes in fate allocation between the two lineages in normal aging and certain diseases. The goal of this study was to determine when during lineage progression ObL cells are susceptible to an AdL fate switch by activation of endogenous peroxisome proliferator-activated receptor (PPAR)gamma.
Methodology/principal findings:
Multiple rat calvaria cells within the ObL developmental hierarchy were isolated by either fractionation on the basis of expression of alkaline phosphatase or retrospective identification of single cell-derived colonies, and treated with BRL-49653 (BRL), a synthetic ligand for PPARgamma. About 30% of the total single cell-derived colonies expressed adipogenic potential (defined cytochemically) when BRL was present. Profiling of ObL and AdL markers by qRT-PCR on amplified cRNA from over 160 colonies revealed that BRL-dependent adipogenic potential correlated with endogenous PPARgamma mRNA levels. Unexpectedly, a significant subset of relatively mature ObL cells exhibited osteo-adipogenic bipotentiality. Western blotting and immunocytochemistry confirmed that ObL cells co-expressed multiple mesenchymal lineage determinants (runt-related transcription factor 2 (Runx2), PPARgamma, Sox9 and MyoD which localized in the cytoplasm initially, and only Runx2 translocated to the nucleus during ObL progression. Notably, however, some cells exhibited both PPARgamma and Runx2 nuclear labeling with concomitant upregulation of expression of their target genes with BRL treatment.
Conclusions/significance:
We conclude that not only immature but a subset of relatively mature ObL cells characterized by relatively high levels of endogenous PPARgamma expression can be switched to the AdL. The fact that some ObL cells maintain capacity for adipogenic fate selection even at relatively mature developmental stages implies an unexpected plasticity with important implications in normal and pathological bone development.
... IL-11 was identified as an adipogenesis inhibitor factor early since 1990s [79][80][81][82]. In in vitro study, it significantly inhibited lipoprotein lipase activity and adipogenesis in 3T3-L1 cells. ...
Interleukin-(IL)-11 is a cytokine involved in hematopoiesis, cancer metastasis, and inflammation. IL-11 belongs to the IL-6 cytokine family, binding to the complex of receptors glycoprotein gp130 and the ligand-specific-receptor subunits (IL-11Rα or their soluble counterpart sIL-11R). IL-11/IL-11R signaling enhances osteoblast differentiation and bone formation and mitigates osteoclast-induced bone resorption and cancer bone metastasis. Recent studies have shown that systemic and osteoblast/osteocyte-specific IL-11 deficiency leads to reduced bone mass and formation, but also adiposity, glucose intolerance, and insulin resistance. In humans, mutations of IL-11 and the receptor IL-11RA genes are associated with height reduction, osteoarthritis, and craniosynostosis. In this review, we describe the emerging role of IL-11/IL-11R signaling in bone metabolism by targeting osteoblasts, osteoclasts, osteocytes, and bone mineralization. Furthermore, IL-11 promotes osteogenesis and suppresses adipogenesis, thereby influencing the fate of osteoblast/adipocyte differentiation derived from pluripotent mesenchymal stem cells. We have newly identified IL-11 as a bone-derived cytokine that regulates bone metabolism and the link between bone and other organs. Thus, IL-11 is vital in bone homeostasis and could be considered a potential therapeutic strategy.
... The cascade of the IL-11 stimulation to osteoblast differentiation is modulated by other factors. Parathyroid hormone acts as a positive hormone, while glucocorticoid and aging are negative factors 14,15 . Those past ndings look favorable in the effect of IL-11 on bone formation, but the in vivo role of IL-11 in regulating osteoblast differentiation and bone formation remains elusive. ...
Exercise offers mechanical loading to the bone, while it stimulates energy expenditure in the adipose tissue. Thus, bone may secrete a factor to communicate with adipose tissue in response to mechanical loading. Interleukin (IL)-11 is expressed in the bone, upregulated by mechanical loading, enhances osteogenesis and suppresses adipogenesis. Systemic IL-11 deletion (IL-11 −/− ) exhibited reduced bone mass, suppressed bone formation response to mechanical loading, enhanced expression of Wnt inhibitors, and suppressed Wnt signaling. Enhancement of bone resorption under mechanical unloading was unaffected. Unexpectedly, IL-11 −/− mice showed increased systemic adiposity and glucose intolerance. Osteoblast/osteocyte-specific IL-11 deletion in osteocalcin-Cre;IL-11 fl/fl mice showed reduced serum IL-11, blunted bone formation under mechanical loading, and increased systemic adiposity similar to IL-11 −/− mice. Adipocyte-specific IL-11 deletion in adiponectin-Cre; IL-11 fl/fl mice exhibited no abnormality. Thus, IL-11 from osteoblast/osteocyte controls both osteogenesis and systemic adiposity in response to mechanical loading. These findings may bring new therapeutic approaches to osteoporosis and metabolic syndrome.
... Verma et al 39 have demonstrated that the bone marrow adipocytes in osteoporosis patients can be significantly increased, indicating that a decrease in osteoblasts is usually accompanied by an increase in adipocytes. Similarly, in a mouse model of aging, bone formation can be significantly reduced when adipocytes in the bone marrow are increased 40 . Moreover, GLP-1 was found to directly interact with osteoblasts to affect its Osteoblast differentiation is regulated by a variety of signaling proteins and transcription factors. ...
Objective:
This study aims to investigate whether liraglutide can affect proliferation, osteogenic differentiation and serum deprivation-induced apoptosis of preosteoblast cell line MC3T3-E1 through the Notch, Wnt/β-catenin, and Hedgehog (Hh) signaling pathways.
Materials and methods:
MC3T3-E1 cells were exposed to different treatments (via Notch inhibitor DAPT, an Hh inhibitor cyclopamine, or serum deprivation) or transfections of different siRNAs (targeting glucagon-like peptide-1 receptor (GLP-1R), β-catenin, or Gli1) in the presence or absence of 100 nM liraglutide. Cell proliferation, mRNA levels of osteogenic differentiation-related genes, mRNA and protein levels of the Notch and Hh signaling pathway proteins, and apoptosis-related proteins were assessed.
Results:
Liraglutide significantly increased proliferation of MC3T3-E1 cells, expression levels of the Notch and Hh signaling pathway proteins and β-catenin, and mRNA levels of osteogenic differentiation-related genes and TCF7L2. Moreover, liraglutide promoted a translocation of β-catenin, increased a ratio of Bcl-2/Bax proteins, reduced serum deprivation-induced apoptosis of MC3T3-E1 cells, and a ratio of caspase-3/procaspase-3. However, a cotreatment with liraglutide and DAPT reversed the alterations. A cyclopamine treatment and knockdowns of GLP-1R, Gli1, and β-catenin significantly reduced the expression of Notch proteins. Furthermore, the knockdown of GLP-1R, β-catenin, or Gli1 significantly increased apoptosis, which could be inhibited by liraglutide.
Conclusions:
In summary, liraglutide can promote proliferation and differentiation of MC3T3-E1 cells, and inhibit their serum deprivation-induced apoptosis by activating both the Notch and Hh signaling pathways involving β-catenin and Gli1. These results provide a therapeutic foundation that patients with diabetes and osteoporosis may be cured with treatments of liraglutide.
... Senile osteoporosis is caused by aging and affects both men and women. The evidences showed that (1) senescence of bone marrow stromal cells (BMSCs) was more likely to differentiate into adipocytes rather than osteoblasts [117,118]; ...
During the aging process, senescent cells gradually accumulate in the organs; they secrete proinflammatory cytokines and other factors, collectively known as the senescence-associated secretory phenotype (SASP). SASP secretions contribute to “inflammaging,” which is a state of chronic, systemic, sterility, low-grade inflammatory microenvironment and a key risk factor in the development of aging-related diseases. Fructus psoraleae is a traditional Chinese medical herb best known for delaying aging and treating osteoporosis. Prenylflavonoids from fructus psoraleae are the main bioactive compounds responsible for its pharmacological applications, such as beaching, bavachinin, bavachalcone, isobavachalcone, and neobavaisoflavone. In previous decades, there have been some promising studies on the pharmacology of fructus psoraleae. Here, we focus on the anti-inflammatory and antiaging diseases of five psoralea prenylflavonoids, such as cardiovascular protection, diabetes and obesity intervention, neuroprotection, and osteoporosis, and discuss the mechanism of these active ingredients for better understanding the material basis and drug application of fructus psoraleae in Chinese medicine.
... (221,222) Indeed, mounting data in both animals and humans suggest that bone is progressively replaced with adipose tissue with advancing chronological age, resulting in a reciprocal relationship between marrow adiposity and skeletal parameters such as bone density and strength. (223)(224)(225) This is also a consistent observation inmurine modelsof accelerated aging, suchastheP6 strain of senescence-accelerated mice (SAMP6) (226) and Hdac null mice. (227) Although the underlying mechanisms explaining this phenomenon are not completely understood, it has been hypothesized that age-related related changes in transcription factors result in altered levels of several adipokines in the marrow milieu that cause a shift in lineage commitment of bone marrow multipotent MSCs from the osteoblast to the adipogenic lineage. ...
Aging research has undergone unprecedented advances at an accelerating rate in recent years, leading to excitement in the field as well as opportunities for imagination and innovation. Novel insights indicate that, rather than resulting from a pre-programmed series of events, the aging process is predominantly driven by fundamental non-adaptive mechanisms that are interconnected, linked, and overlap. To varying degrees, these mechanisms also manifest with aging in bone where they cause skeletal fragility. Because these mechanisms of aging can be manipulated, it might be possible to slow, delay, or alleviate multiple age-related diseases and their complications by targeting conserved genetic signaling pathways, controlled functional networks, and basic biochemical processes. Indeed, findings in various mammalian species suggest that targeting fundamental aging mechanisms (e.g., via either loss- or gain-of-function mutations or administration of pharmacological therapies) can extend healthspan - i.e., the healthy period of life free of chronic diseases. In this review, we summarize the evidence supporting the role of the spectrum of fundamental basic science discoveries contributing to organismal aging, with emphasis on mammalian studies and in particular aging mechanisms in bone that drive skeletal fragility. These mechanisms or aging hallmarks include: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. Because these mechanisms are linked, interventions that ameliorate one hallmark can in theory ameliorate others. In the field of bone and mineral research, current challenges include defining the relative contributions of each aging hallmark to the natural skeletal aging process, better understanding the complex interconnections among the hallmarks, and identifying the most effective therapeutic strategies to safely target multiple hallmarks. Based on their interconnections, it may be feasible to simultaneously interfere with several fundamental aging mechanisms to alleviate a wide spectrum of age-related chronic diseases, including osteoporosis. This article is protected by copyright. All rights reserved.
... Fibroblast growth factor 2 (FGF2) -expressed by MSC, osteoblasts and osteocytes -also exerts pro-osteogenic and anti-adipogenic effects: Fgf2-deficient mice exhibit increased BM adiposity and decreased BMD (107, 108); conversely, FGF2 treatment abrogates the loss in bone formation (109) and reduces the adipogenic lineage potential of murine BM-MSC (110). The pro-osteogenic factor oncostatin produced by osteoblasts and osteocytes also has anti-adipogenic property on differentiating BM-MSCs (111). ...
Bone marrow adipocytes (BMA) constitute an original and heterogeneous fat depot whose development appears interlinked with bone status throughout life. The gradual replacement of the hematopoietic tissue by BMA arises in a well-ordered way during childhood and adolescence concomitantly to bone growth and continues at a slower rate throughout the adult life. Importantly, BM adiposity quantity is found well associated with BMD (Bone Mineral Density) loss at different skeletal sites in primary osteoporosis such as in ageing or menopause but also in secondary osteoporosis consecutive to anorexia nervosa. Since BMA and osteoblasts originate from a common mesenchymal stem cell, adipogenesis is considered as a competitive process that disrupts osteoblastogenesis. Besides, most factors secreted by bone and bone marrow cells (ligands and antagonists of the Wnt/β-catenin pathway, BMP and others) reciprocally regulate the two processes. Hormones such as oestrogens, glucocorticoids, parathyroid and growth hormones that control bone remodelling also modulate the differentiation and the activity of BMA. Actually, BMA could also contribute to bone loss through the release of paracrine factors altering osteoblast and /or osteoclast formation and function. Based on clinical and fundamental studies, this review aims at presenting and discussing these current arguments that support but also challenge the involvement of BMA in the bone mass integrity.
... As an inside factor, Runx2, a key transcription factor for osteoblast differentiation, is the most probable candidate, since the expression is reported to be suppressed during cellular ageing of osteoblasts [9]; however, there is no in vivo evidence of its contribution to age-related bone loss. The local environment outside osteoblasts is mainly controlled by cytokines/growth factors such as insulin-like growth factor-I (IGF-I) [10][11][12], interleukin-11 [13], transforming growth factor-β [14] and bone morphogenetic proteins [15]. ...
... This indicated that reduction of osteoblasts is often accompanied by an increase in adipocyte ratio. Similarly, in the ageing mouse model, when fat cells in bone marrow increased, bone formation decreased significantly [61]. Therefore, the direction of differentiation of BMSCs plays a crucial role in the pathogenesis of osteoporosis [62][63][64]. ...
This review summarizes current knowledge about glucagon-like peptide 1 receptor agonists (GLP-1 RA) and their effects on bone metabolism and fracture risk. Recent in vivo and in vitro experiments indicated that GLP-1 RA could improve bone metabolism. GLP-1 could affect fat-bone axis by promoting osteogenic differentiation and inhibiting adipogenic differentiation of bone mesenchymal precursor cells (BMSCs), which express GLP-1 receptor. GLP-1 RA may also influence the balance between osteoclasts and osteoblast, thus lead to more bone formation and less bone resorption. Wnt/β-catenin signaling is involved in this course. Mature osteocytes, which also express GLP-1 receptor, produce sclerostin which inhibits Wnt/β-catenin signaling by binding to low-density lipoprotein receptor-related protein (LRP) 5 and preventing the binding of Wnt. GLP-1 RA also decreases the expression of SOST/sclerostin and circulating levels of sclerostin. In addition, GLP-1 receptors are expressed in thyroid C cells, where GLP-1 induces calcitonin release and thus indirectly inhibits bone resorption. Furthermore, GLP-1 RA influences the osteoprotegerin(OPG)/receptor activator of nuclear factor-κB ligand (RANKL) /receptor activator of nuclear factor-κB (RANK) system by increasing OPG gene expression, thus reverses the decreased bone mass in rats models. However, a recent Meta-analysis and a cohort study did not show a significant relationship between GLP-RA use and fracture risk. Future clinical trials will be necessary to deeply investigate the relationship between GLP-1 RA use and fracture risk in diabetic patients.
... In vitro, IL-11 stimulates osteoblast differentiation of mouse mesenchymal progenitors (Taguchi et al. 1998, Suga et al. 2001. In vivo, IL-11 administration is able to recover impaired osteoblastogenesis and osteoclastogenesis seen in the P6 strain of senescence-accelerated mice (Kodama et al. 1998). Transgenic mice overexpressing IL-11 show increased cortical thickness and are protected from age-related cortical bone loss, without any apparent alteration of osteoclastogenesis and bone resorption (Takeuchi et al. 2002). ...
It has recently been suggested that interleukin (IL)-11 plays a role in the pathogenesis of glucocorticoid (GC)-induced osteoporosis. IL-11 belongs to the gp130 cytokine family, which includes also IL-6. We have previously investigated GC-IL-6 interplay, showing that GC inhibits IL-6 release and IL-6 up-regulates GC receptor (GR) numbers in the human osteoblast-like cell lines Saos-2 and MG-63, which constitutively have an opposite pattern of expression for GR, IL-11, IL-6, alkaline phosphatase and osteoprotegerin (OPG). The aim of this study was to investigate GC-IL-11 interplay in the same two cell lines. First, cells were incubated with cortisol (0.01-1 microM) for 20 h in the presence and in the absence of a known IL-11 secretagogue (IL-1beta); cell media were assayed for IL-11 by ELISA. Secondly, cells were incubated with IL-11 (0.1-100 ng/ml) or specific anti-IL-11 monoclonal antibody for 20 h, and then assayed for GR by a radioligand binding assay. Similar to IL-6, both constitutive and IL-1beta-inducible IL-11 release were dose-dependently inhibited by cortisol (P<0.01); at variance with IL-6, exogenous IL-11 dose-dependently decreased GR numbers in MG-63 cells (P<0.05), while anti-IL-11 antibody significantly increased GR numbers in both cell lines (P<0.05). IL-11-induced reduction of GR in MG-63 cells was confirmed by Western blot analysis. While exerting opposite effects on GR numbers, neither IL-6 nor IL-11 significantly modified GC-dependent inhibition of OPG release. Our data indicate that even physiological concentrations of cortisol negatively modulate IL-11 secretion and demonstrate, for the first time, an inhibitory effect of the cytokine on GR. Thus, the concept of autocrine-paracrine loops that modulate GC action and involve gp130 cytokines is corroborated. These loops could have clinical relevance for the dynamics of bone loss in patients given GC and having high concentrations of these cytokines in the bone microenvironment.
... In SAMP6, reduced cortical and trabecular volumes and fragmented trabeculae were observed in the mandible as well as in the femur. The femoral findings in the present study are supported by a number of previous studies that with that of SAMR1, consistent with previous findings concerning this bone of SAMP6 in which osteoblastogenesis was disturbed 29) but adipogenesis and myelopoiesis were accelerated 30,31) . In the mandible of this strain, however, the number of osteoblasts was not reduced as was observed in the femur. ...
Histomorphometric analyses of the mandible and femur were performed in senescence-accelerated mice (SAM) using SAMP6 as a senile osteoporosis (OP) model, SAMP8 as a temporomandibular joint osteoarthritis (TMJOA) model, and SAMR1 as control. Thirty-six male mice at 2 or 4 months of age (6 for each strain and age) were used. The cortical thickness index (CTI), bone area (B.Ar/T.Ar), trabecular width (Tb.Wi), trabecular number (Tb.N), trabecular separation (Tb.Sp), osteoblast perimeter (N.Ob/B.Pm), and osteoclast perimeter (N.Oc/B.Pm) were assessed in the distal femoral metaphysis and in the mandibular ramus including the alveolar bone. Compared with SAMR1, SAMP6 showed lower B.Ar/T.Ar and Tb.Wi in the femur and mandible at 2 and 4 months of age. This strain showed lower CTI in both bones and higher Tb.N and Tb.Sp in the femur at 4 months of age. SAMP8 showed higher CTI in both bones at 2 months of age and maintained a high index in the mandible but not in the femur at 4 months of age; however trabecular bone mass was not reduced. SAMP6 exhibited lower N.Ob/B.Pm in the femur but not in the mandible at 4 months of age, while SAMP8 showed lower N.Oc/B.Pm in the mandible but not in the femur at 2 and 4 months of age. The differential histomorphometric features of the mandible in different SAM strains may imply a difference in mandibular bone property between SAM mice with senile OP and TMJOA genetic background.
... It is notable that development of osteopenia in SAMP6 mice was prevented by transplantation of bone marrow from normal allogeneic mice [8]; this suggests that the skeletal defect may be due to dysregulated bone progenitor cells, their products, or other components of marrow. There has been some research comparing in vitro osteoblast potential in SAMP6 and SAMR1 marrow cells, with evidence for no differences, and for less, or for more with SAMP6 marrow depending on the bone and the age of the mice [9][10][11]. ...
The senescence accelerated-prone mouse variant 6 (SAMP6) shows normal growth followed by rapid aging, development of osteopenia, and shortened lifespan, compared with control R1 mice. Because oxidative stress is a fundamental mechanism of tissue aging, we tested whether cellular parameters that are associated with oxidative stress are impaired with marrow from SAMP6 mice. We compared in vitro hematopoiesis, irradiation sensitivity, proliferative potential, and osteoblastogenesis with marrow cells from SAMP6 and R1 mice. Marrow cells from SAMP6 mice showed shortened in vitro hematopoiesis; their stromal cells showed greater radiation sensitivity and decreased proliferation. Consistent with those properties, there was constitutive upregulation of transforming growth factor-β(1), an inhibitor of hematopoiesis, and of cell cycle inhibitory genes, p16(INK4A) and p19(ARF). Paradoxically, there was constitutive expression of osteoblast genes in stromal cells from SAMP6 mice, but in vitro matrix mineralization was impaired. These studies and data included in other reports indicate that impaired proliferation of osteoblast progenitors in SAMP6 marrow may be a major factor contributing to accelerated loss of bone mass. In sum, marrow from SAMP6 mice had diminished capacity for long-term hematopoiesis, increased radiosensitivity, and reduced proliferative capacity.
... A connection between agerelated bone loss due to low bone remodeling osteoporosis was associated with significant increases in marrow fat infiltration, using the inbred albino Louvain rats, as a model of healthy aging (66). The senescence accelerated mouse-P6 also demonstrated low bone turnover osteopenia due to decreased osteoblastogenesis with a parallel increase in adipogenesis, a phenotype that may result from reduced expression of the gp130-cytokine interleukin-11 (IL-11) (67)(68)(69). In the present study, Zfp467 mRNA expression was not altered by IL-11 treatment (data not shown), and IL-11 receptor knock-out mice do not demonstrate reciprocal adipocyte and osteoblast differentiation (70). ...
Osteoblasts and adipocytes are derived from common mesenchymal progenitor cells. The bone loss of osteoporosis is associated
with altered progenitor differentiation from an osteoblastic to an adipocytic lineage. cDNA microarrays and quantitative real-time
PCR (Q-PCR) were carried out in a differentiating mouse stromal osteoblastic cell line, Kusa 4b10, to identify gene targets
of factors that stimulate osteoblast differentiation including parathyroid hormone (PTH) and gp130-binding cytokines, oncostatin
M (OSM) and cardiotrophin-1 (CT-1). Zinc finger protein 467 (Zfp467) was rapidly down-regulated by PTH, OSM, and CT-1. Retroviral overexpression and RNA interference for Zfp467 in mouse stromal cells showed that this factor stimulated adipocyte formation and inhibited osteoblast commitment compared
with controls. Regulation of adipocyte markers, including peroxisome proliferator-activated receptor (PPAR) γ, C/EBPα, adiponectin, and resistin, and late osteoblast/osteocyte markers (osteocalcin and sclerostin) by Zfp467 was confirmed by Q-PCR. Intra-tibial injection of calvarial cells transduced with retroviral Zfp467 doubled the
number of marrow adipocytes in C57Bl/6 mice compared with vector control-transduced cells, providing in vivo confirmation of a pro-adipogenic role of Zfp467. Furthermore, Zfp467 transactivated a PPAR-response element reporter construct and recruited a histone deacetylase complex. Thus Zfp467 is a novel co-factor that promotes
adipocyte differentiation and suppresses osteoblast differentiation. This has relevance to therapeutic interventions in osteoporosis,
including PTH-based therapies currently available, and may be of relevance for the use of adipose-derived stem cells for tissue
engineering.
... We have previously demonstrated that phosphorylated CREB binds to fosB gene promoter, causing an enhancement of fosB gene transcription and an increase in DFosB expression [14], and that DFosB forms a heterodimer with JunD on Interleukin (IL)- 11 gene promoter to enhance IL-11 expression [13]. The expression of IL-11 in osteoblastic cells is reduced by mechanical unloading [13] and aging [15], and is enhanced by mechanical loading [13]. Furthermore, transgenic mice overexpressing IL-11 show high bone mass with continued increase of bone mineral density with aging due to an enhanced bone formation without an increase in bone resorption [16]. ...
Mechanical stress rapidly induces ΔFosB expression in osteoblasts, which binds to interleukin (IL)-11 gene promoter to enhance IL-11 expression, and IL-11 enhances osteoblast differentiation. Because bone morphogenetic proteins (BMPs) also stimulate IL-11 expression in osteoblasts, there is a possibility that BMP-Smad signaling is involved in the enhancement of osteoblast differentiation by mechanical stress. The present study was undertaken to clarify whether mechanical stress affects BMP-Smad signaling, and if so, to elucidate the role of Smad signaling in mechanical stress-induced enhancement of IL-11 gene transcription.
Mechanical loading by fluid shear stress (FSS) induced phosphorylation of BMP-specific receptor-regulated Smads (BR-Smads), Smad1/5, in murine primary osteoblasts (mPOBs). FSS rapidly phosphorylated Y311 of protein kinase C (PKC)δ, and phosphorylated PKCδ interacted with BR-Smads to phosphorylate BR-Smads. Transfection of PKCδ siRNA or Y311F mutant PKCδ abrogated BR-Smads phosphorylation and suppressed IL-11 gene transcription enhanced by FSS. Activated BR-Smads bound to the Smad-binding element (SBE) of IL-11 gene promoter and formed complex with ΔFosB/JunD heterodimer via binding to the C-terminal region of JunD. Site-directed mutagenesis in the SBE and the AP-1 site revealed that both SBE and AP-1 sites were required for full activation of IL-11 gene promoter by FSS.
These results demonstrate that PKCδ-BR-Smads pathway plays an important role in the intracellular signaling in response to mechanical stress, and that a cross-talk between PKCδ-BR-Smads and ΔFosB/JunD pathways synergistically stimulates IL-11 gene transcription in response to mechanical stress.
... A large body of in vitro and animal literature indicates that, depending on the interplay of molecular, biochemical, and physical stimuli, mesenchymal stem cells (MSCs) differ-entiate into the cell lineages responsible for bone and fat formation through alternative activation of mutually exclusive transcriptional programs (1)(2)(3)(4)(5). Multiple basic studies have examined the roles of hormones and their receptors (6 -14), cytokines and other serum factors (15)(16)(17)(18)(19), and mechanical stimuli (20,21) in the commitment of MSCs isolated from bone marrow stroma toward adipocytic or osteoblastic lineages. ...
Considerable evidence indicates that osteoblasts and adipocytes share a common progenitor cell in the bone marrow that is capable of mutually exclusive differentiation into the cell lineages responsible for bone and fat formation.
The purpose of this study was to examine the relation between bone acquisition and changes in marrow adiposity.
This was a longitudinal study. OUTCOME MEASURES AND SUBJECTS: Computed tomography measurements of femoral cortical bone area (CBA), cross-sectional area (CSA), and marrow density, and dual-energy x-ray absorptiometry (DXA) measurements of total body fat and lean mass (LM) were obtained in 39 healthy females (15-20 yr of age) at baseline and 18-24 months later.
Marrow adiposity was inversely related to CBA at baseline and follow-up (r = 0.39 and 0.33; P = 0.015 and 0.039, respectively) but was not associated to CSA (r = 0.19 and 0.17; P = 0.24 and 0.32, respectively). The association between marrow fat and CBA persisted, even after controlling for body mass and DXA values of LM and femoral CSA. Gains in CBA during the course of the study were related to decreases in marrow fat (r = 0.41; P = 0.009), a relation that persisted, even after accounting for changes in bone size. Marrow fat was not associated to anthropometric measures or DXA values of body fat and LM (all r's between -0.15 and 0.19; P > 0.05).
Bone acquisition in the appendicular skeleton of healthy young females is inversely related to changes in marrow adiposity. These results provide support for the growing body of evidence indicating an inversely coupled relationship between osteogenesis and adipogenesis in the skeleton.
... The characteristics of osteoporosis in SAMP6 are low peak bone mass during the maturation period and early onset of bone marrow adipogenesis. Reduced expression of interleukin 11 (IL-11), which is an inhibitory factor for adipogenesis, in femoral bone marrow cells was reported as a cause of bone marrow adipogenesis in SAMP6 mice (21). On the other hand, PTH induced the expression of IL-11 via a protein kinase A (PKA) pathway in human bone marrow cells (22), and as such, it was considered that intermittent PTH treatment would promote ...
We examined trabecular and cortical bone in the senescence-accelerated mouse prone 6 (SAMP6) murine model of senile osteoporosis after treatment with human PTH 1-34. Sixteen-week-old female SAMP6 mice were assigned to control and PTH groups. PTH (20 microg/kg) was administered sc 3 times a week for 12 weeks. The control mouse strain, senescence-accelerated mouse resistant 1 (SAMR1), was used for comparison. The femoral metaphysis and diaphysis were used to measure bone mineral density (BMD), analyze the trabecular and the cortical structure by micro-computed tomography, and for conducting the bone strength test. PTH significantly attenuated the loss of BMD, improved the trabecular bone microstructure, and increased the bone strength in the femoral metaphysis. We did not find any differences in the bone strength of the femoral diaphysis after PTH treatment, although the cortical bone volume and cortical thickness were improved. Although the cortical thickness increased, the cortical bone density decreased, likely because of the increase of cortical porosity in the distal metaphysis after administration of PTH.
... However, PPARg insufficiency leads to high trabecular bone volume and low body fat (5). Senescence-accelerated mice also have increased body fat and decreased bone mass (6,7). ...
Obesity induces a low-grade systemic chronic inflammatory condition for which macrophages are responsible. We hypothesized that obesity affects osteoclastogenesis by acting on bone marrow-derived macrophages (BMM). Male mice were fed a high-fat diet (45% of energy) or a standard diet (10% of energy) for 13 wk. We found that the density of the femurs of obese mice was significantly lower than that of the femurs of lean mice. Osteoclastogenesis was enhanced in the BMM from obese mice. Lower levels of interleukin (IL)-10 were generated by the BMM from obese mice than by those from lean mice upon stimulation of receptor activator of nuclear factor-kappaB ligand. Neutralization of IL-10 in the BMM from obese mice was not as effective in increasing osteoclast (OC) formation as that in those from lean mice. Exogenous IL-10 inhibited OC formation more strongly in the BMM from obese mice than those from lean mice. The elevated level of OC formation in the BMM from obese mice may thus be due to in part to the lower level of IL-10, a negative regulator of osteoclastogenesis. Our results suggest that obesity is associated with bone loss via enhanced osteoclastogenesis due to reduced IL-10 production by the BMM from obese mice.
... Cultures were fed every 3 days by replacing 0.4 ml old medium with fresh medium. After culture of 8–10 days, cells were fi xed in 10% formalin neutral buffer solution for 10 min and stained for tartrate-resistant acid phosphatase (TRACP) [17,18] to confi rm the formation of osteoclast-like cells. A preosteoclastic MLC-7 cell line, expressing fully differentiated osteoclast markers, such as calcitonin receptors, vitronectin receptors, TRACP, and vacuolar H + -ATPase without losing undifferentiated osteoclast precursor cell phenotypes [19], was maintained in McCoy's 5A medium supplemented with 20% FCS. ...
Silicon has been known as an essential element for bone formation. The silicon contents of sea water increase with increasing of depth: 1.8 ppm Si in deep-sea water (DW) at 612 m in depth versus 0.06 ppm in surface sea water (SW). The effects of soluble silicon (Si) and DW from which NaCl was eliminated were studied in comparison with tap water (TW) and SW in cell cultures and in animal experiments using the control strain of senescence accelerated mouse, SAMR1. Si at 10 ppm as sodium metasilicate or 10% DW in the alpha-MEM medium stimulated cellular viability, marker enzymes of osteoblast and osteoclast cell lines, and the (45)CaCl(2) uptake in those cells in comparison with the medium control. After weanling SAMR1 were maintained for 6 months on a diet containing 200 ppm Si and 39% of DW and SW, DW and Si improved bone biochemical indices such as femoral weight, mineral and collagen content, and marker enzymes of bone formation and resorption as well as mechanical properties as compared to TW. In the femoral bone marrow of SAMR1, the mRNA expression of bone morphogenetic protein-2 (BMP-2), interleukin-11 (IL-11), and runt-related transcription factor 2 (Runx 2), which stimulate osteoblast development as well as type I procollagen (COL1A1) mRNA, were significantly increased in both DW and Si groups. The expressions of both osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) were also elevated, resulting in distinct increases of the OPG/RANKL ratio in both DW and Si groups. The results indicated that a soluble silicate and deep-sea water as its natural material stimulated cell growth in both osteoblasts and osteoclasts in cell culture and promoted bone metabolic turnover in favor of bone formation through stimulation of the related mRNA expression in animal experiments.
Bone metabolism is the basis for maintaining the normal physiological state of bone, and imbalance of bone metabolism can lead to a series of metabolic bone diseases. As a member of the IL-6 family, IL-11 acts primarily through the classical signaling pathway IL-11/Receptors, IL-11 (IL-11R)/Glycoprotein 130 (gp130). The regulatory role of IL-11 in bone metabolism has been found earlier, but mainly focuses on the effects on osteogenesis and osteoclasis. In recent years, more studies have focused on IL-11’s roles and related mechanisms in different bone metabolism activities. IL-11 regulates osteoblasts, osteoclasts, BM stromal cells, adipose tissue-derived mesenchymal stem cells, and chondrocytes. It’s involved in bone homeostasis, including osteogenesis, osteolysis, bone marrow (BM) hematopoiesis, BM adipogenesis, and bone metastasis. This review exams IL-11’s role in pathology and bone tissue, the cytokines and pathways that regulate IL-11 expression, and the feedback regulations of these pathways.
Exercise results in mechanical loading of the bone and stimulates energy expenditure in the adipose tissue. It is therefore likely that the bone secretes factors to communicate with adipose tissue in response to mechanical loading. Interleukin (IL)−11 is known to be expressed in the bone, it is upregulated by mechanical loading, enhances osteogenesis and suppresses adipogenesis. Here, we show that systemic IL-11 deletion (IL-11−/−) results in reduced bone mass, suppressed bone formation response to mechanical loading, enhanced expression of Wnt inhibitors, and suppressed Wnt signaling. At the same time, the enhancement of bone resorption by mechanical unloading was unaffected. Unexpectedly, IL-11−/− mice have increased systemic adiposity and glucose intolerance. Osteoblast/osteocyte-specific IL-11 deletion in osteocalcin-Cre;IL-11fl/fl mice have reduced serum IL-11 levels, blunted bone formation under mechanical loading, and increased systemic adiposity similar to IL-11−/− mice. Adipocyte-specific IL-11 deletion in adiponectin-Cre;IL-11fl/fl did not exhibit any abnormalities. We demonstrate that osteoblast/osteocyte-derived IL-11 controls both osteogenesis and systemic adiposity in response to mechanical loading, an important insight for our understanding of osteoporosis and metabolic syndromes.
Aging is a natural physiological process that features various and variable challenges, associated with loss of homeostasis within the organism, often leading to negative consequences for health. Cellular senescence occurs when cells exhaust the capacity to renew themselves and their tissue environment as the cell cycle comes to a halt. This process is influenced by genetics, metabolism and extrinsic factors. Immunosenescence, the aging of the immune system, is a result of the aging process, but can also in turn act as a secondary inducer of senescence within other tissues. This review aims to summarize the current state of knowledge regarding hallmarks of aging in relation to immunosenescence, with a focus on aging-related imbalances in the medullary environment, as well as the components of the innate and adaptive immune responses. Aging within the immune system alters its functionality, and has consequences for the person's ability to fight infections, as well as for susceptibility to chronic diseases such as cancer and cardiovascular disease. The senescence-associated secretory phenotype is described, as well as the involvement of this phenomenon in the paracrine induction of senescence in otherwise healthy cells. Inflammaging is discussed in detail, along with the comorbidities associated with this process. A knowledge of these processes is required in order to consider possible targets for the application of senotherapeutic agents - interventions with the potential to modulate the senescence process, thus prolonging the healthy lifespan of the immune system and minimizing the secondary effects of immunosenescence.
Senile osteoporosis increases the risk of skeletal fractures with age. Cheonggukjang (CGJ), a traditional Korean dry fermented soybean product, has numerous therapeutic effects; however, its effects on bone mineral density (BMD) and bone metabolism in senile osteoporosis are unclear. In this study, we treated the senescence-accelerated mouse prone 6 (SAMP6) model of senile osteoporosis with CGJ to determine its potential for ameliorating and preventing osteoporosis progression. High-performance liquid chromatography analysis for isoflavone profiles revealed that short-term fermentation significantly increased the isoflavone aglycone content in soybeans. Thereafter, we fed 6-week-old SAMP6 mice with experimental diets containing 5% or 10% CGJ for 15 weeks. Microcomputed tomography revealed that CGJ supplementation effectively increased the BMD and relative bone length. In vitro, CGJ increased the osteopontin reactivity and upregulated the expression of Alp, Col1a1, Fak, Bmp2/4, Smad1/5/8, and Runx2 in osteoblasts, and decreased Cathepsin K reactivity and downregulated Rankl and Nfatc1 expression in osteoclasts. In addition, CGJ increased the osteoprotegerin/Rankl ratio. Collectively, these results demonstrate that CGJ can ameliorate the detrimental effects of senile osteoporosis by improving osteogenesis and decreasing osteoclast activity.
The SAMP6 mouse spontaneously develops osteoporosis early in life and is therefore a useful model for examining the mechanisms underlying osteoporosis. We have recently established a new bone marrow transplantation (BMT) method: the bone marrow cells (BMCs) of normal allogeneic mice are directly injected into the bone marrow cavity of irradiated recipients (IBM-BMT). Using IBM-BMT, we attempted to prevent osteoporosis in SAMP6 mice. The hematolymphoid system was completely reconstituted with donor-type cells after IBM-BMT. Thus-treated SAMP6 mice showed marked increases in trabecular bone even at 12 months of age, and the bone mineral density (BMD) remained similar to that of normal B6 mice. Urinary deoxypiridinoline (DPD) also remained continuously low until 10 months of age. In addition, bone marrow stromal cells in the treated SAMP6 mice were replaced by donor stromal cells. The message levels of both IL-11 (known as an important factor of bone remodeling) and IL-6 (known to enhance osteoclastogenesis) were restored to normal. These results indicate that the bone marrow microenvironment is normalized after IBM-BMT, and that the increased production of IL-11 and IL-6 ameliorates the imbalance between bone absorption and formation, resulting in the prevention of osteoporosis in SAMP6 mice.
Bone resorption and formation are complicated and highly regulated cellular processes. The circuitry of this regulation is not completely understood, but it is clear that one important aspect of this control is mediated by cytokines. Cytokines play an important role in the regulation of both osteoblast and osteoclast differentiation and function. With aging, bone mass is lost and this loss is associated with a significant increase in fractures and associated morbidity and mortality. Although complicated by specific pathologies like tumors and metabolic bone disease, age-related bone loss can be accounted for by at least two general mechanisms. First, the number of osteoblasts and their ability to make new bone appear to decrease with age, resulting in less new bone tissue. Second, the number of osteoclasts and their activity increase, resulting in accelerated bone loss. These two related mechanisms work in concert to weaken the skeleton. This chapter examines how cytokines regulate these two functions in the aging skeleton. Furthermore, it provides a summary of the effects of prostaglandins on bone cells and the regulation of endogenous prostaglandin production in bone.
Glucocorticoid medication is one of the most common causes of atraumatic osteonecrosis of the femoral head (ONFH), and vitamin K2 (VK2) has been shown to play an important and beneficial role in bone metabolism. In this study, we hypothesized that VK2 could decrease the incidence of glucocorticoid-induced ONFH in a rat model. Using in vitro studies, we investigated how bone marrow-derived stem cells in the presence of methylprednisolone proliferate and differentiate, specifically examining osteogenic-related proteins, including Runx2, alkaline phosphatase and osteocalcin. Using in vivo studies, we established glucocorticoid-induced ONFH in rats and investigated the preventive effect of VK2. We employed micro-CT scanning, angiography of the femoral head, and histological and immunohistochemical analyses, which demonstrated that VK2 yielded beneficial effects for subchondral bone trabecula. In conclusion, VK2 is an effective antagonist for glucocorticoid on osteogenic progenitors. The underlying mechanisms include acceleration of BMSC propagation and promotion of bone formation-associated protein expression, which combine and contribute to the prevention of glucocorticoid-induced ONFH in rats.
Mechanical loading to bone causes mainly two types of stress to bone forming cells, fluid shear stress (FSS) and tensile stress. FSS appears to be a major stress signal that activates osteoblastic/osteocytic intracellular signaling to enhance bone formation. FSS to osteoblasts causes Ca2+ influx by activating a stress-activated cation channel, which activates extracellular signal-regulated kinase (ERK) to the phosphorylate cyclic AMP response element-binding protein (CREB). Phosphorylated CREB binds to the fosB gene promoter to enhance its transcriptional activity, and upregulates the expression of mainly ΔFosB, a C-terminal truncated splice variant of FosB. The increased ΔFosB binds to the 5′AP-1 site on the IL-11 gene promoter and forms a complex with JunD. FSS to osteoblasts also stimulates tyrosine phosphorylation of protein kinase Cδ (PKCδ), which activates Smad1/5. Activated Smad1/5 is bound to the Smad-binding element of the IL-11 gene promoter, and forms a complex with ΔFosB/JunD heterodimer. Thus, Ca2+–ERK–CREB–ΔFosB and PKCδ-Smad1/5 pathways merge together on the IL-11 gene promoter, and the two pathways cooperatively stimulate IL-11 gene expression in response to mechanical stress. The increase in interleukin (IL)-11 then enhances Wnt/β-catenin signaling by suppressing the expression of its inhibitors, dickkopf 1 and 2, in osteoblasts. Thus, the enhanced IL-11 expression by the cooperative AP-1 and Smad1/5 pathways is the upstream signal for stimulation of the main osteogenic pathway, Wnt/β-catenin signaling.
In bone remodeling during orthodontic tooth movement, osteoblasts are closely related to bone matrix formation and osteoclast differentiation. Interleukin (IL)-11 is known to be a regulating factor of bone remodeling. Our previous study showed that compressive force (CF) induced IL-11 production in osteoblastic Saos-2 cells. Therefore, we examined the effect of CF on the expression of IL-11 receptor (IL-11r), IL-11, Runx2, bone morphogenetic protein (BMP)-2, receptor activator of NF-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) using MC3T3-E1 cells as osteoblasts. To confirm the participation of autocrine mechanisms by CF-induced IL-11, we also examined the effect of CF on expression in the presence and absence of IL-11 antagonist. MC3T3-E1 cells were cultured with or without continuous CF (1.0 or 2.0 g/ cm2) in the presence or absence of IL-11 antagonist for up to 24 h. The expression of the IL-11r, IL-11, Runx2, BMP-2, RANKL, M-CSF, and OPG genes was estimated by determining mRNA levels by real-time PCR; protein expression was examined by ELISA or Western blotting. CF increased the expression of IL-11r and IL-11; the maximum level of these expressions was achieved with CF at 2.0 g/cm2. CF also increased the expression of Runx2 and BMP-2; the maximum level of these expressions was achieved in CF at 1.0 g/cm2. IL-11 antagonist blocked the stimulatory effects of CF at 1.0 or 2.0 g/cm2 on the expression of Runx2, BMP-2, RANKL, and M-CSF but not on the expression of IL-11r, IL-11, and OPG. These results suggest that CF at 1.0 g/cm2 increases the expression of proteins related to bone formation, such as Runx2 and BMP-2, via the autocrine mechanism of CF-induced IL-11 in osteoblasts, whereas CF at 2.0 g/cm2 increases the expression of RANKL and M-CSF that promotes osteoclast formation via CF-induced IL-11. These results also suggest that the balance of bone remodeling changes according to the strength of CF.
Background and objective:
The periodontal ligament (PDL) is continually exposed to mechanical loading caused by mastication or occlusion. Physiological loading is thus considered a key regulator of PDL tissue homeostasis; however, it remains unclear how this occurs. We recently reported that an appropriate magnitude of mechanical stretch can maintain PDL tissue homeostasis via the renin-angiotensin system. In the present study, we investigated the expression of interleukin-11 (IL-11) in human primary PDL cells (HPDLCs) exposed to stretch loading, the contribution of angiotensin II (Ang II) to this event and the effects of IL-11 on osteoblastic/cementoblastic differentiation of human PDL progenitor cells (cell line 1-17).
Material and methods:
Human primary PDL cells, derived from human tissues, with or without antagonists against the Ang II receptors AT1 or AT2, were subjected to cyclical stretch loading with 8% elongation for 1 h. Expression of IL-11 was measured by ELISA in these cultures and by immunohistochemistry in the sectioned maxillae of rats. The osteoblastic/cementoblastic potential of cell line 1-17 was determined using cell proliferation, gene expression and Alizarin Red staining.
Results:
Positive staining for IL-11 was observed in the PDL of rat maxillae and in cultures of HPDLCs. In HPDLCs exposed to stretch, expression of the IL11 gene and the IL-11 protein were up-regulated, concomitant with an increase in Ang II and via AT2. Recombinant human IL-11 (rhIL-11) stimulated an increase in expression of mRNA for the cementoblast-specific marker, CP-23, and for the osteoblastic markers, osteopontin and bone sialoprotein, and promoted proliferation in cell line 1-17. In addition, rhIL-11 also increased the degree of mineralized nodule formation in cell line 1-17 cultures treated with CaCl2 .
Conclusion:
Mechanical loading appears to control proliferation and osteoblastic/cementoblastic differentiation of human PDL stem/progenitor cells through the regulation of Ang II and AT2 by IL-11.
Since a reduction of the insulin/IGF-1 signaling cascade extends life span in many species and IGF-1 signaling might partly mediate the effects of caloric restriction (CR), an experimental intervention for increasing longevity, the purpose of the present study was to use quantitative trait loci (QTL) analysis, an unbiased genetic approach, to identify particular regions of the genome influencing plasma IGF-1 levels in an F2 intercross between F344 and LOU/C rats; the latter being an inbred strain of Wistar origin, considered as a model of healthy aging since it resists to age (and diet)-induced obesity.
Histomorphometric analyses of the mandible and femur were performed in senescence-accelerated mice (SAM) using SAMP6 as a senile osteoporosis (OP) model, SAMP8 as a temporomandibular joint osteoarthritis (TMJOA) model, and SAMR1 as control. Thirty-six male mice at 2 or 4 months of age (6 for each strain and age) were used. The cortical thickness index (CTI), bone area (B.Ar/T.Ar), trabecular width (Tb.Wi), trabecular number (Tb.N), trabecular separation (Tb.Sp), osteoblast perimeter (N.Ob/B.Pm), and osteoclast perimeter (N.Oc/B.Pm) were assessed in the distal femoral metaphysis and in the mandibular ramus including the alveolar bone. Compared with SAMR1, SAMP6 showed lower B.Ar/T.Ar and Tb.Wi in the femur and mandible at 2 and 4 months of age. This strain showed lower CTI in both bones and higher Tb.N and Tb.Sp in the femur at 4 months of age. SAMP8 showed higher CTI in both bones at 2 months of age and maintained a high index in the mandible but not in the femur at 4 months of age; however trabecular bone mass was not reduced. SAMP6 exhibited lower N.Ob/B.Pm in the femur but not in the mandible at 4 months of age, while SAMP8 showed lower N.Oc/B.Pm in the mandible but not in the femur at 2 and 4 months of age. The differential histomorphometric features of the mandible in different SAM strains may imply a difference in mandibular bone property between SAM mice with senile OP and TMJOA genetic background.
IL-6 and IL-11 are two cytokines that increase osteoclast for- mation and augment bone resorption. PTH stimulates the pro- duction of both cytokines by human osteoblast-like cells. Cir- culating levels of IL-6 are elevated in patients with states of PTH excess and correlate strongly to markers of bone resorp- tion. In contrast, serum levels of IL-11 were significantly re- duced in patients with primary hyperparathyroidism com- pared with values in euparathyroid controls. Further, after successful parathyroid adenomectomy, circulating levels of IL-6 fell, whereas IL-11 levels increased. Five-day infusions of human PTH-(1- 84) in rodents resulted in a significant decline in mean circulating levels of IL-11, whereas IL-6 levels signif- icantly increased. Pretreatment of cells and mice with neu- tralizing serum to IL-6 enhanced PTH-induced IL-11 produc- tion compared with the effect of pretreatment with nonimmune sera. These data indicate that IL-6 negatively reg- ulates IL-11 production in vivo and in vitro. Analysis of steady state mRNA levels in SaOS-2 cells indicated that this effect is posttranscriptional. As both IL-6 and IL-11 stimulate oste- oclast formation, down-regulation of IL-11 by IL-6 may help modulate the resorptive response to PTH. (Endocrinology 142: 3850 -3856, 2001)
Glucocorticoid (GC) excess causes a rapid loss of bone with a reduction in bone formation. Intermittent PTH (1-34) administration stimulates bone formation and counteracts the inhibition of bone formation by GC excess. We have previously demonstrated that mechanical strain enhances interleukin (IL)-11 gene transcription by a rapid induction of ΔFosB expression and protein kinase C (PKC)-δ-mediated phosphorylation of phosphorylated mothers against decapentaplegic (Smad)-1. Because IL-11 suppresses the expression of dickkopf-1 and -2 and stimulates Wnt signaling, IL-11 appears to mediate at least a part of the effect of mechanical strain on osteoblast differentiation and bone formation. The present study was undertaken to examine the effect of PTH(1-34) and GCs on IL-11 expression in murine primary osteoblasts (mPOBs). PTH(1-34) treatment of mPOBs enhanced IL-11 expression in a time- and dose-dependent manner. PTH(1-34) also stimulated ΔFosB expression and Smad1 phosphorylation, which cooperatively stimulated IL-11 gene transcription. PTH(1-34)-induced Smad1 phosphorylation was mediated via PKCδ and was abrogated in mPOBs from PKCδ knockout mice. Dexamethasone suppressed IL-11 gene transcription enhanced by PTH(1-34) without affecting ΔFosB expression or Smad1 phosphorylation, and dexamethasone-GC receptor complex was bound to JunD, which forms heterodimers with ΔFosB. High doses of PTH(1-34) counteracted the effect of dexamethasone on apoptosis of mPOBs, which was blunted by neutralizing anti-IL-11 antibody or IL-11 small interfering RNA. These results demonstrate that PTH(1-34) and GCs interact to regulate IL-11 expression in parallel with osteoblast differentiation and apoptosis and suggest that PTH(1-34) and dexamethasone may regulate osteoblast differentiation and apoptosis via their effect on IL-11 expression.
A consistent observation in osteoporosis is bone volume reduction accompanied by increased marrow adipose tissue. No single
cause linking the two phenomena has yet been identified. In a human progenitor cell clone (hOP 7) derived from bone marrow,
however, we have demonstrated that rabbit serum can direct differentiation away from an osteoblast lineage to one of adipocytes.
We now report on whether human serum has a similar effect. Serum was collected from 10 pre- and 10 postmenopausal women and
from the 10 postmenopausal women before and following 6-week hormone replacement therapy (HRT). hOP 7 cells were cultured
with the various sera, and after 7-14 days adipocytogenesis was determined by oil red O staining and lipoprotein lipase (LPL)
and glycerol 3-phosphate dehydrogenase (G3PDH) expression. Incubation with 10% premenopausal serum led to labeling of 10.9%
of cells (P<0.05) with oil red O, whereas application of 10% postmenopausal serum led to a much larger effect, 43.5% labeling (P<0.001 with respect to premenopausal serum). Oil red O positivity was accompanied by loss of type I collagen expression
and increased LPL and G3PDH expression. HRT did not reverse the adipocytogenic effect of postmenopausal serum. In conclusion,
serum from postmenopausal women contains factors that steer hOP 7 bone progenitor cells toward an adipocytic phenotype, irrespective
of HRT. The study suggests a role for serum factors in the development of fatty marrow in postmenopausal osteoporosis.
SAMP6 is a substrain of senescence-accelerated mice (SAM) that exhibits decreased bone mass due to impaired bone formation. Bipotential bone marrow stromal cells from SAMP6 showed enhanced adipogenesis and suppressed osteoblastogenesis. These abnormalities of stromal cells were cured by addition of an anti-adipogenic cytokine, interleukin (IL)-11, expression of which was decreased in SAMP6. We further demonstrated that IL-11 transcription was largely dependent on AP-1 transcription factors and that DNA binding activity of AP-1, especially Jun D, was reduced in SAMP6 marrow stromal cells. These results suggest that reduced AP-1-mediated transcription of IL-11 is a mechanism causing impaired bone formation in SAMP6. Interestingly, reduced AP-1 activity and a resultant decrease in IL-11 expression was also observed in aged mice of ordinary ICR strain. Moreover, transgenic mice over-expressing IL-11 showed increased bone mass due to enhanced bone formation and were protected from aging-associated bone loss. Based on these findings, we propose that IL-11 is a physiological stimulator of bone formation and that the aging-associated decline in stromal expression of IL-11 may be a general mechanism of impaired bone formation in the aged.
SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. We investigated the morphology of the parathyroid gland and thyroid C cell, together with the serum parathyroid hormone (PTH) and calcitonin (CT) in SAMP6 and age-matched normal mice SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 month of age with regard to the serum PTH level and the morphology of the parathyroid glands. As compared with SAMR1, the serum PTH level was significantly higher in SAMP6 at 2, 5 and 12 months of age. In the parathyroid chief cells of SAMP6 at 2, 5 and 12 months of age, the Golgi complexes and the cisternae of the granular endoplasmic reticulum were well developed. Numerous secretory granules were located near the plasma membranes and mitoses were sometimes observed. There was no marked difference between SAMR1 and SAMP6 regarding the morphology of the thyroid C cells and the serum CT level. These findings suggest that the secretory activity of the parathyroid gland is stimulated in SAMP6 at 2, 5 and 12 months of age. The parathyroid follicle was sometimes found in SAMP6, and the significance of this structure was also discussed.
Anemia induced by inflammation is well known to be more serious in the elderly than in non-elderly adults; however, the reason why this is so remains unclear. Neopterin produced by monocytes during inflammation promotes myelopoiesis but suppresses B-lymphopoiesis and erythropoiesis, by activating stromal cells in mice. Here, age-related changes in the erythropoietic response to neopterin were determined using senescence accelerated mice (SAMP1) with senescence stromal-cell impairment. Intravenous injection of neopterin into young mice (8-12 weeks old) resulted in a decrease in erythroid progenitor cell number in the bone marrow (BM), concomitant with an increase in myeloid progenitor cell number over one week. Intravenous injection of neopterin into aged mice (30-36 weeks old) resulted in a prolonged decrease in erythroid progenitor cell number in the BM over three weeks and a limited increase in myeloid progenitor cell number over one day. Neopterin treatment induced a decrease in serum erythropoietin concentrations in young mice but not in aged mice. The gene expression of tumor necrosis factor α (TNF-α), a negative regulator of erythropoiesis, was up-regulated in the BM of both young and aged mice, and the degree of TNF-α up-regulation was the same in both groups. The gene expression of interleukin (IL)-11, a positive regulator of erythropoiesis, was also up-regulated over one day in both young and aged mice. However, IL-11 gene expression remained up-regulated thereafter in young mice, whereas it was rapidly down-regulated in aged mice. These data suggest that prolonged suppression of erythropoiesis in aged mice may be due to a decrease in the production of positive regulators rather than to an increase in the production of negative regulators. Our combined data suggest that age-related impairment of stromal cells induces serious anemia in the elderly during inflammation.
Mesechymal stem cells as pluripotent cells are involved in the differentiation of adipocytes under regulation of genes and transcription factors. The plasticity observed between adipocytes and osteoblasts differentiation is the basis of transdifferentiation, observed in both experimental and clinical level. This review analyzes not only the adipose tissue as an endocrine organ but also the underlying mechanism of trans-differentiation between adipocytes and osteoblasts. Fat and bone tissue interaction is altered by activation or silencing of genes, signaling molecules and transcription factors. Disorders of this interaction include ectopic ossification syndromes and other bone disorders like osteoporosis and multiple myeloma. Further research will reveal the instinct mechanisms of this imbalance in the pathophysiology of many metabolic disorders such as diabetes mellitus, atherogenesis e.t.c.
Senescence-accelerated mouse prone 6 (SAMP6) is a model of senile osteoporosis. From 10 to 22 wk of age, SAMP6 mice were heavier than age-matched AKR/J and SAMR1 mice. Body mass indices of 10- and 25-wk-old SAMP6 mice were higher than those of age-matched AKR/J and SAMR1 mice, indicating obesity in the SAMP6 animals. We compared the blood biochemical values among SAMP6, SAMR1, and AKR/J mice to assess whether the SAMP6 strain has abnormal obesity-related parameters. Plasma glucose, triglyceride, insulin, and leptin levels were higher in 10-wk-old SAMP6 mice than in age-matched SAMR1 and AKR/J mice, whereas plasma glucagon and adiponectin levels in 25-wk-old SAMP6 were lower compared with those in age-matched SAMR1 and AKR/J. Total cholesterol levels in SAMR1 and SAMP6 mice at 10 and 25 wk of age were higher than those in AKR/J mice. Hepatic lipid levels were higher in 10- and 25-wk-old SAMP6 mice compared with age-matched AKR/J and SAMR1 animals. These results indicate that SAMP6 mice exhibit obesity and hyperlipidemia, suggesting that the SAMP6 strain is a potential tool for the study of hyperlipidemia.
Molecular mechanism of mechanical stress-induced bone formation remains unclear. We demonstrate that mechanical unloading suppresses and reloading enhances Interleukin (IL)-11 gene expression in the hindlimb of mice in vivo. Mechanical stress to osteoblasts by fluid shear stress (FSS) in vitro rapidly and transiently enhances fosB gene transcription, stimulates binding of DeltaFosB/JunD complex to activator protein (AP)-1 site of the IL-11 gene promoter, and enhances IL-11 gene transcription. Anti-IL-11 antibody blocks mechanical stress-induced enhancement of osteoblastogenesis and suppression of adipogenesis, suggesting the requirement of IL-11 for the stimulation of osteoblast differentiation by mechanical stress. Down-regulation of DeltaFosB/JunD by small interfering RNA (siRNA) suppresses and overexpression of DeltaFosB/JunD enhances IL-11 gene promoter activity. Consistent with our previous observations that up-regulation of DeltaFosB depends upon activation of cyclic AMP response element-binding protein (CREB) via Ca(2+)-dependent activation of extracellular signal-regulated kinase (ERK) to phosphorylate CREB, mechanical stress-induced activation of IL-11 gene transcription is dependent upon Ca(2+)-ERK pathway. Present results also demonstrated that FSS to osteoblasts enhances canonical Wnt signaling in vitro, and that mechanical unloading induces and reloading suppresses the expression of a canonical Wnt signal inhibitor, dickkopf2 (Dkk2), in vivo. In addition, IL-11 siRNA enhances Dkk2 expression suppressed by FSS, and osteoblasts from IL-11 transgenic mice show reduced Dkk2 mRNA expression than those from wild-type mice. These observations are consistent with the notion that mechanical stress stimulates IL-11 gene transcription via an enhanced DeltaFosB/JunD binding to the IL-11 gene promoter, and that increased IL-11 enhances canonical Wnt signal at least in part via a reduction in Dkk2 expression to stimulate osteoblast differentiation.
At present, little is known about the age-related changes in jaw bones. The aim of this study was to characterize the mandibles of 6 month-old senile osteoporotic mice, SAMP6, and compare with those of age-matched controls, SAMR1. In comparison to SAMR1, SAMP6 showed thinner cortical bone, lower bone volume, and poorly organized collagen matrix. The collagen fibril diameter in SAMP6 was significantly smaller than that of SAMR1. In SAMP6 both collagen content and cross-links were lower than those of SAMR1, but the ratio of the major mature cross-link (pyridinoline) to its precursor reducible cross-link (dehydrodihydroxylysinonorleucine/its ketoamine) was higher in comparison to SAMR1. In addition, the extent of lysine hydroxylation of collagen was higher in SAMP6 than that of SAMR1. These results indicate that not only the quantity of collagen but also its quality are altered in SAMP6 and may result in the age-associated osteoporotic defects of mandibles.
Cancer cells metastasized to bone induce osteoclastogenesis for bone destruction. Coculture of either mouse melanoma B16 or breast cancer Balb/c-MC cells with mouse bone marrow cells (BMCs) induced osteoclast-like cells, which were not observed when cancer cells were segregated from BMCs. Osteoclast differentiation factor (ODF), also known as receptor activator of NF-kappaB ligand (RANKL), is a direct mediator of many osteotropic factors. Neither BMCs, B16 nor Balb/c-MC cells alone expressed ODF mRNA. However, coculture of these cancer cells with BMCs induced ODF expression, which was prevented by indomethacin. Moreover, the coculture with cancer cells inhibited secretion of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF), an inhibitory decoy receptor for ODF, from BMCs. Thus, enhanced osteoclastogenesis in the presence of cancer cells might be due to an increase in ODF activity. These results suggest that interactions between cancer cells and BMCs induce ODF expression and suppress OPG/OCIF level in metastatic foci resulting in pathological osteoclastogenesis for bone destruction.
The development over the last few years of genetically modified mouse models has provided a wealth of new information on the intimate cellular mechanisms involved in bone physiology. This article reviews some of the new insights gained into non-collagenous bone proteins. The bone matrix is no longer viewed as a passive support for bone cells, but rather as a key factor in the regulation of cell recruitment, proliferation, and differentiation. Studies using genetically modified mouse models have demonstrated the central importance of transcription factors such as Cbfa1, c-Fos, and c-Src in the differentiation of osteoblasts or osteoclasts from bone marrow stem cells. They have also allowed to identify the main cytokines involved in the regulation of bone cell activities, particularly in estrogen-deprived individuals. A discussion is provided in this article of the studies that identified the main communication pathway between osteoblasts and osteoclasts, in which the mediators are osteoprotegerin and its ligand, and that demonstrated the central position of these two factors in the regulation of osteoclast differentiation and activity.
In vitro osteoclast differentiation is supported by stromal cells. In order to isolate a stromal cell line that can support osteoclast differentiation, 22 cell lines were cloned from mouse bone marrow. One of these clones, TMS-14, is a line of preadipocytes that supports osteoclast-like cell formation without any bone resorbing factors; and another, TMS-12, is a line of preosteoblasts that supports osteoclast-like cell formation with bone resorbing factors such as prostaglandin E(2)(PGE(2)). The difference of these two lines for osteoclast formation was not related with their abilities of PGE(2)production, but with the expression of osteoclast differentiation factor (ODF, also called OPGL, RANKL, and TRANCE), which detected with RT-PCR, in both cell lines. In TMS-14 cells, ODF mRNA was detected with or without PGE(2). In TMS-12 cells, ODF expression was detected in the PGE(2)-treated cells alone. When TMS-14 cells were induced to undergo adipogenic differentiation in response to treatment with thiazolidinedione, a ligand and activator of peroxisome proliferator-activated receptor gamma (PPARgamma), the ability of TMS-14 cells to support osteoclast-like cell formation was prevented in the presence or absence of 1,25(OH)(2)D(3). The gene expression of ODF in TMS-14 cells was also inhibited by treatment with thiazolidinedione. These results suggest that adipogenesis in bone marrow cells is related to the ability to support osteoclast differentiation. This is the first report of a cloned stromal cell line that can support osteoclastogenesis without the treatment with any osteotropic factors. Furthermore, this murine clonal preadipose cell line may be useful for studying senescence-dependent osteoporosis.
It has been reported that soluble interleukin (IL)-6 receptor (sIL-6R) is detected in the serum of healthy individuals and its level is increased in patients with multiple myeloma and human immunodeficiency virus infection. Although several reports have suggested that sIL-6R potentiates IL-6 action, its physiological role remains unclear. In this study, we examined the role of sIL-6R on osteoclast formation by IL-6, using a coculture of mouse osteoblasts and bone marrow cells. Neither recombinant mouse IL-6 (mIL-6) nor mouse sIL-6R (smIL-6R) induced osteoclast-like multinucleated cell (MNC) formation when they were added separately. In contrast, simultaneous treatment with mIL-6 and smIL-6R strikingly induced MNC formation. These MNCs satisfied major criteria of authentic osteoclasts, such as tartrate-resistant acid phosphatase (TRAP) activity, calcitonin receptors, and pit formation on dentine slices. The MNC formation induced by mIL-6 and smIL-6R was dose-dependently inhibited by adding monoclonal anti-mouse IL-6R antibody (MR16-1). It is likely that osteoblasts and osteoclast progenitors are capable of transducing a signal from a complex of IL-6 and sIL-6R through gp130, even though they may have no or a very small number of IL-6Rs. Factors such as IL-11, oncostatin M, and leukemia inhibitory factor, which are known to exert their functions through gp130 (the signal-transducing chain of IL-6R), also induced MNC formation in our coculture system. These results suggest that increased circulating or locally produced sIL-6R induces osteoclast formation in the presence of IL-6 mediated by a mechanism involving gp130. This may play an important physiological or pathological role in conditions associated with increased osteoclastic bone resorption.
Age-related changes of the femoral bone mass in several strains of the senescence-accelerated mouse (SAM) were investigated. Microdensitometrically, all strains exhibited essentially the same patterns of age changes, that is, bone mass corrected by the diameter of the shaft reached the peak value when the mice were 4 or 5 months of age and then fell linearly with age up to over 20 months of age. Two strains, SAM-R/3 and SAM-P/6, which originated from the same ancestry on pedigree, had a significantly lower peak bone mass than other strains (SAM-R/1, SAM-R/2, SAM-P/1, and SAM-P/2). On the other hand, the strains with a low peak bone mass had the same rate of decrease as other strains. Mineral and collagen contents per dry weight of bone showed little difference among the strains. Histologic studies of tibia, femur, and lumbar spine revealed that the osteopenia was not due to osteomalacia but, rather, to osteoporosis. The elderly mice in these two strains were prone to fracture, thus should be important models for study of senile osteoporosis seen clinically.
Interleukin-11 (IL-11) is a stromal cell-derived cytokine with multiple biologic activities on lymphohematopoietic cells. It belongs to a family of pleiotropic and redundant cytokines that use the gp 130 transducing subunit in their high affinity receptors. By amplifying human cDNA libraries with oligonucleotide primers corresponding to the conserved WSXWS motif found in the hematopoietic cytokine receptor family, a novel cytokine receptor cDNA was identified that, based on high (82%) sequence homology with the recently cloned murine IL-11 receptor, appears to encode the human IL-11 receptor. This receptor is a 422-amino acid protein containing a signal peptide followed by extracellular, transmembrane, and cytoplasmic domains. The extracellular region has a two-domain structure homologous to those of the IL-6 and ciliary neurotrophic factor (CNTF) receptors: an immunoglobulin-like domain and a cytokine receptor-like domain. In addition, an isoform of the human IL-11 receptor that lacks the cytoplasmic domain was also identified. In agreement with the pleiotropic effects of IL-11 on different hematopoietic lineages and bone cells, IL-11 receptor transcripts were found to be expressed by the myelogenous leukemia cell line K562, the megakaryocytic leukemia cell line Mo7E, the erythroleukemia cell line TF1, and the osteosarcoma cell lines MG-63 and Saos-2.
Wall thickness, a major determinant of trabecular thickness, falls with age and falls further in osteoporosis. To estimate the importance of defective osteoblast recruitment in the pathogenesis of this defect, we compared various histologic indices of bone formation in iliac bone biopsies in three groups of subjects--healthy premenopausal women, healthy postmenopausal women, and patients with postmenopausal osteoporosis and at least one non-traumatic vertebral compression fracture. Indices that reflect the frequency of activation of bone remodeling and consequent birth rate of new teams of osteoblasts (osteoid surface, mineralizing surface, osteoblast surface, and bone formation rate, all expressed per unit of bone surface) were each higher in healthy subjects who were postmenopausal than in those who were premenopausal, but lower in osteoporotic than in normal postmenopausal women. In each group, the primary surface measurements were significantly correlated with each other, but the correlation was less close in those with osteoporosis. Indices that reflect the average collective performance of individual teams of osteoblasts (mineralizing surface and osteoblast surface per unit of osteoid surface, mineral apposition rate, adjusted apposition rate, and wall thickness) were all lower in postmenopausal than in premenopausal normal subjects, and even lower in those with postmenopausal osteoporosis. The parameters of the regression lines relating bone formation rate to osteoblast surface were essentially the same in each group, indicating that bone formation rate per unit of osteoblast surface was unaffected by age or menopause, and was the same in osteoporosis as in healthy subjects of similar age.(ABSTRACT TRUNCATED AT 250 WORDS)
An adult mouse liver cDNA library was screened with oligonucleotides corresponding to the conserved WSXWS motif of the haemopoietin receptor family. Using this method, cDNA clones encoding a novel receptor were isolated. The new receptor, named NR1, was most similar in sequence and predicted structure to the alpha-chain of the IL-6 receptor and mRNA was expressed in the 3T3-L1 pre-adipocytic cell line and in a range of primary tissues. Expression of NR1 in the factor-dependent haemopoietic cell line Ba/F3 resulted in the generation of low affinity receptors for IL-11 (Kd approximately 10 nM). The capacity to bind IL-11 with high affinity (Kd = 300-800 pM) appeared to require coexpression of both NR1 and gp130, the common subunit of the IL-6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) receptors. The expression of both NR1 and gp130 was also necessary for Ba/F3 cells to proliferate and M1 cells to undergo macrophage differentiation in response to IL-11.
We analyzed the stimulatory effect of oncostatin M (OSM), leukemia inhibitory factor (LIF), interleukin 6 (IL-6), IL-11, and the inhibitory effect of anti-IL-6 antibody (Ab), anti-IL-6 receptor monoclonal antibody (mAb), and anti-gp130 mAb on the growth of human plasmacytoma cells freshly isolated from a patient with multiple myeloma. The purified cells showed a plasmacytoid morphology and expressed CD38, CD54, and CD56 antigens but no CD3, CD5, CD10, CD19, CD20, or very late antigen 5. IL-6 receptor (IL-6R) and its signal transducer, gp130, were expressed on their cell surface at a low level. Dose-dependent proliferation of the cells in response to OSM, LIF, and IL-6, but not to IL-11, was observed using [3H]TdR incorporation in vitro. Both anti-IL-6 Ab and anti-IL-6R mAb inhibited the growth of the cells in the presence or absence of exogenous IL-6. These cells release IL-6 but not OSM or LIF into the culture supernatant during short-term culture. Therefore, an autocrine growth mechanism mediated by IL-6, but not by OSM or LIF, was confirmed. Furthermore, anti-gp130 mAb completely inhibited the proliferation of the cells induced by OSM, LIF, as well as IL-6. These data indicate that OSM, LIF, and IL-6 can act as growth factors of human plasmacytoma cells through a common signal transducer, gp130, on their cell surface, and also suggest the potential therapeutic application of anti-gp130 mAb, as well as anti-IL-6R mAb against myeloma/plasmacytomas.
Interleukin 6 (IL-6) is a major growth factor for tumor plasma cells involved in human multiple myeloma (MM). In particular, human myeloma cell lines (HMCL), whose growth is completely dependent on addition of exogenous IL-6, can be obtained reproducibly from every patient with terminal disease. Four cytokines, ciliary neurotropic factor (CNTF), IL-11, leukemia inhibitory factor (LIF), and oncostatin M (OM), use the same transducer chain (signal transducer gp130) as IL-6 and share numerous biological activities with this IL. We found that these four cytokines stimulated proliferation and supported the long-term growth of two out of four IL-6-dependent HMCL obtained in our laboratory. Half-maximal proliferation was obtained with cytokine concentrations ranging from 0.4 to 1.2 ng/ml for IL-11, LIF, and OM. CNTF worked at high concentrations only (90 ng/ml), but addition of soluble CNTF receptor increased sensitivity to CNTF 30-fold. The growth-promoting effect of these four cytokines was abrogated by anti-gp130 antibodies, contrary to results for anti-IL-6 receptor or anti-IL-6 antibodies. No detectable changes in the morphology and phenotype were found when myeloma cells were cultured with one of these four cytokines instead of IL-6. Concordant with their IL-6-dependent growth, the four HMCL expressed membrane IL-6R and gp130 detected by FACS analysis. LIF-binding chain gene (LIFR) was expressed only in the two HMCL responsive to LIF and OM.
Stromal cells of the bone marrow control the development of osteoclasts through the production of cytokines capable of promoting the proliferation and differentiation of hematopoietic progenitors. Moreover, the deregulated production of the cytokine IL-6 in the bone marrow mediates an increase in osteoclastogenesis after estrogen loss. IL-6, however, does not influence osteoclastogenesis in the estrogen-replete state, suggesting that other cytokines might be responsible for osteoclast development under physiologic circumstances. We report here that IL-11, a newly discovered cytokine that is produced by marrow stromal cells, induced the formation of osteoclasts exhibiting an unusually high degree of ploidy in cocultures of murine bone marrow and calvarial cells. Osteoclasts formed in the presence of IL-11 were capable of bone resorption, as evidenced by the formation of resorption pits, as well as the release of 45Ca from prelabeled murine calvaria. Further, an antibody neutralizing IL-11 suppressed osteoclast development induced by either 1,25-dihydroxyvitamin D3, parathyroid hormone, interleukin-1, or tumor necrosis factor; whereas inhibitors of IL-1 or TNF had no effect on IL-11-stimulated osteoclast formation. The effects of IL-11 on osteoclast development were blocked by indomethacin; more important, however, they were independent of the estrogen status of the marrow donors.
The functional receptor complexes assembled in response to interleukin-6 and -11 (IL-6 and IL-11), leukemia inhibitory factor (LIF), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF), all involve the signal transducer gp130: IL-6 and IL-11 induce homodimerization of gp130, while the rest heterodimerize gp130 with other gp130-related beta subunits. Some of these cytokines (IL-6, IL-11, and CNTF) also require a specificity-determining alpha subunit not directly involved in signaling. We have searched for functional receptor complexes for these cytokines in cells of the bone marrow stromal/osteoblastic lineage, using tyrosine phosphorylation of the beta subunits as a detection assay. Collectively, murine calvaria cells, bone marrow-derived murine cell lines (+/+LDA11 and MBA13.2), as well as murine (MC3T3-E1) and human (MG-63) osteoblast-like cell lines displayed all the previously recognized alpha and beta subunits of this family of receptors. However, individual cell types had different constellations of alpha and beta subunits. In addition and in difference to the other cell types examined, MC3T3-E1 cells expressed a heretofore unrecognized form of gp130; and MG-63 displayed an alternative form (type II) of the OSM receptor. These findings establish that stromal/osteoblastic cells are targets for the actions of all the members of the cytokine subfamily that shares the gp130 signal transducer; and suggest that different receptor repertoires may be expressed at different stages of differentiation of this lineage.
The differentiation of adipocytic and osteogenic cells has been investigated in cultures of adult rat marrow stromal cells. Adipocytic differentiation was assessed using morphological criteria, changes in expression of procollagen mRNAs, consistent with a switch from the synthesis of predominantly fibrillar (types I and III) to basement membrane (type IV) collagen, and the induction of expression of aP2, a specific marker for differentiation of adipocytes. Osteogenic differentiation was assessed on the basis of changes in the abundance of the mRNAs for the bone/liver/kidney isozyme of alkaline phosphatase and the induction of mRNAs for bone sialoprotein and osteocalcin. In the presence of foetal calf serum and dexamethasone (10(−8) M) there was always differentiation of both adipocytic and osteogenic cells. When the steroid was present throughout primary and secondary culture the differentiation of osteogenic cells predominated. Conversely, when dexamethasone was present in secondary culture only, the differentiation of adipocytes predominated. When marrow stromal cells were cultured in the presence of dexamethasone in primary culture and dexamethasone and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 10(−8) M) in secondary culture, the differentiation of adipocytes was inhibited whereas the differentiation of osteogenic cells was enhanced, as assessed by an increase in expression of osteocalcin mRNA. The results, therefore, demonstrate an inverse relationship between the differentiation of adipocytic and osteogenic cells in this culture system and are consistent with the possibility that the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo.
Interleukin-11 (IL-11) is a stromal cell-derived cytokine with multiple biologic activities on lymphohematopoietic cells. It belongs to a family of pleiotropic and redundant cytokines that use the gp 130 transducing subunit in their high affinity receptors. By amplifying human cDNA libraries with oligonucleotide primers corresponding to the conserved WSXWS motif found in the hematopoietic cytokine receptor family, a novel cytokine receptor cDNA was identified that, based on high (82%) sequence homology with the recently cloned murine IL-11 receptor, appears to encode the human IL-11 receptor. This receptor is a 422-amino acid protein containing a signal peptide followed by extracellular, transmembrane, and cytoplasmic domains. The extracellular region has a two-domain structure homologous to those of the IL-6 and ciliary neurotrophic factor (CNTF) receptors: an immunoglobulin-like domain and a cytokine receptor-like domain. In addition, an isoform of the human IL-11 receptor that lacks the cytoplasmic domain was also identified. In agreement with the pleiotropic effects of IL-11 on different hematopoietic lineages and bone cells, IL-11 receptor transcripts were found to be expressed by the myelogenous leukemia cell line K562, the megakaryocytic leukemia cell line Mo7E, the erythroleukemia cell line TF1, and the osteosarcoma cell lines MG-63 and Saos-2.
Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.
We investigated the effect of recombinant human interleukin-11 (rhIL- 11) on human megakaryocytopoiesis. Nonadherent and T-cell-depleted human bone marrow (BM) mononuclear cells were cultured in a serum-free agar culture system. rhIL-11 alone did not stimulate the growth of human megakaryocyte colonies. However, when rhIL-11 was combined with optimal or suboptimal doses of rhIL-3, the number and size of the megakaryocyte colonies increased. The same results were obtained when highly purified BM CD34-positive cells were used as target cells. Next, we investigated the effect of rhIL-11 on the ploidy of megakaryocytes. The ploidy distribution of individual cells in megakaryocyte colonies obtained by rhIL-11 in combination with rhIL-3 was significantly shifted towards higher values. Furthermore, when highly purified CD41- positive BM cells were cultured in the presence of rhIL-11, the ploidy distribution was shifted towards higher values. This effect was not suppressed by anti-IL-6 antibody. These results suggest that rhIL-11 acts directly as a megakaryocyte potentiator and may play a role in regulating human megakaryocytopoiesis.
Osteoclast-mediated bone resorption plays a crucial role in osseous remodeling. Osteoblasts are important regulators of this activity, in part through their ability to produce osteoclast-regulating soluble factors such as interleukin-6 (IL-6). IL-11 is a newly appreciated pleotropic cytokine whose spectrum of biological activities overlaps with that of IL-6. As a result, we hypothesized that osteoblasts are an important skeletal source of this cytokine. To test this hypothesis, we characterized the IL-11 production of unstimulated and stimulated SaOS-2 human osteosarcoma cells. Unstimulated cells produced modest amounts of IL-11. The osteotropic agents recombinant IL-1 (0.25-5 ng/ml), transforming growth factor-beta 1 (0.1-10 ng/ml), PTH (10(-8)-10(-11) M), and PTH-related peptide ((10(-8)-10-11 M) further increased SaOS-2 cell IL-11 protein production and messenger RNA accumulation. These stimulatory effects were dose and time dependent, and the IL-11 that was produced was bioactive, as demonstrated by i...
A unique population of rat adipocyte precursor cells was derived from normal rat bone marrow. The epitheloid-like preadipocytes were isolated from a mixed culture of bone marrow cells by a combination of differential trypsinization, enrichment by Ficoll gradient centrifugation, and differential seeding. This cell line, designated RBM-Ad, can be fully differentiated into multilocular adipocytes morphologically resembling brown adipose tissue. No changes in the differentiation pattern are observed during propagation of these cells, and they have been successfully carried and differentiated up to passage 49. Histological staining of differentiated cells with Sudan black, Sudan IV, and oil red O indicates the presence of lipids in intracellular vesicles. The nonselective beta-adrenergic agonist isoproterenol stimulates adenylyl cyclase activity in both preadipocytes and differentiated adipocytes. In contrast, BRL-37344, a beta 3-adrenergic receptor-specific agonist, stimulates adenylyl cyclase activity and gl...
We analyzed the stimulatory effect of oncostatin M (OSM), leukemia inhibitory factor (LIF), interleukin 6 (IL-6), IL-11, and the inhibitory effect of anti-IL-6 antibody (Ab), anti-IL-6 receptor monoclonal antibody (mAb), and anti-gp130 mAb on the growth of human plasmacytoma cells freshly isolated from a patient with multiple myeloma. The purified cells showed a plasmacytoid morphology and expressed CD38, CD54, and CD56 antigens but no CD3, CD5, CD10, CD19, CD20, or very late antigen 5. IL-6 receptor (IL-6R) and its signal transducer, gp130, were expressed on their cell surface at a low level. Dose-dependent proliferation of the cells in response to OSM, LIF, and IL-6, but not to IL-11, was observed using [3H]TdR incorporation in vitro. Both anti-IL-6 Ab and anti-IL-6R mAb inhibited the growth of the cells in the presence or absence of exogenous IL-6. These cells release IL-6 but not OSM or LIF into the culture supernatant during short-term culture. Therefore, an autocrine growth mechanism mediated by IL-6, but not by OSM or LIF, was confirmed. Furthermore, anti-gp130 mAb completely inhibited the proliferation of the cells induced by OSM, LIF, as well as IL-6. These data indicate that OSM, LIF, and IL-6 can act as growth factors of human plasmacytoma cells through a common signal transducer, gp130, on their cell surface, and also suggest the potential therapeutic application of anti-gp130 mAb, as well as anti-IL-6R mAb against myeloma/plasmacytomas.
Interleukin 6 (IL-6) is a major growth factor for tumor plasma cells involved in human multiple myeloma (MM). In particular, human myeloma cell lines (HMCL), whose growth is completely dependent on addition of exogenous IL-6, can be obtained reproducibly from every patient with terminal disease. Four cytokines, ciliary neurotropic factor (CNTF), IL-11, leukemia inhibitory factor (LIF), and oncostatin M (OM), use the same transducer chain (signal transducer gp130) as IL-6 and share numerous biological activities with this IL. We found that these four cytokines stimulated proliferation and supported the long-term growth of two out of four IL-6-dependent HMCL obtained in our laboratory. Half-maximal proliferation was obtained with cytokine concentrations ranging from 0.4 to 1.2 ng/ml for IL-11, LIF, and OM. CNTF worked at high concentrations only (90 ng/ml), but addition of soluble CNTF receptor increased sensitivity to CNTF 30-fold. The growth-promoting effect of these four cytokines was abrogated by anti-gp130 antibodies, contrary to results for anti-IL-6 receptor or anti-IL-6 antibodies. No detectable changes in the morphology and phenotype were found when myeloma cells were cultured with one of these four cytokines instead of IL-6. Concordant with their IL-6-dependent growth, the four HMCL expressed membrane IL-6R and gp130 detected by FACS analysis. LIF-binding chain gene (LIFR) was expressed only in the two HMCL responsive to LIF and OM.
We investigated the effect of recombinant human interleukin-11 (rhIL-11) on human megakaryocytopoiesis. Nonadherent and T-cell-depleted human bone marrow (BM) mononuclear cells were cultured in a serum-free agar culture system. rhIL-11 alone did not stimulate the growth of human megakaryocyte colonies. However, when rhIL-11 was combined with optimal or suboptimal doses of rhIL-3, the number and size of the megakaryocyte colonies increased. The same results were obtained when highly purified BM CD34-positive cells were used as target cells. Next, we investigated the effect of rhIL-11 on the ploidy of megakaryocytes. The ploidy distribution of individual cells in megakaryocyte colonies obtained by rhIL-11 in combination with rhIL-3 was significantly shifted towards higher values. Furthermore, when highly purified CD41-positive BM cells were cultured in the presence of rhIL-11, the ploidy distribution was shifted towards higher values. This effect was not suppressed by anti-IL-6 antibody. These results suggest that rhIL-11 acts directly as a megakaryocyte potentiator and may play a role in regulating human megakaryocytopoiesis.
The differentiation of adipocytic and osteogenic cells has been investigated in cultures of adult rat marrow stromal cells. Adipocytic differentiation was assessed using morphological criteria, changes in expression of procollagen mRNAs, consistent with a switch from the synthesis of predominantly fibrillar (types I and III) to basement membrane (type IV) collagen, and the induction of expression of aP2, a specific marker for differentiation of adipocytes. Osteogenic differentiation was assessed on the basis of changes in the abundance of the mRNAs for the bone/liver/kidney isozyme of alkaline phosphatase and the induction of mRNAs for bone sialoprotein and osteocalcin. In the presence of foetal calf serum and dexamethasone (10(-8) M) there was always differentiation of both adipocytic and osteogenic cells. When the steroid was present throughout primary and secondary culture the differentiation of osteogenic cells predominated. Conversely, when dexamethasone was present in secondary culture only, the differentiation of adipocytes predominated. When marrow stromal cells were cultured in the presence of dexamethasone in primary culture and dexamethasone and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 10(-8) M) in secondary culture, the differentiation of adipocytes was inhibited whereas the differentiation of osteogenic cells was enhanced, as assessed by an increase in expression of osteocalcin mRNA. The results, therefore, demonstrate an inverse relationship between the differentiation of adipocytic and osteogenic cells in this culture system and are consistent with the possibility that the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo.
A causal role in age-related bone loss has been attributed to alterations in vitamin D status, the bone mineral regulating hormones, and/or renal function. We assessed biochemical parameters of bone metabolism and renal function in healthy subsets of young and old men (n = 191) and women (n = 120) and evaluated the relationships between these parameters and bone mineral density (BMD) in the radius, spine, and femur. There were no significant associations between BMD at any site and serum 25-OHD, 1,25-(OH)2D, PTH, or creatinine clearance in either young men or in young or old women, after controlling for age. In old men, however, lower radius BMD was significantly related to higher PTH and higher 1,25-(OH)2D and marginally related to lower 25-OHD values. In young men, there were unexpected but significant associations between lower femoral neck BMD and higher serum osteocalcin and urinary calcium/creatinine excretion after age adjustment. In old women, lower spine and radius BMD was also significantly correlated with higher serum osteocalcin. In this healthy, vitamin D-replete population, there were significant cross-sectional declines in BMD in the femur in young and old men and at all sites in old women. Elevated remodeling may be an important feature that contributes to reduced femoral BMD in young men and reduced spine and radius BMD in old women. However, compromised renal function or levels of 1,25-(OH)2D or elevated PTH appear to be neither necessary nor relevant as determinants of osteopenia in the spine or femur in these normal, healthy men and women.
A cDNA encoding a novel adipogenesis inhibitory factor (AGIF) that inhibits the process of adipogenesis in mouse 3T3-L1 preadipocytes was cloned from a cDNA library of the human bone marrow-derived stromal cell line KM-102. The cloned cDNA contains an open reading frame coding for an AGIF precursor of 199 amino acids. Analysis of the sequence of this cDNA revealed identity of this factor with a recently reported novel cytokine, designated interleukin-11 (IL-11). AGIF/IL-11 may play an important role in stromal cell-associated hematopoeisis through its regulatory action on adipocyte differentiation in the bone marrow microenvironment.
We studied the mechanism of impaired calcium absorption with aging in 51 healthy women whose ages ranged from 26 to 88 years. Serum concentrations of 1,25-dihydroxyvitamin D [1,25-(OH)2D, mean of four measurements per subject] increased with age by 22% (P less than 0.05) but, by split-point analysis, plateaued or decreased slightly after age 65. In a subset of 20 subjects, [3H]1,25-(OH)2D3 kinetic analysis showed that this increase with age resulted from both increased production and decreased metabolic clearance of 1,25-(OH)2D. Despite the increase in serum 1,25-(OH)2D concentration, true calcium absorption did not change with age. The expected inverse correlation between true fractional calcium absorption and dietary calcium intake, however, was easily demonstrated (r = 0.66, P less than 0.001). Serum intact parathyroid hormone (PTH) increased with age by 35% (P less than 0.02) and serum bone gla protein (BGP, osteocalcin) increased by 47% (P less than 0.001); the increases in serum PTH and serum BGP were directly correlated (r = 0.32, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Although vitamin D is essential for mineralization of bone, it is as yet unclear whether vitamin D has a direct stimulatory effect on the bone mineralization process. In the present study, the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on in vitro mineralization mediated by osteoblast-like MC3T3-E1 cells was examined. MC3T3-E1 cells continued to grow after they reached confluency, and DNA content and alkaline phosphatase activity increased linearly until about 16 days of culture, whereas 45Ca accumulation into cell and matrix layer remained low. After this period, DNA content plateaued, and 45Ca accumulation increased sharply. Histological examination by von Kossa staining revealed that calcium was accumulated into extracellular matrix. In addition, needle-shaped mineral crystals similar to hydroxyapatite crystals could be demonstrated in between collagen fibrils by electron microscopy. Thus, MC3T3-E1 cells differentiate in vitro into cells with osteoblastic phenotype and exhibit mineralization. When MC3T3-E1 cells were treated with 1,25(OH)2D3 at this stage of culture, there was a dose-dependent stimulation of 45Ca accumulation by 1,25(OH)2D3, and a significant stimulation of 45Ca accumulation was observed with 3 x 10(-10) M 1,25(OH)2D3. Although 1,25(OH)2D3 enhanced alkaline phosphatase activity and collagen synthesis at the early phase of culture, it did not affect any of these parameters at the late phase when 1,25(OH)2D3 stimulated mineralization. Neither 24,25-dihydroxyvitamin D3 nor human PTH(1-34) affected mineralization in the presence or absence of 1,25(OH)2D3. These results demonstrate that 1,25(OH)2D3 stimulates matrix mineralization induced by osteoblastic MC3T3-E1 cells, and are consistent with the possibility that 1,25(OH)2D3 has a direct stimulatory effect on bone mineralization process.(ABSTRACT TRUNCATED AT 250 WORDS)
Hematopoiesis occurs in close association with a complex network of cells loosely termed the hematopoietic microenvironment. Analysis of the mechanisms of microenvironmental regulation of hematopoiesis has been hindered by the complexity of the microenvironment as well as the heterogeneity of hematopoietic stem cells and early progenitor cells. We have established immortalized primate bone marrow-derived stromal cell lines to facilitate analysis of the interactions of hematopoietic cells with the microenvironment in a large animal species. One such line, PU-34, was found to produce a variety of growth factors, including an activity that stimulates the proliferation of an interleukin 6-dependent murine plasmacytoma cell line. A cDNA encoding the plasmacytoma stimulatory activity was isolated through functional expression cloning in mammalian cells. The nucleotide sequence contained a single long reading frame of 597 nucleotides encoding a predicted 199-amino acid polypeptide. The amino acid sequence of this cytokine, designated interleukin 11 (IL-11), did not display significant similarity with any other sequence in the GenBank data base. Preliminary biological characterization indicates that in addition to stimulating plasmacytoma proliferation, IL-11 stimulates the T-cell-dependent development of immunoglobulin-producing B cells and synergizes with IL-3 in supporting murine megakaryocyte colony formation. These properties implicate IL-11 as an additional multifunctional regulator in the hematopoietic microenvironment.
The effects of aging on calcium and bone metabolism have not been systematically examined in men. To identify age-related alterations in vitamin D and PTH physiology and to assess their impact on skeletal health, we studied 62 normal men, aged 30-92 yr. The men were in excellent health, and none had any evidence of metabolic bone disease and/or known risk factors for osteopenia. Serum 25-hydroxyvitamin D (25OHD) concentrations declined steadily with advancing age (r = -0.47; P less than 0.001), and there was a corresponding decline in serum 24,25-dihydroxyvitamin D [24,25-(OH)2D] levels (r = -0.41; P less than 0.001). Serum 1,25-(OH)2D concentrations, however, did not vary over this age range (r = -0.07; P = NS). Plasma PTH levels increased with aging (r = -0.24; P less than 0.001), and there was a concomitant increase in urinary cAMP excretion (r = 0.38; P less than 0.001). Renal function (creatinine clearance) clearly declined with increasing age (r = -0.71; P less than 0.001). In conjunction with these changes in calcium metabolism, radial and vertebral bone mineral content declined. Whereas the fall in radial bone mineral content (single photon absorptiometry) at both proximal and distal sites was slight, there was a marked decrease in vertebral bone mineral content, as measured by quantitative computed tomography (r = -0.72; P less than 0.0001). The fall in vertebral bone mineral content correlated well with the declines in serum 25OHD and 24,25-(OH)2D concentrations (r = 0.47; P less than 0.001 and r = 0.51; P less than 0.001, respectively) and with the decline in renal function (r = 0.46; P less than 0.001). Multiple regression analysis revealed that the effects of aging on bone mineral content could be accounted for in large part by concomitant changes in mineral metabolism. Both the decline in renal function and the fall in serum 24,25-(OH)2D levels were closely associated with the fall in bone mineral content. These results indicate that a decline in renal function and alterations in vitamin D metabolism occur with aging in normal men. These changes contribute to, if not cause, the associated decline in skeletal mineral content in aging men.
We developed a mouse bone marrow culture system to examine the process of osteoclast-like multinucleated cell formation from its progenitors. When mouse marrow cells were cultured for 8 days with 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3, 10(-10) to 10(-7) M] or human PTH (1-34) (25-100 ng/ml), tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells formed. No TRACP-positive multinucleated cells appeared in the absence of these hormones. 1 alpha,25-(OH)2D3 and PTH also increased the number of the clusters of TRACP-positive mononuclear cells. Time course studies showed that these TRACP-positive mononuclear cell clusters appeared before the formation of TRACP-positive multinucleated cells, suggesting that the TRACP-positive mononuclear cells are precursors of the multinucleated cells. Salmon calcitonin markedly inhibited the formation of TRACP-positive multinucleated cells but not TRACP-positive mononuclear cell clusters induced by 1 alpha,25-(OH)2D3 or PTH. TRACP-positive mononuclear cells and multinucleated cells were rarely stained for nonspecific esterase, but some mononuclear cells were positively stained for both nonspecific esterase and TRACP. More that 90% of the TRACP-positive mononuclear cell clusters and multinucleated cells were found near colonies of alkaline phosphatase-positive mononuclear cells (possibly osteoblasts). When marrow mononuclear cells were cultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 or PTH, numerous resorption lacunae were formed. These results suggest that 1) TRACP-positive multinucleated cells formed in response to osteotropic hormones in mouse marrow cultures satisfy most of the criteria of osteoclasts, and 2) osteoblasts may play an important role in osteoclast formation.
Five senescence-prone series of mice (P-1, P-2, P-3, P-4 and P-5) and three senescence-resistant series (R-1, R-2 and R-3) were obtained by continuous sister-brother breeding from five original litters of AKR mice with severe deterioration, and the three original litters of AKR mice with normal aging, respectively. A grading score system was adopted to evaluate the degree of senescence of these mice and a steady and irreversible increase in this grading score was seen with advancing age in both the R and P series. The high grading score in the P series was due to an earlier onset of loss of passivity and reactivity, loss of skin glossiness and increased coarseness, hair loss, periophthalmic lesions, increased lordokyphosis of the spine and a more marked increase in their severity with advancing age as compared to the R series. Among the P series, P-2 showed a 100% incidence of systemic amyloidosis after 6 months of age and P-3 a 70% incidence of cataract over 16 months of age. The life span in the P series was shortened by about 26% of that of the R series. In view of the evidence obtained from the survivors, the growth rate and Gompertz function, the aging pattern in the P series was considered to be an acceleration of senescence. The P series has been named "SAM" ("Senescence Accelerated Mouse").
A unique population of rat adipocyte precursor cells was derived from normal rat bone marrow. The epitheloid-like preadipocytes were isolated from a mixed culture of bone marrow cells by a combination of differential trypsinization, enrichment by Ficoll gradient centrifugation, and differential seeding. This cell line, designated RBM-Ad, can be fully differentiated into multilocular adipocytes morphologically resembling brown adipose tissue. No changes in the differentiation pattern are observed during propagation of these cells, and they have been successfully carried and differentiated up to passage 49. Histological staining of differentiated cells with Sudan black, Sudan IV, and oil red O indicates the presence of lipids in intracellular vesicles. The nonselective beta-adrenergic agonist isoproterenol stimulates adenylyl cyclase activity in both preadipocytes and differentiated adipocytes. In contrast, BRL-37344, a beta 3-adrenergic receptor-specific agonist, stimulates adenylyl cyclase activity and glycerol release in differentiated adipocytes, but not preadipocytes. In addition, differentiated adipocytes contain messenger RNA encoding the brown adipose-specific protein, thermogenin. Thus, this rat preadipocyte cell line can be differentiated into adipocytes that histologically and functionally resemble brown adipose tissue.
The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-beta gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.
Osteoclast-mediated bone resorption plays a crucial role in osseous remodeling. Osteoblasts are important regulators of this activity, in part through their ability to produce osteoclast-regulating soluble factors such as interleukin-6 (IL-6). IL-11 is a newly appreciated pleotropic cytokine whose spectrum of biological activities overlaps with that of IL-6. As a result, we hypothesized that osteoblasts are an important skeletal source of this cytokine. To test this hypothesis, we characterized the IL-11 production of unstimulated and stimulated SaOS-2 human osteosarcoma cells. Unstimulated cells produced modest amounts of IL-11. The osteotropic agents recombinant IL-1 (0.25-5 ng/ml), transforming growth factor-beta 1 (0.1-10 ng/ml), PTH (10(-8)-10(-11) M), and PTH-related peptide ((10(-8)-10-11 M) further increased SaOS-2 cell IL-11 protein production and messenger RNA accumulation. These stimulatory effects were dose and time dependent, and the IL-11 that was produced was bioactive, as demonstrated by its ability to stimulate the proliferation of T10D plasmacytoma cells. The protein kinase-C activator, 12-O-Tetra-decanoylphorbol 13-acetate, and a variety of cAMP agonists [forskolin, prostaglandin E1, prostaglandin E2, and (Bu)2AMP] also stimulated osteoblast IL-11 protein production and messenger RNA accumulation. In contrast, recombinant IL-4, recombinant interferon-gamma, and endotoxin did not stimulate SaOS-2 cells in a similar fashion. Importantly, the ability to produce IL-11 was not a unique property of SaOS-2 cells, because primary human trabecular bone osteoblasts also produced significant amounts of bioactive IL-11 when stimulated with transforming growth factor-beta 1. These studies demonstrate that appropriately stimulated human osteoblasts and osteoblast-like cells are potent producers of IL-11 and suggest that osteoblast-derived IL-11 may be an important component of the cytokine network mediating osteoblast-osteoclast communication in normal and pathological bone remodeling.
The bone marrow stroma consists of a heterogeneous population of cells which participate in osteogenic, adipogenic, and hematopoietic events. The murine stromal cell line, BMS2, exhibits the adipocytic and osteoblastic phenotypes in vitro. BMS2 differentiation was examined in response to cytokines which share the gp130 signal transducing protein within their receptor complex. Four of the cytokines (interleukin 6, interleukin 11, leukemia inhibitory factor, and oncostatin M) inhibited hydrocortisone-induced adipocyte differentiation in a dose dependent manner based on lipid accumulation and lipoprotein lipase enzyme activity. Inhibition occurred only when the cytokines were present during the initial 24 h of the induction period; after 48 h their effects were diminished. Likewise, these cytokines increased alkaline phosphatase enzyme activity twofold in preadipocyte BMS2 cells. Both leukemia inhibitory factor and oncostatin M induced early active gene expression in resting preadipocyte BMS2 cells and decreased the steady state mRNA level of a unique osteoblastic gene marker, osteocalcin. A fifth cytokine whose receptor complex shares the gp130 protein, ciliary neurotrophic factor, did not significantly regulate stromal cell differentiation when added by itself. However, with the addition of a missing component of its receptor complex, ciliary neurotrophic factor receptor alpha protein, this cytokine also inhibited BMS2 adipogenesis. Together, these data indicate that the cytokines whose receptors share the gp130 protein can modulate stromal cell commitment to the adipocyte and osteoblast differentiation pathways.
IL-11 and IL-6 are fibroblast-derived cytokines with overlapping biologic properties. To determine whether IL-11 and IL-6 are similarly regulated, we characterized the effects of rIL-1 and TGF-beta (beta 1 and beta 2) on human lung fibroblast IL-11 production and compared this regulation with that of IL-6. Unstimulated fibroblasts did not produce significant amounts of IL-11, whereas rIL-1 alpha and TGF-beta were dose-dependent stimulators of IL-11 protein production, mRNA accumulation, and gene transcription. rIL-1 alpha and TGF-beta also interacted in a synergistic fashion to further increase IL-11 protein production and mRNA accumulation. The effects of rIL-1 and TGF-beta individually were not altered by the cyclic nucleotide-dependent protein kinase inhibitor HA1004, protein kinase C (PKC) inhibition with staurosporine, or chronic phorbol ester preincubation, or the calmodulin antagonists W7 and TFP. The effects of rIL-1 alpha and TGF-beta in combination were also unaltered by HA1004, staurosporine, and chronic phorbol ester exposure. A23187, however, did induce IL-11 mRNA accumulation and W7 and TFP did reverse the synergistic stimulation caused by rIL-1 and TGF-beta in combination. In contrast with the regulation of IL-11, TGF-beta did not effectively stimulate IL-6 mRNA accumulation, rIL-1 alpha was a more potent stimulator of IL-6 than IL-11 production, and rIL-1-induced IL-6 mRNA accumulation was augmented by W7 and TFP. These studies demonstrate that: 1) rIL-1, TGF-beta, and agents that increase intracellular calcium stimulate lung fibroblast IL-11; 2) the IL-11 stimulatory effects of rIL-1 and TGF-beta are, at least partially, transcriptionally mediated and are the result of signal transduction pathways that are largely PKC, cyclic nucleotide, and calmodulin independent; and 3) rIL-1 and TGF-beta interact in a synergistic fashion to further increase fibroblast IL-11 production and that this synergy is mediated by a largely PKC- and cyclic nucleotide-independent and calmodulin-dependent activation pathway. Importantly, they also demonstrate that rIL-1 and TGF-beta stimulate lung fibroblast IL-6 and IL-11 production via distinct and differentially regulatable activation pathways.
Genetic control of bone mass in the spontaneously osteoporotic mouse (SAM-P/6) was examined by classical cross-mating experiments, in which SAM-P/2 with a high bone mass was used as a control. Femoral bone mass corrected by size (= bone density) was assayed photometrically. F1 hybrids exhibited intermediate values between those of the two parental strains, i.e. higher than SAM-P/6 and lower than SAM-P/2. F2 hybrids showed a more widely distributed pattern with a mean value quite similar to that of F1 hybrids. The values for backcrosses were between those of F1 and the respective parental strains. They did not show segregations expected in cases of a single-gene control. Numerical analysis of data distribution in each generation suggested that strain-specific bone mass in these strains of mice is inherited as a polygenic characteristic and controlled by a relatively small number of genes.
IL-11 is a novel cytokine with a variety of biofunctions which overlap with those of IL-6. We have previously identified IL-11 specific binding protein which is distinct from that of IL-6 in a number of cell lines. The similarities in biofunctions and differences in ligand binding proteins between IL-11 and IL-6 prompted us to investigate whether IL-11 shares common signal transduction mechanisms with IL-6. We have examined early signals triggered by IL-11 or IL-6 in a multifactor-dependent human erythroleukemic cell line, TF-1. The results showed that IL-11 and IL-6 can both stimulate cell proliferation, induce similar pattern of protein tyrosine phosphorylation, and activate the same proto-oncogene (junB) expression in TF-1 cells. These findings imply that IL-11 and IL-6 share similar early signaling events with the possibility of using the same signal transducer, gp130. We next tested whether IL-11 induced signaling can be inhibited by anti-gp130 antibodies which blocked IL-6-mediated functions. It was observed that anti-gp130 antibodies abolished cell proliferation, protein tyrosine phosphorylation, and junB gene expression elicited by IL-11 or IL-6 in TF-1 cells. The same antibodies, however, had no effect on granulocyte-macrophage colony stimulating factor or erythropoietin-induced TF-1 cell proliferation. Finally, anti-IL-6R antibody inhibited the ability of IL-6, but not IL-11, to transduce early signals in TF-1 cells. These results demonstrate that IL-11 and IL-6 utilize different ligand binding proteins, but share common signal transducer, gp130, in TF-1 cells.
We have examined the effects of recombinant human interleukin-11 (rhIL-11) on the recovery of peripheral blood cell counts and proliferation of progenitors and hematopoietic stem cells (day 12 colony-forming units-spleen-CFU-S12) in vivo using a mouse bone marrow (BM) and spleen cell transplantation model. Recovery of leukocytes was accelerated in animals receiving daily administration of rhIL-11 (100 micrograms/kg/d) and reached normal levels by day 14 posttransplantation. This increased total leukocyte count reflected mainly an increase in neutrophils. Neutropenia (absolute neutrophil count [ANC] < 1,500) was present in control transplant mice for 14 to 15 days, while in the rhIL-11-treated group, neutrophils recovered to normal by days 8 to 10 and continued to increase until day 19. Animals treated with rhIL-11 had only 1 day with ANC demonstrated < 500. Correspondingly, rhIL-11 treatment increased granulocyte-macrophage progenitors (CFU-GM) derived from both spleen and BM cells. Higher doses of IL-11 increased CFU-GM nearly threefold and CFU-Mix fourfold to fivefold, while increasing burst-forming units-erythroid to a lesser degree. BM and spleen cellularity were both increased in IL-11-treated mice, but no increase in CFU-S12 was noted. In addition, in vivo daily administration of IL-11 increased peripheral platelet counts by threefold over control transplant mice at day 10 posttransplantation during the post-irradiation platelet nadir. Further treatment led to platelet counts higher than normal 18 days posttransplantation when control animals had just attained normal platelet counts. IL-11 can accelerate the recovery of the peripheral blood leukocytes, mainly neutrophils, and platelets in transplant mice, effects that may be clinically useful in future applications for BM transplantation and chemotherapy-related cytopenias.
Bone marrow is the principal site for osteoclastogenesis and osteoblastogenesis; and an increase in the former has been linked with bone loss caused by acute loss of gonadal steroids. We have now used an established murine model of accelerated senescence and osteopenia (SAMP6) to test the hypothesis that reduced osteoblastogenesis is linked with decreased bone mass. At 1 mo of age, the number of osteoblast progenitors in SAMP6 marrow was indistinguishable from controls; however a threefold decrease was found at 3-4 mo of age. Impaired osteoblast formation was temporally associated with decreased bone formation and decreased bone mineral density, as determined by histomorphometric analysis of tetracycline-labeled cancellous bone and dual-energy x-ray absorptiometry, respectively. Osteoclastogenesis determined in ex vivo bone marrow cultures was also decreased in these mice, as was the number of osteoclasts in histologic sections. Moreover, unlike controls, senescence-accelerated mice failed to increase osteoclast development after gonadectomy. The osteoclastogenesis defeat was secondary to impaired osteoblast formation as evidenced by the fact that osteoclastogenesis could be restored by addition of osteoblastic cells from normal mice. These findings provide the first demonstration of a link between low bone mineral density and decreased osteoblastogenesis in the bone marrow and validate the senescence-accelerated mouse as a model of involutional osteopenia.
Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the alpha-chain (IL-11R alpha). Two genes potentially encode the IL-11R alpha: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11R alpha, we have generated mice with a null mutation of IL11Ra (IL11Ra-/-) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra-/- bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra-/- mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra-/- mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra-/- and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra-/ - and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.
Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. However, little is known about the interrelationship between these two processes. We have previously shown that the senescence accelerated mouse-P6 (SAMP6) exhibits decreased osteoblastogenesis in the bone marrow that is temporally linked with a low rate of bone formation and decreased bone mineral density. Here we report that in contrast to decreased osteoblastogenesis, ex vivo bone marrow cultures from SAMP6 mice exhibited an increase in the number of colony-forming unit adipocytes, as well as an increase in the number of fully differentiated marrow adipocytes, compared with SAMR1 (nonosteopenic) controls. Further, long-term bone marrow cultures from SAMP6 produced an adherent stromal layer more rapidly, generated significantly more myeloid progenitors and produced more IL-6 and colony-stimulating activity. Consistent with this, the number of myeloid cells in freshly isolated marrow from SAMP6 mice was increased, as was the number of granulocytes in peripheral blood. The evidence that SAMP6 mice exhibit decreased osteoblastogenesis, and increased adipogenesis and myelopoiesis, strongly suggests that a switch in the differentiation program of multipotential mesenchymal progenitors may underlie the abnormal phenotype manifested in the skeleton and other tissues of these animals. Moreover, these observations support the contention for the existence of a reciprocal relationship between osteoblastogenesis and adipogenesis that may explain the association of decreased bone formation and the resulting osteopenia with the increased adiposity of the marrow seen with advancing age in animals and humans.
Identity of PCR products was confirmed by fluorescence-based dideoxy sequencing of each PCR product using an ABI PRISM 310 genetic analyzer (Perkin-Elmer Corp A set of primers for PCR of perox-isome proliferator activator receptor-␥2 (PPAR␥2) was 5Ј
- Norwalk
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Identity of PCR products was confirmed by fluorescence-based dideoxy sequencing of each PCR product using an ABI PRISM 310 genetic analyzer (Perkin-Elmer Corp., Norwalk, CT, U.S.A.). A set of primers for PCR of perox-isome proliferator activator receptor-␥2 (PPAR␥2) was 5Ј-GTG AAC CAC TGA TAT TCA GG-3Ј as 5Ј primer and 5Ј-GGG TCA GCT CTT GTG AAT GG-3Ј as 3Ј primer. REFERENCES
Hill MR 1994 Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 protein
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Gimble JM, Wanker F, Wang CS, Bass H, Wu X, Kelly K, Yancopoulos GD, Hill MR 1994 Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 protein. J Cell Biochem 54:122–133.
First Department of Internal Medicine University of Tokushima School of Medicine 3–18 –15 Kuramotocho Tokushima 770, Japan Received in original form November 18
- Address
- Requests
Address reprint requests to:
Toshio Matsumoto, M.D.
First Department of Internal Medicine
University of Tokushima School of Medicine
3–18 –15 Kuramotocho
Tokushima 770, Japan
Received in original form November 18, 1997; in revised form
April 24, 1998; accepted May 20, 1998.
Interleu- kin-11: A new cytokine critical for osteoclast development
- G Girasole
- G Passeri
- Rl Jilka
- Sc Manolagas
Girasole G, Passeri G, Jilka RL, Manolagas SC 1994 Interleu-
kin-11: A new cytokine critical for osteoclast development.
J Clin Invest 93:1516 –1524.
Interleukin-11: A new cytokine critical for osteoclast development
- Girasole