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Chondroitin-4-sulfate (A and B) and cell surface-associated chondroitin sulfate (Cand D) localization as determined by antibodies 2-B-6 and CS-56, respectively, in epiphyses of normal (,4 and C) and mutant (B and D) litter mates. Immunolocalization of chondroitin4-sulfate displayed uniform reactivity throughout the cartilage matrix of both wild-type and mutant epipbyses. Immunoreactivity to cell surface-associated chondroitin sulfate (arrows) was demonstrated on NHC in the mutant epiphysis (D) while no staining was evident in the normal hyperti'ophic zone (C). Specimens were counterstained with methyl green. P, proliferative zone; H, hypertrophic zone. Bar: (A-D) 15 #m.
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![Figure 3. Percentage of [3H]Thymidine-labeled cells...](profile/Hershey-Warshawsky/publication/15110440/figure/fig1/AS:601649965957149@1520455938775/Percentage-of-3HThymidine-labeled-cells-3Hthymidine-labeling-index-in-the_Q320.jpg)
To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18-19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphys...
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... proteoglycan were used to determine the differential distribution of 4-sulfated (2-B-6) and oversul- fated (7-D-4) chondroitin sulfate in normal and mutant epi- physeal cartilage. The distribution of chondroitin-4-sulfate was uniform throughout the extracellular cartilage matrix in all zones of the epiphyses of both normal and homozygous mice (Fig. 6, A and B). A similar immunohistochemical dis- tribution was observed with antibody 7-D-4 (data not ...
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... proximity to the articular sur- face (data not shown). However, differences in staining were evident in the mutant hypertrophic zone when compared to its normal counterpart. In the mutant, immunoreactivity was present and was distinctly associated with the cell surface of the non-hypertrophic chondrocytes clustered in the hyper- trophi c zone (Fig. 6 D). No staining with this antibody was detected in the hypertrophic zone of the PTHrP-normal mice (Fig. 6 ...
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... the mutant hypertrophic zone when compared to its normal counterpart. In the mutant, immunoreactivity was present and was distinctly associated with the cell surface of the non-hypertrophic chondrocytes clustered in the hyper- trophi c zone (Fig. 6 D). No staining with this antibody was detected in the hypertrophic zone of the PTHrP-normal mice (Fig. 6 ...
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Skeletal development is tightly regulated through the processes of chondrocyte proliferation and differentiation. Although the involvement of transcription and growth factors on the regulation of skeletal development has been extensively studied, the role of cell cycle regulatory proteins in this process remains elusive. To date, through cell-speci...
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... Parathyroid hormone (PTH) can stimulate the formation of trabecular bone and participate in bone remodeling, enhancing osteoblast activity and promoting the production of ALP [65,66]. Parathyroid hormone-related protein (PTHrP) is essential for the production of trabecular bone mass and cortical bone [67]. PTHrP, originating from osteocytes, is transported through the network of lacunar-canalicular. ...
Heterotopic ossification (HO) is a pathological process characterized by the aberrant formation of bone in muscles and soft tissues. It is commonly triggered by traumatic brain injury, spinal cord injury, and burns. Despite a wide range of evidence underscoring the significance of neurogenic signals in proper bone remodeling, a clear understanding of HO induced by nerve injury remains rudimentary. Recent studies suggest that injury to the nervous system can activate various signaling pathways, such as TGF-β, leading to neurogenic HO through the release of neurotrophins. These pathophysiological changes lay a robust groundwork for the prevention and treatment of HO. In this review, we collected evidence to elucidate the mechanisms underlying the pathogenesis of HO related to nerve injury, aiming to enhance our understanding of how neurological repair processes can culminate in HO.
... Several early lines of evidence have revealed that PTHrP opposes chondrogenic differentiation both in vitro and in vivo [41][42][43]. Ito et al. [44] reported that in mouse chondrogenic embryonal carcinoma ATDC5 cells PTHrP negatively modulated the mRNA levels of bone morphogenetic protein-4 (BMP4), thus decreasing the amplitude of the chondrogenic signal. Moreover, in chick upper sternal chondrocytes co-treated with BMP2, PTHrP abrogated the expression of RUNX2, which in the cartilage acts as an inducer of chondrocyte maturation both in vitro and in vivo [45]. ...
Parathyroid-hormone-related protein (PTHrP) is encoded by PTHLH gene which, by alternative promoter usage and splicing mechanisms, can give rise to at least three isoforms of 139, 141 and 173 amino acids with distinct C-terminals. PTHrP is subjected to different post-translational processing that generates smaller bioactive forms, comprising amino terminus, midregion (containing a nuclear/nucleolar targeting signal) and carboxy terminus peptides. Both the full-length protein and the discrete peptides are key controllers of viability, proliferation, differentiation and apoptosis in diverse normal and pathological biological systems via the reprogramming of gene expression and remodulation of PKA or PKC-mediated signalization mechanisms. The aim of this review is to pick up selected studies on PTHrP-associated signatures as revealed by molecular profiling assays, focusing on the available data about exemplary differentiating, differentiated or non-tumoral cell and tissue models. In particular, the data presented relate to adipose, bone, dental, cartilaginous and skin tissues, and also intestinal, renal, hepatic, pulmonary and pancreatic epithelia, with a focus on hepatic fibrosis-, pancreatitis- and diabetes-related changes as diseased states. Whether reported, the biochemical and/or physiological aspects associated with the specific molecular modulation of gene expression and signal transduction pathways in the target model systems under examination will be also briefly commented.
... In the experimental conditions used here, the in vivo intraperitoneal treatment of mice (tibially-injected with Pthlh WT cells) with our anti-PTHrP did not induce osteoporotic-like features in terms of osteoblast and osteoclast numbers as well as bone volume loss in non-injected bone as had been observed in homozygous or heterozygous Pthlh-ablated mice. (75,76) The in vivo mAb , and CD49 f , enhances E-cadherin and lowers CD44/CD24 ratio and mammosphere formation, while our anti-PTHrP mAb inhibits KI67 and cell survival and motility. In vivo, the mAb inhibits vimentin, Slug, and ALDH1 in TNBC xenografts and increases Ecadherin, leading to growth inhibition of established TNBC bone tumors. ...
Parathyroid hormone‐related protein (PTHrP) plays a major role in skeletal metastasis but its action mechanism has not been fully defined. We previously demonstrated the crucial importance of PTHrP in promoting mammary tumor initiation, growth and metastasis in a mouse model with a mammary epithelium‐targeted Pthlh gene ablation. We demonstrate here a novel mechanism for bone invasion involving PTHrP induction of epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs) regulation. Crispr‐mediated Pthlh gene ablation was used to study EMT markers, phenotype and invasiveness in two triple‐negative breast cancer (TNBC) cell types (established MDA‐MB‐231 and patient‐derived PT‐TNBC cells). In vitro, Pthlh ablation in TNBC cells reduced EMT markers, mammosphere‐forming ability and CD44high/CD24low cells ratio. In vivo, cells were injected intra‐tibially into athymic nude mice, and therapeutic treatment with our anti‐PTHrP blocking antibody was started 2 weeks after skeletal tumors were established. In vivo, compared to control, lytic bone lesion from Pthlh ‐ablated cells decreased significantly over two weeks by 27% for MDA‐MB‐231 and by 75% for PT‐TNBC‐injected mice (P<0.001). Micro‐CT analyses also showed that antibody therapy reduced bone lytic volume loss by 52% and 48% for non‐ablated MDA‐MB‐231 and PT‐TNBC respectively (P<0.05). Antibody therapy reduced skeletal tumor burden by 45% and 87% for non‐ablated MDA‐MB‐231 and PT‐TNBC respectively (P<0.002) and caused a significant decrease of CSC/EMT markers ALDH1, vimentin and Slug and an increase in E‐cadherin in bone lesions. We conclude that PTHrP is a targetable EMT molecular driver and suggest that its pharmacological blockade can provide a potential therapeutic approach against established TNBC‐derived skeletal lesions. This article is protected by copyright. All rights reserved.
... Resting (nonproliferating) chondrocytes, specifically expressing parathyroid-hormone-related protein (PTHrP), are skeletal stem cells that undergo chondrocyte differentiation and proliferation (10). PTHrP maintains chondrocytes in a proliferative state, which is indispensable for endochondral bone growth (11,12). As the distance from PTHrP-expressing resting chondrocytes increases, the columnar chondrocytes stop proliferating and differentiate into type X collagen (COL X, Col10a1)-expressing hypertrophic chondrocytes, mineralizing their surrounding matrix. ...
Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common pathological condition that causes intractable myelopathy and radiculopathy, mainly the result of an endochondral ossification-like process. Our previous genome-wide association study identified six susceptibility loci for OPLL, including the CDC5L gene region. Here, we found CDC5L to be expressed in type II collagen-producing chondrocyte-like fibroblasts in human OPLL specimens, as well as in differentiating ATDC5 chondrocytes. Cdc5l siRNA transfection in murine chondrocytes decreased the expression of the early chondrogenic genes Sox9 and Col2a1, diminished the cartilage matrix production, and enhanced the expression of parathyroid hormone-related protein (a resting chondrocyte marker). We also showed that Cdc5l shRNA suppressed the growth of cultured murine embryonal metatarsal cartilage rudiments, and that Cdc5l knockdown suppressed the growth of ATDC5 cells. Fluorescence-activated cell sorting analysis revealed that the G2/M cell cycle transition was blocked; our data showed that Cdc5l siRNA transfection enhanced expression of Wee1, an inhibitor of the G2/M transition. Cdc5l siRNA also decreased the pre-mRNA splicing efficiency of Sox9 and Col2a1 genes in both ATDC5 cells and primary chondrocytes; conversely, loss of Cdc5l resulted in enhanced splicing of Wee1 pre-mRNA. Finally, an RNA-binding protein immunoprecipitation assay revealed that Cdc5l bound directly to these target gene transcripts. Overall, we conclude that Cdc5l promotes both early chondrogenesis and cartilage growth, and may play a role in the etiology of OPLL, at least in part by fine-tuning the pre-mRNA splicing of chondrogenic genes and Wee1, thus initiating the endochondral ossification process.
... The first evidence of a physiological role for PTHrP came with the dramatic phenotype in mice null for the Pthlh gene following homologous recombination (113,114,123,124). Global deletion of Pthlh gene was lethal, causing the mice to die immediately after birth due to inadequate rib cage development and what has been thought to result in respiratory failure (114). ...
... The first evidence of a physiological role for PTHrP came with the dramatic phenotype in mice null for the Pthlh gene following homologous recombination (113,114,123,124). Global deletion of Pthlh gene was lethal, causing the mice to die immediately after birth due to inadequate rib cage development and what has been thought to result in respiratory failure (114). Abnormalities were evident throughout the endochondral skeleton in Pthlh null mice, but none were detected in bone that developed through intramembranous ossification, thus indicating that the defect was driven by deficiency of PTHrP in chondrocytes. ...
... Although genetic ablation of Pthlh was neonatal lethal in the mouse (114), the heterozygous (Pthlh+/-) mice were otherwise healthy and had normal adult skeletal size, but demonstrated a significant deficiency in the amount of bone that was detected by 3 months of age (36). This indicated that loss of 1 functional allele of Pthlh results in bone loss without affecting skeletal development. ...
The hormone, parathyroid hormone (PTH) and the paracrine factor, parathyroid hormone-related protein (PTHrP) have preserved in evolution sufficient identities in their amino-terminal domains to share equivalent actions upon a common G protein coupled receptor, PTH1R, that predominantly uses the cyclic AMP (cAMP)-protein kinase A signaling pathway. Such a relationship between a hormone and local factor poses questions about how their common receptor mediates pharmacological and physiological actions of the two. Mouse genetic studies shown that PTHrP is essential for endochondral bone lengthening in the fetus and is essential for bone remodeling. In contrast, the main postnatal function of PTH is hormonal control of calcium homeostasis, with no evidence that PTHrP contributes. Pharmacologically, amino-terminal PTH and PTHrP peptides (teriparatide and abaloparatide) promote bone formation when administered by intermittent (daily) injection. This anabolic effect is remodeling-based with a lesser contribution from modeling. The apparent lesser potency of PTHrP than PTH peptides as skeletal anabolic agents could be explained by lesser bioavailability to PTH1R. By contrast, prolongation of PTH1R stimulation by excessive dosing or infusion, converts the response to a predominantly resorptive one by stimulating osteoclast formation. Physiologically, locally generated PTHrP is better equipped than the circulating hormone to regulate bone remodeling, which occurs asynchronously at widely distributed sites throughout the skeleton where it is needed to replace old or damaged bone. While it remains possible that PTH, circulating within a narrow concentration range, could contribute in some way to remodeling and modeling, its main physiological role is in regulating calcium homeostasis.
... [42,43] Moreover, the effects of PTHrP are partly mediated by paracrine and/or autocrine activation of the PTH1R, Furthermore, our results suggested that PTHrP could not enhance the proliferation of osteoblasts, which was not consistent with evidence that supports the positive correlation betweenPTHrP and osteoblast proliferation. [48] PTHrP has been shown to target cyclin D1, CDK1 p21, p27 and p16 protein in differentiated osteoblasts, induced G1 phase growth arrest, [49] and the proliferation is a consequence of an action on the maturation stage of the osteoblast cell cycle. ...
Osteoporosis is a severe health problem causing bone fragility and consequent fracture. Titanium (Ti) implants, used in patients with osteoporotic fractures, are prone to failure because of the decreased bone mass and strength. Therefore, it is of utmost importance to fabricate implants possessing osteogenic properties to improve implant osseointegration. To improve the long-term survival rate of Ti implants in osteoporotic patients, hyaluronic acid/ε-polylysine multilayers containing the parathyroid hormone (PTH)-related protein (PTHrP) were deposited on Ti implants by a layer-by-layer (LBL) electro assembly technique. The murine pre-osteoblast cell line MC3T3-E1 possessing a high potential of osteoblast differentiation, was used to evaluate the osteo-inductive effects of Ti-LBL-PTHrP in vitro. In addition, the performance of the Ti (Ti-LBL-PTHrP) implant was evaluated in vivo in a femoral intramedullary implantation in Sprague Dawley rats. The Ti-LBL-PTHrP implant regulated the release of the loaded PTHrP to increase bone formation in the early stage of implantation. The in vitro results revealed that cells on Ti-LBL-PTHrP did not show any evident proliferation, but a high level of alkaline phosphatase (ALP) activity and osteoblast-related protein expression was found, compared to uncoated Ti group (p < 0. 05). In addition, in vivo micro-CT and histological analysis demonstrated that the Ti-LBL-PTHrP implants could significantly promote the formation and remodeling of new bone in osteoporotic rats at 14 days after implantation. Overall, this study established a profound and straightforward methodology for the manufacture of biofunctional Ti implants for the treatment of osteoporosis.
... As far as we know, this was the first demonstration of statistically significant post-transplantational effects of an agent used for the treatment of tissue-engineered cartilage at a pre-transplantational stage. PTHrP is also known to maintain immature chondrocytes in a proliferative state in growth plate [213,214]. In fact, our 5-ethynyl-2ʹ-deoxyuridine (EdU) incorporation study revealed that forskolin significantly increased the proportion of DNA-replicated chondrocytes (SOX9 + EdU + cells) in the cartilage pellets. ...
The cartilage of joints, such as meniscus and articular cartilage, is normally long lasting (i.e., permanent). However, once damaged, especially in large animals and humans, joint cartilage is not spontaneously repaired. Compensating the lack of repair activity by supplying cartilage-(re)forming cells, such as chondrocytes or mesenchymal stromal cells, or by transplanting a piece of normal cartilage, has been the basis of therapy for biological restoration of damaged joint cartilage. Unfortunately, current biological therapies face problems on a number of fronts. The joint cartilage is generated de novo from a specialized cell type, termed a ‘joint progenitor’ or ‘interzone cell’ during embryogenesis. Therefore, embryonic chondroprogenitors that mimic the property of joint progenitors might be the best type of cell for regenerating joint cartilage in the adult. Pluripotent stem cells (PSCs) are expected to differentiate in culture into any somatic cell type through processes that mimic embryogenesis, making human (h)PSCs a promising source of embryonic chondroprogenitors. The major research goals toward the clinical application of PSCs in joint cartilage regeneration are to (1) efficiently generate lineage-specific chondroprogenitors from hPSCs, (2) expand the chondroprogenitors to the number needed for therapy without loss of their chondrogenic activity, and (3) direct the in vivo or in vitro differentiation of the chondroprogenitors to articular or meniscal (i.e., permanent) chondrocytes rather than growth plate (i.e., transient) chondrocytes. This review is aimed at providing the current state of research toward meeting these goals. We also include our recent achievement of successful generation of “permanent-like” cartilage from long-term expandable, hPSC-derived ectomesenchymal chondroprogenitors.
... it was noted by albright in 1941, 21 and it has since been found in many tissues. 22 PTHrP plays a key role during endochondral ossification; 23 it is expressed by perichondrial cells and early proliferating chondrocytes and then diffuses to act on PTH/PTHrP receptor-bearing cells to inhibit expression of ruNX2 and suppress chondrocyte hypertrophy. 24 in addition to regulating the proliferation and differentiation of chondrocytes by limiting ihh expression, a recent study showed that PTHrP signalling promotes HDaC4 localization to the nucleus and increases HDaC4-induced inhibition of chondrocyte hypertrophy. ...
Chondrocyte hypertrophy represents a crucial turning point during endochondral bone development. This process is tightly regulated by various factors, constituting a regulatory network that maintains normal bone development. Histone deacetylase 4 (HDAC4) is the most well-characterized member of the HDAC class IIa family and participates in different signalling networks during development in various tissues by promoting chromatin condensation and transcriptional repression. Studies have reported that HDAC4-null mice display premature ossification of developing bones due to ectopic and early-onset chondrocyte hypertrophy. Overexpression of HDAC4 in proliferating chondrocytes inhibits hypertrophy and ossification of developing bones, which suggests that HDAC4, as a negative regulator, is involved in the network regulating chondrocyte hypertrophy. Overall, HDAC4 plays a key role during bone development and disease. Thus, understanding the role of HDAC4 during chondrocyte hypertrophy and endochondral bone formation and its features regarding the structure, function, and regulation of this process will not only provide new insight into the mechanisms by which HDAC4 is involved in chondrocyte hypertrophy and endochondral bone development, but will also create a platform for developing a therapeutic strategy for related diseases.
Cite this article: Bone Joint Res. 2020;9(2):82–89.
... With such evidence of production of PTHrP in many tissues throughout fetal life in a number of species, it was perhaps to be expected that functional evidence of a role for PTHrP and its receptor in mammalian fetal development might come from studies of animals that are transgenic or have gene-targeted knockouts; these models, including later cell-specific models, are summarized in Table 25.1. The first evidence of a physiological role for PTHrP came soon after the discovery of PTHrP, with a dramatic phenotype in mice null for the Pthlh gene following homologous recombination (Amizuka et al., 1994;Karaplis et al., 1994;Karaplis and Kronenberg, 1996;Lee et al., 1996). Neonatal mice homozygous for Pthlh gene ablation exhibited severe skeletal abnormalities at birth and died within 24 h, with their abnormal ribcage development not allowing adequate respiration and this respiratory distress accentuated by reduced surfactant production and impaired type II alveolar cell development. ...
... Major insights into the role of PTHrP in bone were obtained from further studies carried out in heterozygous Pthlh þ/À mice. Although homozygous Pthlh À/À mice died soon after birth (Amizuka et al., 1994), heterozygous Pthlh þ/À mice survived and, although phenotypically normal at birth, by 3 months of age exhibited a markedly lower trabecular thickness and connectivity, and histomorphometry revealed low bone formation rate and osteoclast surface (Amizuka et al., 1996). In Pthlh þ/À mice the number of apoptotic osteoblasts was greater than in controls, and ex vivo culture of bone marrow cells resulted in markedly fewer alkaline phosphataseepositive colonies, indicating impairment of osteogenic cell recruitment and survival. ...
... Further investigations of the PTHrP mutant mice provided evidence that PTHrP is equally important for the orderly commitment of precursor cells toward the osteogenic lineage and their subsequent maturation and/or function (see Fig. 25.3). In the Pthlh þ/À mice, osteoblastic progenitor cells (as with chondrocytes) were observed to contain inappropriate accumulations of glycogen, indicative of a defect in cells of the osteogenic lineage arising as a consequence of PTHrP deficiency (Amizuka et al., 1994). The overall conclusion was that PTHrP haploinsufficiency resulted in a low-bone-turnover state with low bone mass resulting from impaired bone formation. ...
... In contrast, when ACs are induced to cartilage formation in vitro under the same conditions (re-differentiation culture), they form phenotypically stable cartilage that maintains low expression of hypertrophic markers and does not form ectopic bone in vivo. In line with developmental mouse studies (Amizuka et al., 1994;Vortkamp et al., 1996), suppression of prohypertrophic Indian hedgehog (IHH) signaling by parathyroid hormone related protein was proposed as mechanism how ACs maintain phenotype stability in vitro (Fischer et al., 2010). In contrast, MSCs upregulate IHH-activity along with bone morphogenetic proteins (BMPs), all of which is supported by WNT-signaling ( Fischer et al., 2010;Dexheimer et al., 2016;Diederichs et al., 2019). ...
A major problem with chondrocytes derived in vitro from stem cells is undesired hypertrophic degeneration, to which articular chondrocytes (ACs) are resistant. As progenitors of all adult tissues, induced pluripotent stem cells (iPSCs) are in theory able to form stable articular cartilage. In vitro differentiation of iPSCs into chondrocytes with an AC-phenotype and resistance to hypertrophy has not been demonstrated so far. Here, we present a novel protocol that succeeded in deriving chondrocytes from human iPSCs without using pro-hypertrophic bone-morphogenetic-proteins. IPSC-chondrocytes had a high cartilage formation capacity and deposited two-fold more proteoglycans per cell than adult ACs. Importantly, cartilage engineered from iPSC-chondrocytes had similar marginal expression of hypertrophic markers (COL10A1, PTH1R, IBSP, ALPL mRNAs) like cartilage from ACs. Collagen X was barely detectable in iPSC-cartilage and 30-fold lower than in hypertrophic cartilage derived from mesenchymal stromal cells (MSCs). Moreover, alkaline phosphatase (ALP) activity remained at basal AC-like levels throughout iPSC chondrogenesis, in contrast to a well-known significant upregulation in hypertrophic MSCs. In line, iPSC-cartilage subjected to mineralizing conditions in vitro showed barely any mineralization, while MSC-derived hypertrophic cartilage mineralized strongly. Low expression of Indian hedgehog (IHH) like in ACs but rising BMP7 expression like in MSCs suggested that phenotype stability was linked to the hedgehog rather than the bone morphogenetic protein (BMP) pathway. Taken together, unlimited amounts of AC-like chondrocytes with a high proteoglycan production reminiscent of juvenile chondrocytes and resistance to hypertrophy and mineralization can now be produced from human iPSCs in vitro. This opens new strategies for cartilage regeneration, disease modeling and pharmacological studies.
