Cartilage abnormalities are associated with abnormal Phex expression and with altered matrix protein and MMP-9 localization in Hyp mice

X-linked hypophosphatemic rickets (HYP) in humans is caused by mutations in the PHEX gene. This gene mutation is also found in Hyp mice, the murine homologue of the human disease. At present, it is unknown why loss of Phex function leads to cartilage abnormalities in Hyp mice. In the present study, we compared in wild-type and Hyp mice Phex protein localization in cartilage of developing long bone as well as localization of skeletal matrix proteins and matrix metalloproteinase-9 (MMP-9). Also compared were chondrocyte apoptosis in the growth plate, mineralization and cartilage remnant retention in the metaphysis, and chondroclast/osteoclast characteristics in the primary spongiosa. Phex protein was detected in proliferating and hypertrophic chondrocytes in growth plate cartilage of wild-type mice, but not in Hyp mice. Hyp mice exhibited a widened and irregular hypertrophic zone in growth plate cartilage showing hypomineralization, increased cartilage remnants from the growth plate in both metaphyseal trabecular and cortical bone, and fewer and smaller chondroclasts/osteoclasts in the primary spongiosa. Increased link protein and C-propeptide of type II procollagen of Hyp mice reflected the increase in chondrocytes and matrix in the cartilaginous growth plate and in bone. In addition, growth plate osteocalcin and bone sialoprotein levels were decreased, while osteonectin was increased, in hypertrophic chondrocytes and cartilage matrix in Hyp mice. MMP-9 in hypertrophic chondrocytes was also reduced in Hyp mice and fewer apoptotic hypertrophic chondrocytes were detected. These findings suggest that Phex may control mineralization and removal of hypertrophic chondrocytes and cartilage matrix in growth plate by regulating the synthesis and deposition of certain bone matrix proteins and proteases such as MMP-9.     

PTH Receptor Signaling in Osteoblasts Regulates Endochondral Vascularization in Maintenance of Postnatal Growth Plate

Longitudinal growth of postnatal bone requires precise control of growth plate cartilage chondrocytes and subsequent osteogenesis and bone formation. Little is known about the role of angiogenesis and bone remodeling in maintenance of cartilaginous growth plate. Parathyroid hormone (PTH) stimulates bone remodeling by activating PTH receptor (PTH1R). Mice with conditional deletion of PTH1R in osteoblasts showed disrupted trabecular bone formation. The mice also exhibited postnatal growth retardation with profound defects in growth plate cartilage, ascribable predominantly to a decrease in number of hypertrophic chondrocytes, resulting in premature fusion of the growth plate and shortened long bones. Further characterization of hypertrophic zone and primary spongiosa revealed that endochondral angiogenesis and vascular invasion of the cartilage were impaired, which was associated with aberrant chondrocyte maturation and cartilage development. These studies reveal that PTH1R signaling in osteoblasts regulates cartilaginous growth plate for postnatal growth of bone. © 2014 American Society for Bone and Mineral Research.     

Immunohistochemical localization of interleukin 1 in human growth cartilage

Although it has been reported that interleukin 1 (IL-1) stimulate chondrocytes to produce collagenase and proteoglycanase in vitro, IL-1 producing cells and the function of IL-1 have not been demonstrated in osteocartilaginous tissue in vivo. Immunohistochemical studies of human cartilaginous epiphysis and growth cartilage demonstrated that IL-1 was detected in: (1) chondrocytes surrounding cartilage canal, (2) hypertrophic chondrocytes in cartilaginous epiphysis, (3) chondrocytes at the hypertrophic and calcified zones in the growth cartilage of actively growing bone. In contrast, few hypertrophic chondrocytes showed positive reactions to IL-1 in growth plates nearing physiologic closure. Furthermore, IL-1 was detected in chondrocytes cultured from human growth cartilage. These results show that IL-1 is produced by matured chondrocytes of human growth cartilage in vivo. Chondrocyte-derived IL-1 might play a key role in the hypertrophy of chondrocytes, in the vascularization of cartilage and in the formation of bone.     

The critical role of the epidermal growth factor receptor in endochondral ossification

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.     

Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone

A connection has been suggested between glucocorticoid-induced osteopenia and an increase in the apoptosis of bone cells, and between the dimerization of the glucocorticoid receptor (GR) and the development of apoptosis. On this basis, a study has been carried out on the relationships between the occurrence of apoptotic cells and their detectable GR content, and between apoptosis frequency and changes in histomorphometric variables, in the growth plate and secondary spongiosa of rat long bones after the high-dose (10 mg/day) administration of corticosterone (CORT) and after recovery. The main results of the CORT treatment were: a significant increase in apoptotic osteoblasts, and a concomitant decrease in the histomorphometric variables of bone formation, with a reversal of both values during recovery; a nonsignificant increase in the apoptosis of osteoclasts, without changes in the histomorphometric variables of bone resorption; a significant increase in apoptotic terminal hypertrophic chondrocytes; the presence of GR in all types of skeletal cells in control rats, with different (cytoplasmic and/or nuclear) immunohistochemical detection in the same type of cell; a decrease in GR detection in proliferative chondrocytes and osteocytes in CORT and recovery groups, and in the maturative/hypertrophic chondrocytes of the recovery group; a fall in growth cartilage width, possibly due to the reduced proliferation of proliferative chondrocytes and increased apoptosis in terminal hypertrophic chondrocytes. In conclusion, pharmacological doses of CORT reduce bone formation by increasing osteoblast apoptosis; they reduce growth cartilage width, probably by inhibiting chondrocyte proliferation and increasing the apoptosis of terminal hypertrophic chondrocytes, and they reduce osteocyte GR. Although these effects appear to be mediated by the presence of GR in all skeletal cells, no precise correlation between GR immunohistochemical detection and apoptosis induction has been found.     

Parathyroid hormone-related protein regulates proliferation of condylar hypertrophic chondrocytes.

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, e.g., it shows delayed appearance in development relative to the limb bud cartilage, originates from the periosteum rather than from undifferentiated mesenchymal cells, and shows rapid differentiation into hypertrophic chondrocytes as opposed to the epiphyseal growth plate cartilage, which has resting and proliferative zones. Recently, attention has been focused on the role of parathyroid hormone-related protein (PTHrP) in modulating the proliferation and differentiation of chondrocytes. To investigate further the characteristic modes of growth and differentiation of this cartilage, we used mice with a disrupted PTHrP allele. Immunolocalization of type X collagen, the extracellular matrix specifically expressed by hypertrophic chondrocytes, was greatly reduced in the condylar cartilage of homozygous PTHrP-knockout mice compared with wild-type mice. In contrast, immunolocalization of type X collagen of the tibial cartilage did not differ. In wild-type mice, proliferative chondrocytes were mainly located in both the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but were limited to the proliferative zone of the tibial cartilage. The number of proliferative chondrocytes was greatly reduced in both cartilages of homozygous PTHrP-knockout mice. Moreover, apoptotic chondrocytes were scarcely observed in the condylar hypertrophic cell layer, whereas a number of apoptotic chondrocytes were found in the tibial hypertrophic zone. Expression of the type I PTH/PTHrP receptor was localized in the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but was absent from the tibial hypertrophic chondrocytes. It is therefore concluded that, unlike tibial hypertrophic chondrocytes, condylar hypertrophic chondrocytes have proliferative activity in the late embryonic stage, and PTHrP plays a pivotal role in regulating the proliferative capacity and differentiation of these cells.     

Signalling by fibroblast growth factor receptor 3 and parathyroid hormone-related peptide coordinate cartilage and bone development

Bone development is regulated by conserved signalling pathways that are linked to multifunctional growth factors and their high affinity receptors. Parathyroid hormone-related peptide (PTHrP) and fibroblast growth factor receptor 3 (FGFR3) have been shown to play pivotal, and sometimes complementary, roles in the replication, maturation and death of chondrocytes during endochondral bone formation. To gain further insight into how these pathways coordinate cartilage and bone development, we generated mice lacking expression of both PTHrP and FGFR3. The phenotype of compound mutant mice resembled that of their PTHrP-deficient littermates with respect to neonatal lethality, facial dysmorphism and foreshortening of the limbs. The absence of PTHrP in the developing epiphyseal cartilage of PTHrP-/- and PTHrP-/-/FGFR3-/- mice resulted in a dominant hypo-proliferative phenotype. However, abnormalities such as the presence of nonhypertrophic cells among hypertrophic chondrocytes and excessive apoptosis seen in the hypertrophic zone of PTHrP-/- mice were absent in the PTHrP-/-/FGFR3-/- mice. Furthermore, the absence of FGFR3 in single and compound mutant mice led to decreased expression of vascular endothelial growth factor (VEGF) and an increase in depth of hypertrophic chondrocytes. These observations indicate that FGFR3 deficiency can rescue some of the defects seen in PTHrP-deficient mice and that it plays an important role in the regulation of chondrocyte differentiation and hypertrophy. These studies support a dominant role for PTHrP in regulating the pool of proliferating cells during limb development and suggest that signalling by FGFR3 plays a more prominent role in cartilage maturation and vascular invasion at the chondro-osseous junction.     

Misexpression of Dickkopf-1 in endothelial cells, but not in chondrocytes or hypertrophic chondrocytes, causes defects in endochondral ossification

Developing cartilage serves as a template for long-bone development during endochondral ossification. Although the coupling of cartilage and bone development with angiogenesis is an important regulatory step for endochondral ossification, the molecular mechanisms are poorly understood. One possible mechanism involves the action of Dickkopf (DKK), which is a family of soluble canonical Wnt antagonists with four members (DKK1-4). We initially observed opposite expression patterns of Dkk1 and Dkk2 during angiogenesis and chondrocyte differentiation: downregulation of Dkk1 and upregulation of Dkk2. We examined the in vivo role of Dkk1 and Dkk2 in linking cartilage/bone development and angiogenesis by generating transgenic (TG) mice that specifically express Dkk1 or Dkk2 in chondrocytes, hypertrophic chondrocytes, or endothelial cells. Despite specific expression pattern during cartilage development, chondrocyte- and hypertrophic chondrocyte-specific Dkk1 and Dkk2 TG mice showed normal developmental phenotypes. However, Dkk1 misexpression in endothelial cells resulted in defects of endochondral ossification and reduced skeletal size. The defects are caused by the inhibition of angiogenesis in developing bone and subsequent inhibition of apoptosis of hypertrophic chondrocytes and cartilage resorption.     

P-glycoprotein is expressed in the mineralizing regions of the skeleton

Using oligonucleotide primers specific for the human MDR 1 gene, we were able to identify a specific amplicon using RT-PCR from total bovine growth plate chondrocyte RNA. The identification of MDR mRNA in growth plate chondrocytes led us to examine the precise distribution of MDR P-glycoprotein in bone and cartilage. We applied two monoclonal antibodies (C219 and C494) to human fetal, neonatal, and childhood growth plates and bone. In growth plates, P-glycoprotein was detected at high levels in a perilacunar distribution in the calcifying zone and at lower levels in hypertrophic, but not proliferative or reserve zone, chondrocytes. P-glycoprotein was also observed in perichondrial chondrocytes, in perivascular chondrocytes and matrix in the fetal cartilage anlage, and in osteoblasts and the surface osteoid matrix of newly formed bone trabeculae in the primary spongiosa. The recently described chloride channel of P-glycoprotein suggests a potential role of P-glycoprotein in growth plate chondrocyte hypertrophy. D. C. Mangham is supported by the Wechsler fellowship     

Morphometric Changes in the Epiphyseal Plate of the Growing and Young Adult Male Rat After Long-Term Salmon Calcitonin Administration

The function of the epiphyseal plate is related to the differentiation and maturation of the chondrocytes, especially of the hypertrophic zone. Salmon calcitonin exerts a positive effect on chondrocytes of different types of cartilage, e.g., articular cartilage, osteochondral callus formation, and the epiphyseal plate. In the present study, the effect of long-term daily salmon calcitonin treatment upon epiphyseal plate function was examined in 80 male Wistar rats aged 12 weeks at the beginning of the experiment. A daily dose of 6 IU of salmon calcitonin enhanced the number of the chondrocytes of the hypertrophic zone of the upper tibial epiphyseal plate, increased the mean thickness of the epiphyseal plate, and accelerated the longitudinal growth of long bones. It was found that the peripheral growth of the epiphyseal plate was delayed after calcitonin treatment in comparison with the placebo-treated animals. The most effective period for calcitonin treatment on epiphyseal plate function seems to be the late accelerated period of growth, i.e., puberty. In conclusion, long-term salmon calcitonin treatment has a beneficial effect on longitudinal skeletal growth and this effect remains throughout the adult life of the animal. Salmon calcitonin does not enlarge the surface of the epiphyseal plate.     

Expression of p21CIP1/WAF1 in Chondrocytes

During endochondral ossification, proliferative activity of chondrocytes is arrested and the cells undergo terminal hypertrophic differentiation. We examined the expression of the cyclin-dependent kinase inhibitor, p21^CIP1/WAF1 in permanent cartilage (xyphoid and articular cartilage) and in cartilage undergoing endochondral ossification (growth plate, epiphyseal ossification centers, and costochondral junctions) to determine if p21 is up-regulated in chondrocytes during hypertrophic differentiation. Northern blot analyses demonstrated expression of p21 in chondrocytes undergoing endochondral ossification and from sites of permanent cartilage. Quantitative analyses of Northern data showed an association between expression of the hypertrophic-specific marker, collagen type X, and the level of 21 expression. In situ hybridization of rodent femoropatellar joints and costochondral junctions localized p21 mRNA to chondrocytes within both the proliferative and hypertrophic zones of the growth plates, in chondrocytes involved in formation of the epiphyseal ossification centers, and in articular chondrocytes. Immunohistochemical analyses of p21 expression in the same tissues demonstrated comparatively higher levels of p21 protein in postmitotic chondrocytes. These data suggest that p21 is active in cell cycle regulation in chondrocytes, and that increased p21 expression is associated with hypertrophic differentiation. Received: 11 October 1996 / Accepted: 23 April 1997     

Histopathological features of unilateral stapling in animal experiments

Summary Blount stapling of the proximal medial tibial growth was performed in 10-week-old domestic pigs. A total of 37 animals for up to 17 weeks were followed up. The histological evaluation showed a different reaction of the growth plate in the medial (stapled), central and lateral area. Up to 6 days after stapling the morphological features were characterized by deviations of the cell column from the axial alignment. Herniations of mature chondrocytes as well as isles of epiphyseal cartilage splintered into the adjacent metaphyseal cancellous bone were seen. From the 10th to the 11th postoperative day on there was a decline in the number of distal hypertrophic and degenerating cartilage cells due to impaired proliferation. Cell atrophy due to disturbed maturation or cell degeneration was evident. Local clusters of chondrocytes with loss of columnar arrangement indicated impaired chondrogenesis. Twenty-eight days after the operation coarses structures and changed alignment of cancellous bone characterized the morphological picture. The postoperative follow-up showed epiphyseal plates bounded by mature bone blocks or even a distinct sheet of horizontally oriented bone plates on the metaphyseal side. Many metaphyseal findings, such as augmented hypertrophic cells in the early postoperative period, or platelike limitations of the epiphyseal plate complete the morphological picture.Supported by the Deutsche Forschungsgemeinschaft     

Expression of cartilage oligomeric matrix protein (COMP) by embryonic and adult osteoblasts.

Cartilage oligomeric matrix protein has been implicated as an important component of endochondral ossification because of its direct effects on chondrocytes. The importance of this protein for skeletal development and growth has been recently illustrated by the identification of mutations in cartilage oligomeric protein genes in two types of inherited chondrodysplasias and osteoarthritic phenotypes: multiple epiphyseal dysplasia and pseudoachondroplasia. In the present study, we report the presence of cartilage oligomeric protein in embryonic and adult osteoblasts. A foot from a 21-week-old human fetus, subchondral bone obtained from knee replacement surgery in an adult patient, and a limb from a 19-day-postcoital mouse embryo were analyzed with immunostaining and in situ hybridization. In the human fetal foot, cartilage oligomeric protein was localized to osteoblasts of the bone collar and at the newly formed bone at the growth plate and bone diaphyses. Immunostaining was performed on the adult subchondral bone and showed positive intracellular staining for cartilage oligomeric protein of the osteoblasts lining the trabecular bone. There was no staining of the osteocytes. Immunostaining of the mouse limb showed the most intense staining for cartilage oligomeric protein in the hypertrophic chondrocytes and in the surrounding osteoblast cells of the developing bone. Cartilage oligomeric protein mRNA and protein were detected in an osteoblast cell line (MG-63), and cartilage oligomeric protein mRNA was detected from human cancellous bone RNA. These results suggest that the altered structure of cartilage oligomeric protein by the mutations seen in pseudoachondroplasia and multiple epiphyseal dysplasia may have direct effects on osteoblasts, contributing to the pathogenesis of these genetic disorders.     

Expression of mouse HtrA1 serine protease in normal bone and cartilage and its upregulation in joint cartilage damaged by experimental arthritis

Levels of HtrA1 protein in cartilage have been reported to elevate in joints of human osteoarthritis patients. To understand roles of HtrA1 in normal osteogenesis as well as in pathogenesis of arthritis, we examine HtrA1 expression pattern during bone and cartilage development and in articular cartilage affected by experimental arthritis. HtrA1 is not expressed in mesenchymal or cartilage condensations before initiation of ossification. When ossification begins in the condensations, the expression of HtrA1 starts in chondrocytes undergoing hypertrophic differentiation near the ossification center. Hypertrophic chondrocytes found in adult articular cartilage and epiphyseal growth plates also express HtrA1. When arthritis is induced by injection of anti-collagen antibodies and lipopolysaccharide, resting chondrocytes proceed to terminal hypertrophic differentiation and start expressing HtrA1. These data suggest that hypertrophic change induces HtrA1 expression in chondrocytes both in normal and pathological conditions. HtrA1 has been reported to inhibit TGF-beta signaling. We show that HtrA1 digests major components of cartilage, such as aggrecan, decorin, fibromodulin, and soluble type II collagen. HtrA1 may, therefore, promote degeneration of cartilage by inducing terminal hypertrophic chondrocyte differentiation and by digesting cartilage matrix though its TGF-beta inhibitory activity and protease activity, respectively. In bone, active cuboidal osteoblasts barely express HtrA1, but osteoblasts which flatten and adhere to the bone matrix and osteocytes embedded in bone are strongly positive for HtrA1 production. The bone matrix shows a high level of HtrA1 protein deposition akin to that of TGF-beta, suggesting a close functional interaction between TGF-beta and HtrA1.     

Transforming growth factor alpha controls the transition from hypertrophic cartilage to bone during endochondral bone growth

We have recently identified transforming growth factor alpha (TGFα) as a novel growth factor involved in the joint disease osteoarthritis. The role of TGFα in normal cartilage and bone physiology however, has not been well defined.PurposeThe objective of this study was to determine the role of TGFα in bone development through investigation of the Tgfa knockout mouse.MethodsThe gross skeletons as well as the cartilage growth plates of Tgfa knockout mice and their control littermates were examined during several developmental stages ranging from newborn to ten weeks old.ResultsKnockout mice experienced skeletal growth retardation and expansion of the hypertrophic zone of the growth plate. These phenotypes were transient and spontaneously resolved by ten weeks of age. Tgfa knockout growth plates also had fewer osteoclasts along the cartilage/bone interface. Furthermore, knockout mice expressed less RUNX2, RANKL, and MMP13 mRNA in their cartilage growth plates than controls did.ConclusionsTgfa knockout mice experience a delay in bone development, specifically the conversion of hypertrophic cartilage to true bone. The persistence of the hypertrophic zone of the growth plate appears to be mediated by a decrease in MMP13 and RANKL expression in hypertrophic chondrocytes and a resulting reduction in osteoclast recruitment. Overall, TGFα appears to be an important growth factor regulating the conversion of cartilage to bone during the process of endochondral ossification.     

Evaluation of 9.4-T MR microimaging in assessing normal and defective fetal bone development: comparison of MR imaging and histological findings

We evaluated 9.4-T magnetic resonance (MR) microimaging in assessing normal and defective bone development in mouse embryos. For this purpose, we performed 9.4-T MR microimaging on developing bones in normal embryos, and also in Runx2/Cbfa1-/- embryos with severely defective bone development. MR images were compared with the histological and histochemical features of these fetal bones. MR microimaging delineate successfully the normal long bone development in embryos. The T1- and T2-weighted MR microimaging demonstrated chondrocyte maturation in different regions of growing cartilage, such as epiphysis, physis, hypertrophic cartilage, and zone of provisional calcification. These developmental changes were detectable in as early as E14.5 embryos. The MR microimaging clearly demonstrated defective bone development in Runx2/Cbfa1-/- embryos. The femur from E18.5 homozygous Runx2/Cbfa1-/- embryos lacked MR signal intensity patterns including the hypertrophic cartilage, which are characteristic of the bone from the age-matched Runx2/Cbfa1+/+ embryos. Interestingly, however, the tibia from the same mutants was associated with MR signal patterns indicative of hypertrophic cartilage but not of the primary spongiosa and ossifying perichondrium, suggesting that bone development is differently regulated in these two long bones. On the other hand, the bones from heterozygous Runx2/Cbfa1+/- embryos exhibited an MR phenotype intermediate between the Runx2/Cbfa1+/+ and Runx2/Cbfa1-/- embryos; the primary spongiosa and ossifying perichondrium formation occurred normally even in the absence of preceding organized maturation of chondrocytes, a phenotype that was not detected by histological examinations. We concluded that MR microimaging is useful in assessing the bone development.     

Inactivation of Pten in osteo-chondroprogenitor cells leads to epiphyseal growth plate abnormalities and skeletal overgrowth.

To study the role of the Pten tumor suppressor in skeletogenesis, we generated mice lacking this key phosphatidylinositol 3'-kinase pathway regulator in their osteo-chondroprogenitors. A phenotype of growth plate dysfunction and skeletal overgrowth was observed. INTRODUCTION: Skeletogenesis is a complex process relying on a variety of ligands that activate a range of intracellular signal transduction pathways. Although many of these stimuli are known to activate phosphatidylinositol 3'-kinase (PI3K), the function of this pathway during cartilage development remains nebulous. To study the role of PI3K during skeletogenesis, we used mice deficient in a negative regulator of PI3K signaling, the tumor suppressor, Pten. MATERIALS AND METHODS: Pten gene deletion in osteo-chondrodroprogenitors was obtained by interbreeding mice with loxP-flanked Pten exons with mice expressing the Cre recombinase under the control of the type II collagen gene promoter (Pten(flox/flox):Col2a1Cre mice). Phenotypic analyses included microcomputed tomography and immunohistochemistry techniques. RESULTS: MicroCT revealed that Pten(flox/flox):Col2a1Cre mice exhibited both increased skeletal size, particularly of vertebrae, and massive trabeculation accompanied by increased cortical thickness. Primary spongiosa development and perichondrial bone collar formation were prominent in Pten(flox/flox):Col2a1Cre mice, and long bone growth plates were disorganized and showed both matrix overproduction and evidence of accelerated hypertrophic differentiation (indicated by an altered pattern of type X collagen and alkaline phosphatase expression). Consistent with increased PI3K signaling, Pten-deficient chondrocytes showed increased phospho-PKB/Akt and phospho-S6 immunostaining, reflective of increased mTOR and PDK1 activity. Interestingly, no significant change in growth plate proliferation was seen in Pten-deficient mice, and growth plate fusion was found at 6 months. CONCLUSIONS: By virtue of its ability to modulate a key signal transduction pathway responsible for integrating multiple stimuli, Pten represents an important regulator of both skeletal size and bone architecture.     

Chondrocyte-specific knockout of the G protein G(s)alpha leads to epiphyseal and growth plate abnormalities and ectopic chondrocyte formation.

G(s)alpha is a ubiquitously expressed G protein alpha-subunit that couples receptors to adenylyl cyclase. Mice with chondrocyte-specific ablation of the G(s)alpha gene had severe epiphyseal and growth plate abnormalities and ectopic cartilage formation within the metaphyseal region of the tibia. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTH-rP receptor in growth plate chondrocytes. INTRODUCTION: G(s)alpha is a ubiquitously expressed G protein alpha-subunit that mediates signaling through G protein-coupled receptors to activate the cAMP/protein kinase A signaling pathway. Although studies suggest an important role for G(s)alpha in regulating growth plate development, direct in vivo results examining this role are lacking. MATERIALS AND METHODS: The G(s)alpha gene was ablated in murine cartilage by mating mice with loxP sites surrounding the G(s)alpha promoter and first exon with collagen 2a1 promoter-Cre recombinase transgenic mice. Skeletal tissues were studied by gross and microscopic pathology, and gene expression was determined by in situ hybridization. RESULTS AND CONCLUSIONS: Mice with complete chondrocyte-specific G(s)alpha deficiency (homozygotes) died within minutes after birth and had severe epiphyseal and growth plate defects with shortening of the proliferative zone and accelerated hypertrophic differentiation of growth plate chondrocytes, a phenotype similar to that of PTH/PTH-related peptide (PTHrP) receptor knockout mice. Indian hedgehog and PTH/PTHrP receptor expression in prehypertrophic chondrocytes was unaffected in mutant mice. PTHrP expression in periarticular cartilage was increased in the mutant mice, probably because of the closer proximity of Ihh-secreting chondrocytes to the periarticular zone. In addition, these mice developed ectopic cartilage at the anterior side of the metaphyseal region in the tibia. Mice with partial G(s)alpha deficiency (heterozygotes) exhibited no phenotype. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTHrP receptor in epiphyseal and growth plate chondrocytes.