Parathyroid Hormone and Transforming Growth Factor-β1 Coregulate Chondrocyte Differentiation In Vitro (original) (raw)
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Bone, 1997
The aims of this study were to clarify the role of cell maturation stage on chondrocyte response to parathyroid hormone (PTH) by examining the effect of PTH(1-34) on alkaline-phosphatase-specific activity (ALPase) of chondrocyte cultures at two distinct stages of maturation, and to determine the signaling p~tthways used by the cells to mediate this effect. Confluent, fourth passage rat costochondral resting zone (RC) and growth zone (GC) chondrocytes were used. ALPase was measured in the cell layer, as well as in matrix vesicles (MV) and plasma membranes (PM), after the addition of 10-7 to 10-I1 moVE bovine PTH(1-34), the active peptide, or bovine PTH(3-34), the inactive peptide, to the cultures. PTH(1-34) :increased ALPase in the GC cultures at two separate times: between 5 and 180 min, with maximal stimulation at 10 min, and 36 to 48 h. In contrast, PTH(3-34) had no effect. At 10 min and 48 h, PTH(1-34) produced a dose-dependent increase in ALPase of both MV and PM isolated from GC cultures. Addition of forskolin and IBMX to increase cAMP increased ALPase in GC cultures to a level similar to that seen after addition of PTH(1-34). In contrast, the addition of PTH(1-34) to RC cells only increased ALPase between 5 and 60 min, with peak activity at 10 rain. As with GC, PTH increased ALPase in both MV and PM. Moreover, the addition of PTH(3-34) or forskolin and IBMX had no effect on ALPase in RC. PTH(1-34) had no effect on GC protein kinase C (PKC) activity; however, the addition of PTH(1-34) to RC caused a dose-dependent increase in PKC activity. H8, an inhibitor of PKA, had no effect on PTHstimulated ALPase in RC cells, but inhibited the PTH-dependent response in GC cells. In contrast, chelerythrine, an inhibitor of PKC activity, inhibited PTH-stimulated ALPase in RC cells, but had no effect on PTH-stimulated ALPase in GC cells. This study shows that the effect of PTH(1-34) on RC and GC cells is maturation dependent in terms of time course and mechanism. Whereas both cell types exhibit a rapid response to PTH, only GC cells show a long-term response. In GC, the effects of PTH are associated with
Journal of Biological Chemistry
Resting zone and growth zone (GC) costochondral chondrocytes constitutively release latent, but not active, transforming growth factor+ (TGF-P) into the culture medium. When exogenous TGF-P is added to the culture medium, no autocrine effect is observed. However, when 1,25-(OH),D, is added, a dose-dependent inhibition of latent TGF-P release is found. Messenger RNAlevels for TGF-/3l are unchanged by treatment with either 1,25-(OH),D, or TGF-P1. Since active growth factor was not observed in the conditioned medium, we tested the hypothesis that latent TGF-P could be activated in the matrix. GC matrix vesicles, extracellular organelles associated with matrix calcification, were able to activate latent TGF-Pl and TGF-PB when preincubated with 1,25-(OH),D,. In contrast, GC plasma membranes activated latent TGF-P, and addition of 1,25-(OH),D, inhibited this activation. The 1,25-(OH),D,-dependent decrease in latent TGF-P in the medium, with no detectable change in mRNA level, and the inhibition of plasma membrane activation of latent TGF-/3 by 1,25-(OH),D, suggest that 1,25-(0H),D, may act through post-transcriptional and/or nongenomic mechanisms. The resulta also suggest that latent TGF-P is activated in the matrix and that 1,25-(0H)& regulates this activation by a direct, nongenomic action on the matrix vesicle membrane. Transforming growth factor p (TGF-p)' is an important regulator of cartilage development and chondrocyte differentiation (1-3). It is synthesized by chondrocytes and appears to act in an autocrine manner (4-6). TGF-p production vanes with stage of chondrocyte differentiation. Chondrocytes isolated from avian growth plates produce latent, but not active, TGF-P, and hypertrophic chondrocytes isolated from this tissue release almost twice as much growth factor as reserve zone cells into their culture medium (4). Similarly, rat growth zone chondrocytes produce 5 times as much latent TGF-p as do resting zone chondrocytes, with little or no active TGF-p detected in the culture medium (5). * These studies were supported by United States Public Health Service Grants DE05937, DE08603, DE08569, AGO68872, andAR39529, the Department of Veterans Mairs, and the United Statesfisrael Binational Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; 'The abbreviations used are: TGF, transforming growth factor; RC, resting zone chondrocytes; G C , growth zone chondrocytes.
Experimental Cell Research, 2000
Although PTHrP has been identified as a key regulator of chondrocyte differentiation in the growth plate, the factors directly regulating PTHrP expression have not been identified. Furthermore, while cells from the epiphysis are considered the physiologic source of PTHrP, the relative expression of PTHrP in epiphyseal and growth plate chondrocytes has not been defined. PTHrP expression was examined in chondrocytes isolated from 3-to 5-week-old chick long bones. The expression of PTHrP mRNA was 10-fold higher in epiphyseal chondrocytes compared to cells from the growth plate. Growth plate chondrocytes were isolated into populations with distinct maturational characteristics by countercurrent centrifugal elutriation and analyzed for PTHrP expression. The expression was highest in the least mature cells and progressively declined with the onset of maturation. The regulation of PTHrP expression was further examined in epiphyseal chondrocytes. Both TGF-1 and cis-retinoic acid stimulation markedly increased PTHrP mRNA levels, while BMP-2 and PTHrP stimulation decreased the expression of this transcript. The effects of TGF-1 (8.9-fold stimulation) and TGF-3 (9.2-fold) were slightly greater than the effects of TGF-2 (4.9-fold). The effect of TGF- was dose-dependent and increases could be detected after 68 h of treatment. To analyze the paracrine effect of epiphyseal and growth plate chondrocytes on each other, these cells were placed in coculture and the mRNA from each of the populations was harvested separately after 24 h. Following coculture the PTHrP mRNA levels increased in the epiphyseal cells while the expression of type X collagen and Indian hedgehog transcripts decreased in growth plate chondrocytes. The results demonstrate potentially important paracrine interactions between these cell populations, possibly mediated by TGF- and PTHrP.
Effects of parathyroid hormone-related peptide on chick growth plate chondrocytes
Journal of Orthopaedic Research, 1993
Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH) have similar biological effects in vitro that are mediated through the PTH receptor. PTH receptors have been demonstrated in the zone of provisional calcification and the hypertrophic zone of the cartilaginous growth plate. The current study examined the biological effects of PTHrP on chick growth plate chondrocytes. Chondrocytes were exposed to varying doses of PTHrP for 24 h, and the incorporation of radioactive thymidine into DNA was used as an index of proliferation. A dose-dependent stimulation of proliferation was seen, with a maximal 27-fold increase at 50 nM PTHrP. A dose-dependent stimulation of CAMP was seen, with a maximal effect at a dose of 50 nM. Proteoglycan synthesis, measured by incorporation of radioactive sulfate, was stimulated, with a maximal effect of 65% at 1 nM. Collagen synthesis and alkaline phosphatase activity from both cellular and matrix vesicle sources decreased in a dose-dependent fashion, with a maximal inhibition of -50% of the control value. The physiologic significance of the PTH and PTHrP-responsiveness of growth plate chondrocytes is uncertain at the present time. It is possible that PTH or PTHrP, or both, act as a systemic, developmental modulator of cellular proliferation and differentiation in the growth plate.
Journal of Bone and Mineral Research, 1999
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.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2002
Transforming growth factor beta 1 (TGF-h1) affects growth plate chondrocytes through Smad-mediated mechanisms and has been shown to increase protein kinase C (PKC). This study determined if PKC mediates the physiological response of rat costochondral growth zone (GC) chondrocytes to TGF-h1; if the physiological response occurs via type II or type III TGF-h receptors, and, if so, which receptor mediates the increase in PKC; and the signal transduction pathways involved. Treatment of confluent GC cells with TGF-h1 stimulated [ 3 H]thymidine and [ 35 S]sulfate incorporation as well as alkaline phosphatase (ALPase) and PKC specific activities. Inhibition of PKC with chelerythrine, staurosporine, or H-7 caused a dose-dependent decrease in these parameters, indicating that PKC signaling was involved. TGF-h1-dependent PKC and the physiological response of GC cells to TGF-h1 was reversed by anti-type II TGF-h receptor antibody and soluble type II TGF-h receptor, showing that TGF-h1 mediates these effects through the type II receptor. The increase in [ 3 H]thymidine incorporation and ALPase specific activity were also regulated by protein kinase A (PKA) signaling, since the effects of TGF-h1 were partially blocked by the PKA inhibitor H-8. The mechanism of TGF-h1 activation of PKC is through phospholipase A 2 (PLA 2) and not through phospholipase C (PLC). Arachidonic acid increased PKC in control cultures and was additive with TGF-h1. Prostanoids are required, as indomethacin blocked the effect of TGF-h1, and Cox-1, but not Cox-2, is involved. TGF-h1 stimulates prostaglandin E 2 (PGE 2) production and exogenous PGE 2 stimulates PKC, but not as much as TGF-h1, suggesting that PGE 2 is not sufficient for all of the prostaglandin effect. In contrast, TGF-h1 was not regulated by diacylglycerol; neither dioctanoylglycerol (DOG) nor inhibition of diacylglycerol kinase with R59022 had an effect. G-proteins mediate TGF-h1 signaling at different levels in the cascade. TGF-h1-dependent increases in PGE 2 levels and PKC were augmented by the G protein activator GTPgS, whereas inhibition of G-protein activity via GDPhS, pertussis toxin, or cholera toxin blocked stimulation of PKC by TGF-h1, indicating that both G i and G s are involved. Inhibition of PKA with H-8 partially blocked TGF-h1-dependent PKC, suggesting that PKA inhibition on the physiological response was via PKA regulation of PKC signaling. This indicates that multiple interacting signaling pathways are involved: TGF-h1 stimulates PLA 2 and prostaglandin release via the action of Cox-1 on arachidonic acid. PGE 2 activates the EP2 receptor, leading to G-protein-dependent activation of PKA. PKA signaling results in increased PKC activity and PKC signaling regulates proliferation, differentiation, and matrix synthesis.
Kidney International, 1997
Parathyroid hormone prevents 1,25 (OH)2D3 induced down-regulation of the vitamin D receptor in growth plate chondrocytes in vitro. I,25(OH)2D1 has an antiproliferative effect on growth plate chondrocytes when given in high doses, whereas low doses stimulate chondrocyte proliferation. In the present in vitro study we investigated the effects of parathyroid hormone (PTI-l) when given concomitantly with 1,25(OH)2D3 on cell proliferation and vitamin D receptor (VDR) regulation. Freshly isolated rat tibial chondrocytes were grown in monolayer cultures or in agarose stabilized suspension cultures (10% charcoal-treated FCS). VDR expression was determined by RT-PCR generating a 297 hp fragment and by binding assays (Scatchard analysis) with [3H]-1,25(OH)2D3. Cell proliferation was measured by [3H]-thymidinc incorporation, growth curves in monolayer cultures and by colony formation in agarose-stabilized suspension cultures. Optimal concentration of 1,25(OH)2D3 (10_12 M) and of PTH fragments [hPTI-I(1-34) or hPTH(28-48), lO° M] showed additive effects on DNA synthesis of and colony formation by growth plate chondrocytes. This may be explained in part by an up-regulation of VDR by PTH: PTH increased both mRNA expression of VDR and binding capacity. 1,25(OH)2D3 (10-12 M)
Kidney International, 1998
Interaction of IGF-I and 1␣,25(OH) 2 D 3 on receptor expression and growth stimulation in rat growth plate chondrocytes. Growth plate cartilage cells express receptors for, and are affected by both IGF-I and 1␣,25(OH) 2 D 3. The studies were undertaken to investigate interaction between these two hormone systems, that is, (i) to study effects of 1␣,25(OH) 2 D 3 on IGF-type 1 receptors (IGFIR), on IGF-I stimulated cell replication, colony formation, and on alkaline phosphatase activity (AP), and conversely, (ii) to study the effect of IGF-I on vitamin D receptor (VDR) expression on 1␣,25(OH) 2 D 3 stimulated growth parameters and on AP activity. Freshly isolated rat tibial chondrocytes were grown in monolayer cultures, (serum-free) or in agarose stabilized suspension cultures (0.1% FCS). Vitamin D receptor and IGFIR were visualized by immunostaining with the monoclonal antibody (mAb) 9A7␥ and mAb ␣IR3, respectively, and quantitated by RT-PCR for mRNA and by Scatchard analysis using [ 3 H]-1,25(OH) 2 D 3 and [ 125 I]-␣IR3. Cell proliferation was measured by [ 3 H]-thymidine incorporation, growth curves in monolayer cultures, and by colony formation in agarose-stabilized suspension cultures. IGF-I dose-dependently increased [ 3 H]-thymidine incorporation. 1␣,25(OH) 2 D 3 , but not 1,25(OH) 2 D 3 was stimulatory at low (10 Ϫ12 M) and slightly inhibitory at high (10 Ϫ8 M) concentrations. The effect of IGF-I was additive to that of 1␣,25(OH) 2 D 3 [IGF-I 60 ng/ml, 181 Ϯ 12.7; 1␣,25(OH) 2 D 3 10 Ϫ12 M, 181 Ϯ 9.8%, IGF-I ϩ 1␣,25(OH) 2 D 3 , 247 Ϯ 16.7%; P Ͻ 0.05 by ANOVA] and specifically obliterated by polyclonal IGF-I antibody (AB-1). Interaction could also be confirmed in suspension cultures. IGFIR mRNA and [ 125 I]-␣IR3 binding was increased by low (10 Ϫ12 M) but not by high (10 Ϫ8 M) concentrations of 1␣,25(OH) 2 D 3. Homologous up-upregulation by IGF-I (60 ng/ml) was specifically inhibited by AB-1 and markedly amplified by coincubation with 1␣,25(OH) 2 D 3 (10 Ϫ12 M). Immunostaining with ␣IR3 showed specific IGFIR expression in rat growth cartilage, but not liver tissue. Stimulation of chondrocytes with 1␣,25(OH) 2 D 3 or IGF-I suggested some increase of receptor expression in single cells, but the predominant effect was increased recruitment of receptor positive cells. Vitamin D receptor expression was markedly stimulated (fourfold) by IGF-I (60 ng/ml), but not IGF-II and inhibited by actinomycin D. This study shows that IGF-I and 1␣,25(OH) 2 D 3 mutually up-regulate their respective receptors in growth plate chondrocytes. In parallel, they have additive effects on cell proliferation and colony formation suggesting independent effector pathways. METHODS Materials 1␣,25(OH) 2-[26,27-methyl-3 H]cholecalciferol (158 Ci/mmol) was obtained from Amersham Buchler (Braunschweig, Germany); unlabeled 1␣,25(OH) 2 D 3 and 1,25(OH) 2 D 3 , were gifts from
In Vitro Cellular & Developmental Biology - Animal, 1992
We have developed a serum-free system to culture postembryonic growth plate chondrocytes while maintaining some important phenotypic characteristics of their tissue of origin. This serum-free medium was as effective as medium containing 10% newborn bovine serum (NBS) for recovering the cells from enzymatic isolation. Surface secretory activity of ehondrocytes cultured in monolayer, assessed through scanning electron microscopy, was also comparable to cells grown in medium containing serum. The effects of growth hormone (GH) and insulinlike growth factor-I (IGF-I) were also studied using the serum-free medium. GH had no effect on cell density and morphology of the cells compared to the control without the hormone. In contrast, ehondrocytes grown in medium containing IGF-I had a marked increase in cell density after 3 days and presented similar morphologic characteristics to cells grown in the presence of NBS. The growth factors required for proliferation of chondrocytes cultured in the serum-free medium are IGF-I and fibroblast growth factor (100 ng/ml, respectively). Addition of ascorbic acid to the serum-free medium (0 to 50 #g/ml) produced a dose-dependant decrease in cell proliferation. This medium should provide a useful tool for studying the effects of different growth factors/hormones in the regulation of longitudinal bone growth and their interactions.