Cellular and subcellular distribution of galectin-3 in the epiphyseal cartilage and bone of fetal and neonatal mice.

Galectin-3 is a 30 kDa beta-galactoside binding protein that belongs to the galectin family of animal lectins. By immunocytochemistry we show the presence of galectin-3 protein in the differentiated chondrocytes of the epiphyseal plate cartilage of long bones of both fetal and neonatal mice. The highest concentrations of galectin-3 are found in the cytoplasm of mature and early hypertrophic chondrocytes. Very little protein is detected in the late hypertrophic chondrocytes undergoing terminal maturation and cell death. Galectin-3 has also been found in osteoblasts and osteocytes of the woven bone of the metaphysis and the cortical bone of the diaphysis, as well as in osteoclasts and mononuclear cells within bone marrow cavities. Galectin-3 is never detected extracellularly, the protein seems restricted to the cytoplasm of chondrocytes and bone cells, although it is occasionally detected in the nuclei of dense non-hypertrophic chondrocytes in the zone of calcification and in young osteoblasts. The results indicate that galectin-3 is a marker of both chondrogenic and osteogenic cell lineages. They also suggest that galectin-3 could be involved in the process of endochondral bone formation, possibly as a regulator of chondrocyte survival.     

Parathyroid hormone-related peptide expression in the epiphyseal growth plate of the juvenile chicken: evidence for the origin of the parathyroid hormone-related peptide found in the epiphyseal growth plate

Parathyroid hormone-related peptide (PTHrP) has been shown to be essential for normal endochondral bone formation. Along with Indian hedgehog (Ihh), it forms a paracrine regulatory loop that governs the pace of chondrocyte differentiation. However, the source of PTHrP for this regulatory loop is not clear. While one hypothesis has suggested the periarticular perichondrium as the source of PTHrP for growth plate regulation, other data utilizing immunohistochemistry and in situ hybridization would indicate that growth plate chondrocytes themselves are the source of this peptide. The data described in this report supports the view that postnatal growth plate chondrocytes have the ability to synthesize this important regulatory peptide. Immunohistochemistry of tissue sections showed that PTHrP protein was evident throughout the chick epiphysis. PTHrP was seen in chondrocytes in the periarticular perichondrium, the perichondrium adjacent to the growth plate, the prehypertrophic zone of the growth plate, and the hypertrophic zone of the growth plate. However, cells in the proliferative zone, as well as some chondrocytes in the deeper layers of articular cartilage were predominantly negative for PTHrP. PTHrP was detected by Western blotting as a band of 16,400 Da in extracts from hypertrophic chondrocytes, but not from proliferative cells. RT-PCR detected PTHrP mRNA in both proliferative and hypertrophic growth plate chondrocytes, as well as in articular chondrocytes. PTH/PTHrP receptor mRNA was detected by Northern blotting in growth plate, but not articular chondrocytes. Thus, we conclude that most of the PTHrP present in the epiphyseal growth plate of the juvenile chick originates in the growth plate itself. Furthermore, the presence of large amounts of PTHrP protein in the hypertrophic zone supports the concept that PTHrP has other functions in addition to regulating chondrocyte differentiation.     

Galectin-3 is a downstream regulator of matrix metalloproteinase-9 function during endochondral bone formation

Endochondral bone formation is characterized by the progressive replacement of a cartilage anlagen by bone at the growth plate with a tight balance between the rates of chondrocyte proliferation, differentiation, and cell death. Deficiency of matrix metalloproteinase-9 (MMP-9) leads to an accumulation of late hypertrophic chondrocytes. We found that galectin-3, an in vitro substrate of MMP-9, accumulates in the late hypertrophic chondrocytes and their surrounding extracellular matrix in the expanded hypertrophic cartilage zone. Treatment of wild-type embryonic metatarsals in culture with full-length galectin-3, but not galectin-3 cleaved by MMP-9, mimicked the embryonic phenotype of Mmp-9 null mice, with an increased hypertrophic zone and decreased osteoclast recruitment. These results indicate that extracellular galectin-3 could be an endogenous substrate of MMP-9 that acts downstream to regulate hypertrophic chondrocyte death and osteoclast recruitment during endochondral bone formation. Thus, the disruption of growth plate homeostasis in Mmp-9 null mice links galectin-3 and MMP-9 in the regulation of the clearance of late chondrocytes through regulation of their terminal differentiation.     

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.     

Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy.

Longitudinal bone growth, and hence stature, are functions of growth plate chondrocyte proliferation and hypertrophy. Insulin-like growth factor 1 (Igf1) is reputed to augment longitudinal bone growth by stimulating growth plate chondrocyte proliferation. In this study, however, we demonstrate that chondrocyte numbers and proliferation are normal in Igf1 null mice despite a 35% reduction in the rate of long bone growth. Igf1 null hypertrophic chondrocytes differentiate normally in terms of expressing specialized proteins such as collagen X and alkaline phosphatase, but are smaller than wild-type at all levels of the hypertrophic zone. The terminal hypertrophic chondrocytes, which form the scaffold on which long bone growth extends, are reduced in linear dimension by 30% in Igf1 null mice, accounting for most of their decreased longitudinal growth. The expression of the insulin-sensitive glucose transporter, GLUT4, is significantly decreased and the insulin-regulated enzyme glycogen synthase kinase 3beta (GSK3) is hypo-phosphorylated in Igf1 null chondrocytes. Glycogen levels were significantly decreased and ribosomal RNA levels were reduced by almost 75% in Igf1 null chondrocytes. These data suggest that Igf1 promotes longitudinal bone growth by 'insulin-like' anabolic actions which augment chondrocyte hypertrophy.     

Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival

Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.     

SIK3 is essential for chondrocyte hypertrophy during skeletal development in mice

Chondrocyte hypertrophy is crucial for endochondral ossification, but the mechanism underlying this process is not fully understood. We report that salt-inducible kinase 3 (SIK3) deficiency causes severe inhibition of chondrocyte hypertrophy in mice. SIK3-deficient mice showed dwarfism as they aged, whereas body size was unaffected during embryogenesis. Anatomical and histological analyses revealed marked expansion of the growth plate and articular cartilage regions in the limbs, accumulation of chondrocytes in the sternum, ribs and spine, and impaired skull bone formation in SIK3-deficient mice. The primary phenotype in the skeletal tissue of SIK3-deficient mice was in the humerus at E14.5, where chondrocyte hypertrophy was markedly delayed. Chondrocyte hypertrophy was severely blocked until E18.5, and the proliferative chondrocytes occupied the inside of the humerus. Consistent with impaired chondrocyte hypertrophy in SIK3-deficient mice, native SIK3 expression was detected in the cytoplasm of prehypertrophic and hypertrophic chondrocytes in developing bones in embryos and in the growth plates in postnatal mice. HDAC4, a crucial repressor of chondrocyte hypertrophy, remained in the nuclei in SIK3-deficient chondrocytes, but was localized in the cytoplasm in wild-type hypertrophic chondrocytes. Molecular and cellular analyses demonstrated that SIK3 was required for anchoring HDAC4 in the cytoplasm, thereby releasing MEF2C, a crucial facilitator of chondrocyte hypertrophy, from suppression by HDAC4 in nuclei. Chondrocyte-specific overexpression of SIK3 induced closure of growth plates in adulthood, and the SIK3-deficient cartilage phenotype was rescued by transgenic SIK3 expression in the humerus. These results demonstrate an essential role for SIK3 in facilitating chondrocyte hypertrophy during skeletogenesis and growth plate maintenance.     

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.     

Tissue and cellular morphological changes in growth plate explants under compression

The mechanisms by which mechanical loading may alter bone development within growth plates are still poorly understood. However, several growth plate cell or tissue morphological parameters are associated with both normal and mechanically modulated bone growth rates. The aim of this study was to quantify in situ the three-dimensional morphology of growth plate explants under compression at both cell and tissue levels. Growth plates were dissected from ulnae of immature swine and tested under 15% compressive strain. Confocal microscopy was used to image fluorescently labeled chondrocytes in the three growth plate zones before and after compression. Quantitative morphological analyses at both cell (volume, surface area, sphericity, minor/major radii) and tissue (cell/matrix volume ratio) levels were performed. Greater chondrocyte bulk strains (volume decrease normalized to the initial cell volume) were found in the proliferative (35.4%) and hypertrophic (41.7%) zones, with lower chondrocyte bulk strains (24.7%) in the reserve zone. Following compression, the cell/matrix volume ratio decreased in the reserve and hypertrophic zones by 24.3% and 22.6%, respectively, whereas it increased by 35.9% in the proliferative zone. The 15% strain applied on growth plate explants revealed zone-dependent deformational states at both tissue and cell levels. Variations in the mechanical response of the chondrocytes from different zones could be related to significant inhomogeneities in growth plate zonal mechanical properties. The ability to obtain in situ cell morphometry and monitor the changes under compression will contribute to a better understanding of mechanisms through which abnormal growth can be triggered.     

Transforming growth factor-beta and Wnt signals regulate chondrocyte differentiation through Twist1 in a stage-specific manner

We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-beta and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, whereas growth plate-extracted cells displayed a hypertrophic phenotype. TGF-beta significantly induced beta-catenin in immature chondrocytes, whereas it repressed it in mature chondrocytes. TGF-beta further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it inhibited Topflash activity in a time-dependent manner in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-beta induced Sma- and Mad-related protein 3 interaction with T-cell factor 4 in immature chondrocytes, whereas it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin immunoprecipitation showing that TGF-beta differentially shifts T-cell factor 4 occupancy on the Runx2 promoter in lower sternal chondrocytes vs. growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-beta and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, whereas TGF-beta induced Twist1 in mature chondrocytes, it inhibited Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment, which predominates over TGF-beta effects on these cells. Finally, overexpression of chick Twist1 in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression toward terminal maturation in response to TGF-beta and canonical Wnt signaling.     

Smad4 is required for the normal organization of the cartilage growth plate

Smad4 is the central intracellular mediator of transforming growth factor-beta (TGF-beta) signals. To study the role of Smad4 in skeletal development, we introduced a conditional mutation of the gene in chondrocytes using Cre--loxP system. We showed that Smad4 was expressed strongly in prehypertrophic and hypertrophic chondrocytes. The abrogation of Smad4 in chondrocytes resulted in dwarfism with a severely disorganized growth plate characterized by expanded resting zone of chondrocytes, reduced chondrocyte proliferation, accelerated hypertrophic differentiation, increased apoptosis and ectopic bone collars in perichondrium. Meanwhile, Smad4 mutant mice exhibited decreased expression of molecules in Indian hedgehog/parathyroid hormone-related protein (Ihh/PTHrP) signaling. The cultured mutant metatarsal bones failed to response to TGF-beta1, while the hypertrophic differentiation was largely inhibited by Sonic hedgehog (Shh). This indicated that Ihh/PTHrP inhibited the hypertrophic differentiation of chondrocytes independent of the Smad4-mediated TGF-beta signals. All these data provided the first genetic evidence demonstrating that Smad4-mediated TGF-beta signals inhibit the chondrocyte hypertrophic differentiation, and are required for maintaining the normal organization of chondrocytes in the growth plate.     

Expression of Bcl-2 protein in the epiphyseal plate cartilage and trabecular bone of growing rats

 The protooncogene protein, Bcl-2, protects cells from apoptosis and ensures their survival in vitro by inhibiting the action of the apoptosis-inducer, Bax. Its expression in proliferative and long-lived cells in vivo also indicates that it protects against cell death. The chondrocytes of the epiphyseal plate cartilage undergo a series of maturation steps and deposit mineral in the cartilage matrix before dying. The possibility that Bcl-2 helps protect chondrocytes until mineral deposition is completed was investigated by determining the distribution of Bcl-2 immunoreactivity in the epiphyseal plate cartilage of growing rats and its subcellular localization, using a specific antibody. The involvement of Bax in the triggering of chondrocyte death was checked by immunocytochemistry. Bcl-2 expression in the osteoblasts and the final result of their evolution, the osteocytes, was also examined in trabecular bone. Bcl-2 immunoreactivity was non-uniformly distributed throughout the epiphyseal cartilage. It was maximal in proliferative chondrocytes, decreased in mature chondrocytes, and low in hypertrophic chondrocytes, whereas there was Bax immunoreactivity in all chondrocytes examined. Immunolabeling was intense in osteoblasts but considerably lower in fully differentiated osteocytes. Bcl-2 immunoreactivity was mainly in the cytoplasm of chondrocytes, osteoblasts, and early osteocytes; the nuclei appeared clear. The subcellular distribution of Bcl-2 immunolabeling in chondrocytes, revealed by gold particles in the electron microscope, showed that gold particles were frequently concentrated in the mitochondria in all the cartilage zones and lay mainly within the organelles, not at their periphery. The endoplasmic reticulum contained moderate immunoreactivity and there were few gold particles in the cytoplasm and nuclei. The number of gold particles decreased in all the subcellular compartments from proliferative to hypertrophic chondrocytes. In contrast, Bax immunoreactivity changed little during chondrocyte terminal evolution, and its subcellular distribution mirrored that of Bcl-2. These immunocytochemical data indicate that Bcl-2 helps maintain chondrocytes and osteoblasts until their terminal maturation. Accepted: 19 February 1997     

Suppression of mammalian bone growth by membrane transport inhibitors

Bone lengthening during skeletal growth is driven primarily by the controlled enlargement of growth plate (GP) chondrocytes. The cellular mechanisms are unclear but membrane transporters are probably involved. We investigated the role of the Na⁺/H⁺ antiporter (NHE1) and anion exchanger (AE2) in bone lengthening and GP chondrocyte hypertrophy in Sprague–Dawley 7‐day‐old rat (P7) bone rudiments using the inhibitors EIPA (5‐(N‐ethyl‐N‐isopropyl)amiloride) and DIDS (4,4‐diidothiocyano‐2,2‐stilbenedisulphonate), respectively. We have also determined cell‐associated levels of these transporters along the GP using fluorescent immunohistochemistry (FIHC). Culture of bones with EIPA or DIDS inhibited rudiment growth (50% at approx. 250 and 25 µM, respectively). Both decreased the size of the hypertrophic zone (P < 0.05) but had no effect on overall length or cell density of the GP. In situ chondrocyte volume in proliferative and hypertrophic zones was decreased (P < 0.01) with EIPA but not DIDS. FIHC labeling of NHE1 was relatively high and constant along the GP but declined steeply in the late hypertrophic zone. In contrast, AE2 labeling was relatively low in proliferative zone cells but increased (P < 0.05) reaching a maximum in the early hypertrophic zone, before falling rapidly in the late hypertrophic zone suggesting AE2 might regulate the transition phase of chondrocytes between proliferative and hypertrophic zones. The inhibition of bone growth by EIPA may be due to a reduction to chondrocyte volume set‐point. However the effect of DIDS was unclear but could result from inhibition of AE2 and blocking of the transition phase. These results demonstrate that NHE1 and AE2 are important regulators of bone growth. J. Cell. Biochem. 114: 658–668, 2013. © 2012 Wiley Periodicals, Inc.     

Immunolocation analysis of glycosaminoglycans in the human growth plate.

Monoclonal antibodies were used in this study to immunolocate glycosaminoglycans throughout the human growth plate. Chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate were observed in the extracellular matrix of all zones of the growth plate and persisted into the cartilage trabeculae of newly formed metaphyseal bone. Also present in the extracellular matrix was an oversulfated chondroitin/dermatan sulfate glycosaminoglycan which appeared to be specific to the proliferative and hypertrophic zones of the growth plate. As with the other extracellular matrix molecules, this epitope persisted into the cartilage trabeculae of the metaphyseal bone. Zonal differences between the extracellular and pericellular or lacunae matrix were also observed. The hypertrophic chondrocytes appeared to synthesize chondroitin sulfate chains containing a non-reducing terminal 6-sulfated disaccharide, which were located in areas immediately adjacent to the cells. This epitope was not found to any significant extent in the other zones. The pericellular region around hypertrophic chondrocytes also contained a keratan sulfate epitope which was also observed in the resting zone but not in the proliferative zone. These cell-associated glycosaminoglycans were not found in the cartilage trabeculae of metaphyseal bone, indicating their removal as the terminal hypertrophic chondrocytes and their lacunae are removed by invading blood vessels. These changes in matrix glycosaminoglycan content, both in the different zones and within zones, indicate constant subtle alterations in chondrocyte metabolic products as they proceed through their life cycle of proliferation, maturation, and hypertrophy.     

Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation

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 epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.     

Phosphate-induced Apoptosis of Hypertrophic Chondrocytes Is Associated with a Decrease in Mitochondrial Membrane Potential and Is Dependent upon Erk1/2 Phosphorylation

Growth plate abnormalities, associated with impaired hypertrophic chondrocyte apoptosis, are observed in humans and animals with abnormalities of vitamin D action and renal phosphate reabsorption. Low circulating phosphate levels impair hypertrophic chondrocyte apoptosis, whereas treatment of these cells with phosphate activates the mitochondrial apoptotic pathway. Because phosphate-mediated apoptosis of chondrocytes is differentiation-dependent, studies were performed to identify factors that contribute to hypertrophic chondrocyte apoptosis. An increase in the percentage of cells with low mitochondrial membrane potential, evaluated by JC-1 fluorescence, was observed during hypertrophic differentiation of primary murine chondrocytes in culture. This percentage was further increased by treatment of hypertrophic, but not proliferative, chondrocytes with phosphate. Phosphate-mediated apoptosis was observed as early as 30 min post-treatment and was dependent upon Erk1/2 phosphorylation. Inhibition of Erk1/2 phosphorylation in vivo confirmed an important role for this signaling pathway in regulating hypertrophic chondrocyte apoptosis in growing mice. Murine embryonic metatarsals cultured under phosphate-restricted conditions demonstrated a 2.5-fold increase in parathyroid hormone-related protein mRNA expression accompanied by a marked attenuation in phospho-Erk immunoreactivity in hypertrophic chondrocytes. Thus, these investigations point to an important role for phosphate in regulating mitochondrial membrane potential in hypertrophic chondrocytes and growth plate maturation by the parathyroid hormone-related protein signaling pathway.     

The osmotic sensitivity of rat growth plate chondrocytes in situ; clarifying the mechanisms of hypertrophy

Bone elongation is predominantly driven by the volume expansion of growth plate chondrocytes. This mechanism was initially believed to be "hypertrophy", describing a proportional increase of cell water and organelles. However, morphometrical analysis subsequently assumed the increase to be "swelling", resulting in a disproportionate increase of cell water (osmotically active fraction). Histological approaches were performed on fixed tissue, and for the "swelling" assumption to be valid, the osmotic sensitivity of living cells before and during volume increase should differ. To test this, analysis of images acquired by 2-photon laser scanning microscopy (2PLSM) were used to determine the osmotic sensitivity, and osmotically active/inactive proportions of in situ chondrocytes from 15 living rat growth plates exposed to varying media osmolarities ( approximately 0-580 mOsm). The dimensions of cell volume swelling in hypotonic media were different to the preferential lengthening seen in vivo, confirming the complexity of directional cell volume increase. Boyle-van't Hoff analysis of cell volume over the range of media osmolarity indicated no significant difference (Student's t-test) in the osmotically inactive fraction, 39.5 +/- 2.9% and 47.0 +/- 4.3% (n = 13) for proliferative and hypertrophic zones, respectively, or the sensitivity of volume to changes in media osmolarity (proliferative 15.5 +/- 0.8 and hypertrophic zone 15.5 +/- 1.2%volume . Osm). The osmotic fractions did not change as chondrocytes progress from proliferative to hypertrophic regions of the growth plate. Our data suggest cell volume increase by hypertrophy may play a greater role in cell enlargement than swelling, and should be re-evaluated as a mechanism responsible for growth plate chondrocyte volume increase and hence bone elongation.     

Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes

Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C alpha (PKCalpha), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKCalpha play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKCalpha was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKCalpha led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKCalpha stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis.