Glucocorticoid effects on chondrogenesis,differentiation and apoptosis in the murine ATDC5 chondrocyte cell line

Glucocorticoids (GC) are used extensively in children and may cause growth retardation, which is in part due to the direct effects of GC on the growth plate. We characterised the ATDC5 chondrocyte cell line, which mimics the in vivo process of longitudinal bone growth, to examine the effects of dexamethasone (Dex) and prednisolone (Pred) during two key time points in the chondrocyte life cycle - chondrogenesis and terminal differentiation. Additionally, we studied the potential for recovery following Dex exposure. During chondrogenesis, Dex and Pred exposure at 10(-8) M, 10(-7) M and 10(-6) M resulted in a significant mean reduction in cell number (28% vs 20%), cell proliferation (27% vs 24%) and proteoglycan synthesis (47% vs 43%) and increased alkaline phosphatase (ALP) activity (106% vs 62%), whereas the incidence of apoptosis was unaltered. Minimal effects were noted during terminal differentiation with both GC although all concentrations of Dex lowered apoptotic cell number. To assess catch-up growth the cells were incubated for a total of 14 days which included 1, 3, 7, 10 or 14 days exposure to 10(-6) M Dex, prior to the recovery period. Recovery of proteoglycan synthesis was irreversibly impaired following just one day exposure to Dex. Although cell number showed a similar pattern, significant impairment was only achieved following 14 days exposure. Irreversible changes in ALP activity were only noticed following 10 days exposure to Dex. In conclusion, GC have maximal effects during chondrogenesis; Dex is more potent than Pred and cells exposed to Dex recover but this may be restricted due to differential effects of GC on specific chondrocyte phenotypes.     

Insulin-like growth factor-I augments chondrocyte hypertrophy and reverses glucocorticoid-mediated growth retardation in fetal mice metatarsal cultures

The study aims were to improve our understanding of the mechanisms of glucocorticoid-induced growth retardation at the growth plate and determine whether IGF-I could ameliorate the effects. Fetal mouse metatarsals were cultured for up to 10 d with dexamethasone (Dex; 10(-6) m) and/or IGF-I and GH (both at 100 ng/ml). Both continuous and alternate-day Dex treatment inhibited bone growth to a similar degree, whereas IGF-I alone or together with Dex caused an increase in bone growth. GH had no effects. These observations may be explained at the cellular level; cell proliferation within the growing bone was decreased by Dex and increased by IGF-I and these effects were more marked in the cells of the perichondrium than those in the growth plate. However, the most prominent observation was noted in the hypertrophic zone where all treatments containing IGF-I significantly increased (3-fold) the length of this zone, whereas Dex alone had no significant effect. In conclusion, Dex impaired longitudinal growth by inhibiting chondrocyte proliferation, whereas IGF-I stimulated chondrocyte hypertrophy and reversed the growth-inhibitory Dex effects. However, the IGF-I-mediated improvement in growth was at the expense of altering the balance between proliferating and hypertrophic chondrocytes within the metatarsal.     

IGF-I signalling in bone growth: inhibitory actions of dexamethasone and IL-1beta.

OBJECTIVE: To determine if glucocorticoids and proinflammatory cytokines inhibit bone growth through a common mechanism involving impaired IGF-I signalling. DESIGN: IGF-I (100 ng/ml), dexamethasone (dex) (10(-6)M) and IL-1beta (10 ng/ml) with inhibitors of the PI3K (LY294002) and Erk 1/2 (PD98059 and UO126) IGF-I pathways (all 10 microM) were studied using the ATDC5 chondrocyte cell line and murine fetal metatarsal cultures. RESULTS: IGF-I stimulated ATDC5 chondrocyte proliferation (322%; P < 0.001 versus control). Addition of PD or LY individually to IGF-I supplemented ATDC5 cultures partially reduced proliferation by 32% (P < 0.001), and 66% (P < 0.001), respectively. PD and LY in combination blocked all IGF-I stimulated ATDC5 proliferation. LY significantly reversed IGF-I stimulatory effects on metatarsal growth (P < 0.001), whereas PD and UO treatment had no effect. IGF-I induced ATDC5 proliferation was further decreased when Dex (24%; P < 0.01) or IL-1beta (33%; P < 0.001) were added to PD but not LY cultures. Metatarsal growth inhibition by LY was unaltered by Dex or IL-1beta addition. CONCLUSIONS: Both the PI3K and Erk 1/2 pathways contributed independently to IGF-I mediated ATDC5 proliferation. However in metatarsal cultures, the Erk 1/2 pathway was not required for IGF-I stimulated growth. Dex and IL-1beta may primarily inhibit IGF-I induced bone growth through the PI3K pathway.     

Short-term effects on linear growth and bone turnover in children randomized to receive prednisolone or dexamethasone

AIM: To compare the relative potency of prednisolone (Pred) and dexamethasone (Dex) on short-term growth and bone turnover. METHOD: Prospective study over 16 weeks of children randomized to receive Pred (40 mg/m2) or Dex (6.5 mg/m2) for the first 5 weeks as part of the MRC-ALL97/99 induction chemotherapy for acute lymphoblastic leukaemia (ALL). MEASUREMENTS: Lower leg length velocity (LLLV) and weight, serum IGF-I, serum bone alkaline phosphatase (bALP) levels and creatinine-adjusted, urinary excretion of deoxypyridinoline cross-links (DPD). SUBJECTS: Nineteen children (eight boys, 11 girls) with a median age of 5.9 years (range 2.6-13) and with a diagnosis of ALL. RESULTS: At week 2 of therapy, median LLLV in the Dex group was -1.5 mm/week (range 0.7 to -2.1) and significantly lower than the LLLV in the Pred group which was -0.1 mm/week (range 0.20 to -0.28; P < 0.05). In the Dex group, LLLV remained lower at week 8 (med LLLV, -0.3 mm/week, range 0 to -1.3) compared to LLLV in the Pred group at 0.3 mm/week (range 0.2-1.0; P < 0.05). Body weight showed an increase after week 2 and reached a peak in both groups of children at week 6. The change in weight from baseline was greater in the Dex group than the Pred group reaching a maximum change by week 5 of 17.5% (range 5-25) and 8.7% (range -3 to 18), respectively (P < 0.05). At presentation, median IGF-I level for the whole group was 83.5 micro g/l (range 31.8-293). IGF-I levels fell markedly during Dex therapy and continued to remain lower than baseline. At weeks 4, 6 and 8, median change in IGF-I from baseline was lower in the Dex group than the Pred group. From week 1 to week 3, median change in bALP was 72% (range -8 to 304) in the Pred group, whereas in the Dex group change in bALP was -1% (range 23 to -28; P < 0.005). By week 3, median bALP was higher in the Pred group at 65 U/l (range 36-187) than in the Dex group at 39 U/l (range 26-60; P < 0.05) but by week 6 median bALP in the Pred group had fallen to a similar level to the Dex group. At presentation, median DPD was 22 nmol/l (range 17-38) and 20 nmol/l (range 12-26) in the Pred and Dex groups, respectively (ns), reaching a nadir between weeks 3 and 6. The median percentage change in DPD in the Pred and Dex group from week 1 to week 3 was -34% (range -7 to 14) and -53% (range -6 to -69), respectively (ns). By week 8, DPD excretion had started to rise more dramatically in the Pred group such that the median DPD was 35 nmol/l (range 10-53) in the Pred group and 22 (range 9-30) in the Dex group (P < 0.05). On average, between weeks 2 and 8, LLLV was three times lower, percentage gain in weight was three times higher, bALP was 1.3 times lower and DPD was 1.5 times lower in the Dex group than the Pred group. CONCLUSION: Pred and Dex both affect short-term growth and bone turnover. The mechanism of the effect on bone formation may be different between the two drugs. Dex may be about 18 times more potent than Pred at suppressing short-term linear growth and stimulating weight gain, and about nine times more potent at suppressing bone turnover. Glucocorticoids have a variable effect on different parameters of growth and bone turnover and the intensity may depend on the steroid used.     

Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent

Proinflammatory cytokines inhibit growth plate development. However, their underlying mechanisms of action are unclear. These effects may be mediated by ceramide, a sphingosine-based lipid second messenger, which is elevated in a number of chronic inflammatory diseases. To test this hypothesis, we determined the effects of C2-ceramide, a cell permeable ceramide analogue, on the growth of the ATDC5 chondrogenic cell line and on cultured fetal mice metatarsals. In ATDC5 cells, C2-ceramide significantly induced apoptosis at both 40 (82%; P < 0.05) and 25 microM (53%; P < 0.05). At 40 microM, C2-ceramide significantly reduced proliferation ([3H]-thymidine uptake/mg protein) (62%; P < 0.05). C2-ceramide did not markedly alter the differentiation state of the cells as judged by the expression of markers of chondrogenesis and differentiation (sox 9, collagen II and collagen X). The IGF-I signalling pathway is the major autocrine/paracrine regulator of bone growth. Both in the presence and absence of IGF-I, C2-ceramide (25 microM) induced an equivalent reduction in proliferation (60%; P < 0.001). Similarly, C2-ceramide (40 microM) induced a 31% reduction in fetal metatarsal growth both in the presence and absence of IGF-I (both P < 0.001). Furthermore, C2-ceramide reduced ADCT5 proliferation in the presence of AG1024, an IGF-I and insulin receptor blocker. Therefore, C2-ceramide-dependent inhibition appears to be independent of IGF-mediated stimulation of bone growth. Indeed, biochemical studies demonstrated that C2-ceramide (25 microM) pretreatment did not alter IGF-I-stimulated phosphorylation of insulin receptor substrate-1, Akt or P44/42 MAP kinase. In conclusion, C2-ceramide inhibits proliferation and induces apoptosis in growth plate chondrocytes through an IGF-I independent mechanism.     

The restricted potential for recovery of growth plate chondrogenesis and longitudinal bone growth following exposure to pro-inflammatory cytokines

Childhood chronic inflammatory disease can be associated with transient and permanent growth retardation. This study examined the potential for spontaneous growth recovery following pro-inflammatory cytokine exposure. Murine ATDC5 chondrogenic cells and postnatal metatarsals were exposed to interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha (TNFalpha), and their growth and proliferative capacity were determined following recovery. TNFalpha and IL-1beta reduced chondrocyte proliferation and aggrecan and collagen types II and X expression at minimum concentrations of 10 ng/ml and 0.1 ng/ml respectively. TNFalpha but not IL-1beta exposure led to increased caspase-3 activity and altered cellular morphology, consistent with reduced viability. Cytokine exposure particularly inhibited proteoglycan synthesis. This effect was dose and duration dependent. Compared with the control, IL-1beta and TNFalpha led to a 71% and 45% reduction in metatarsal growth after 8 days of exposure respectively (P < 0.05). An additive effect of IL-1beta combined with TNFalpha was observed (110% decrease; P < 0.05). Metatarsals exposed to IL-1beta or TNFalpha individually for a 2-day period, and allowed to recover spontaneously in the absence of cytokines for a further 6 days, showed normal growth trajectories. In combination, growth was 59% lower (P < 0.01) compared with control metatarsals at the end of the recovery period. Exposure to the combination for 4 days followed by a 4-day recovery period resulted in 87% decrement compared with controls (P < 0.05). IL-6 did not alter any parameter studied. IL-1beta and TNFalpha exert diverse inhibitory effects on ATDC5 chondrocyte dynamics and metatarsal growth. The extent of recovery following cytokine exposure depends on the duration of exposure, and may be incomplete following longer periods of exposure.     

Insulin-like growth factor-I signaling is modified during chondrocyte differentiation

Insulin-like growth factor-I (IGF-I) is a critical regulator of skeletal growth. While IGF-I has been shown to be a potent chondrocyte mitogen in vitro, its role in chondrocyte differentiation is less well characterized. We chose to study the action of IGF-I on an accepted model of chondrocyte differentiation, the ATDC5 cell line. Insulin concentrations sufficiently high to interact with the IGF-I receptor are routinely used to induce ATDC5 cells to differentiate. Therefore, we first examined the ability of IGF-I to promote chondrocyte differentiation at physiological concentrations. IGF-I could induce differentiation of these cells at concentrations below 10 nM. However, increasing IGF-I concentrations were less potent at inducing differentiation. We hypothesized that mitogenic effects of IGF-I might inhibit its differentiating effects. Indeed, the extracellular-signal-regulated kinase (ERK)-pathway inhibitor PD98059 inhibited ATDC5 cell DNA synthesis while enhancing differentiation. This suggested that the ability of IGF-I to promote both proliferation and differentiation might require that its signaling be modulated through the differentiation process. We therefore compared IGF-I-mediated ERK activation in proliferating and hypertrophic chondrocytes. IGF-I potently induced ERK activation in proliferating cells, but minimal ERK response was seen in hypertrophic cells. In contrast, IGF-I-mediated Akt activation was unchanged by differentiation, indicating intact upstream IGF-I receptor signaling. Similar findings were observed in the RCJ3.1C5.18 chondrogenic cell line and in primary chick chondrocytes. We conclude that IGF-I promotes both proliferation and differentiation of chondrocytes and that the differentiation effects of IGF-I may require uncoupling of signaling to the ERK pathway.     

Phosphate stimulates matrix Gla protein expression in chondrocytes through the extracellular signal regulated kinase signaling pathway

Whereas increasing evidence suggests that inorganic phosphate (Pi) may act as a signaling molecule in mineralization-competent cells, its mechanisms of action remain largely unknown. The aims of the present work were to determine whether Pi regulates expression of matrix Gla protein (MGP), a mineralization inhibitor, in growth plate chondrocytes and to identify the involved signaling pathways. Chondrogenic ATDC5 cells and primary growth plate chondrocytes were used. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting, respectively. The activation and role of MAPKs were, respectively, determined by Western blotting and the use of specific inhibitors. Immunohistological detection of ERK1/2 was performed in rib organ cultures from newborn mice. The results indicate that Pi markedly stimulates expression of MGP in ATDC5 cells and primary growth plate chondrocytes. Investigation of the involved intracellular signaling pathways reveals that Pi activates ERK1/2 in a cell-specific manner, because the stimulation was observed in ATDC5 and primary chondrocytes, MC3T3-E1 osteoblasts, and ST2 stromal cells, but not in L929 fibroblasts or C2C12 myogenic cells. Accordingly, immunohistological detection of ERK1/2 phosphorylation in rib growth plates revealed a marked signal in chondrocytes. Finally, a specific ERK1/2 inhibitor, UO126, blocks Pi-stimulated MGP expression in ATDC5 cells, indicating that ERK1/2 mediates, mainly, the effects of Pi. These data demonstrate, for the first time, that Pi regulates MGP expression in growth plate chondrocytes, thereby suggesting a key role for Pi and ERK1/2 in the regulation of bone formation.     

The role of insulin in chondrogenesis

The ATDC5 chondrogenic cell line is typically induced to differentiate by exposure to insulin at high concentration (10 microg/ml, approximately 1600 nM). Differentiation can also be induced by physiological concentrations of insulin-like growth factor-I (IGF-I). Unlike previous reports, we observed a stimulation of differentiation, as measured by collagen X expression and Alcian Blue staining for proteoglycan synthesis, upon exposure to insulin at concentrations (10-50 nM) consistent with signaling via the insulin receptor. Analysis of lysates from proliferating and hypertrophic ATDC5 cells demonstrated that exposure to 50 nM insulin induced tyrosine phosphorylation of insulin receptors but not IGF-I receptors or hybrid receptors. In contrast to the potent effects of IGF-I to stimulate both ATDC5 proliferation and differentiation, insulin was not as potent as IGF-I as a proliferating agent but more selectively a differentiating agent. Consistent with this result, insulin was less potent than IGF-I in inducing activation of the Erk1/Erk2 mitogenic signaling pathway. Furthermore, Erk pathway inhibition did not enhance the differentiating effects of insulin as it does in the case of IGF-I exposure. Extending our observations to fetal rat metatarsal explants, we observed significant stimulation of bone growth by 50 nM insulin. This could be accounted for by a disproportionate stimulatory effect on growth of the hypertrophic zone. The proliferative zone was not significantly affected. Based on our results in both ATDC5 cells and metatarsal explants, we conclude that the insulin functioning through insulin receptor has a dominant effect as an inducer of chondrocyte differentiation. These results support assignment of a physiological role for this hormone in linear bone growth.     

DHEA suppresses longitudinal bone growth by acting directly at growth plate through estrogen receptors

Dehydroepiandrosterone (DHEA) is produced by the adrenal cortex and is the most abundant steroid in humans. Although in some physiological and pathological conditions the increased secretion of DHEA and its sulfated form is associated with accelerated growth rate and skeletal maturation, it is unclear whether DHEA can affect longitudinal bone growth and skeletal maturation by acting directly at the growth plate. In our study, DHEA suppressed metatarsal growth, growth plate chondrocyte proliferation, and hypertrophy/differentiation. In addition, DHEA increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, DHEA reduced chondrocyte proliferation and induced apoptosis. The DHEA-induced inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was nullified by culturing metatarsals with DHEA in the presence of ICI 182,780, an inhibitor of estrogen receptor, but not in the presence of Casodex, an inhibitor of androgen receptor. Lastly, nuclear factor-κB DNA binding activity was inhibited by the addition of DHEA in the medium of cultured chondrocyte. Our findings indicate that DHEA suppressed bone growth by acting directly at growth plate through estrogen receptor. Such growth inhibition is mediated by decreased chondrocyte proliferation and hypertrophy/differentiation and by increased chondrocyte apoptosis.     

Monocarboxylate transporter 10 functions as a thyroid hormone transporter in chondrocytes

Thyroid hormone is essential for normal proliferation and differentiation of chondrocytes. Thus, untreated congenital hypothyroidism is marked by severe short stature. The monocarboxylate transporter 8 (MCT8) is a highly specific transporter for thyroid hormone. The hallmarks of Allan-Herndon-Dudley syndrome, caused by MCT8 mutations, are severe psychomotor retardation and elevated T(3) levels. However, growth is mostly normal. We therefore hypothesized that growth plate chondrocytes use transporters other than MCT8 for thyroid hormone uptake. Extensive analysis of thyroid hormone transporter mRNA expression in mouse chondrogenic ATDC5 cells revealed that monocarboxylate transporter 10 (Mct10) was most abundantly expressed among the transporters known to be highly specific for thyroid hormone, namely Mct8, Mct10, and organic anion transporter 1c1. Expression levels of Mct10 mRNA diminished with chondrocyte differentiation in these cells. Accordingly, Mct10 mRNA was expressed most abundantly in the growth plate resting zone chondrocytes in vivo. Small interfering RNA-mediated knockdown of Mct10 mRNA in ATDC5 cells decreased [(125)I]T(3) uptake up to 44% compared with negative control (P < 0.05). Moreover, silencing Mct10 mRNA expression abolished the known effects of T(3), i.e. suppression of proliferation and enhancement of differentiation, in ATDC5 cells. These results suggest that Mct10 functions as a thyroid hormone transporter in chondrocytes and can explain at least in part why Allan-Herndon-Dudley syndrome patients do not exhibit significant growth impairment.     

Effect of adiponectin on ATDC5 proliferation,differentiation and signaling pathways

Adiponectin, an adipose-secreted adipocytokine, exhibits various metabolic functions but has no known effect on bone development through the growth plate and specifically, in chondrocytes. Using the mouse ATDC5 cell line, a widely used in vitro model of chondrogenesis, we demonstrated the expression of adiponectin and its receptors during chondrogenic differentiation. Adiponectin at 0.5 μg/ml increased chondrocyte proliferation, proteoglycan synthesis and matrix mineralization, as reflected by upregulation of the expression of type II collagen, aggrecan, Runx2 and type X collagen, and of alkaline phosphatase activity. Quantitative RT-PCR and gelatin zymography showed a significant increase in the matrix metalloproteinase MMP9's expression and activity following adiponectin treatment. We therefore concluded that adiponectin can directly stimulate chondrocyte proliferation and differentiation. To evaluate the underlying mechanisms, we examined the effect of adiponectin on the expression of chondrogenic signaling molecules: Ihh, PTHrP, Ptc1, FGF18, BMP7, IGF1 and p21 were all upregulated while FGF9 was downregulated. This study reveals novel and direct activity of adiponectin in chondrocytes, suggesting its positive effects on bone development.     

Interleukin-1β promotes proliferation and inhibits differentiation of chondrocytes through a mechanism involving down-regulation of FGFR-3 and p21

The proinflammatory cytokine IL-1β is elevated in many childhood chronic inflammatory diseases as well as obesity and can be associated with growth retardation. Here we show that IL-1β affects bone growth by directly disturbing the normal sequence of events in the growth plate, resulting in increased proliferation and widening of the proliferative zone, whereas the hypertrophic zone becomes disorganized, with impaired matrix structure and increased apoptosis and osteoclast activity. This was also evident in vitro: IL-1β increased proliferation and caused a G1-to-S phase shift in the cell cycle in ATDC5 chondrocytes, accompanied by a reduction in fibroblast growth factor receptor-3 (FGFR-3) and its downstream gene, the cell-cycle inhibitor p21 and its family member p57, whereas the cell-cycle promoter E2F-2 was increased. The reduction in FGFR-3, p21, and p57 was followed by delayed cell differentiation, manifested by decreases in proteoglycan synthesis, mineralization, alkaline phosphatase activity, and the expression of Sox9, RunX2, collagen type II, collagen type X, and other matrix proteins. Taken together, we suggest that IL-1β alters normal chondrogenesis and bone growth through a mechanism involving down-regulation of FGFR-3 and p21.     

Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvaria cells in vitro

Isolated rat calvaria cells plated at low density in medium supplemented with ascorbic acid and organic phosphate form discrete three-dimensional mineralized nodules having the characteristics of bone. We have studied the effects of glucocorticoids on the formation of bone nodules by these cell populations. Cells isolated from 21-day-old fetal rat calvaria were maintained in vitro for up to 27 days. Dexamethasone (Dex) induced a dose-related increase in the number of nodules formed, with a peak at 10 nM and a half-maximal response at about 1 nM. Dex (10 nM) also significantly increased the size of bone nodules formed (P less than 0.002). High concentrations of Dex (1 microM) did not increase nodule number. In cells in primary culture maintained in medium containing 10 nM Dex, the increase in nodule number was 50-100% over the control value. The effect of Dex was much greater in first subculture cells, where the number of nodules was 600-800% higher than the control value. Dishes collected and quantitated from 12-27 days showed that nodule formation ceased between 15 and 18 days in cultures without Dex, whereas in the presence of Dex the number of nodules increased up to 27 days. Addition of 10 nM Dex only during specific periods resulted in significantly more nodules than in control cultures, but significantly fewer nodules than in cultures constantly exposed to Dex. Cell population doubling times during log phase growth were unaltered, but a significant increase in saturation density (P less than 0.001) was observed with 10 nM Dex. Hydrocortisone also caused an increase in the number of nodules formed, with a maximal effect of 50 nM and a half-maximal response at 8 nM. The results indicate that physiological levels of glucocorticoids stimulate bone nodule formation in long term cell culture by increasing the number of cells forming bone nodules and that maximization of the stimulatory effect of glucocorticoids on bone formation may require constant exposure to low levels of the hormone.     

BIG-3, a novel WD-40 repeat protein, is expressed in the developing growth plate and accelerates chondrocyte differentiation in vitro

Among the local signaling pathways that regulate the sequential steps of chondrocyte differentiation is the bone morphogenetic protein (BMP) signaling pathway. We have identified a novel gene, named BIG-3 (BMP-2-induced gene 3 kb) that is expressed in a BMP-regulated fashion in the prechondroblastic cell line MLB13MYC clone 17. BIG-3 is also expressed in proliferating and hypertrophic chondrocytes in the developing growth plate in vivo. We undertook studies to address whether BIG-3 played a functional role in chondrocyte differentiation, using mouse clonal chondrogenic ATDC5 cells. BIG-3 protein levels increased during ITS (insulin, transferrin, sodium selenite)-induced ATDC5 differentiation and in response to BMP-2 treatment. To determine whether stable expression of BIG-3 could alter the program of chondrocytic differentiation, ATDC5 cells were stably transfected with the full-length coding region of BIG-3 (ATDC5-BIG-3) or with the empty vector (ATDC5-EV). Accelerated matrix proteoglycan synthesis was observed in the pooled ATDC5-BIG-3 clones. Alkaline phosphatase and osteopontin mRNA levels were also increased in ATDC5-BIG-3 clones compared with ATDC5-EV clones. Stable expression of BIG-3 also accelerated mineralized matrix formation in both the presence and absence of ITS. These findings, which demonstrate that BIG-3 accelerates chondrocyte differentiation in vitro, combined with the observation that BIG-3 is expressed in the growth plate during embryonic development, suggest that this novel protein is likely to play an in vivo regulatory role in the developing growth plate.     

Transforming growth factor-beta stimulates inorganic phosphate transport and expression of the type III phosphate transporter Glvr-1 in chondrogenic ATDC5 cells

Members of the transforming growth factor (TGF)-beta family are important regulators of skeletal development. In this study, we investigated the effect of TGF-beta1 on inorganic phosphate (Pi) transport and on expression of the type III Pi carriers Glvr-1 and Ram-1 in murine ATDC5 chondrocytes. TGF-beta1 induced a selective, dose- and time-dependent increase in sodium-dependent Pi transport in ATDC5 cells. This response was dependent on RNA and protein synthesis and reflected a change in the maximal rate of the transport system, suggesting that TGF-beta1 induces the synthesis of new Pi carriers and their insertion into the plasma membrane. Consistently, Northern blotting analysis showed a dose-dependent increase in Glvr-1 messenger RNA expression in response to TGF-beta1, which preceded the maximal stimulation of Pi transport by several hours. Glvr-1 thus likely mediates at least part of the increase in Pi uptake induced by TGF-beta1. Ram-1 messenger RNA expression was not affected by TGF-beta1. TGF-beta1 activated the Smad signaling pathway and the mitogen-activated protein kinases ERK and p38 in ATDC5 cells. Unlike the regulation of Pi transport by receptor tyrosine kinase agonists in osteoblasts, the effect of TGF-beta1 on Pi uptake in ATDC5 cells did not involve protein kinase C or mitogen-activated protein kinases, suggesting that a specific, possibly Smad-dependent, signal mediates this response. In conclusion, TGF-beta1 stimulates Pi transport and Glvr-1 expression in chondrocytes, suggesting that, like proliferation, differentiation, and matrix synthesis, Pi handling is subject to regulation by TGF-beta3 family members in bone-forming cells.     

Estrogen stimulates leptin receptor expression in ATDC5 cells via the estrogen receptor and extracellular signal-regulated kinase pathways

Regulation of the physiological processes of endochondral bone formation during long bone growth is controlled by various factors including the hormones estrogen and leptin. The effects of estrogen are mediated not only through the direct activity of estrogen receptors (ERs) but also through cross talk with other signaling systems implicated in chondrogenesis. The receptors of both estrogen and leptin (OBR (LEPR)) are detectable in growth plate chondrocytes of all zones. In this study, the expression of mRNA and protein of OBR in chondrogenic ATDC5 cells and the effect of 17β-estradiol (E(2)) stimulation were assessed using quantitative PCR and western blotting. We have found that the mRNA of Obr was dynamically expressed during the differentiation of ATDC5 cells over 21 days. Application of E(2) (10(-7) M) at day 14 for 48?h significantly upregulated OBR mRNA and protein levels (P<0.05). The upregulation of Obr mRNA by E(2) was shown to take place in a concentration-dependent manner, with a concentration of 10(-7) M E(2) having the greatest effect. Furthermore, we have confirmed that E(2) affected the phosphorylation of ERK1/2 (MAPK1/MAPK3) in a time-dependent manner where a maximal fourfold change was observed at 10?min following application of E(2). Finally, pretreatment of the cells with either U0126 (ERK1/2 inhibitor) or ICI 182?780 (ER antagonist) blocked the upregulation of OBR by E(2) and prevented the E(2)-induced phosphorylation of ERK. These data demonstrate, for the first time, the existence of cross talk between estrogen and OBR in the regulation of bone growth whereby estrogen regulates the expression of Obr in growth plate chondrocytes via ERs and the activation of ERK1/2 signaling pathways.