Expression of parathyroid hormone-related peptide and insulin-like growth factor I during rat fracture healing.

Parathyroid hormone-related peptide (PTHrP) and insulin-like growth factor I (IGF-I) are both involved in the regulation of bone and cartilage metabolisms and their interaction has been reported in osteoblasts. To investigate the interaction of PTHrP and IGF-I during fracture healing, the expression of mRNA for PTHrP and IGF-I, and receptors for PTH/PTHrP and IGF were examined during rat femoral fracture healing using an in situ hybridization method and an immunohistochemistry method, respectively. During intramembranous ossification, PTHrP mRNA, IGF-I mRNA and IGF receptors were detected in preosteoblasts, differentiated osteoblasts and osteocytes in the newly formed trabecular bone. PTH/PTHrP receptors were markedly detected in osteoblasts and osteocytes, but only barely so in preosteoblasts. During cartilaginous callus formation, PTHrP mRNA was expressed by mesenchymal cells and proliferating chondrocytes. PTH/PTHrP receptors were detected in proliferating chondrocytes and early hypertrophic chondrocytes. IGF-I mRNA and IGF receptor were co-expressed by mesenchymal cells, proliferating chondrocytes, and early hypertrophic chondrocytes. At the endochondral ossification front, osteoblasts were positive for PTHrP and IGF-I mRNA as well as their receptors. These results suggest that IGF-I is involved in cell proliferation or differentiation in mesenchymal cells, periosteal cells, osteoblasts and chondrocytes in an autocrine and/or paracrine fashion. Furthermore, PTHrP may be involved in primary callus formation presumably co-operating with IGF-I in osteoblasts and osteocytes, and by regulating chondrocyte differentiation in endochondral ossification.     

Thyroid hormones regulate hypertrophic chondrocyte differentiation and expression of parathyroid hormone-related peptide and its receptor during endochondral bone formation.

Hypothyroidism in children causes developmental abnormalities in bone and growth arrest, while thyrotoxicosis accelerates growth rate and advances bone age. To determine the effects of thyroid hormones on endochondral bone formation, we examined epiphyseal growth plates in control, hypothyroid, thyrotoxic, and hypothyroid-thyroxine (hypo-T4)-treated rats. Hypothyroid growth plates were grossly disorganized, contained an abnormal matrix rich in heparan sulfate, and hypertrophic chondrocyte differentiation failed to progress. These effects correlated with the absence of collagen X expression and increased parathyroid hormone-related protein (PTHrP) messenger RNA (mRNA) expression. In thyrotoxic growth plates, histology essentially was normal but PTHrP receptor (PTHrP-R) mRNA was undetectable. PTHrP is a potent inhibitor of hypertrophic chondrocyte differentiation that acts in a negative feedback loop with the secreted factor Indian hedgehog (Ihh) to regulate endochondral bone formation. Thyroid hormone receptor alpha1(TRalpha1), TRalpha2, and TRbeta1 proteins were localized to reserve zone progenitor cells and proliferating chondrocytes in euthyroid rat cartilage; regions in which PTHrP and PTHrP-R expression were affected by thyroid status. Thus, dysregulated Ihh/PTHrP feedback loop activity may be a key mechanism that underlies growth disorders in childhood thyroid disease.     

Inhibition of terminal chondrocyte differentiation by bone morphogenetic protein 7 (OP-1) in vitro depends on the periarticular region but is independent of parathyroid hormone-related peptide.

Bone morphogenetic protein-7, or BMP-7 (OP-1), is highly expressed in the perichondrium of embryonic long bones and is thought to play a role in endochondral ossification. Previously we have shown that BMP-7 inhibits terminal chondrocyte differentiation; that is, chondrocyte hypertrophy and mineralization in cultured explants of embryonic mouse metatarsals. However, the mechanism of this inhibition and the target cells of BMP-7 are still unknown. In this study we show that BMP-7 inhibits terminal chondrocyte differentiation indirectly, via an interaction with the periarticular region of the explants. This region also expresses parathyroid hormone-related peptide (PTHrP). PTHrP regulates terminal chondrocyte differentiation by inhibiting hypertrophic differentiation of prehypertrophic chondrocytes. The differentiating center in turn regulates PTHrP expression via a feedback loop involving Indian hedgehog (Ihh), which is expressed in the prehypertrophic chondrocytes. Ihh is thought to act on perichondrial cells, which in turn start to express an as yet unknown mediator that stimulates PTHrP expression in the periarticular region. It has been suggested that this factor belongs to the BMP-family. We investigated whether the inhibition of terminal chondrocyte differentiation by BMP-7 was due to upregulation of the PTHrP-Ihh feedback loop and whether BMP-7 was the unknown factor in the loop. Here we show that exogenous BMP-7 did not upregulate the mRNA expression of PTHrP, Ihh, or the PTH/PTHrP receptor in cultured wild-type embryonic metatarsals. Furthermore, BMP-7 could still inhibit terminal chondrocyte differentiation in the metatarsals of PTHrP-deficient (PTHrP-/-) mouse embryos. These data indicate that the BMP-7-mediated inhibition of terminal chondrocyte differentiation in vitro is independent of the PTHrP-Ihh feedback loop. We concluded that BMP-7 modulates terminal chondrocyte differentiation and cartilage mineralization of fetal bone explants in vitro via as yet unknown inhibitory factor(s) produced in the periarticular region.     

PTH ameliorates acidosis-induced adverse effects in skeletal growth centers: the PTH-IGF-I axis

BACKGROUND: Chronic metabolic acidosis (CMA) exerts profound adverse effects on bone metabolism thereby leading to impaired skeletal linear growth. We have recently shown that CMA in vitro causes distinct morphological changes in skeletal growth centers along with inhibition of endochondral differentiation. In addition, CMA causes an end organ resistance to the anabolic effects of growth hormone (GH) and locally produced insulin-like growth factor-I (IGF-I) in skeletal growth centers. Given the effects of parathyroid hormone (PTH) and PTH related protein (PTHrP) on the development of cartilaginous bone, we sought to determine whether PTH has any effects on the changes induced by CMA in skeletal growth centers. The interaction between PTH and IGF-I in growth centers during neutral or acidic conditions were studied specifically. METHODS: An in vitro organ culture system using the murine mandibular condyle was employed as a model for endochondral active growth center. Condyles from six-day-old mice were cultured in BGJb medium of either neutral pH (pH approximately 7.4) or acidic pH (pH approximately 7.15) in the presence or absence of 10-10 mol/L [1-34] PTH. After 24, 48, 72 and 96 hours of culture, the condyles were washed, fixed in formaldehyde, and processed for paraffin embedding. Histologic markers of the growth center were assessed. In addition, the protein level and mRNA expression for various markers of cartilage differentiation were evaluated by immunohistochemistry and in situ hybridization, respectively. The abundance and expression levels of IGF-I and IGF-I receptor (IGF-I-R) were assessed also. RESULTS: Following incubation for 72 hours in acidic conditions, there was a marked attenuation of the chondroblastic zone, suggesting a defect in the process of cellular differentiation. Acidosis also down-regulated endochondral differentiation markers (cartilage specific proteoglycans, collagen type II). This was accompanied by a reduction in the expression of IGF-1, IGF-1 receptor and PTH receptors. PTH (10-10 mol/L) added to acidic cultures prevented the adverse effects of CMA on endochondral differentiation and increased the overall condylar growth, when compared to acidic conditions without PTH. PTH also up-regulated its own receptor in control as well as during acidic conditions, and increased the expression levels of IGF-1 and IGF-1 receptor in the acidotic condyle. Acidosis increased the expression of IGF-I binding protein-4 (IGFBP-4, an inhibitor of IGF-I activity), whereas coincubation with PTH during acidic conditions abrogated the up-regulation of IGFBP-4. Addition of a neutralizing antibody to IGF-I-R during PTH treatment under acidic conditions resulted in the abrogation of the ameliorative effect of PTH on endochondral differentiation. The protein kinase C (PKC) signaling pathway was modulated negatively by CMA. However, PTH activated PKC-alpha under both control and acidic conditions. The phorbol ester, PMA (phorbol 12-myristate 13-acetate), a PKC activator, mimicked the effect of PTH on chondrocyte differentiation. CONCLUSION: Parathyroid hormone at low concentration stimulates the differentiation and proliferation of cartilage cells and prevents the suppressive effect of acidosis on endochondral bone differentiation and on the IGF-I/IGF-I-R system in skeletal growth centers. Increased local production of IGF-I by PTH, which takes place even during acidotic conditions, mediates, at least in part, the ameliorative effect of PTH. Protein kinase C is probably one of the signaling pathways mediating the salutary effects of PTH on chondrocyte differentiation in growth centers. This study lends further credence to the notion that under certain conditions, PTH or PTHrP can exert anabolic effects in the skeleton. These findings may be of clinical-therapeutic significance in children and patients with CMA.     

Growth retardation in children with chronic renal failure.

Growth retardation is a major obstacle to full rehabilitation of children with chronic renal failure (CRF). Several factors have been identified as contributors to impaired linear growth and they include protein and calorie malnutrition, metabolic acidosis, growth hormone resistance, anemia, and renal osteodystrophy. Although therapeutic interventions such as the use of recombinant human growth hormone, recombinant human erythropoietin, and calcitriol have made substantial contributions, the optimal therapeutic strategy remains to be defined. Indeed, growth failure persists in a substantial proportion of children with renal failure and those treated with maintenance dialysis. In addition, the increasing prevalence of adynamic lesions of renal osteodystrophy and its effect on growth have raised concern about the continued generalized use of calcitriol in children with CRF. Recent studies have shown the critical roles of parathyroid hormone-related protein (PTHrP) and the PTH/PTHrP receptor in the regulation of endochondral bone formation. The PTH/PTHrP receptor mRNA expression has been shown to be down-regulated in kidney and growth plate cartilage of animals with renal failure. Differences in the severity of secondary hyperparathyroidism influence not only growth plate morphology but also the expression of selected markers of chondrocyte proliferation and differentiation in these animals. Such findings suggest potential molecular mechanisms by which cartilage and bone development may be disrupted in children with CRF, thereby contributing to diminished linear growth.     

Modulation of endochondral bone formation: roles of growth hormone, 1,25-dihydroxyvitamin D and hyperparathyroidism

Impairment of linear growth occurs invariably in children with chronic renal failure. Recombinant human growth hormone and 1,25-dihydroxyvitamin D (calcitriol) are widely utilized to improve linear growth in children. Large doses of calcitriol, however, have been shown to suppress chondrocyte proliferation and may lead to the development of adynamic bone. Substantial reductions of growth have been shown in children with chronic renal failure treated with intermittent calcitriol therapy. These findings suggest that calcitriol can modify chondrocyte proliferation and/or differentiation in epiphyseal growth plate cartilage and may counteract the effects of growth hormone therapy in increasing linear growth in children with chronic renal failure. Parathyroid hormone related peptide (PTHrP) and its receptor (PTH/PTHrP receptor) play critical roles in regulating chondrocyte differentiation in the growth plate. The expression of PTH/PTHrP receptor mRNA is downregulated in animals with chronic renal failure and advanced secondary hyperparathyroidism; calcitriol and growth hormone therapy may modify the expression of PTH/PTHrP receptor. This article summarizes the separate and combined effects of growth hormone and calcitriol on endochondral bone formation in chronic renal failure and secondary hyperparathyroidism. Received: 10 September 1999 / Revised: 23 December 1999 / Accepted: 30 December 1999     

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.     

Runx2 Regulates Endochondral Ossification Through Control of Chondrocyte Proliferation and Differentiation

Synthesis of cartilage by chondrocytes is an obligatory step for endochondral ossification. Global deletion of the Runx2 gene results in complete failure of the ossification process, but the underlying cellular and molecular mechanisms are not fully known. Here, we elucidated Runx2 regulatory control distinctive to chondrocyte and cartilage tissue by generating Runx2 exon 8 floxed mice. Deletion of Runx2 gene in chondrocytes caused failure of endochondral ossification and lethality at birth. The limbs of Runx2^ΔE8/ΔE8 mice were devoid of mature chondrocytes, vasculature, and marrow. We demonstrate that the C‐terminus of Runx2 drives its biological activity. Importantly, nuclear import and DNA binding functions of Runx2 are insufficient for chondrogenesis. Molecular studies revealed that despite normal levels of Sox9 and PTHrP, chondrocyte differentiation and cartilage growth are disrupted in Runx2^ΔE8/ΔE8 mice. Loss of Runx2 in chondrocytes also impaired osteoprotegerin‐receptor activator of NF‐κB ligand (OPG‐RANKL) signaling and chondroclast development. Dwarfism observed in Runx2 mutants was associated with the near absence of proliferative zone in the growth plates. Finally, we show Runx2 directly regulates a unique set of cell cycle genes, Gpr132, Sfn, c‐Myb, and Cyclin A1, to control proliferative capacity of chondrocyte. Thus, Runx2 is obligatory for both proliferation and differentiation of chondrocytes. © 2014 American Society for Bone and Mineral Research.     

Lead alters parathyroid hormone-related peptide and transforming growth factor-beta1 effects and AP-1 and NF-kappaB signaling in chondrocytes.

The skeletal system is an important target for lead toxicity. One of the impacts of lead in the skeleton, the inhibition of axial bone development, is likely due to its effect on the normal progression of chondrocyte maturation that is central to the process of endochondral ossification. Since little is known about the effect of lead on chondrocyte function/maturation, its impact on (1) growth factor-induced proliferation, (2) expression of maturation-specific markers type X collagen and BMP-6, and (3) the activity of AP-1 and NF-kappaB was examined in chick growth plate and sternal chondrocyte models. Exposure to lead alone (1-30 microM) resulted in a dose-dependent inhibition of thymidine incorporation in growth plate chondrocytes. Lead also blunted the stimulation of thymidine incorporation by parathyroid hormone-related peptide (PTHrP) and transforming growth factor-beta1 (TGF-beta1), two critical regulators of chondrocyte maturation. Lead (1 and 10 microM), TGF-beta1 (3 ng/ml) and PTHrP (10(-7) M) all significantly inhibited the expression of type X collagen, a marker of chondrocyte terminal differentiation. However, when in combination, lead completely reversed the inhibition of type X collagen by PTHrP and TGF-beta1. The effect of lead on BMP-6. an inducer of terminal differentiation. was also examined. Independently, lead and TGF-beta1 were without effect on BMP-6 expression, but PTHrP significantly suppressed it. Comparatively, lead did not alter PTHrP-mediated suppression of BMP-6, but in combination with TGF-beta1. BMP-6 expression was increased 3-fold. To determine if lead effects on signaling might play a role in facilitating these events, the impact of lead on NF-kappaB and AP-1 signaling was assessed using luciferase reporter constructs in sternal chondrocytes. Lead had no effect on the AP-1 reporter, but it dose-dependently inhibited the NF-kappaB reporter. PTHrP, which signals through AP-1, did not activate the NF-kappaB reporter and did not affect inhibition of this reporter by lead. In contrast, PTHrP activation of the AP-1 reporter was dose-dependently enhanced by lead. These findings, which establish that chondrocytes are important targets for lead toxicity, suggest that the effects of lead on bone growth are derived from its impact on the modulation of chondrocyte maturation by growth factors and second messenger signaling responses.     

NDST-1 modulates BMPR and PTHrP signaling during endochondral bone formation in a gene knockout model

GlcNAc N-deacetylase/N-sulfotransferase-1 (NDST-1), a member of the enzyme family catalyzing the first modification step in the biosynthesis of heparan sulfate (HS), was knocked out in mice to investigate its role in embryonic development. NDST-1 null mice exhibited delayed endochondral bone formation including shortened calcified zones in limbs, delayed chondrocyte and osteogenetic differentiation, and increased chondrocyte proliferation. In situ HS binding assay revealed that the binding ability of bone morphogenetic protein (BMP) -2, -4, and -6 to endogenous HS was decreased in mutant phalanges, while that of fibroblast growth factor-1 (FGF-1) was not affected. Up-regulation of BMPR-IA, Phospho-Smad1 (P-Smad1) and parathyroid-hormone related protein (PTHrP), but not the Indian hedgehog, Gli1, Gli3, Patched, and FGFR-3, was observed. Furthermore, block of BMPR signaling with noggin rescued the delayed chondrocyte hypertrophic differentiation in NDST-1 (−/−) mice and recovered the expression of both P-Smad1 and PTHrP proteins. These results suggested that NDST-1-dependent heparan sulfate might negatively modulate BMP and its downstream PTHrP signaling, and thus affect endochondral bone development.     

Parathyroid hormone [PTH(1-34)] and parathyroid hormone-related protein [PTHrP(1-34)] promote reversion of hypertrophic chondrocytes to a prehypertrophic proliferating phenotype and prevent terminal differentiation of osteoblast-like cells.

The effects of parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) on late events in chondrocyte differentiation were investigated by a dual in vitro model where conditions of suspension versus adhesion culturing are permissive either for apoptosis or for the further differentiation of hypertrophic chondrocytes to osteoblast- like cells. Chick embryo hypertrophic chondrocytes maintained in suspension synthesized type II and type X collagen and organized their extracellular matrix, forming a tissue highly reminiscent of true cartilage, which eventually mineralized. The formation of mineralized cartilage was associated with the expression of alkaline phosphatase (ALP), arrest of cell growth, and apoptosis, as observed in growth plates in vivo. In this system, PTH/PTHrP was found to repress type X collagen synthesis, ALP expression, and cartilage matrix mineralization. Cell proliferation was resumed, whereas apoptosis was blocked. Hypertrophic chondrocytes cultured in adherent conditions in the presence of retinoic acid underwent further differentiation to osteoblast-like cells (i.e., they resumed cell proliferation, switched to type I collagen synthesis, and produced a mineralizing bone-like matrix). In this system, PTH addition to culture completely inhibited the expression of ALP and matrix mineralization, whereas cell proliferation and expression of type I collagen were not affected. These data indicate that PTH/PTHrP inhibit both the mineralization of a cartilage-like matrix and apoptosis (mimicked in the suspension culture) and the production of a mineralizing bone-like matrix, characterizing further differentiation of hypertrophic chondrocytes to osteoblasts like cells (mimicked in adhesion culture). Treatment of chondrocyte cultures with PTH/PTHrP reverts cultured cells in states of differentiation earlier than hypertrophic chondrocytes (suspension), or earlier than mineralizing osteoblast-like cells (adhesion). However, withdrawal of hormonal stimulation redirects cells toward their distinct, microenvironment-dependent, terminal differentiation and fate.     

Mice expressing a constitutively active PTH/PTHrP receptor in osteoblasts show reduced callus size but normal callus morphology during fracture healing

Background The parathyroid hormone-/parathyroid hormone-related protein (PTH/PTHrP) receptor plays a crucial role in endochondral bone formation and possibly also in fracture healing. Patients with Jansen's metaphysial chondrodysplasia (JMC) have a gain-of-function mutation in the PTH/PTHrP receptor. Transgenic mice expressing JMC PTH/PTHrP receptor mutants in osteoblasts are characterized by increased trabecular bone formation and reduced osteoblastic activity at periosteal sites. We have analyzed the bone phenotype and studied the fracture healing process in this model.

Methods We performed bone density analysis of tibiae from 17-week-old transgenic mice and controls. Also, tibial fractures were produced in 14-week-old mice. Fracture healing was examined by radiographic and histological analysis.

Results Transgenic mice had a lower total bone mineral content (BMC), by a factor of one-third. The changes were bone compartment-specific with an increase in trabecular bone volume and a decrease in cortical thickness. The calluses in the transgenic mice were smaller, with a reduction in BMC and mean crosssectional area by a factor of one-half. Despite the smaller size, however, the morphology and progression through the healing process were similar in both transgenic and wild-type littermates.

Interpretation We conclude that the constitutively active PTH/PTHrP receptor has compartment-specific effects on bone formation when expressed in osteoblasts. During fracture healing, however, both the periosteal and the endochondral processes are activated, leading to fracture healing that is temporally and morphologically normal, although the callus tissue is less prominent.     

Nuclear localization of type I parathyroid hormone/parathyroid hormone-related protein receptors in deer antler osteoclasts: evidence for parathyroid hormone-related protein and receptor activator of NF-kappaB-dependent effects on osteoclast formation in regenerating mammalian bone.

Parathyroid hormone-related protein (PTHrP) is not required for osteoclastogenesis during embryonic development; however, after birth it has been shown to regulate osteoclast formation during tooth eruption. Our study explores the hypothesis that PTHrP also may regulate osteoclast differentiation in the regenerating skeletal tissues of deer antlers, bones capable of complete regeneration. Osteoclast-like multinucleated cells (MNCs) formed spontaneously in micromass cultures derived from antler cartilage and these cells had the phenotypic characteristics of osteoclasts. PTHrP and receptor activator of NF-kappaB ligand (RANKL) stimulated antler osteoclast formation although the effect of RANKL was less marked than that of PTHrP. The addition of osteoprotegerin (OPG) only partially decreased (by approximately 65%) the number of osteoclasts in PTHrP-treated cultures. To determine whether PTHrP also potentially could have direct effects on antler osteoclasts, we studied, by confocal microscopy, the expression of the type I PTH/PTHrP receptor (PTH1R) in MNCs cultured on glass and found the receptor protein to have a nuclear localization. In situ hybridization showed that antler MNCs also expressed PTH1R and PTHrP messenger RNAs (mRNAs). PTHrP was immunolocalized in MNCs cultured on glass but was undetectable in cells resorbing a dentine substrate. In tissue sections of antler cartilage, PTHrP and PTH1R were expressed in vitronectin receptor-positive (VNR+) osteoclast-like cells localized in the perivascular stroma. Thus, these data show that PTHrP plays a role in the regulation of osteoclast differentiation in regenerating skeletal tissues and that PTHrP can have effects on osteoclastogenesis that are independent of RANKL synthesis. Ours is the first study to describe the expression of the type I PTH/PTHrP receptor in mammalian osteoclasts at a protein and mRNA level, which indicates that PTHrP also may have a direct effect on osteoclasts. This also is the first study to show a nuclear localization of the PTHIR in cells of the osteoclast lineage, although the functional significance of this observation has yet to be established.     

Parathyroid hormone-related protein is required for normal intramembranous bone development.

It is well established that parathyroid hormone-related protein (PTHrP) regulates chondrocytic differentiation and endochondral bone formation. Besides its effect on cartilage, PTHrP and its major receptor (type I PTH/PTHrP receptor) have been found in osteoblasts, suggesting an important role of PTHrP during the process of intramembranous bone formation. To clarify this issue, we examined intramembranous ossification in homozygous PTHrP-knockout mice histologically. We also analyzed phenotypic markers of osteoblasts and osteoclasts in vitro and in vivo. A well-organized branching and anastomosing pattern was seen in the wild-type mice. In contrast, marked disorganization of the branching pattern of bone trabeculae and irregularly aligned osteoblasts were recognized in the mandible and in the bone collar of the femur of neonatal homozygous mutant mice. In situ hybridization showed that most of the osteoblasts along the bone surfaces of the wild-type mice and some of the irregularly aligned osteoblastic cells in the homozygous mice expressed osteocalcin. Alkaline phosphatase (ALP) activity and expression of osteopontin messenger RNA (mRNA) in primary osteoblastic cells did not show significant differences between cultures derived from the mixture of heterozygous mutant and wild-type mice (+/? mice) and those from homozygous mutant mice. However, both mRNA and protein levels of osteocalcin in the osteoblastic cells of homozygous mutant mice were lower than those of +/? mice, and exogenous PTHrP treatment corrected this suppression. Immunohistochemical localization of characteristic markers of osteoclasts and ruffled border formation did not differ between genotypes. Cocultures of calvarial osteoblastic cells and spleen cells of homozygous mutant mice generated an equivalent number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear and multinucleated cells and of pit formation to that of +/? mice, suggesting that osteoclast differentiation is not impaired in the homozygous mutant mice. These results suggest that PTHrP is required not only for the regulation of cartilage formation but also for the normal intramembranous bone development.     

Type 1 parathyroid hormone receptor expression level modulates renal tone and plasma renin activity in spontaneously hypertensive rat

These studies examine whether PTHrP(1-36), a vasodilator, modulates BP and renal vascular resistance (RVR) in spontaneously hypertensive rat (SHR). Within the kidney of normotensive rats, PTHrP(1-36) was enriched in vessels. In vessels of SHR, PTHrP was upregulated by 40% and type 1 PTH receptor (PTH1R) was downregulated by 65% compared with normotensive rats. To investigate the role of endogenous PTHrP in the regulation of BP and RVR, SHR were subjected to somatic human (h)PTH1R gene delivery. Three weeks after a single intravenous injection of pcDNA1.1 plasmid containing the hPTH1R gene under the control of the cytomegalovirus promoter, hPTH1R mRNA was detected in all of the main organs. Within the kidney, the transgene was enriched in vessels. In the isolated perfused kidney, RVR was reduced by 23% and PTHrP(1-36)-induced vasodilation, which is depressed in SHR, was restored and a vasoconstrictory response to PTH(3-34), a PTH1R antagonist, was revealed. These effects were not observed in control SHR treated with empty plasmid. BP remained unchanged, and plasma renin activity increased by 60%. Thus, in SHR renal vessels, a reduced number of PTH1R contributes to the high RVR, despite the higher expression of vasodilatory PTHrP. Moreover, these studies provide evidence for a direct link between the density of PTH1R and plasma renin activity, which might be responsible for the absence of effect of PTH1R gene delivery on BP in SHR. Overall, PTHrP significantly contributes to the homeostasis of renal and systemic hemodynamics in SHR.     

Leptin regulates chondrocyte differentiation and matrix maturation during endochondral ossification

Leptin has been suggested to mediate a variety of actions, including bone development, via its ubiquitously expressed receptor (Ob-Rb). In this study, we investigated the role of leptin in endochondral ossification at the growth plate. The growth plates of wild-type and ob/ob mice were analyzed. Effects of leptin on chondrocyte gene expression, cell cycle, apoptosis and matrix mineralization were assessed using primary chondrocyte culture and the ATDC5 cell differentiation culture system. Immunohistochemistry and in situ hybridization showed that leptin was localized in prehypertrophic chondrocytes in normal mice and that Ob-Rb was localized in hypertrophic chondrocytes in normal and ob/ob mice. Growth plates of ob/ob mice were more fragile than those of wild-type mice in a mechanical test and were broken easily at the chondro-osseous junction. The growth plates of ob/ob mice showed disturbed columnar structure, decreased type X collagen expression, less organized collagen fibril arrangement, increased apoptosis and premature mineralization. Leptin administration in ob/ob mice led to an increase in femoral and humeral lengths and decrease in the proportional length of the calcified hypertrophic zone to the whole hypertrophic zone. In primary chondrocyte culture, the matrix mineralization in ob/ob chondrocytes was stronger than that of wild-type mice; this mineralization in both types of mice was abolished by the addition of exogenous leptin (10 ng/ml). During ATDC5 cell differentiation culture, exogenous leptin at a concentration of 1-10 ng/ml (equivalent to the normal serum concentration of leptin) altered type X collagen mRNA expression and suppressed apoptosis, cell growth and matrix calcification. In conclusion, we demonstrated that leptin modulates several events associated with terminal differentiation of chondrocytes. Our finding that the growth plates of ob/ob mice were fragile implies a disturbance in the differentiation/maturation process of growth plates due to depletion of leptin signaling in ob/ob mice. These findings suggest that peripheral leptin signaling plays an essential role in endochondral ossification at the growth plate.     

Expression of parathyroid hormone-related protein in rat articular cartilage

Expression and localization of parathyroid hormone-related protein (PTHrP) in rat articular cartilage during fetal and postnatal periods were investigated by immunohistochemistry and in situ hybridization. PHTrP displayed distinct distribution and intensity of staining at different ages. In fetal (18-day-old) and young (3-week-old) rats, articular chondrocytes expressed abundant PTHrP throughout the entire thickness of cartilage. In contrast, in 60-week-old rats, PTHrP was expressed in a few articular chondrocytes of superficial and middle layers. Regulation of PTHrP and PTH/PTHrP receptor mRNA was also studied in cultured rat articular chondrocytes. Northern blot analysis revealed that both transforming growth factor- (TGF-), an important stimulator for chondrocyte proliferation and differentiation, and 10% fetal bovine serum (FBS) stimulated the expression of PTHrP mRNA with down-regulation of its receptor mRNA. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) down-regulated the expression of receptor without changes of PTHrP mRNA level. These results suggest that the changes in abundance and localization of PTHrP and its receptor may be directly involved in the cell growth and differentiation of articular cartilage.