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Wnt but Not BMP Signaling Is Involved in the Inhibitory Action of Sclerostin on BMP-Stimulated Bone Formation
Abstract
Sclerostin is an osteocyte-derived negative regulator of bone formation. It inhibits BMP-stimulated bone formation both in vitro and in vivo but has no direct effect on BMP signaling. Instead, sclerostin inhibits Wnt signaling that is required for BMP-stimulated osteoblastic differentiation.
Sclerostin is a member of the Dan family of glycoproteins of which many members have been reported to antagonize BMP activity. Sclerostin has been shown to inhibit BMP-stimulated bone formation, but its mechanism of action seems to be different from classical BMP antagonists. In this study, we investigated the mechanism by which sclerostin inhibits BMP-stimulated bone formation.
DNA electroporation of calf muscle of mice using expression plasmids for BMP and sclerostin was used to study the effect of sclerostin on BMP-induced bone formation in vivo. Transcriptional profiling using microarrays of osteoblastic cells treated with BMP in the absence or presence of sclerostin was used to find specific growth factor signaling pathways affected by sclerostin. The affected pathways were further studied using growth factor-specific reporter constructs.
BMP-induced ectopic bone formation in calf muscle of mice was prevented by co-expression of sclerostin in vivo. Transcriptional profiling analysis of osteoblastic cultures indicated that sclerostin specifically affects BMP and Wnt signaling out of many other growth signaling pathways. Sclerostin, however, did not inhibit stimulation of direct BMP target genes. Furthermore, we did not obtain any evidence for sclerostin acting as a direct BMP antagonist using a BMP-specific reporter construct. In contrast, sclerostin shared many characteristics with the Wnt antagonist dickkopf-1 in antagonizing BMP-stimulated bone formation and BMP- and Wnt-induced Wnt reporter construct activation.
Sclerostin inhibits BMP-stimulated bone formation but does not affect BMP signaling. Instead, it antagonizes Wnt signaling in osteoblastic cells. High bone mass in sclerosteosis and van Buchem disease may, therefore, result from increased Wnt signaling.
... Sclerostin (SOST) is a glycoprotein that plays an important role in bone formation and the regulation of osteoblastic activity [8] . In mammals, SOST is mainly expressed in mature osteocytes where SOST plays a role in bone regulation [9] . However, SOST mRNA has also been found in the heart, liver, and kidney although the function in these tissues is not known [9] . ...
... In mammals, SOST is mainly expressed in mature osteocytes where SOST plays a role in bone regulation [9] . However, SOST mRNA has also been found in the heart, liver, and kidney although the function in these tissues is not known [9] . In addition, SOST has been proved to inhibit BMP-stimulated bone formation through antagonizes Wnt signaling in osteoblastic cells [10] . ...
... In addition, SOST has been proved to inhibit BMP-stimulated bone formation through antagonizes Wnt signaling in osteoblastic cells [10] . Analysis of SOST knockout mice revealed an inhibitory action of SOST on Wnt signaling in both osteoblasts and osteocytes [9] . ...
Previous studies have demonstrated that sclerostin (SOST) binds to and antagonizes the activity of bone morphogenetic proteins (BMPs). Because of the potential role of BMPs in intermuscular bone (IB) development , we explored the relationship between SOST and IB development. In this study, we identified a cDNA sequence encoding a SOST homologue in barbel steed (Hemibarbus labeo). Sequence analysis revealed that the SOST protein is composed of a signal peptide and a mature peptide which contained a C-terminal cystine knot-like domain. Phylogenetic tree analysis indicated that barbel steed SOST was closely related to goldfish (Carassius auratus) SOST. Expression of SOST transcripts were detected in all the tested tissues, with the highest transcript levels being found in the gill. in situ RNA hybridization results showed that SOST mRNA was obviously distributed in the myosepta. RT-qPCR showed that the transcript levels of the SOST gene significantly changed during the four developmental stages of IBs. This timing overlapped with IB formation, providing support for a role of SOST in IB development.
... Sclerostin expression can be modulated by multiple factors such as mechanical stress, oxygen, hormones, and transcription factors [14][15][16][17]. Sclerostin potently inhibits bone formation by regulating the proliferation, differentiation, mineralization, and apoptosis of preosteoblastic cells and osteoblasts [18][19][20]. Sclerostin deficiency causes high bone mass in humans and experimental animals [3,9,12]. In contrast, sclerostin overexpression in mice causes a remarkable reduction in bone mass and volume [18,21]. ...
... The Wnt proteins are crucial for skeletal formation and development. Sclerostin competitively binds to the lowdensity lipoprotein receptor protein 5/6, decreasing its combination with Wnt proteins and Frizzled receptor, blocking the activation of Wnt/β-catenin pathway and consequently inhibiting bone formation ( Fig. 1) [19,23,24]. Moreover, several studies have proposed that sclerostin also plays an essential role in bone resorption. ...
Sclerostin is the protein product of the SOST gene and is known for its inhibitory effects on bone formation. The monoclonal antibody against sclerostin has been approved as a novel treatment method for osteoporosis. Oral health is one of the essential aspects of general human health. Hereditary bone dysplasia syndrome caused by sclerostin deficiency is often accompanied by some dental malformations, inspiring the therapeutic exploration of sclerostin in the oral and dental fields. Recent studies have found that sclerostin is expressed in several functional cell types in oral tissues, and the expression level of sclerostin is altered in pathological conditions. Sclerostin not only exerts similar negative outcomes on the formation of alveolar bone and bone-like tissues, including dentin and cementum, but also participates in the development of oral inflammatory diseases such as periodontitis, pulpitis, and peri-implantitis. This review aims to highlight related research progress of sclerostin in oral cavity, propose necessary further research in this field, and discuss its potential as a therapeutic target for dental indications and regenerative dentistry.
... Therefore, secretion of RANKL by osteocytes increases bone resorption. Sclerostin is a glycoprotein that causes inhibition of osteoblast precursor differentiation and bone formation [34][35][36]. Sclerostin is an important inhibitor of the Wnt/β-catenin signaling pathway [37,38]. Wnt proteins are able to bind to the low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and the co-receptor Frizzled (FZD) [39]. ...
... Sclerostin competes with Wnt for binding to LRP5/6, as sclerostin replaces the Wnt proteins that are bound to LRP5/6 [39]. This in turn leads to inactivation of the Wnt/β-catenin signaling pathway [34,35]. Furthermore, it is suggested that sclerostin increases bone resorption via regulation of RANKL [42]. ...
Osteoporosis is a highly prevalent bone disease affecting more than 37.5 million individuals in the European Union (EU) and the United States of America (USA). It is characterized by low bone mineral density (BMD), impaired bone quality, and loss of structural and biomechanical properties, resulting in reduced bone strength. An increase in morbidity and mortality is seen in patients with osteoporosis, caused by the approximately 3.5 million new osteoporotic fractures occurring every year in the EU. Currently, different medications are available for the treatment of osteoporosis, including anti-resorptive and osteoanabolic medications. Bisphosphonates, which belong to the anti-resorptive medications, are the standard treatment for osteoporosis based on their positive effects on bone, long-term experience, and low costs. However, not only medications used for the treatment of osteoporosis can affect bone: several other medications are suggested to have an effect on bone as well, especially on fracture risk and BMD. Knowledge about the positive and negative effects of different medications on both fracture risk and BMD is important, as it can contribute to an improvement in osteoporosis prevention and treatment in general, and, even more importantly, to the individual's health. In this review, we therefore discuss the effects of both osteoporotic and non-osteoporotic medications on fracture risk and BMD. In addition, we discuss the underlying mechanisms of action.
... Furthermore, mice with the LRP5 G171V mutation exhibit higher bone mass and bone strength than wild-type mice, which is associated with enhanced bone anabolism mediated by increased osteoblast activity and survival (Gong et al., 2001;Babij et al., 2003). Taken together, the specific mechanism by which LRP5, an important molecule in the Wnt signaling pathway, regulates bone mass by disrupting endogenous LRP antagonists such as DKK1 or sclerostin leads to increased Wnts signaling activity and the ability to stimulate osteogenesis (Balemans et al., 2007;van Bezooijen et al., 2007;Li et al., 2008), further solidifying the central role of LRP5 in Wnt pathwaymediated bone metabolism. Several potential targets for regulating bone mass through the Wnt pathway have been identified, such as antibodies to sclerostin and/or DKK1 (Li et al., 2011), NELL1 (James et al., 2015), Lithium (Amirhosseini et al., 2018), and Sirtuins (Zainabadi et al., 2017). ...
Background: The role of the Wnt pathway in bone and its targets in skeletal disease has garnered interest, but the field lacks a systematic analysis of research. This paper presents a bibliometric study of publications related to the Wnt signaling pathway in bone to describe the current state of study and predict future outlooks.
Methods: All relevant articles and reviews from 1993 to 2022 were collected from the Web of Science Core Collection (WoSCC). Bibliometric analysis and visualization were performed using CiteSpace 6.1 R3, VOSviewer 1.6.15, and the Online Analysis Platform of Literature Metrology ( http://bibliometric.com/ ).
Results: A total of 7,184 papers were retrieved, authored by 28,443 researchers from 89 countries/regions and published in 261 academic journals. The annual publication numbers peaked in 2021. China and United States are the leading countries, with the University of California and Harvard University as the most active institutions. Wang, Yang is the most prolific author. Bone has the most published research, while Proceedings of the National Academy of Sciences of the United States is the most cited journal on average. The main keywords include expression, Wnt, osteoporosis, bone, and osteogenic differentiation. Current and developing research hotspots focus on bone mass, sclerostin antibody, multiple myeloma, and cartilage development.
Conclusion: This paper provides new insights for researchers to delve into the mechanisms of Wnt and bone related diseases and translate into clinical studies. It reveals the development and future research trends in Wnt and skeletal-related studies.
... 10,13 Sclerostin binds to low-density lipoprotein receptor protein (LRP) 5/LRP6 complex, thus blocking Wnt binding and Wnt/β-catenin signaling. [14][15][16] The significance of sclerostin in bone metabolism has been clarified by recent in vitro and in vivo studies. 1,2,6,11,[17][18][19][20] Sclerostin is known to affect periodontal health and disease status. ...
Background:
Sclerostin, a glycoprotein, plays a key role in regulating bone mass. In this study, sclerostin levels in the gingival crevicular fluid (GCF) were assessed in patients with Stage III Grade C generalized periodontitis (SIII-GC) and Stage III Grade B generalized periodontitis (SIII-GB).
Methods:
This cross-sectional study included 30 participants divided equally into three groups: group I (gingival health), group II (SIII-GC), and group III (SIII-GB). Clinical periodontal parameters like plaque index (PI), gingival bleeding index (GBI), probing pocket depth (PD), and clinical attachment level (CAL) were recorded. A sandwich ELISA was used to determine the sclerostin levels in GCF samples. One-way ANOVA and post hoc Tukey tests were used to analyze the clinical parameters and GCF sclerostin levels. The association between GCF sclerostin levels and periodontal parameters was assessed using Pearson's correlation coefficient (r).
Results:
Patients in groups II and III had much higher sclerostin levels in their GCF than in group I (P≤0.05). In contrast, no significant difference in sclerostin levels was observed between the two diseased conditions (P=0.841). Concerning periodontal parameters, a statistically significant difference was observed between the three groups. There was a positive correlation between the periodontal clinical parameters and the expression levels of sclerostin in GCF (P≤0.05).
Conclusion:
Increased expression of sclerostin in GCF in patients with periodontitis indicated that it could be considered a reliable biomarker of periodontal disease activity.
... The importance of Wnt signaling in bone formation was originally discovered in the context of a rare autosomal recessive skeletal disorder, sclerosteosis (OMIM #269500), which is characterized by progressive skeletal overgrowth. A homozygous mutation was found in the SOST gene, which encodes an antagonist of Wnt ligands, in individuals with the disorder (Brunkow et al., 2001;van Bezooijen et al., 2007). Osteoporosis-pseudoglioma syndrome (OMIM #259770) is another rare skeletal disorder characterized by osteoporosis, fractures, osteogenesis imperfecta and low mineral density in the bones and retinoblastoma in the eyes. ...
Endochondral ossification contributes to longitudinal skeletal growth. Osteoblasts, which are bone-forming cells, appear close to terminally differentiated hypertrophic chondrocytes during endochondral ossification. We established mice with conditional knockout (cKO) of Smad4, an essential co-activator for transforming growth factor β family signaling. The mice showed a marked increase in bone volume in the metaphysis as a result of increased bone formation by osteoblasts, in which β-catenin, an effector of canonical Wnt signaling, accumulated. We identified Wnt7b as a factor with increased expression in growth plate cartilage in Smad4 cKO mice. Wnt7b mRNA was expressed in differentiated chondrocytes and suppressed by BMP4 stimulation. Ablation of Wnt7b blunted the increase in bone in adult Smad4 cKO mice and reduced skeletal growth in juvenile mice. Overall, we conclude that Wnt7b is a crucial factor secreted from hypertrophic chondrocytes to initiate endochondral ossification. These results suggest that Smad4-dependent BMP signaling regulates the Wnt7b-β-catenin axis during endochondral ossification.
... Moreover, it is a negative key regulator of osteoblastic functions [6]. It inhibits osteoblast differentiation and bone formation by inhibiting the Wnt signaling pathway after binding with LRP5 and 6 (Wntcoreceptor) [7][8][9][10]. ...
Objective: This study reviewed the literature on local or systemic administration of antisclerostin, presenting results associated with osseointegration of dental/orthopedic implants and stimulation of bone remodeling. Materials and Methods: An extensive electronic search was conducted through MED-LINE/PubMed, PubMed Central, Web of Science databases and specific peer-reviewed journals to identify case reports, case series, randomized controlled trials, clinical trials and animal studies comparing either the systemic or local administration of antisclerostin and its effect in osseointegration and bone remodeling. Articles in English and with no restriction on period were included. Results: Twenty articles were selected for a full-text, and one was excluded. Finally, 19 articles were included in the study (16 animal studies and 3 randomized control trials). These studies were divided into two groups, which evaluated (i) osseointegration and (ii) bone remodeling potential. Initially 4560 humans and 1191 animals were identified. At least 1017 were excluded from the studies (981 humans and 36 animals), totaling 4724 subjects who completed (3579 humans and 1145 animals). (a) Osseointegration: 7 studies described this phenomenon; 4 reported bone-implant contact, which increased in all included studies. Similar results were found for bone mineral density, bone area/volume and bone thickness. (b) Bone remodeling: 13 studies were used for description. The studies reported an increase in BMD with sclerostin antibody treatment. A similar effect was found for bone mineral density/area/volume, trabecular bone and bone formation. Three biomarkers of bone formation were identified: bone-specific alkaline phosphatase (BSAP), osteocalcin and procollagen type 1 N-terminal Pro-peptide (P1NP); and markers for bone resorption were: serum C-telopeptide (sCTX), C-terminal telopeptides of type I collagen (CTX-1), β-isomer of C-terminal telopeptides of type I collagen (β-CTX) and tartrate-resistant acid phosphatase 5b (TRACP-5b). There were limitations: low number of human studies identified; high divergence in the model used (animal or human); the variance in the type of Scl-Ab and doses of administration; and the lack of reference quantitative values in the parameters analyzed by authors' studies (many articles only reported qualitative information). Conclusion: Within the limitations of this review and carefully observing all data, due to the number of articles included and the heterogeneity existing, more studies must be carried out to better evaluate the action of the antisclerostin on the osseointegration of dental implants. Otherwise, these findings can accelerate and stimulate bone remodeling and neoformation.
... TOP-Wnt-induced luciferase reporter assay. To study the role of sclerostin and its loops in regulating Wnt signaling pathway and the inhibitory effect of aptscl56 to sclerostin's antagonistic effect on Wnt signaling pathway for OI, a TOP-Wnt induced luciferase reporter assay was used in the primary osteoblasts from Col1a2 +/G610C mice [51,52]. The primary osteoblasts (OI) were seeded in 24-well plates and were transfected with corresponding reporter plasmids, Wnt3a plasmid and sclerostin plasmids (FL SOST or truncated SOST) as necessary in the following day. ...
Rationale: Sclerostin inhibition demonstrated bone anabolic potential in osteogenesis imperfecta (OI) mice, whereas humanized therapeutic sclerostin antibody romosozumab for postmenopausal osteoporosis imposed clinically severe cardiac ischemic events. Therefore, it is desirable to develop the next generation sclerostin inhibitors to promote bone formation without increasing cardiovascular risk for OI.
Methods and Results: Our data showed that sclerostin suppressed inflammatory responses, prevented aortic aneurysm (AA) and atherosclerosis progression in hSOSTki.Col1a2+/G610C.ApoE-/- mice. Either loop2&3 deficiency or inhibition attenuated sclerostin's suppressive effects on expression of inflammatory cytokines and chemokines in vitro, whilst loop3 deficiency maintained the protective effect of sclerostin on cardiovascular system both in vitro and in vivo. Moreover, loop3 was critical for sclerostin's antagonistic effect on bone formation in Col1a2+/G610C mice. Accordingly, a sclerostin loop3-specific aptamer aptscl56 was identified by our lab. It could recognize both recombinant sclerostin and sclerostin in the serum of OI patients via targeting loop3. PEG40k conjugated aptscl56 (Apc001PE) demonstrated to promote bone formation, increase bone mass and improve bone microarchitecture integrity in Col1a2+/G610C mice via targeting loop3, while did not show influence in inflammatory response, AA and atherosclerosis progression in Col1a2+/G610C.ApoE-/- mice with Angiotensin II infusion. Further, Apc001PE had no influence in the protective effect of sclerostin on cardiovascular system in hSOSTki.Col1a2+/G610C.ApoE-/- mice, while it inhibited the antagonistic effect of sclerostin on bone formation in hSOSTki.Col1a2+/G610C mice via targeting loop3. Apc001PE was non-toxic to healthy rodents, even at ultrahigh dose. Apc001PE for OI was granted orphan drug designation by US-FDA in 2019 (DRU-2019-6966).
Conclusion: Sclerostin loop3-specific aptamer Apc001PE promoted bone formation without increasing cardiovascular risk in OI mice.
... BMP I and II receptors inhibit BMP signaling and mineralization in osteoblasts. Sclerostin is an antagonist of BMPs; however, its mechanism of action remains unclear (Van Bezooijen et al. 2007). The osteoinductivity of BMP-2 is well documented (Wei et al. 2019). ...
We investigated the effects of caffeic acid phenethyl ester (CAPE) and low-dose doxycycline (LDD) on sclerostin and bone morphogenic protein (BMP)-2 expression in experimental periodontitis. We used male rats in groups as follows: control group (C), periodontitis + CAPE group (PC), periodontitis + LDD group (PD), periodontitis + LDD + CAPE group (PCD) and periodontitis group (P). We administered 10 µmol/kg/day CAPE by an intraperitoneal (i.p.) injection and 10 mg/kg/day LDD by oral gavage. Histopathological changes among groups were evaluated and compared. Sclerostin and BMP-2 expression was analyzed using immunohistochemistry. LDD and/or CAPE treatment ameliorated pathology. The highest sclerostin and lowest BMP-2 expressions were found in P group. Group PC exhibited the highest BMP-2 expression scores and the most significant improvement among the treatment groups. The lowest sclerostin expression was observed in the PD group. We found that preventing sclerostin activity may be a useful treatment alternative for bone resorption, especially in cases of periodontitis and peri-implantitis. We found that CAPE and/or LDD may act as anti-sclerostin agents.
... Sclerostin and SOSTdc1 share 55% homology, with a very similar C-terminal domain. Both have been described as inhibitors of the BMP pathway and as WNT antagonists [325,[369][370][371][372][373][374]. ...
The Wnt pathway is involved in several processes essential for bone development and homeostasis. For proper functioning, the Wnt pathway is tightly regulated by numerous extracellular elements that act by both activating and inhibiting the pathway at different moments. This review aims to describe, summarize and update the findings regarding the extracellular modulators of the Wnt pathway, including co-receptors, ligands and inhibitors, in relation to bone homeostasis, with an emphasis on the animal models generated, the diseases associated with each gene and the bone processes in which each member is involved. The precise knowledge of all these elements will help us to identify possible targets that can be used as a therapeutic target for the treatment of bone diseases such as osteoporosis.
... Продукти активації Wnt-сигнального шляху потрібні для ВМР-індукованої активації кісткової лужної фосфатази в декількох клітинних лініях остеобластів. Отже, СКС надає опосередкований вплив на ВМР-стимульоване утворення кісткової тканини та виступає як негативний регулятор остеобластогенезу [15]. ...
Bones are not inert structures inside the human body; they respond dynamically and with high plasticity to exo- and endogenous factors by changes of their content, structure, characteristics of solidity, etc. This process of skeletal changes known as bone remodeling provides structural integrity of the osseous system and promotes metabolic balance of calcium and phosphorus; remodeling causes resorption of the old or damaged bone followed by the formation of new osseous material. Bone morphogenetic proteins (BMP) constituting a group of morphogenetic signal growth factors (known as cytokines as well) first were described as molecules stimulating formation of the endochondrial osseous tissue. Osteoprotegerin (OPG) is a representative of the super-family of soluble factors to tumor necrosis factor-α (TNF-α) and belongs to secretory low molecular glycoproteins which trans-membrane receptors are located on the surface of osteoblasts, immune cells and precursors of osteoclasts. Transforming growth factor-β1 (TGFβ1) is a representative of cytokines protein in nature, released into the intercellular matrix by the osseous tissue cells and macrophages. It controls a life cycle of cells from the osteoid line, that is, their proliferation, cellular differentiation and functional activity. Sclerostin is produced by osteocytes, mineralized hypertrophic chondrocytes and cementocytes (dental cells) only. It belongs to the components of DAN glycoprotein family (differential screening-selected aberrant genes of neuroblastoma).
... The high affinity binding to heparin (K D~3 6-77 n M) is chain length dependent, with preferred binding to full length heparin or large oligosaccharides (octadecasaccharide) and is enhanced by higher sulphation levels [100]. SOST acts as both as inhibitor of BMP [101] and Wnt/ b-catenin [102] signal transduction, but its interaction with heparin/ HS may facilitate a connection between both pathways. For instance, it was found that osteoblastic HS controls bone remodelling by regulating Wnt signalling and the crosstalk between bone surface and marrow cells [103]. ...
Bone morphogenic proteins (BMPs) are important growth regulators in embryogenesis and postnatal homeostasis. Their tight regulation is crucial for successful embryonal development as well as tissue homeostasis in the adult organism. BMP inhibition by natural extracellular biologic antagonists represents the most intensively studied mechanistic concept of BMP growth factor regulation which was shown to be critical for numerous developmental programs, including germ layer specification and spatiotemporal gradients required for the establishment of the dorsal-ventral axis and organ formation. The importance of BMP antagonists for extracellular matrix homeostasis is illustrated by the numerous human connective tissue disorders caused by their mutational inactivation. Thereby, we will also focus on the known functional interactions targeting BMP antagonists to the ECM and how these interactions influence BMP antagonist activity. Here, we will provide an overview about the current concepts and investigated molecular mechanisms modulating BMP inhibitor function in the context of development and disease.
... In addition, although sclerostin could bind to BMPs in vitro, the binding affinities were weak (26,29). In order to address the unclear mechanism by which sclerostin antagonizes BMPs, van Bezooijen et al. discovered that sclerostin exerted its function through blocking Wnt signaling pathway but not acting as a BMPs antagonist (30). Mechanically, sclerostin was proven to inhibit Wnt signaling pathway through binding competitively to Wnt co-receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) (4,5). ...
Wnt signaling is one of the fundamental pathways that play a major role in almost every aspect of biological systems. In addition to the well-known influence of Wnt signaling on bone formation, its essential role in the immune system also attracted increasing attention. Sclerostin, a confirmed Wnt antagonist, is also proven to modulate the development and differentiation of normal immune cells, particularly B cells. Aptamers, single-stranded (ss) oligonucleotides, are capable of specifically binding to a variety of target molecules by virtue of their unique three-dimensional structures. With in-depth study of those functional nucleic acids, they have been gradually applied to diagnostic and therapeutic area in immune diseases due to their various advantages over antibodies. In this review, we focus on several issues including the roles of Wnt signaling and Wnt antagonist sclerostin in the immune system. For the sake of understanding, current examples of aptamers applications for the immune diseases are also discussed. At the end of this review, we propose our ideas for the future research directions.
... MiR-21 is robustly expressed in osteoblasts and our results show a novel role for miR-21 in controlling the expression of SOST and thereby in the regulation of adult bone mass. MiR-21KO mice have activated SOST expression leading to the secretion of SOST, which inhibits osteoblast-mediated bone formation [33,34]. ...
MicroRNAs are emerging as critical post-transcriptional modulators in bone remodeling, regulating the functions of osteoblasts and osteoclasts. Intercellular crosstalk between osteoblasts and osteoclasts is mediated by miR-21 that controls the bone homeostasis response, providing potential targets for the maintenance of osteoblast function. The aim of this study was to investigate the effects of miR-21 on osteoblast function, and to explore the underlying mechanism. Increased alkaline phosphatase (ALP) activity and accelerated matrix mineralization was observed in mouse pre-osteoblast MC3T3-E1 cells compared with the non-induction (control) group. MiR-21 positively regulates osteogenic differentiation and mineralization by facilitating the expression of key osteo-genic factors (ALP, Runx2, Osteopontin (OPN), Osterix (OSX) and Mef2c) in MC3T3-E1 cells. Furthermore, a deficiency of miR-21 suppresses the expression of those factors at both the mRNA and protein levels, indicating that miR-21 is a positive regulator of osteoblastic differentiation. HE staining, Azan staining, Masson's Tri-chrome staining and Toluidine blue staining were performed in jaw and femur tissues of miR-21 knockout (miR-21KO) and wild-type (WT) mice. Immunohistochemical staining revealed substantially lower levels of ALP, Runx2 and OSX expression in jaw and femur tissues of miR-21KO mice. A similar trend was observed in femur tissues using quantitative real-time (RT) PCR. A total of 17 osteogenesis-related mRNAs were found to be differentially expressed in miR-21KO femur tissues using Mouse Gene Expression Microarray analysis. Gene-Spring and Ingenuity Pathway Analysis revealed several potential target genes that are involved in bone remodeling, such as IL-1β and HIF-1α. Several important pathways were determined to be facilitators of miR-21, which provides a reliable reference for future studies to elucidate the biological mechanisms of osteoblast function. Taken together, these results lead us to hypothesize a potential role for miR-21 in regulating osteoblast function, thus representing a potential biomarker of osteogenesis.
... GSK-3β is mostly known for its inhibitory role in the Wnt signaling pathway, in which it phosphorylates β-catenin which targets the latter for proteasomal degradation and thereby prevents Wnt signaling [14]. To confirm this in our cells, PC-3 cells were transiently transfected with a bioluminescent canonical Wnt-reporter construct, BAT-Firefly luciferase [19,20], and CAGGSpromotor-Renilla luciferase as a control for transfection efficiency. As expected, GSK-3β inhibition led to a strong, 5-fold, induction of Firefly luciferase/ Renilla luciferase ratio, indicating enhanced Wnt signaling (Fig. 1, left). ...
Cancer cells with stem or progenitor properties play a pivotal role in the initiation, recurrence and metastatic potential of solid tumors, including those of the human prostate. Cancer stem cells are generally more resistant to conventional therapies thus requiring the characterization of key pathways involved in the formation and/or maintenance of this malignant cellular subpopulation.
To this end, we identified Glycogen Synthase Kinase-3β (GSK-3β) as a crucial kinase for the maintenance of prostate cancer stem/progenitor-like cells and pharmacologic inhibition of GSK-3β dramatically decreased the size of this cellular subpopulation. This was paralleled by impaired clonogenicity, decreased migratory potential and dramatic morphological changes. In line with our in vitro observations, treatment with a GSK-3β inhibitor leads to a complete loss of tumorigenicity and a decrease in metastatic potential in preclinical in vivo models. These observed anti-tumor effects appear to be largely Wnt-independent as simultaneous Wnt inhibition does not reverse the observed antitumor effects of GSK-3β blockage. We found that GSK-3β activity is linked to cytoskeletal protein F-actin and inhibition of GSK-3β leads to disturbance of F-actin polymerization. This may underlie the dramatic effects of GSK-3β inhibition on prostate cancer migration. Furthermore, GSK-3β inhibition led to strongly decreased expression of several integrin types including the cancer stem cell-associated α2β1 integrin. Taken together, our mechanistic observations highlight the importance of GSK-3β activity in prostate cancer stemness and may facilitate the development of novel therapy for advanced prostate cancer.
... [1][2][3][4] Sclerostin, coded by the gene SOST, is produced by osteocytes and can inhibit osteoblast activity 5,6 and suppress bone formation by inhibiting Wnt signaling. 7,8 Sclerostin knockout mice show a high bone mass and transgenic mice overexpressing sclerostin in osteocytes develop osteoporosis. 9,10 A previous study revealed that romosozumab treatment increased a circulating marker of bone formation and decreased a marker of bone resorption. 1 Many studies showed that romosozumab treatment reduced vertebral fracture risk and clinical fracture. ...
Anti‐sclerostin monoclonal antibody romosozumab, a treatment for osteoporosis, reduced vertebral fracture risk and clinical fracture. Laser irradiation triggers various effects, including bio‐stimulation, which can induce beneficial therapeutic effects and biological responses. Originally, we performed in vivo experiments to clarify the mechanism of better bone healing in laser‐ablated bone. Here, we evaluated comprehensive and sequential gene expression in Er:YAG laser‐ablated, bur‐drilled, and nontreated control bones, and found laser irradiation suppressed Sost (coding protein: sclerostin) expression in the bone, possibly via stimulation of mechanotransducers. Surprisingly, bio‐stimulation effect of laser suppressed Sost expression in the primary osteogenic cells. Decreased sclerostin expression after laser irradiation was also validated both in vivo and in vitro. In addition, sequential microarray analysis revealed that the gene expression pattern was clearly different at 24 hours after bone ablation between bur‐drilled and laser‐ablated bones. The Hippo signaling pathway was significantly enriched, whereas inflammation‐related pathways were not affected at 6 hours after the laser ablation, indicating that laser irradiation caused mechanical stimulation. Only bur‐drilled bone showed enriched inflammation‐related gene sets and pathways at 24 hours, not in the laser‐ablated bone. Our study suggests that laser irradiation may become a new treatment modality for osteoporosis, by inhibiting sclerostin expression without inducing inflammation.
... In C2C12 cells, Wnt3a was found to enhance the expression of myoD and myogenin and the size of the myotubes and therefore to promote myogenesis (Romero- Suarez et al., 2011). Sclerostin which is a suppressor of the canonical Wnt/β-catenin signal which is mainly produced by osteocytes but also by osteoblasts and impacts osteoblastic differentiation (Semenov et al., 2005;van Bezooijen et al., 2007;Winkler et al., 2003) has thus been identified as a factor which could link bone to muscle during exercise (Kaji, 2013), even if Mo showed that it did not impact C2C12 myogenesis. As a matter of fact, during weight loss in obese adults, the increase in sclerostin levels which occurs in volunteers subjected only to diet was prevented by adding exercise (Armamento-Villareal et al., 2012). ...
... Sclerostin (SOST) is an important regulator of bone homeostasis and is secreted by several cell types, in particular osteocytes (mature mineralized bone cells (OCYs)). This expression inhibits the bone formation through blocking canonical Wnt signaling in osteoblasts (OBs) and preventing OB maturation into OCYs (1)(2)(3)(4). In mice and humans, inhibition of SOST through gene-targeting or monoclonal antibodies results in enhanced Wnt signaling in OBs and increased bone mass (5,6). ...
Purpose of Review
We reviewed recent progress on the role of sclerostin (SOST) and its effects on the immune system in order to summarize the current state of knowledge in osteoimmunology, in regard to hematopoiesis, lymphopoiesis, and inflammation.
Recent Findings
Changes in sclerostin levels affect distinct niches within the bone marrow that support hematopoietic stem cells and B cell development. Sclerostin’s regulation of adipogenesis could also be important for immune cell maintenance with age. Surprisingly, B cell development in the bone marrow is influenced by Sost produced by mesenchymal stem cells and osteoblasts, but not by osteocytes. Additionally, extramedullary hematopoiesis in the spleen and increased pro-inflammatory cytokine levels in the bone marrow are observed in global Sost−/− mice.
Summary
In addition to changes in bone marrow density, sclerostin depletion affects B lymphopoiesis and myelopoiesis, as well as other changes within the bone marrow cavity that could affect hematopoiesis. It is therefore important to monitor for hematopoietic changes in patients receiving sclerostin-depleting therapies.
... SOST reportedly interacts with a number of proteins including bone morphogenetic proteins (BMPs) -2, -4, -5, -6, -7 and low-density lipoprotein receptor-related protein (LRP) -5 and -6 [3][4][5], which are co-receptors in Wnt signaling system. These SOST involved interactions provide the important mechanisms of bone growth and remodeling through the modulation of Wnt/β-catenin signaling [6]. SOST has attracted considerable interest as a target for therapeutics to treat bone diseases, such as osteoporosis and sclerosteosis [1,2]. ...
Sclerostin (SOST) is a glycoprotein having many important functions in the regulation of bone formation as a key negative regulator of Wnt signaling in bone. Surface plasmon resonance (SPR), which allows for a direct quantitative analysis of the label-free molecular interactions in real-time, has been widely used for the biophysical characterization of glycosaminoglycan (GAG)-protein interactions. In the present study, we report kinetics, structural analysis and the effects of physiological conditions (e.g., salt concentrations, Ca2+ and Zn2+concentrations) on the interactions between GAGs and recombinant human (rh) and recombinant mouse (rm) SOST using SPR. SPR results revealed that both SOSTs bind heparin with high affinity (rhSOST-heparin, KD~36 nM and rmSOST-heparin, KD~77 nM) and the shortest oligosaccharide of heparin that effectively competes with full size heparin for SOST binding is octadecasaccharide (18mer). This heparin binding protein also interacts with other highly sulfated GAGs including, disulfated-dermatan sulfate and chondroitin sulfate E. In addition, liquid chromatography-mass spectrometry was used to characterize the structure of sulfated GAGs that bound to SOST.
... In the presence of sclerostin, Wnt-receptor interaction is inhibited, and glycogen synthase kinase 3 phosphorylates β-catenin and targets it for ubiquitination and degradation via the proteasome pathway (Semenov et al., 2005). Sclerostin impacts osteoblastic differentiation (van Bezooijen et al., 2007) and is a factor, which could link bone to muscle during exercise (Kaji, 2013). Sclerostin could function through the Wnt/β-catenin signaling pathway to regulate muscle myogenesis (Huang et al., 2017). ...
... PDL cells have been shown to possess osteoblastic phenotypes for the continued remodeling of alveolar bone (45), and osteoblasts transduce mechanical stimuli into biochemical signals via intracellular signaling cascades, including the Wnt (46), GTPase (47), and Ca 2+ pathways (48). Wnt/ b-catenin signaling plays a key role in osteogenesis by promoting the osteoblastic differentiation and mineralization, and Sost/sclerostin has been reported to mediate mechanical cues in bone by antagonizing Wnt/b-catenin signaling (49,50). Past studies suggested that Osx has the ability to modulate Sost/sclerostin via a negative feedback loop of bone formation through Wnt (51,52). ...
Human periodontal ligament (hPDL) fibroblasts are thought to receive mechanical stress (MS) produced by orthodontic tooth movement, thereby regulating alveolar bone remodeling. However, the role of intracellular calcium ([Ca²⁺]i)‐based mechanotransduction is not fully understood. We explored the MS‐induced [Ca²⁺]i responses both in isolated hPDL fibroblasts and in intact hPDL tissue and investigated its possible role in alveolar bone remodeling. hPDL fibroblasts were obtained from healthy donors' premolars that had been extracted for orthodontic reasons. The oscillatory [Ca²⁺]i activity induced by static compressive force was measured by a live‐cell Ca²⁺ imaging system and evaluated by several feature extraction method. The spatial pattern of cell‐cell communication was investigated by Moran's I, an index of spatial autocorrelation and the gap junction (GJ) inhibitor. The Ca²⁺‐transporting ionophore A23187 was used to further investigate the role of [Ca²⁺]i up‐regulation in hPDL cell behavior. hPDL fibroblasts displayed autonomous [Ca²⁺]i responses. Compressive MS activated this autonomous responsive behavior with an increased percentage of responsive cells both in vitro and ex vivo. The integration, variance, maximum amplitude, waveform length, and index J in the [Ca²⁺]i responses were also significantly increased, whereas the mean power frequency was attenuated in response to MS. The increased Moran's I after MS indicated that MS might affect the pattern of cell‐cell communication via GJs. Similar to the findings of MS‐mediated regulation, the A23187‐mediated [Ca²⁺]i uptake resulted in the up‐regulation of receptor activator of NF‐κB ligand (Rankl) and Sost along with increased sclerostin immunoreactivity, suggesting that [Ca²⁺]i signaling networks may be involved in bone remodeling. In addition, A23187‐treated hPDL fibroblasts also showed the suppression of osteogenic differentiation and mineralization. Our findings suggest that augmented MS‐mediated [Ca²⁺]i oscillations in hPDL fibroblasts enhance the production and release of bone regulatory signals via Rankl/Osteoprotegerin and the canonical Wnt/β‐catenin pathway as an early process in tooth movement–initiated alveolar bone remodeling.—Ei Hsu Hlaing, E., Ishihara, Y., Wang, Z., Odagaki, N., Kamioka, H. Role of intracellular Ca²⁺‐based mechanotransduction of human periodontal ligament fibroblasts. FASEB J. 33, 10409–10424 (2019). www.fasebj.org
... Human SOST gene comprises three exons and two introns, mapping to chromosomal region 17q12-q21. It inhibits osteoblastic bone formation by inhibiting the Wnt signaling pathway, which is critically important for the development and function of osteoblasts [2]. ...
Romosozumab treatment reduces the rate of hip fractures and increases hip bone density, increasing bone formation by inhibiting sclerostin protein. We studied the normal pattern of bone formation and osteocyte expression in the human proximal femur because it is relevant to both anti‐sclerostin treatment effects and fracture. Having visualized and quantified buds of new bone formation in trabeculae, we hypothesized that they would coincide with areas of a) higher mechanical stress and b) low sclerostin expression by osteocytes. In patients with hip fracture, we visualised each bud of active modeling‐based formation, (Forming Minimodeling Structure, FMiS) in trabecular cores taken from different parts of the femoral head. Trabecular bone structure was also measured with high resolution imaging.
More buds of new bone formation (by volume) were present in the higher stress supero‐medial zone (FMiS density, N.FMiS/T.Ar) than lower stress supero‐lateral (p < 0.05), and inferomedial (p < 0.001) regions. There were fewer sclerostin expressing osteocytes close to, or within FMiS. FMiS density correlated with greater amount, thickness, number and connectivity of trabeculae (bone volume BV/TV, r = 0.65, p < 0.0001; bone surface BS/TV, r = 0.47, p < 0.01; trabecular thickness Tb.Th, r = 0.55, p < 0.001; trabecular number Tb.N, r = 0.47, p < 0.01; and connectivity density Conn.D, r = 0.40, p < 0.05) and lower trabecular separation (Tb.Sp, r = −0.56, p < 0.001).
These results demonstrate modeling‐based bone formation in femoral trabeculae from patients with hip fracture as a potential therapeutic target to enhance bone structure.
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Purpose:
To assess the alterations in bone mineral density and bone turnover marker concentrations following the administration of denosumab and romosozumab therapies in patients with osteoporosis.
Methods:
PubMed was searched for studies published until January 28, 2023, that investigated the clinical efficacy and bone turnover marker changes of denosumab and romosozumab in the treatment of osteoporosis, with a minimum follow-up of 3 months in each study. Studies were screened, and data on changes in bone mineral density (BMD), P1NP, and TRACP-5b levels after treatment were extracted and included in the analysis.
Results:
Six studies were analyzed. At 3 months after treatment, the romosozumab group showed greater changes in lumbar BMD and bone turnover markers. BMD of total hip and femoral neck was relatively delayed. Beginning at 6 to 12 months, romosozumab showed greater changes in bone mineral density and markers of bone turnover.
Conclusion:
Both romosozumab and denosumab have antiosteoporotic effects, with greater effects on BMD and bone turnover markers observed within 12 months of romosozumab treatment.
Systematic review registration:
https://www.crd.york.ac.uk/prospero, identifier CRD42023395034.
Değerli okuyucular, Diş hekimliği alanında değerli meslektaşlarımızın dikkat çekici başarılarının yer aldığı son kitabımızın heyecan verici haberini sizlerle paylaşmanın heyecanını yaşıyoruz. Diş hekimliği, disiplinler arası araştırma ve işbirliği gerektiren çok yönlü bir alandır ve kitabımız, çenelerin ilaca bağlı osteonekrozu (mronj), dil hastalıkları ve tedavi yaklaşımları, odontojenik ve gelişimsel çene kistleri, sonlu elemanlar stres analizi yönteminin implant cerrahisinde kullanımı, kemik dokusunun potansiyel biyobelirteçleri, endodontik patolojilerin diş eti oluğu sıvısında bulunan enflamatuar mediyatörlere etkisi, irreversible pulpitisli dişlerde vital pulpa tedavileri, döner eğelerde performans ve dayanıklılığı artırmaya yönelik işlemler gibi diş hekimliğinde geniş bir yelpazeyi kapsamaktadır. Bu kitabı hayata geçirmek için yorulmak bilmez çabaları ve kararlılıkları için seçkin yazarlarımıza ve yayın ekibimize yürekten şükranlarımızı sunmak isteriz. Umudumuz, bu kitabın, insanlığın geleceği için diş hekimliği alanını ilerletmeye çabalayan diğer bilim insanlarına ilham vermesi ve onları aydınlatmasıdır. İçtenlikle, Dr. Öğr. Üy. Fatma Pertek Hatipoğlu
Since the proposal of Paul Ehrlich's magic bullet concept over 100 years ago, tremendous advances have occurred in targeted therapy. From the initial selective antibody, antitoxin to targeted drug delivery that emerged in the past decades, more precise therapeutic efficacy is realized in specific pathological sites of clinical diseases. As a highly pyknotic mineralized tissue with lessened blood flow, bone is characterized by a complex remodeling and homeostatic regulation mechanism, which makes drug therapy for skeletal diseases more challenging than other tissues. Bone-targeted therapy has been considered a promising therapeutic approach for handling such drawbacks. With the deepening understanding of bone biology, improvements in some established bone-targeted drugs and novel therapeutic targets for drugs and deliveries have emerged on the horizon. In this review, we provide a panoramic summary of recent advances in therapeutic strategies based on bone targeting. We highlight targeting strategies based on bone structure and remodeling biology. For bone-targeted therapeutic agents, in addition to improvements of the classic denosumab, romosozumab, and PTH1R ligands, potential regulation of the remodeling process targeting other key membrane expressions, cellular crosstalk, and gene expression, of all bone cells has been exploited. For bone-targeted drug delivery, different delivery strategies targeting bone matrix, bone marrow, and specific bone cells are summarized with a comparison between different targeting ligands. Ultimately, this review will summarize recent advances in the clinical translation of bone-targeted therapies and provide a perspective on the challenges for the application of bone-targeted therapy in the clinic and future trends in this area.
Context:
Female athletes, particularly runners, with insufficient caloric intake for their energy expenditure (low energy availability or relative energy deficiency) are at risk for impaired skeletal integrity. Data are lacking in male runners.
Objective:
To determine whether male runners at risk for energy deficit have impaired bone mineral density (BMD), microarchitecture, and estimated strength.
Design:
Cross-sectional.
Setting:
Clinical research center.
Participants:
39 men (20 runners, 19 controls), ages 16-30y.
Main outcome measures:
Areal BMD (DXA); tibia and radius volumetric BMD and microarchitecture (high resolution peripheral quantitative CT); failure load (microfinite element analysis); serum testosterone, estradiol, leptin; energy availability (EA).
Results:
Mean age (24.5 ± 3.8y), lean mass, testosterone, and estradiol levels were similar; BMI, percent fat mass, leptin, and lumbar spine BMD Z-score (-1.4 ± 0.8 vs. -0.8 ± 0.8) lower (p < 0.05); and calcium intake and running mileage higher (p ≤ 0.01) in runners vs. controls. Runners with EA < median had lower lumbar spine (-1.5 ± 0.7, p = 0.028), while runners with EA ≥ median had higher hip (0.3 ± 0.7 vs. -0.4 ± 0.5, p = 0.002), BMD Z-scores vs. controls. After adjusting for calcium intake and running mileage, runners with EA < median had lower mean tibial total and trabecular volumetric BMD, trabecular bone volume fraction, cortical porosity, and apparent modulus vs. controls (p < 0.05). Appendicular lean mass and serum estradiol (R ≥ 0.45, p ≤ 0.046), but not testosterone, were positively associated with tibial failure load among runners.
Conclusions:
Despite weight-bearing activity, skeletal integrity is impaired in male runners with lower caloric intake relative to exercise energy expenditure, which may increase bone stress injury risk. Lower estradiol and lean mass are associated with lower tibial strength in runners.
Osteogenic differentiation plays important roles in the pathogenesis of osteoporosis. In this study, we explored the regulatory mechanism of histone methyltransferase SET domain bifurcated 1 (SETDB1) underlying the osteogenic differentiation in osteoporosis. The common osteoporosis-related genes were retrieved from the GeneCards, CTD, and Phenolyzer databases. The enrichment analysis was conducted on the candidate osteoporosis-related genes using the PANTHER software, and the binding site between transcription factors and target genes predicted by hTFtarget. The bioinformatics analyses suggested 6 osteoporosis-related chromatin/chromatin binding protein or regulatory proteins (HDAC4, SIRT1, SETDB1, MECP2, CHD7, and DKC1). Normal and osteoporosis tissues were collected from osteoporosis patients to examine the expression of SETDB1. It was found that SETDB1 was poorly expressed in osteoporotic femoral tissues, indicating that SETDB1 might be involved in the development of osteoporosis. We induced SETDB1 overexpression/knockdown, orthodenticle homeobox 2 (OTX2) overexpression, activation of Wnt/β-catenin or BMP-Smad pathways alone or in combination in osteoblasts or ovariectomized mice. The data indicated that SETDB1 methylation regulated H3K9me3 in the OTX2 promoter region and inhibited the expression of OTX2. Besides, the BMP-Smad and Wnt/β-catenin pathways were inhibited by OTX2, thereby resulting in inhibited osteogenic differentiation. Animal experiments showed that overexpressed SETDB1 could promote the increase of calcium level and differentiation of femoral tissues. In conclusion, upregulation of SETDB1 promotes osteogenic differentiation by inhibiting OTX2 and activating the BMP-Smad and Wnt/β-catenin pathways in osteoporosis.
Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. The average lifetime risk of a 50-year-old woman to suffer a fracture of the spine, hip, proximal humerus, or distal forearm has been estimated at close to 50%. In general, pharmacological treatment is recommended in patients who suffered a fragility fracture because their risk of suffering a subsequent fracture is increased dramatically. Therefore, many guidelines recommend pharmacological treatment in patients without a prevalent fracture if their fracture probability is comparable to or higher than that of a person of the same age with a prevalent fracture. The present review aims to highlight currently available pharmacological treatment options and their antifracture efficacy including safety aspects. Drug classes discussed comprise bisphosphonates, selective estrogen receptor modulators, parathyroid hormone peptides and derivatives, humanized monoclonal antibodies, and estrogens and gestagens and their combinations. Furthermore, a brief glimpse is provided into a potentially promising treatment option that involves mesenchymal stem cells.
Purpose
Baduanjin (BDJ) exercise is a traditional exercise that combines breathing, body movement, meditation and awareness to help delay the onset and progression of senile degenerative musculoskeletal diseases, such as osteoporosis (OP). The aim of this meta-analysis is to evaluate the efficacy of BDJ exercise, and preliminarily infer its effective mechanism in the treatment of OP.
Methods
We identified relevant randomized controlled trials (RCTs) through eight databases, and compared BDJ exercise with the control groups (including blank control and conventional treatment intervention). The main outcome measure was bone mineral density (BMD), the additional outcome measures were visual analogue scale (VAS), Berg balance scale (BBS), serum Calcium (Ca), serum Phosphorus (P), serum Alkaline phosphatase (ALP), and serum bone gla protein (BGP). Meta-analysis and trial sequence analysis (TSA) were performed using RevMan 5.4, Stata 16.0, and TSA 0.9.
Results
In total, 13 RCTs involving 919 patients were included in the analysis. For postmenopausal osteoporosis, BDJ exercise alone and BDJ exercise combined with conventional treatment can improve the BMD of lumbar spine. BDJ exercise alone can influence serum Ca and ALP. BDJ exercise combined with conventional treatment can improve balance (BBS) and influence serum BGP. For senile osteoporosis, BDJ exercise alone and BDJ exercise combined with conventional treatment can improve balance (BBS). BDJ exercise combined with conventional treatment can improve the BMD of hip and pain relieve (VAS). For primary osteoporosis, BDJ exercise combined with conventional treatment can improve the BMD of lumbar spine and femoral neck.
Conclusion
Baduanjin exercise may be beneficial to improve BMD, relieve pain, improve balance ability, influence serum BGP and serum ALP in patients with OP, but differences occur due to various types of OP. Due to the low quality of research on the efficacy and mechanism of BDJ exercise in the treatment of OP, high-quality evidence-based research is still needed to provide reliable supporting evidence.
Systematic Review Registration
[ http://www.crd.york.ac.uk/PROSPERO ], identifier [CRD42022329022].
Sclerostin negatively regulates bone formation by antagonizing Wnt signalling. An antibody targeting sclerostin for the treatment of postmenopausal osteoporosis was approved by the U.S. Food and Drug Administration, with a boxed warning for cardiovascular risk. Here we demonstrate that sclerostin participates in protecting cardiovascular system and inhibiting bone formation via different loops. Loop3 deficiency by genetic truncation could maintain sclerostin’s protective effect on the cardiovascular system while attenuating its inhibitory effect on bone formation. We identify an aptamer, named aptscl56, which specifically targets sclerostin loop3 and use a modified aptscl56 version, called Apc001PE, as specific in vivo pharmacologic tool to validate the above effect of loop3. Apc001PE has no effect on aortic aneurysm and atherosclerotic development in ApoE−/− mice and hSOSTki.ApoE−/− mice with angiotensin II infusion. Apc001PE can promote bone formation in hSOSTki mice and ovariectomy-induced osteoporotic rats. In summary, sclerostin loop3 cannot participate in protecting the cardiovascular system, but participates in inhibiting bone formation. Antibodies targeting sclerostin can ameliorate postmenopausal osteoporosis but present some cardiovascular risk. Here the authors show that the cardiovascular and skeletal effects of sclerostin are mediated by different loops, suggesting ways to preserve the positive effects on bone formation while avoiding the negative cardiovascular consequences.
Objectives:
The glycoprotein sclerostin is mostly expressed in osteocytes and plays a central role in human bone metabolism. However, sclerostin and the corresponding SOST gene have been found in periodontal ligament cells under mineralizing conditions as well. The present study aimed to investigate, whether there was a correlation between endogenous SOST expression, the corresponding gene, and mineralization potential in human periodontal ligament cells and to identify different sclerostin expression and secretion patterns in cells derived from individual donors.
Material and methods:
Primary human periodontal ligament cells of three different donors were cultivated under control or mineralizing conditions for 6, 13, 15 and 18 days, respectively. Calcium deposits were stained with alizarin red and quantified afterwards. Quantitative expression analysis of the SOST gene encoding sclerostin was performed using quantitative reverse transcription polymerase chain reaction (RT-PCR). Additionally, intracellular sclerostin expression was analyzed using Western blotting and extracellular sclerostin secretion was quantified using Enzyme-linked Immunosorbent Assay (ELISA).
Results:
Alizarin red staining identified calcium deposits in periodontal ligament cells under mineralizing conditions beginning from day 13, relative SOST expression occurred on day 6. Whereas staining continued to increase in donor 1 on day 15, it remained stable in donors 2 and 3. Conversely, baseline SOST expression was significantly lower in donor 1 compared to donors 2 and 3. Western blotting and ELISA revealed increased intra- and extracellular sclerostin expression at day 13 under mineralizing conditions. Donor 3 exhibited the highest overall sclerostin levels.
Conclusions:
Our data emphasize donor-specific characteristics in differentiation potential and sclerostin expression patterns in primary human periodontal ligament cells. Sclerostin might play a central role in modulating osteogenic differentiation in periodontal ligament cells as part of a negative feedback mechanism in avoiding excessive mineralization.
Osteoporosis (OP) is known as a silent disease in which the loss of bone mass and bone density does not cause obvious symptoms, resulting in insufficient treatment and preventive measures. The losses of bone mass and bone density become more severe over time and an only small percentage of patients are diagnosed when OP-related fractures occur. The high disability and mortality rates of OP-related fractures cause great psychological and physical damage and impose a heavy economic burden on individuals and society. Therefore, early intervention and treatment must be emphasized to achieve the overall goal of reducing the fracture risk. Anti-OP drugs are currently divided into three classes: antiresorptive agents, anabolic agents, and drugs with other mechanisms. In this review, research progress related to common anti-OP drugs in these three classes as well as targeted therapies is summarized to help researchers and clinicians understand their mechanisms of action and to promote pharmacological research and novel drug development.
Bones play several roles in the human body, providing structure, protecting organs, anchoring muscles, and storing calcium. Therefore, it is fundamental to keep the bone health as close as possible to the optimum. While it is important to build strong and healthy bones during childhood and adolescence, every effort should be taken to protect bone health, during adulthood, too. Bones are continuously changing — new bone is made, and old bone is broken down. At young ages, the body makes new bone faster than it breaks down the old bones, consequently bone mass increases. Most people reach their peak bone mass around age 30. After that, bone remodeling continues, but bones start to lose slightly more bone mass than what they gain. This paves the way for osteoporosis or what is known as “thinning of the bones,” a condition that causes bones to become weak and brittle. This relies on how much bone mass has been attained during the adulthood and how rapidly the body loses it after that. The higher the peak bone mass, the more bone the person would have “in the bank” and the less likely to develop osteoporosis as he/she gets older. Building healthy bones is the only way to reduce the enormous personal and economic costs of osteoporosis — a major cause of pain, disability, and premature death affecting both women and men.
Osteoporosis is a skeletal disorder in which bone strength is decreased, leading to increased risk of fracture. In recent years, monoclonal antibodies have increasingly been used to treat human diseases, including osteoporosis. In this article, we discuss two monoclonal antibodies currently approved to treat osteoporosis-denosumab, and romosozumab. We aim to give the reader a basic understanding of the basic properties of monoclonal antibodies, the technology used to develop them, and their clinical application in the treatment of osteoporosis.
We investigated the effects of single and repeated exposures to whole-body cryotherapy on biomarkers of bone remodeling and osteo-immune crosstalk: sclerostin, osteocalcin (OC), C-terminal cross-linked telopeptide of type I collagen (CTx-I), osteoprotegerin (OPG) and free soluble receptor activator for nuclear factor κ B ligand (sRANKL). The study included 22 healthy males, grouped in high physical fitness level (HPhL) and low physical fitness level (LPhL), all undergone 10 consecutive sessions in a cryogenic chamber (− 110 °C). We observed a significant time-effect on sclerostin ( p < 0.05), OC ( p < 0.01), CTx-I ( p < 0.001), OC/CTx-I ( p < 0.05), and significant differences in sRANKL between the groups ( p < 0.05) after the 1st cryostimulation; a significant time-effect on OC ( p < 0.001) and OC/CTx-I ( p < 0.001) after the 10th cryostimulation, and a significant time-effect on CTx-I ( p < 0.001) and OC/CTx-I ( p < 0.01) after 10 sessions of WBC. In conclusion, in young men, the first exposure to extreme cold induced significant changes in serum sclerostin. The changes in sRANKL, between groups, suggest that fitness level may modify the body's response to cold. The effects of the first stimulus and the whole session are not identical, probably due to the physiological development of habituation to cold.
BACKGROUND
Periodontitis is a highly prevalent inflammatory disease affecting the periodontium that results from an imbalance between periodontopathogens and host mechanisms. Continuous progression of the disease may lead to tissue and bone destruction, eventually resulting in tooth loss. The extent of bone loss depends on the dysregulated host immune response. Various host-elicited molecules play a major role in disease progression. The discovery of the glycoprotein sclerostin and its role as a regulator of bone mass has led to a better understanding of bone metabolism.
HIGHLIGHT
Sclerostin, which is primarily expressed by osteocytes, is a negative regulator of bone formation. It is a potent antagonist of the canonical Wingless-related integration site (Wnt) pathway, which is actively involved in bone homeostasis. Sclerostin is known to stimulate bone resorption by altering the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa- β ligand (RANKL) balance. Additionally, in periodontitis, activation of the inflammatory cascade also increases the synthesis of sclerostin.
CONCLUSION
The recently discovered sclerostin antibody has emerged as a positive therapeutic tool for the treatment of metabolic bone diseases. It has been reported to improve bone strength, bone formation, osseointegration around implants and lower the risk of bone fractures in various animal and human models. This review describes the properties and action of sclerostin, its role in periodontal diseases, and the advent and efficacy of sclerostin antibodies.
The DAN (differential screening-selected gene aberrative in neuroblastoma) family are a group of secreted extracellular proteins which typically bind to and antagonize BMP (bone morphogenetic protein) ligands. Previous studies have revealed discrepancies between the oligomerization state of certain DAN family members, with SOST (a poor antagonist of BMP signaling) forming a monomer while Grem1, Grem2, and NBL1 (more potent BMP antagonists) form non-disulfide linked dimers. The protein SOSTDC1 (Sclerostin domain containing protein 1) is sequentially similar to SOST, but has been shown to be a better BMP inhibitor. In order to determine the oligomerization state of SOSTDC1 and determine what effect dimerization might have on the mechanism of DAN family antagonism of BMP signaling, we isolated the SOSTDC1 protein and, using a battery of biophysical, biochemical, and structural techniques, showed that SOSTDC1 forms a highly stable non-covalent dimer. Additionally, this SOSTDC1 dimer was shown, using an in vitro cell based assay system, to be an inhibitor of multiple BMP signaling growth factors, including GDF5, while monomeric SOST was a very poor antagonist. These results demonstrate that SOSTDC1 is distinct from paralogue SOST in terms of both oligomerization and strength of BMP inhibition.
Sclerosteosis, a rare autosomal recessive genetic disorder caused by a mutation of the Sost gene, manifests in the facial skeleton by gigantism, facial distortion, mandibular prognathism, cranial nerve palsy, and, in extreme cases, compression of the medulla oblongata. Mice lacking sclerostin reflect some symptoms of sclerosteosis, but this is the first report of the effect on the facial skeleton. We used geometric morphometrics (GMM) to analyze the deformations of the murine facial skeleton from the wild‐type to the Sost gene knockout. Landmark coordinates were obtained by surface reconstructions from micro‐computed tomography. Centroid size, principal component scores in shape space and form space, and asymmetry were computed by the standard GMM formulas, and dental and skeletal jaw lengths were examined as ratios. We show here that, compared to wild type controls, mice lacking Sost have larger centroid size (effect size, p‐value: 4.59, <.001), higher mean asymmetry (1.14, .065), dental and skeletal mandibular prognathism (1.36, .010 and 5.92, <.001), a smaller foramen magnum (−1.71, .015), and calvaria that are more highly curved (form space p = 4.09, .002; shape space p = 12.82, .002). These features of mice lacking sclerostin largely correspond to the changes of the facial skeleton observed in sclerosteosis. This alignment further supports claims that the Sost gene plays a fundamental role in bony facial development in rodents and humans alike.
Parathyroid hormone (PTH) is an essential hormone that plays a vital role in calcium homeostasis in our body. Exogenous recombinant human PTH is widely applicable in health care, and the clinical utilization of PTH is emerging. The biological effects of systemic PTH depend upon the mode of administration: it is bone catabolic when continuously administered but bone anabolic when intermittently administered. Animal studies have shown that intermittent PTH therapy greatly improves the outcome of osseous regeneration procedures. The bone anabolic actions of PTH have been further investigated in human clinical trials to assess its effects on fracture repair, spinal fusion, bone regeneration, implant osseointegration, and healing of periodontal surgery. Findings support the positive effects of intermittent PTH therapy on the outcomes of osseous regenerative procedures. Furthermore, biomaterials through which PTH can be delivered locally have been developed and investigated in order to evaluate the therapeutic potential of PTH at the preclinical level. With advances in our understanding of PTH and hard tissue physiology, PTH provides a new therapeutic modality for osseous regeneration therapy.
Sclerostin is a protein which is involved in bone metabolism and probably also in vessel wall function. This prospective observational cohort study evaluated the prognostic significance of sclerostin in hemodialysis (HD) patients. In total, 106 HD patients and 25 healthy controls participated in the study. HD patients were prospectively followed up for five years. Sclerostin was measured in serum using standard ELISA kits by Biomedica. Sclerostin concentrations in serum were higher in HD patients compared to the controls (89.2±40.3 pmol/l vs. 32.8±13.0 pmol/l, p<0.001). Sclerostin levels were significant for cardiovascular mortality but not for overall mortality and mortality due to infection. A higher cardiovascular risk was connected to sclerostin concentrations above the median (>84 pmol/l), HR (95 % CI): 2.577 (1.0002-10.207), p=0.04. When sclerostin was evaluated together with residual diuresis in Kaplan-Meier analysis the worst prognosis due to cardiovascular events was observed in the group with high sclerostin and zero residual diuresis compared to all other patients (p=0.007). In summary, serum sclerostin levels in HD patients were increased when compared to healthy subjects. High sclerostin levels were demonstrated as a risk factor for cardiovascular mortality. Further studies are required to clarify the pathophysiological mechanisms of sclerostin action in patients with renal failure before therapeutic measures can be established.
We induced chronic kidney disease (CKD) with adenine in WT mice, mice with osteocyte-specific deletion of Cyp27b1, encoding the 25-hydroxyvitamin D 1(OH)ase [Oct-1(OH)ase-/-], and mice with global deletion of Cyp27b1 [global-1α(OH)ase-/-]; we then compared extraskeletal calcification. After adenine treatment, mice displayed increased blood urea nitrogen, decreased serum 1,25(OH)2D, and severe hyperparathyroidism. Skeletal expression of Cyp27b1 and of sclerostin and serum sclerostin all increased in WT mice but not in Oct-1α(OH)ase-/- mice or global-1α(OH)ase-/- mice. In contrast, skeletal expression of BMP2 and serum BMP2 rose in the Oct-1α(OH)ase-/- mice and in the global-1α(OH)ase-/- mice. Extraskeletal calcification occurred in muscle and blood vessels of mice with CKD and was highest in Oct-1α(OH)ase-/-mice. In vitro, recombinant sclerostin (100 ng/mL) significantly suppressed BMP2-induced osteoblastic transdifferentiation of vascular smooth muscle A7r5 cells and diminished BMP2-induced mineralization. Our study provides evidence that local osteocytic production of 1,25(OH)2D stimulates sclerostin and inhibits BMP2 production in murine CKD, thus mitigating osteoblastic transdifferentiation and mineralization of soft tissues. Increased osteocytic 1,25(OH)2D production, triggered by renal malfunction, may represent a "primary defensive response" to protect the organism from ectopic calcification by increasing sclerostin and suppressing BMP2 production.
Introduction: Over 80% of patients with multiple myeloma (MM) develop myeloma bone disease (MBD) during the disease course. The clinical consequences include serious skeletal related events (SRE) that impact survival and quality of life. Bisphosphonates are the mainstay in the treatment of MBD. Currently, new therapeutic strategies are being introduced and broaden the therapeutic options in MBD.
Areas covered: The purpose of this review is to summarize the current clinical management of MBD, and present novel data regarding monoclonal antibodies against the receptor activator of NF-kappa B ligand (RANKL) and sclerostin that may change the clinical practice.
Expert opinion: Our better understanding of the pathophysiology of MBD has identified several factors as potential therapeutic targets. Recent data have shown that the RANKL inhibitor denosumab constitutes a new promising option. The non-inferiority compared with bisphosphonates in terms of SRE prevention, the potential survival benefit, the convenience of subcutaneous administration and the favorable toxicity profile make denosumab a valuable alternative for physicians in the current treatment of MBD. Anti-sclerostin antibodies are currently under clinical development. Further investigations are needed to address open questions in the field including the value of anabolic agents combined with anti-resorptive and anti-MM drugs in MBD.
Bone is a highly metabolic organ that undergoes continuous remodeling to maintain its structural integrity. During development, bones, and in particular osteoblasts, rely on glucose uptake. However, the role of glucose metabolism in osteocytes is unknown. Osteocytes are terminally differentiated osteoblasts orchestrating bone modeling and remodeling. In these cells, parathyroid hormone (PTH) suppresses Sost/sclerostin expression (a potent inhibitor of bone formation) by promoting nuclear translocation of class IIa histone deacetylase (HDAC) 4 and 5, and repression of myocyte enhancer factor 2 (MEF2) type C. Recently, Scriptaid, an HDAC complex co-repressor inhibitor, has been shown to induce MEF2 activation and exercise-like adaptation in mice. In muscles, Scriptaid disrupts the HDAC4/5 co-repressor complex, increases MEF2C function and promotes cell respiration. We hypothesized that Scriptaid, by affecting HDAC4/5 localization and MEF2C activation might affect osteocyte functions. Treatment of the osteocytic Ocy454-12H cells with Scriptaid increased metabolic genes expression, cell respiration and glucose uptake. Similar effects were also seen upon treatment with PTH, suggesting that both Scriptaid and PTH can promote osteocyte metabolism. Similar to PTH, Scriptaid potently suppressed Sost expression. Silencing of HDAC5 in Ocy454-12H cells abolished Sost suppression but not glucose transporter type 4 (Glut4) up-regulation induced by Scriptaid. These results demonstrate that Scriptaid increases osteocyte respiration and glucose uptake by mechanisms independent of HDACs complex inhibition. In osteocytes, Scriptaid, similar to PTH, increases binding of HDAC5 to Mef2c with suppression of Sost whereas only partially increases receptor activator of nuclear factor kappa-B ligand (Rankl) expression, suggesting a potential bone anabolic effect.
In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). We find that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. We demonstrate LRP5 expression by osteoblasts in situ and show that LRP5 can transduce Wnt signaling in vitro via the canonical pathway. We further show that a mutant-secreted form of LRP5 can reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.
One of the main motivations for publishing this paper is to make available a matrix of phone-distance measures which may be useful in dealing with large corpora of conversational speech. The paper reports how this matrix of phone-distances was created from transcriber labeling disagreements, and how it can be used in a dynamic time warping algorithm to align phonetic transcriptions of conversational speech with their citation forms. The weighted string edit distance produced by the phone-distance DTW algorithm may also be useful in calculating neighborhood densities for studies of auditory word recognition.
The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.
Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk-tail development. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures. Here we show that the cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signalling by BMP and Wnt. In order for the head territory to form, we propose that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions.
Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
Sclerosteosis is a progressive sclerosing bone dysplasia with an autosomal recessive mode of inheritance. Radiologically, it is characterized by a generalized hyperostosis and sclerosis leading to a markedly thickened and sclerotic skull, with mandible, ribs, clavicles and all long bones also being affected. Due to narrowing of the foramina of the cranial nerves, facial nerve palsy, hearing loss and atrophy of the optic nerves can occur. Sclerosteosis is clinically and radiologically very similar to van Buchem disease, mainly differentiated by hand malformations and a large stature in sclerosteosis patients. By linkage analysis in one extended van Buchem family and two consanguineous sclerosteosis families we previously mapped both disease genes to the same chromosomal 17q12-q21 region, supporting the hypothesis that both conditions are caused by mutations in the same gene. After reducing the disease critical region to approximately 1 Mb, we used the positional cloning strategy to identify the SOST gene, which is mutated in sclerosteosis patients. This new gene encodes a protein with a signal peptide for secretion and a cysteine-knot motif. Two nonsense mutations and one splice site mutation were identified in sclerosteosis patients, but no mutations were found in a fourth sclerosteosis patient nor in the patients from the van Buchem family. As the three disease-causing mutations lead to loss of function of the SOST protein resulting in the formation of massive amounts of normal bone throughout life, the physiological role of SOST is most likely the suppression of bone formation. Therefore, this gene might become an important tool in the development of therapeutic strategies for osteoporosis.
Transforming growth factor-beta (TGF-beta) family members, which include bone morphogenetic proteins (BMPs) and TGF-betas, elicit their cellular effects by activating specific Smad proteins, which control the transcription of target genes. BMPs and TGF-betas have overlapping as well as specific effects on mesenchymal cell differentiation for which the mechanisms are incompletely understood. Here we report that Id1, a dominant negative inhibitor of basic helix-loop-helix proteins, is a direct target gene for BMP. BMP, but not TGF-beta, strongly activates the Id1 promoter in an Smad-dependent manner. We identified two BMP-responsive regions in the mouse Id1 promoter, which contain three distinct sequence elements; one region contains two Smad binding elements (SBEs), and the other region contains a GGCGCC palindromic sequence flanked by two CAGC and two CGCC motifs. Whereas SBEs and GGCGCC sequence are critically important, the CAGC and CGCC motifs are needed for efficient BMP-induced Id1 promoter activation. Smads are part of nuclear transcription factor complexes that specifically bind to SBEs and GGCGCC sequence in response to BMP but not TGF-beta. Multimerization of the all three distinct sequence motifs is needed to generate a highly sensitive and BMP/Smad-dependent specific enhancer. Our results provide important new insights into how the BMP/Smad pathway can specifically activate target genes.
Van Buchem disease is an autosomal recessive skeletal dysplasia characterised by generalised bone overgrowth, predominantly in the skull and mandible. Clinical complications including facial nerve palsy, optic atrophy, and impaired hearing occur in most patients. These features are very similar to those of sclerosteosis and the two conditions are only differentiated by the hand malformations and the tall stature appearing in sclerosteosis. Using an extended Dutch inbred van Buchem family and two inbred sclerosteosis families, we mapped both disease genes to the same region on chromosome 17q12-q21, supporting the hypothesis that van Buchem disease and sclerosteosis are caused by mutations in the same gene. In a previous study, we positionally cloned a novel gene, called SOST, from the linkage interval and identified three different, homozygous mutations in the SOST gene in sclerosteosis patients leading to loss of function of the underlying protein. The present study focuses on the identification of a 52 kb deletion in all patients from the van Buchem family. The deletion, which results from a homologous recombination between Alu sequences, starts approximately 35 kb downstream of the SOST gene. Since no evidence was found for the presence of a gene within the deleted region, we hypothesise that the presence of the deletion leads to a down regulation of the transcription of the SOST gene by a cis regulatory action or a position effect.
The development of novel anti-cancer strategies requires more sensitive and less invasive methods to detect and monitor in vivo minimal residual disease in cancer models. Bone marrow metastases are indirectly detected by radiography as osteolytic and/or osteosclerotic lesions. Marrow micrometastases elude radiographic detection and, therefore, more sensitive methods are needed for their direct identification. Injection of cancer cells into the left cardiac ventricle of mice closely mimics micrometastatic spread. When luciferase-transfected cells are used, whole-body bioluminescent reporter imaging can detect microscopic bone marrow metastases of approximately 0.5 mm(3) volume, a size below the limit in which tumors need to induce angiogenesis for further growth. This sensitivity translates into early detection of intramedullary tumor growth, preceding the appearance of a radiologically evident osteolysis by approximately 2 weeks. Bioluminescent reporter imaging also enables continuous monitoring in the same animal of growth kinetics for each metastatic site and guides end-point analyses specifically to the bones affected by metastatic growth. This model will accelerate the understanding of the molecular events in metastasis and the evaluation of novel therapies aiming at repressing initial stages of metastatic growth.
The Wnt family of secreted glycoproteins mediate cell cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the beta-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular beta-catenin. Wnt/beta-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. Here we show that the transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/beta-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates.
Patterning of the pre-gastrula embryo and subsequent neural induction post-gastrulation are very complex and intricate processes of which little, until recently, has been understood. The earliest decision in neural development, the choice between epidermal or neural fates, is regulated by bone morphogenetic protein (BMP) signaling within the ectoderm. Inhibition of BMP signaling is sufficient for neural induction. Many secreted BMP inhibitors are expressed exclusively within the organizer of the Xenopus gastrula embryo and therefore are predicted to act as bona fide endogenous neural inducers. Other cell-autonomous inhibitors of the BMP pathway are more widely expressed, such as the inhibitory Smads, Smad6 and Smad7. In this report we describe the biological and biochemical characterization of 51-B6, a novel member of Cerberus/Dan family of secreted BMP inhibitors, which we identified in a screen for Smad7-induced genes. This gene is expressed maternally in an animal to vegetal gradient, and its expression levels decline rapidly following gastrulation. In contrast to known BMP inhibitors, 51-B6 is broadly expressed in the ectoderm until the end of gastrulation. The timing, pattern of expression, and activities of this gene makes it unique when compared to other BMP/TGFbeta/Wnt secreted inhibitors which are expressed only zygotically and maintained post-gastrulation. We propose that a function of 51-B6 is to block BMP and TGFbeta signals in the ectoderm in order to regulate cell fate specification and competence prior to the onset of neural induction. In addition, we demonstrate that 51-B6 can act as a neural inducer and induce ectopic head-like structures in neurula staged embryos. Because of this embryological activity, we have renamed this clone Coco, after the Spanish word meaning head.
The efficiency of plasmid gene transfer to skeletal muscle can be significantly improved by the application of an electrical field to the muscle following injection of plasmid DNA. However, this electrotransfer is associated with significant muscle damage which may result in substantial loss of transfected muscle fibres. Reduction of the voltage used in the technique can result in a decrease in muscle damage, with a concomitant reduction in expression, but without a significant decrease in the number of transfected fibres. Pre-treatment of the muscle with a solution of bovine hyaluronidase greatly increases the efficiency of plasmid gene transfer when used in conjunction with electrotransfer, but not when used alone. This combination treatment results in greatly enhanced levels of transfected muscle fibres without the increases in muscle damage associated with the electrotransfer process.
We have isolated a novel secreted molecule, Wise, by a functional screen for activities that alter the anteroposterior character of neuralised Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of beta-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonises the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor, lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling.
Myeloma cells may secrete factors that affect the function of osteoblasts, osteoclasts, or both.
We subjected purified plasma cells from the bone marrow of patients with newly diagnosed multiple myeloma and control subjects to oligonucleotide microarray profiling and biochemical and immunohistochemical analyses to identify molecular determinants of osteolytic lesions.
We studied 45 control subjects, 36 patients with multiple myeloma in whom focal lesions of bone could not be detected by magnetic resonance imaging (MRI), and 137 patients in whom MRI detected such lesions. Different patterns of expression of 57 of approximately 10,000 genes from purified myeloma cells could be used to distinguish the two groups of patients (P<0.001). Permutation analysis, which adjusts the significance level to account for multiple comparisons in the data sets, showed that 4 of these 57 genes were significantly overexpressed by plasma cells from patients with focal lesions. One of these genes, dickkopf 1 (DKK1), and its corresponding protein (DKK1) were studied in detail because DKK1 is a secreted factor that has been linked to the function of osteoblasts. Immunohistochemical analysis of bone marrow-biopsy specimens showed that only myeloma cells contained detectable DKK1. Elevated DKK1 levels in bone marrow plasma and peripheral blood from patients with multiple myeloma correlated with the gene-expression patterns of DKK1 and were associated with the presence of focal bone lesions. Recombinant human DKK1 or bone marrow serum containing an elevated level of DKK1 inhibited the differentiation of osteoblast precursor cells in vitro.
The production of DKK1, an inhibitor of osteoblast differentiation, by myeloma cells is associated with the presence of lytic bone lesions in patients with multiple myeloma.
Sclerosteosis, a skeletal disorder characterized by high bone mass due to increased osteoblast activity, is caused by loss of the SOST gene product, sclerostin. The localization in bone and the mechanism of action of sclerostin are not yet known, but it has been hypothesized that it may act as a bone morphogenetic protein (BMP) antagonist. We show here that SOST/sclerostin is expressed exclusively by osteocytes in mouse and human bone and inhibits the differentiation and mineralization of murine preosteoblastic cells (KS483). Although sclerostin shares some of the actions of the BMP antagonist noggin, we show here that it also has actions distinctly different from it. In contrast to noggin, sclerostin did not inhibit basal alkaline phosphatase (ALP) activity in KS483 cells, nor did it antagonize BMP-stimulated ALP activity in mouse C2C12 cells. In addition, sclerostin had no effect on BMP-stimulated Smad phosphorylation and direct transcriptional activation of MSX-2 and BMP response element reporter constructs in KS483 cells. Its unique localization and action on osteoblasts suggest that sclerostin may be the previously proposed osteocyte-derived factor that is transported to osteoblasts at the bone surface and inhibits bone formation.
The mechanism by which the high-bone-mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates canonical Wnt signaling
was investigated. The mutation was previously shown to reduce DKK1-mediated antagonism, suggesting that the first YWTD repeat
domain where G171 is located may be responsible for DKK-mediated antagonism. However, we found that the third YWTD repeat,
but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted
the interaction of LRP5 with Mesd, a chaperone protein for LRP5/6 that is required for transport of the coreceptors to cell
surfaces, resulting in fewer LRP5 molecules on the cell surface. Although the reduction in the number of cell surface LRP5
molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity
of coexpressed Wnt in an autocrine paradigm. Together with the observation that osteoblast cells produce autocrine canonical
Wnt, Wnt7b, and that osteocytes produce paracrine DKK1, we think that the G171V mutation may cause an increase in Wnt activity
in osteoblasts by reducing the number of targets for paracrine DKK1 to antagonize without affecting the activity of autocrine
Wnt.
The Bioconductor project is an initiative for the collaborative creation of extensible software for computational biology and bioinformatics. The goals of the project include: fostering collaborative development and widespread use of innovative software, reducing barriers to entry into interdisciplinary scientific research, and promoting the achievement of remote reproducibility of research results. We describe details of our aims and methods, identify current challenges, compare Bioconductor to other open bioinformatics projects, and provide working examples.
High bone mass diseases are caused both by activating mutations in the Wnt pathway and by loss of SOST, a bone morphogenetic protein (BMP) antagonist, leading to the activation of BMP signaling. Given the phenotypic similarity between mutations that activate these signaling pathways, it seems likely that BMPs and Wnts operate in parallel or represent components of the same pathway, modulating osteoblast differentiation. In this study, we show that in C3H10T1/2 cells, Wnt-3A and BMP-6 proteins were inducers of osteoblast differentiation, as measured by alkaline phosphatase (ALP) induction. Surprisingly, sclerostin, noggin, and human BMP receptor 1A (BMPR1A)-FC fusion proteins blocked Wnt-3A-induced ALP as well as BMP-6-induced ALP activity. Dkk-1, a Wnt inhibitor, blocked Wnt-induced ALP activity but not BMP-induced ALP activity. Early Wnt-3A signaling as measured by beta-catenin accumulation was not affected by the BMP antagonists but was blocked by Dkk-1. Wnt-3A induced the appearance of BMP-4 mRNA 12 h prior to that of ALP in C3H10T1/2 cells. We propose that sclerostin and other BMP antagonists do not block Wnt signaling directly. Sclerostin blocks Wnt-induced ALP activity by blocking the activity of BMP proteins produced by Wnt treatment. The expression of BMP proteins in this autocrine loop is essential for Wnt-3A-induced osteoblast differentiation.
Wnts comprise a family of secreted signaling proteins that regulate diverse developmental processes. Activation of Wnt signaling by Wnt10b inhibits differentiation of preadipocytes and blocks adipose tissue development; however, the effect of Wnt10b on other mesenchymal lineages has not been defined. To explore the physiological role of Wnt signaling in bone development, we analyzed FABP4-Wnt10b mice, which express the Wnt10b transgene in marrow. Femurs from FABP4-Wnt10b mice have almost four times as much bone in the distal metaphyses and are mechanically stronger. These mice maintain elevated bone mass at least through 23 months of age. In addition, FABP4-Wnt10b mice are protected from the bone loss characteristic of estrogen deficiency. We used pharmacological and genetic approaches to demonstrate that canonical Wnt signaling stimulates osteoblastogenesis and inhibits adipogenesis of bipotential mesenchymal precursors. Wnt10b shifts cell fate toward the osteoblast lineage by induction of the osteoblastogenic transcription factors Runx2, Dlx5, and osterix and suppression of the adipogenic transcription factors C/EBPα and PPARγ. One mechanism whereby Wnt10b promotes osteoblastogenesis is suppression of PPARγ expression. Finally, Wnt10b-/- mice have decreased trabecular bone and serum osteocalcin, confirming that Wnt10b is an endogenous regulator of bone formation.
• adipogenesis
• development
• stem cells
The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass. In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293T cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by overexpression of Wnt co-receptor low density lipoprotein receptor-related protein (LRP) 5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transduction of canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b. Given the plethora of evidence indicating that canonical Wnt signaling stimulates osteogenesis, we believe that the high bone mass phenotype associated with the loss of sclerostin may be attributed, at least in part, to an increase in canonical Wnt signaling resulting from the reduction in sclerostin-mediated Wnt antagonism.
Sclerosteosis is an autosomal recessive disease that is characterized by overgrowth of bone tissue and is linked to mutations
in the gene encoding the secreted protein SOST. Sclerosteosis shares remarkable similarities with “high bone mass” diseases
caused by “gain-of-function” mutations in the LRP5 gene, which encodes a coreceptor for Wnt signaling proteins. We show here that SOST antagonizes Wnt signaling in Xenopus embryos and mammalian cells by binding to the extracellular domain of the Wnt coreceptors LRP5 and LRP6 and disrupting Wnt-induced
Frizzled-LRP complex formation. Our findings suggest that SOST is an antagonist for Wnt signaling and that the loss of SOST
function likely leads to the hyperactivation of Wnt signaling that underlies bone overgrowth seen in sclerosteosis patients.
The low-density-lipoprotein receptor-related protein 5 (LRP5), a coreceptor in the canonical Wnt signaling pathway, has been
implicated in human disorders of low and high bone mass. Loss-of-function mutations cause the autosomal recessive osteoporosis-pseudoglioma
syndrome, and heterozygous missense mutations in families segregating autosomal dominant high bone mass (HBM) phenotypes have
been identified. We expressed seven different HBM-LRP5 missense mutations to delineate the mechanism by which they alter Wnt
signaling. None of the mutations caused activation of the receptor in the absence of ligand. Each mutant receptor was able
to reach the cell surface, albeit at differing amounts, and transduce exogenously supplied Wnt1 and Wnt3a signal. All HBM
mutant proteins had reduced physical interaction with and reduced inhibition by DKK1. These data suggest that HBM mutant proteins
can transit to the cell surface in sufficient quantity to transduce Wnt signal and that the likely mechanism for the HBM mutations'
physiologic effects is via reduced affinity to and inhibition by DKK1.
Sclerosteosis is an autosomal recessive sclerosing bone disorder due to deficiency of sclerostin, a protein secreted by the osteocytes that inhibits bone formation. In the present study we assessed the effect of variable expression of the genetic defect on bone mineral density (BMD) in patients and carriers of the determinant gene.
We studied 25 individuals (seven patients and 18 phenotypically normal heterozygotes). BMD was measured by dual x-ray absorptiometry at the lumbar spine, total hip, and distal forearm, and lateral radiographs of the skull were obtained.
Individuals with sclerosteosis had markedly increased BMD at all skeletal sites (Z-score ranges: lumbar spine, +7.73 to +14.43; total hip, +7.84 to +11.51; forearm, +4.44 to +9.53). In heterozygotes, BMD was above the mean value of healthy age-matched individuals at all skeletal sites and had a wide range of normal and clearly increased values. Skull radiographs showed the typical hyperostotic changes in affected individuals and mild or no changes in heterozygotes.
Heterozygous carriers of sclerosteosis have BMD values consistently higher than the mean of healthy subjects without any of the bone complications encountered in homozygotes. This finding suggests that the production and/or activity of sclerostin can be titrated in vivo, leading to variable increases in bone mass without any unwanted skeletal effects, a hypothesis of obvious significance for the development of new therapeutics for osteoporosis.
Sclerosteosis is a progressive sclerosing bone dysplasia. Sclerostin (the SOST gene) was originally identified as the sclerosteosis-causing gene. However, the physiological role of sclerostin remains
to be elucidated. Sclerostin was intensely expressed in developing bones of mouse embryos. Punctuated expression of sclerostin was localized on the
surfaces of both intramembranously forming skull bones and endochondrally forming long bones. Sclerostin-positive cells were identified as osteoclasts. Recombinant sclerostin protein produced in cultured cells was efficiently
secreted as a monomer. We examined effects of sclerostin on the activity of BMP2, BMP4, BMP6, and BMP7 for mouse preosteoblastic
MC3T3-E1 cells. Sclerostin inhibited the BMP6 and BMP7 activity but not the BMP2 and BMP4 activity. Sclerostin bound to BMP6
and BMP7 with high affinity but bound to BMP2 and BMP4 with lower affinity. In conclusion, sclerostin is a novel secreted
osteoclast-derived BMP antagonist with unique ligand specificity. We suggest that sclerostin negatively regulates the formation
of bone by repressing the differentiation and/or function of osteoblasts induced by BMPs. Since sclerostin expression is confined to the bone-resorbing osteoclast, it provides a mechanism whereby bone apposition is inhibited in
the vicinity of resorption. Our findings indicate that sclerostin plays an important role in bone remodeling and links bone
resorption and bone apposition.
The increasing amount of genomic and molecular information is the basis for understanding higher-order biological systems,
such as the cell and the organism, and their interactions with the environment, as well as for medical, industrial and other
practical applications. The KEGG resource (http://www.genome.jp/kegg/) provides a reference knowledge base for linking genomes to biological systems, categorized as building blocks in the genomic
space (KEGG GENES) and the chemical space (KEGG LIGAND), and wiring diagrams of interaction networks and reaction networks
(KEGG PATHWAY). A fourth component, KEGG BRITE, has been formally added to the KEGG suite of databases. This reflects our
attempt to computerize functional interpretations as part of the pathway reconstruction process based on the hierarchically
structured knowledge about the genomic, chemical and network spaces. In accordance with the new chemical genomics initiatives,
the scope of KEGG LIGAND has been significantly expanded to cover both endogenous and exogenous molecules. Specifically, RPAIR
contains curated chemical structure transformation patterns extracted from known enzymatic reactions, which would enable analysis
of genome-environment interactions, such as the prediction of new reactions and new enzyme genes that would degrade new environmental
compounds. Additionally, drug information is now stored separately and linked to new KEGG DRUG structure maps.
Patterning of the pre-gastrula embryo and subsequent neural induction post-gastrulation are very complex and intricate processes of which little, until recently, has been understood. The earliest decision in neural development, the choice between epidermal or neural fates, is regulated by bone morphogenetic protein (BMP) signaling within the ectoderm. Inhibition of BMP signaling is sufficient for neural induction. Many secreted BMP inhibitors are expressed exclusively within the organizer of the Xenopus gastrula embryo and therefore are predicted to act as bona fide endogenous neural inducers. Other cell-autonomous inhibitors of the BMP pathway are more widely expressed, such as the inhibitory Smads, Smad6 and Smad7. In this report we describe the biological and biochemical characterization of 51-B6, a novel member of Cerberus/Dan family of secreted BMP inhibitors, which we identified in a screen for Smad7-induced genes. This gene is expressed maternally in an animal to vegetal gradient, and its expression levels decline rapidly following gastrulation. In contrast to known BMP inhibitors, 51-B6 is broadly expressed in the ectoderm until the end of gastrulation. The timing, pattern of expression, and activities of this gene makes it unique when compared to other BMP/TGFβ/Wnt secreted inhibitors which are expressed only zygotically and maintained post-gastrulation. We propose that a function of 51-B6 is to block BMP and TGFβ signals in the ectoderm in order to regulate cell fate specification and competence prior to the onset of neural induction. In addition, we demonstrate that 51-B6 can act as a neural inducer and induce ectopic head-like structures in neurula staged embryos. Because of this embryological activity, we have renamed this clone Coco, after the Spanish word meaning head.
The development of novel anti-cancer strategies requires more sensitive and less invasive methods to detect and monitor in vivo minimal residual disease in cancer models. Bone marrow metastases are indirectly detected by radiography as osteolytic and/or osteosclerotic lesions. Marrow micrometastases elude radiographic detection and, therefore, more sensitive methods are needed for their direct identification. Injection of cancer cells into the left cardiac ventricle of mice closely mimics micrometastatic spread. When luciferase-transfected cells are used, whole-body bioluminescent reporter imaging can detect microscopic bone marrow metastases of ≈0.5 mm 3 volume, a size below the limit in which tumors need to induce angiogenesis for further growth. This sensitivity translates into early detection of intramedullary tumor growth, preceding the appearance of a radiologically evident osteolysis by ≈2 weeks. Bioluminescent reporter imaging also enables continuous monitoring in the same animal of growth kinetics for each metastatic site and guides end-point analyses specifically to the bones affected by metastatic growth. This model will accelerate the understanding of the molecular events in metastasis and the evaluation of novel therapies aiming at repressing initial stages of metastatic growth.
The Bioconductor project is an initiative for the collaborative creation of extensible software for computational biology and bioinformatics. We detail some of the design decisions, software paradigms and operational strategies that have allowed a small number of researchers to provide a wide variety of innovative, extensible, software solutions in a relatively short time. The use of an object oriented programming paradigm, the adoption and development of a software package system, designing by contract, distributed development and collaboration with other projects are elements of this project's success. Individually, each of these concepts are useful and important but when combined they have provided a strong basis for rapid development and deployment of innovative and flexible research software for scientific computation. A primary objective of this initiative is achievement of total remote reproducibility of novel algorithmic research results.
Sclerosteosis is an autosomal recessive sclerosing bone dysplasia characterized by progressive skeletal overgrowth. The majority of affected individuals have been reported in the Afrikaner population of South Africa, where a high incidence of the disorder occurs as a result of a founder effect. Homozygosity mapping in Afrikaner families along with analysis of historical recombinants localized sclerosteosis to an interval of ∼2 cM between the loci D17S1787 and D17S930 on chromosome 17q12-q21. Here we report two independent mutations in a novel gene, termed “SOST.” Affected Afrikaners carry a nonsense mutation near the amino terminus of the encoded protein, whereas an unrelated affected person of Senegalese origin carries a splicing mutation within the single intron of the gene. The SOST gene encodes a protein that shares similarity with a class of cystine knot–containing factors including dan, cerberus, gremlin, prdc, and caronte. The specific and progressive effect on bone formation observed in individuals affected with sclerosteosis, along with the data presented in this study, together suggest that the SOST gene encodes an important new regulator of bone homeostasis.
We have examined 50 persons with sclerosteosis in the Afrikaner community of South Africa and 15 individuals with van Buchem disease in Holland. The clinical and radiographic manifestations of these conditions are very similar, the only notable differences being greater severity and syndactyly in the majority of the patients with sclerosteosis.
The Afrikaners have Dutch antecedants and it seems likely that these autosomal recessive disorders result from homozygosity of the same faulty genes. The phenotypic variation may be due to the epistatic effect of modifying genes in the Afrikaner population.
Wnt glycoproteins have been implicated in diverse processes during embryonic patterning in metazoa. They signal through frizzled-type seven-transmembrane-domain receptors to stabilize beta-catenin. Wnt signalling is antagonized by the extracellular Wnt inhibitor dickkopf1 (dkk1), which is a member of a multigene family. dkk1 was initially identified as a head inducer in Xenopus embryos but the mechanism by which it blocks Wnt signalling is unknown. LDL-receptor-related protein 6 (LRP6) is required during Wnt/beta-catenin signalling in Drosophila, Xenopus and mouse, possibly acting as a co-receptor for Wnt. Here we show that LRP6 (ref. 7) is a specific, high-affinity receptor for Dkk1 and Dkk2. Dkk1 blocks LRP6-mediated Wnt/beta-catenin signalling by interacting with domains that are distinct from those required for Wnt/Frizzled interaction. dkk1 and LRP6 interact antagonistically during embryonic head induction in Xenopus where LRP6 promotes the posteriorizing role of Wnt/beta-catenin signalling. Thus, DKKs inhibit Wnt co-receptor function, exemplifying the modulation of LRP signalling by antagonists.
We outline and describe steps for a statistically rigorous approach to analyzing probe-level Affymetrix GeneChip data. The approach employs classical linear mixed models and operates on a gene-by-gene basis. Forgoing any attempts at gene presence or absence calls, the method simultaneously considers the data across all chips in an experiment. Primary output includes precise estimates of fold change (some as low as 1.1), their statistical significance, and measures of array and probe variability. The method can accommodate complex experiments involving many kinds of treatments and can test for their effects at the probe level. Furthermore, mismatch probe data can be incorporated in different ways or ignored altogether. Data from an ionizing radiation experiment on human cell lines illustrate the key concepts.
Osteoporosis is a major public health problem of largely unknown cause. Loss-of-function mutations in the gene for low-density lipoprotein receptor-related protein 5 (LRP5), which acts in the Wnt signaling pathway, have been shown to cause osteoporosis-pseudoglioma.
We performed genetic and biochemical analyses of a kindred with an autosomal dominant syndrome characterized by high bone density, a wide and deep mandible, and torus palatinus.
Genetic analysis revealed linkage of the syndrome to chromosome 11q12-13 (odds of linkage, >1 million to 1), an interval that contains LRP5. Affected members of the kindred had a mutation in this gene, with valine substituted for glycine at codon 171 (LRP5V171). This mutation segregated with the trait in the family and was absent in control subjects. The normal glycine lies in a so-called propeller motif that is highly conserved from fruit flies to humans. Markers of bone resorption were normal in the affected subjects, whereas markers of bone formation such as osteocalcin were markedly elevated. Levels of fibronectin, a known target of signaling by Wnt, a developmental protein, were also elevated. In vitro studies showed that the normal inhibition of Wnt signaling by another protein, Dickkopf-1 (Dkk-1), was defective in the presence of LRP5V171 and that this resulted in increased signaling due to unopposed Wnt activity.
The LRP5V171 mutation causes high bone density, with a thickened mandible and torus palatinus, by impairing the action of a normal antagonist of the Wnt pathway and thus increasing Wnt signaling. These findings demonstrate the role of altered LRP5 function in high bone mass and point to Dkk as a potential target for the prevention or treatment of osteoporosis.
Van Buchem disease is an autosomal recessive sclerosing bone dysplasia characterized by skeletal hyperostosis, overgrowth of the mandible, and a liability to entrapment of the seventh and eighth cranial nerves. The genetic determinant maps to chromosome 17q12-q21. We refined the critical interval to the < 1-Mb region between D17S2250 and D17S2253 in 15 affected individuals, all of whom shared a common disease haplotype. Furthermore, we report here the identification of a 52-kb deletion located within the interval and encompassing D17S1789 that is 100% concordant with the disorder. Although the deletion itself does not appear to disrupt the coding region of any known or novel gene(s), the closest flanking genes are MEOX1 on the proximal side, and SOST on the distal side of the deletion. MEOX1 is known to be important for the development of the axial skeleton, whereas the SOST gene is the determinant of sclerosteosis, a disorder that shares many features with van Buchem disease, thus raising the possibility that van Buchem disease results from dysregulation of the expression of one or both of these genes.
Sclerosteosis (SCL) is a severe, progressive, autosomal-recessive craniotubular hyperostosis (MIM 269500). The determinant gene (SOST) has been isolated, and genotype-phenotype correlations, as well as the elucidation of pathogenetic mechanisms, are dependent upon the documentation of the natural history of the condition. For this reason, the course and complications in 63 affected individuals in South Africa, seen over a 38-year period, have been analyzed. Thirty-four of these persons died during the course of the survey, 24 from complications related to elevation of intracranial pressure as a result of calvarial overgrowth. The mean age of death in this group of individuals was 33 years, with an even gender distribution. Facial palsy and deafness, as a result of cranial nerve entrapment, developed in childhood in 52 (82%) affected persons. Mandibular overgrowth was present in 46 (73%) adults and syndactyly in 48 (76%). In South Africa in 2002, 29 affected persons were alive, 10 being < or =20 years of age. It is evident that sclerosteosis is a severe disorder which places a considerable burden upon affected individuals and their families.
In this paper we report exploratory analyses of high-density oligonucleotide array data from the Affymetrix GeneChip system with the objective of improving upon currently used measures of gene expression. Our analyses make use of three data sets: a small experimental study consisting of five MGU74A mouse GeneChip arrays, part of the data from an extensive spike-in study conducted by Gene Logic and Wyeth's Genetics Institute involving 95 HG-U95A human GeneChip arrays; and part of a dilution study conducted by Gene Logic involving 75 HG-U95A GeneChip arrays. We display some familiar features of the perfect match and mismatch probe (PM and MM) values of these data, and examine the variance-mean relationship with probe-level data from probes believed to be defective, and so delivering noise only. We explain why we need to normalize the arrays to one another using probe level intensities. We then examine the behavior of the PM and MM using spike-in data and assess three commonly used summary measures: Affymetrix's (i) average difference (AvDiff) and (ii) MAS 5.0 signal, and (iii) the Li and Wong multiplicative model-based expression index (MBEI). The exploratory data analyses of the probe level data motivate a new summary measure that is a robust multi-array average (RMA) of background-adjusted, normalized, and log-transformed PM values. We evaluate the four expression summary measures using the dilution study data, assessing their behavior in terms of bias, variance and (for MBEI and RMA) model fit. Finally, we evaluate the algorithms in terms of their ability to detect known levels of differential expression using the spike-in data. We conclude that there is no obvious downside to using RMA and attaching a standard error (SE) to this quantity using a linear model which removes probe-specific affinities.
TGF-beta family proteins with a cystine knot motif serve as ligands for diverse families of plasma membrane receptors. Bone morphogenetic protein (BMP) antagonists represent a subgroup of these proteins, some of which bind BMPs and antagonize their actions during development and morphogenesis. Availability of completed genome sequences from diverse organisms allows bioinformatic analysis of the evolution of BMP antagonists and facilitates their classification. Using a regular expression algorithm (http://BioRegEx.stanford.edu), an exhaustive search of the human genome identified all cystine knot-containing BMP antagonists. Based on the size of the cystine ring, these proteins were divided into three subfamilies: CAN (eight-membered ring), twisted gastrulation (nine-membered ring), as well as chordin and noggin (10-membered ring). The CAN family can be divided further into four subgroups based on a conserved arrangement of additional cysteine residues-gremlin and PRDC, cerberus and coco, and DAN, together with USAG-1 and sclerostin. We searched for orthologs of human BMP antagonists in the genomes of model organisms and analyzed their phylogenetic relationship. New human paralogs were identified together with the verification of orthologous relationships of known genes. We also discuss the physiological roles of the CAN subfamily of BMP antagonists and the associated genetic defects. Based on the known three-dimensional structure of key cystine knot proteins, we postulated disulfide bondings for eight-membered ring BMP antagonists to predict their potential folding and dimerization.
Wnt/beta-catenin signaling has recently been suggested to be involved in bone biology. The precise role of this cascade in osteoblast differentiation was examined. We show that a Wnt autocrine loop mediates the induction of alkaline phosphatase and mineralization by BMP-2 in pre-osteoblastic cells.
Loss of function of LRP5 leads to osteoporosis (OPPG syndrome), and a specific point mutation in this same receptor results in high bone mass (HBM). Because LRP5 acts as a coreceptor for Wnt proteins, these findings suggest a crucial role for Wnt signaling in bone biology.
We have investigated the involvement of the Wnt/LRP5 cascade in osteoblast function by using the pluripotent mesenchymal cell lines C3H10T1/2, C2C12, and ST2 and the osteoblast cell line MC3T3-E1. Transfection experiments were carried out with a number of elements of the Wnt/LRP5 pathway. Measuring osteoblast and adipocyte differentiation markers addressed the effect of this cascade on osteoblast differentiation.
In mesenchymal cells, only Wnt's capable of stabilizing beta-catenin induced the expression of alkaline phosphatase (ALP). Wnt3a-mediated ALP induction was inhibited by overexpression of either Xddl, dickkopf 1 (dkk1), or LRP5deltaC, indicating that canonical beta-catenin signaling is responsible for this activity. The use of Noggin, a bone morphogenic protein (BMP) inhibitor, or cyclopamine, a Hedgehog inhibitor, revealed that the induction of ALP by Wnt is independent of these morphogenetic proteins and does not require de novo protein synthesis. In contrast, blocking Wnt/LRP5 signaling or protein synthesis inhibited the ability of both BMP-2 and Shh to induce ALP in mesenchymal cells. Moreover, BMP-2 enhanced Wntl and Wnt3a expression in our cells. In MC3T3-E1 cells, where endogenous ALP levels are maximal, antagonizing the Wnt/LRP5 pathway led to a decrease of ALP activity. In addition, overexpression of dkkl reduced extracellular matrix mineralization in a BMP-2-dependent assay.
Our data strongly suggest that the capacity of BMP-2 and Shh to induce ALP relies on Wnt expression and the Wnt/LRP5 signaling cascade. Moreover the effects of BMP-2 on extracellular matrix mineralization by osteoblasts are mediated, at least in part, by the induction of a Wnt autocrine/paracrine loop. These results may help to explain the phenotype of OPPG patients and HBM.
Recent revelations that the canonical Wnt signaling pathway promotes postnatal bone accrual are major advances in our understanding of skeletal biology and bring tremendous promise for new therapeutic treatments for osteoporosis and other diseases of altered bone mass. Wnts are soluble glycoproteins that engage receptor complexes composed of Lrp5/6 and Frizzled proteins. A subgroup of Wnts induces a cascade of intracellular events that stabilize beta-catenin, facilitating its transport to nuclei where it binds Lef1/Tcf transcription factors and alters gene expression to promote osteoblast expansion and function. Natural extracellular Wnt antagonists, Dickkopfs and secreted frizzled-related proteins, impair osteoblast function and block bone formation. In several genetic disorders of altered skeletal mass, mutations in LRP5 create gain-of-function or loss-of-function receptors that are resistant to normal regulatory mechanisms and cause higher or lower bone density, respectively. In this review, we summarize the available molecular, cellular, and genetic data that demonstrate how Lrp5 and other components of the Wnt signaling pathway influence osteoblast proliferation, function, and survival. We also discuss regulatory mechanisms discovered in developmental and tumor models that may provide insights into novel therapies for bone diseases.
HE IDENTIFICATION OF mutations in the human low- density lipoprotein (LDL) receptor-related protein 5 (LRP5) gene that give rise to conditions of low bone mass(1,2) and increased bone mass(3-5) has brought what just a few years ago were two seemingly unrelated fields crash- ing together, namely bone biology and Wnt signaling. It is now clear that the Wnt signaling pathway is another key pathway involved in the regulation of bone mass. This is not to diminish the importance of any other pathway that is known to play a role; rather, this discovery only highlights how little we probably really know about bone mass regu- lation. Certainly, a big challenge we now face will be to integrate the Wnt signaling pathway with all of its complex- ities and subtleties into the already complex nature of the other pathways that have been studied, with the ultimate goal of developing a composite understanding of their col- lective roles and interactions. In this article, we will summarize the main points of our current understanding of LRP5, Wnt's and Wnt signaling, and the role they may play in bone. This is by no means comprehensive, and there are more unanswered questions than answers.
Sclerosteosis and Van Buchem disease are two closely related bone disorders characterized by progressive bone thickening due to increased bone formation. Sclerosteosis is associated with mutations in the SOST gene and Van Buchem disease with a 52 kb deletion downstream of the SOST gene that probably affects transcription of the gene. Expression of the gene product sclerostin in bone is restricted to osteocytes and it is a negative regulator of bone formation. It inhibits BMP-stimulated bone formation, but cannot antagonize all BMP responses. The exclusive bone phenotype of good quality of patients with sclerosteosis and Van Buchem disease and the specific localization of sclerostin make it an attractive target for the development of bone forming therapeutics.
Osteocytes are the most abundant cells in bone and are ideally located to influence bone turnover through their syncytial relationship with surface bone cells. Osteocyte-derived signals have remained largely enigmatic, but it was recently reported that human osteocytes secrete sclerostin, an inhibitor of bone formation. Absent sclerostin protein results in the high bone mass clinical disorder sclerosteosis. Here we report that within adult iliac bone, newly embedded osteocytes were negative for sclerostin staining but became positive at or after primary mineralization. The majority of mature osteocytes in mineralized cortical and cancellous bone was positive for sclerostin with diffuse staining along dendrites in the osteocyte canaliculi. These findings provide for the first time in vivo evidence to support the concept that osteocytes secrete sclerostin after they become embedded in a mineralized matrix to limit further bone formation by osteoblasts. Sclerostin did not appear to influence the formation of osteocytes. We propose that sclerostin production by osteocytes may regulate the linear extent of formation and the induction or maintenance of a lining cell phenotype on bone surfaces. In doing so, sclerostin may act as a key inhibitory signal governing skeletal microarchitecture.
We examined the role of Wnt/beta-catenin signaling in successive stages of osteoblast differentiation. It has been shown that Wnt signaling in mature osteoblasts needs to be downregulated to enable the formation of a mineralized matrix. Using RNA interference, we showed that this is, at least in part, accomplished by upregulation of the Wnt antagonists Dickkopf-1 and -2.
The role of Wnt signaling in the initiation of osteoblast differentiation has been well studied. However, the role during late-stage differentiation is less clear. We have examined the role of Wnt/beta-catenin signaling in successive stages of osteoblast differentiation.
We treated murine bone marrow and mesenchymal stem cell-like KS483 cells with either LiCl or Wnt3A during several stages of osteoblast differentiation. In addition, we generated stable KS483 cell lines silencing either the Wnt antagonist Dkk-1 or -2
Activation of Wnt signaling by LiCl inhibits the formation of a mineralized bone matrix in both cell types. Whereas undifferentiated KS483 cells respond to Wnt3A by inducing nuclear beta-catenin translocation, differentiated cells do not. This is at least in part accomplished by upregulated expression of Dkk-1 and -2 during osteoblast differentiation. Using RNA interference, we showed that Dkk-1 plays a crucial role in blunting the BMP-induced alkaline phosphatase (ALP) response and in the transition of an ALP+ osteoblast in a mineralizing cell. In contrast, Dkk-2 plays a role in osteoblast proliferation and the initiation of osteoblast differentiation.
Our data suggest that Wnt signaling in maturing osteoblasts needs to be downregulated to enable the formation of a mineralized bone matrix. Furthermore, they suggest that Dkk-1 and Dkk-2 may have distinct functions in osteoblast differentiation.