C-type natriuretic peptide stimulates osteoblastic proliferation and collagen-X expression but suppresses fibroblast growth factor-23 expression in vitro.

BACKGROUND: The effects of C-type natriuretic peptide (CNP) and fibroblast growth factor (FGF)-23 appear to oppose each other during the process of bone formation, whereas few studies exist on the interaction between CNP and FGF-23. The main objective of the present study is to probe whether CNP is directly responsible for the regulation of osteoblast or via antagonizing FGF-23. METHODS: Osteoblasts were cultured in the absence or presence of CNP (0, 10, and 100 pmol/L) for 24 h, 48 h and 72 h, respectively. RESULTS: The findings of the present study indicated that: (1) CNP significantly stimulated osteoblastic proliferation and collagen (Col)-X expression; (2) both osteoblastic (osteocalcin, procollagen type I carboxy-terminal propeptide, total alkaline phosphatase and bone-specific alkaline phosphatase) and osteolytic (tartrate-resistant acid phosphatase and cross-linked carboxyterminal telopeptide of type I collagen) bone turnover biomarkers were up-regulated by CNP in osteoblasts; (3) FGF-23 mRNA and protein were significantly down-regulated at 24 h by CNP in osteoblasts, but the expression of FGF receptor-1/Klotho had no significant change. CONCLUSIONS: CNP stimulates osteoblastic proliferation and Col-X expression via the down-regulation of FGF-23 possibly in vitro. However, the specific mechanisms of the interaction between CNP and FGF-23 in osteoblasts are still unclear according to our findings. A further study on osteoblasts cultured with CNP and FGF-23 inhibitor will be undertaken in our laboratory.

Positive Effects of a Young Systemic Environment and High Growth Differentiation Factor 11 Levels on Chondrocyte Proliferation and Cartilage Matrix Synthesis in Old Mice.

OBJECTIVE: To investigate the effects of a young systemic environment and growth differentiation factor 11 (GDF-11) on aging cartilage. METHODS: A heterochronic parabiosis model (2-month-old mouse and 12-month-old mouse [Y/O]), an isochronic parabiosis model (12-month-old mouse and 12-month-old mouse [O/O]), and 12-month-old mice alone (O) were evaluated. Knee joints and chondrocytes from old mice were examined by radiography, histology, cell proliferation assays, immunohistochemistry, Western blotting, and quantitative reverse transcriptase-polymerase chain reaction 16 weeks after parabiosis surgery. GDF-11 was injected into 12-month-old mouse joints daily for 16 weeks. Cartilage degeneration, cell proliferation, and osteoarthritis-related gene expression were evaluated. RESULTS: Osteoarthritis Research Society International scores in old mice were significantly lower in the Y/O group than in the O/O and O groups (both P < 0.05). The percentage of 5-ethynyl-2'-deoxyuridine-positive chondrocytes in old mice was significantly higher in the Y/O group than in the other groups (P < 0.05). Type II collagen (CII) and SOX9 messenger RNA levels differed in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). RUNX-2, CX, and matrix metalloproteinase 13 levels were significantly lower in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). Similar results were obtained for protein expression levels and after GDF-11 treatment in vitro and in vivo. Phosphorylated Smad2/3 (pSmad2/3) levels were higher in the recombinant GDF-11-treated group than in the control group. CONCLUSION: A young systemic environment promotes chondrocyte proliferation and cartilage matrix synthesis in old mice. GDF-11, a "young factor," contributes to these effects through the up-regulation of pSmad2/3.

The protein tyrosine kinase inhibitor, Genistein, delays intervertebral disc degeneration in rats by inhibiting the p38 pathway-mediated inflammatory response.

The treatment for intervertebral disc degeneration (IDD) has drawn great attention and recent studies have revealed that the p38 MAPK pathway is a potential therapeutic target for delaying the degeneration of intervertebral discs. In this study, we analyzed a nature-derived protein tyrosine kinase inhibitor, Genistein, and its function in delaying IDD in rats both in vitro and in vivo via the p38 MAPK pathway. Nucleus pulposus cells treated with Genistein showed better function compared with untreated cells. Further study revealed that Genistein could play a protective role in IDD by inhibiting phosphorylation of p38, consequently inhibiting the p38 pathway-mediated inflammatory response. The rat IDD model also demonstrated that Genistein could effectively delay the degeneration of intervertebral disc tissue. The current study reveals new biological functions of Genistein, further demonstrates the effects of the p38 MAPK pathway on intervertebral disc degeneration, and deepens our understanding of the treatment and prevention of IDD.

Comparison between the effect of kartogenin and TGFß3 on chondrogenesis of human adipose- derived stem cells in fibrin scaffold.

BACKGROUND: Due to very sluggish turnover at the molecular and cellular level, the healing of chondral damages has been considered difficult. In the current study, the effects of the Kartogenin, a small heterocyclic molecule on chondrogenic differentiation of stem cells was compared to TGF-ß3. METHODS: Human Adipose-Derived Stem Cells were extracted during an elective surgery. Cell viability was estimated by MTT assay, differentiated cells evaluated by histological and immunohistochemical techniques. Expression of cartilage specific genes (SOX9, Aggrecan, type II and X collagens) assessed by real-time PCR. RESULTS: The real-time PCR assay has revealed the expression of gene marker of chondrogenesis, SOX9, Aggrecan and type II collagen, both in Kartogenin and TGFß3 groups compared to the control group, significantly (p < 0.05). A low expression level of collagen type X as a hypertrophic marker was seen in cartilage produced by using Kartogenin. Meanwhile, the level of type X collagen protein in Kartogenin group was significantly decreased (p > 0.05) compared to TGF-ß3 group. CONCLUSION: Kartogenin was suitable for successful chondrogenic differentiation of human adipose- derived stem cells and a suppressor of the consequent hypertrophy (Tab. 1, Fig. 5, Ref. 31).

TLR signalling can modify the mineralization of tooth germ.

OBJECTIVE: The aim of this work is to investigate the possible role of Toll-like receptor 4 (TLR4) during the development of mouse tooth germ. TLR4 is well known to inhibit mineralization and cause inflammation in mature odontoblasts and dental pulp cells. However, unlike these pathological functions of TLR4, little is known about the developmental function(s) of TLR4 during tooth development. MATERIALS AND METHODS: TLR4 expression was studied via Western blot in developing lower mouse incisors from E13.5 to E18.5. To generate functional data about the effects of TLR4, a specific agonist (LPS) was applied to the medium of in vitro tooth germ cultures, followed by Western blot, histochemical staining, ELISA assay, in situ hybridization and RT-qPCR. RESULTS: Increased accumulation of biotin-labelled LPS was detected in the enamel organ and in preodontoblasts. LPS treatment induced degradation of the inhibitor molecule (IκB) of the NF-κB signalling pathway. However, no morphological alterations were detected in cultured tissue after LPS addition at the applied dosage. Activation of TLR4 inhibited the mineralization of enamel and dentin, as demonstrated by alizarin red staining and as decreased levels of collagen type X. mRNA expression of ameloblastin was elevated after LPS administration. CONCLUSION: These results demonstrate that TLR4 may decrease the mineralization of hard tissues of the tooth germ and may trigger the maturation of ameloblasts; it can give valuable information to understand better congenital tooth abnormalities.

Restriction of spontaneous and prednisolone-induced leptin production to dedifferentiated state in human hip OA chondrocytes: role of Smad1 and ß-catenin activation.

OBJECTIVE: The aetiology of OA is not fully understood although several adipokines such as leptin are known mediators of disease progression. Since leptin levels were increased in synovial fluid compared to serum in OA patients, it was suggested that joint cells themselves could produce leptin. However, exact mechanisms underlying leptin production by chondrocytes are poorly understood. Nevertheless, prednisolone, although displaying powerful anti-inflammatory properties has been recently reported to be potent stimulator of leptin and its receptor in OA synovial fibroblasts. Therefore, we investigated, in vitro, spontaneous and prednisolone-induced leptin production in OA chondrocytes, focusing on transforming growth factor-ß (TGFß) and Wnt/ß-catenin pathways. DESIGN: We used an in vitro dedifferentiation model, comparing human freshly isolated hip OA chondrocytes cultivated in monolayer during 1 day (type II, COL2A1 +; type X, COL10A1 + and type I collagen, COL1A1 -) or 14 days (COL2A1 -; COL10A1 - and COL1A1+). RESULTS: Leptin expression was not detected in day1 OA chondrocytes whereas day14 OA chondrocytes produced leptin, significantly increased with prednisolone. Activin receptor-like kinase 1 (ALK1)/ALK5 ratio was shifted during dedifferentiation, from high ALK5 and phospho (p)-Smad2 expression at day1 to high ALK1, endoglin and p-Smad1/5 expression at day14. Moreover, inactive glycogen synthase kinase 3 (GSK3) and active ß-catenin were only found in dedifferentiated OA chondrocytes. Smad1 and ß-catenin but not endoglin stable lentiviral silencing led to a significant decrease in leptin production by dedifferentiated OA chondrocytes. CONCLUSIONS: Only dedifferentiated OA chondrocytes produced leptin. Prednisolone markedly enhanced leptin production, which involved Smad1 and ß-catenin activation.

Insulin-Like Growth Factor-Independent Effects of Growth Hormone on Growth Plate Chondrogenesis and Longitudinal Bone Growth.

GH stimulates growth plate chondrogenesis and longitudinal bone growth directly at the growth plate. However, it is not clear yet whether these effects are entirely mediated by the local expression and action of IGF-1 and IGF-2. To determine whether GH has any IGF-independent growth-promoting effects, we generated (TamCart)Igf1r(flox/flox) mice. The systemic injection of tamoxifen in these mice postnatally resulted in the excision of the IGF-1 receptor (Igf1r) gene exclusively in the growth plate. (TamCart)Igf1r(flox/flox) tamoxifen-treated mice [knockout (KO) mice] and their Igf1r(flox/flox) control littermates (C mice) were injected for 4 weeks with GH. At the end of the 4-week period, the tibial growth and growth plate height of GH-treated KO mice were greater than those of untreated C or untreated KO mice. The systemic injection of GH increased the phosphorylation of Janus kinase 2 and signal transducer and activator of transcription 5B in the tibial growth plate of the C and KO mice. In addition, GH increased the mRNA expression of bone morphogenetic protein-2 and the mRNA expression and protein phosphorylation of nuclear factor-κB p65 in both C and KO mice. In cultured chondrocytes transfected with Igf1r small interfering RNA, the addition of GH in the culture medium significantly induced thymidine incorporation and collagen X mRNA expression. In conclusion, our findings demonstrate that GH can promote growth plate chondrogenesis and longitudinal bone growth directly at the growth plate, even when the local effects of IGF-1 and IGF-2 are prevented. Further studies are warranted to elucidate the intracellular molecular mechanisms mediating the IGF-independent, growth-promoting GH effects.

Muscle cell-derived factors inhibit inflammatory stimuli-induced damage in hMSC-derived chondrocytes.

OBJECTIVE: Pro-inflammatory cytokines play an important role in inducing cartilage degradation during osteoarthritis pathogenesis. Muscle is a tissue that lies near cartilage in situ. However, muscle's non-loading biochemical effect on cartilage has been largely unexplored. Here, we tested the hypothesis that muscle cells can regulate the response to pro-inflammatory cytokine-mediated damage in chondrocytes derived from human bone marrow-derived mesenchymal stem cells (hMSCs). METHOD: hMSCs were allowed to undergo chondrogenic differentiation in porous silk scaffolds in the typical chondrogenic medium for 12 days. For the next 9 days, the cells were cultured in chondrogenic medium containing 50% conditioned medium derived from C2C12 muscle cells or fibroblast control cells, and were subject to treatments of pro-inflammatory cytokines IL-1ß or TNFα. RESULTS: Both IL-1ß and TNFα-induced strong expression of multiple MMPs and hypertrophic markers Runx2 and type X collagen. Strikingly, culturing hMSC-derived chondrocytes in C2C12 muscle cell-conditioned medium strongly inhibited the expression of all these genes, a result further confirmed by GAG content and histological evaluation of matrix protein. To determine whether these effects were due to altered chondrocyte growth and survival, we assayed the expression of cell proliferation marker Ki67, cell cycle arrest markers p21 and p53, and apoptosis marker caspase 3. Muscle cell-conditioned medium promoted proliferation and inhibited apoptosis, thereby suggesting a possible decrease in the cellular aging and death that typically accompanies cartilage inflammation. CONCLUSION: Our findings suggest the role of muscle in cartilage homeostasis and provide insight into designing strategies for promoting resistance to pro-inflammatory cytokines in hMSC-derived chondrocytes.

Hydrostatic pressure acts to stabilise a chondrogenic phenotype in porcine joint tissue derived stem cells.

Hydrostatic pressure (HP) is a key component of the in vivo joint environment and has been shown to enhance chondrogenesis of stem cells. The objective of this study was to investigate the interaction between HP and TGF-ß3 on both the initiation and maintenance of a chondrogenic phenotype for joint tissue derived stem cells. Pellets generated from porcine chondrocytes (CCs), synovial membrane derived stem cells (SDSCs) and infrapatellar fat pad derived stem cells (FPSCs) were subjected to 10 MPa of cyclic HP (4 h/day) and different concentrations of TGF-ß3 (0, 1 and 10 ng/mL) for 14 days. CCs and stem cells were observed to respond differentially to both HP and TGF-ß3 stimulation. HP in the absence of TGF-ß3 did not induce robust chondrogenic differentiation of stem cells. At low concentrations of TGF-ß3 (1 ng/mL), HP acted to enhance chondrogenesis of both SDSCs and FPSCs, as evident by a 3-fold increase in Sox9 expression and a significant increase in glycosaminoglycan accumulation. In contrast, HP had no effect on cartilage-specific matrix synthesis at higher concentrations of TGF-ß3 (10 ng/mL). Critically, HP appears to play a key role in the maintenance of a chondrogenic phenotype, as evident by a down-regulation of the hypertrophic markers type X collagen and Indian hedgehog in SDSCs irrespective of the cytokine concentration. In the context of stem cell based therapies for cartilage repair, this study demonstrates the importance of considering how joint specific environmental factors interact to regulate not only the initiation of chondrogenesis, but also the development of a stable hyaline-like repair tissue.

Gene expression profiling concerning mineralization in human dental pulp cells treated with mineral trioxide aggregate.

INTRODUCTION: This study investigated changes in gene expressions related to mineralization when mineral trioxide aggregate (MTA) is applied in vitro to human dental pulp cells (HDPCs). METHODS: MTA in a Teflon tube (diameter 10 mm, height 2 mm) was applied to HDPCs. Empty tube-applied HDPCs were used as negative control. Total RNA was extracted at 6, 24, and 72 hours after MTA application for microarray analysis. The results were confirmed selectively by performing reverse transcriptase-polymerase chain reaction for genes that showed changes of more than 2-fold or less than half. RESULTS: Of the 24,546 genes, 109 genes were up-regulated more than 2-fold (eg, THBS1, VCAN, BHLHB2, FN1, COL10A1, TUFT1, and HMOX1), and 69 genes were down-regulated below 50% (eg, DCN, SOCS2, and IL8). CONCLUSIONS: These results suggest that rather than being a bio-inert material, MTA affects pulp cells in various ways. MTA appears to affect mineralization and induces slight inflammation and protective role against slight inflammation.

Chloride channels regulate chondrogenesis in chicken mandibular mesenchymal cells.

Voltage gated chloride channels (ClCs) play an important role in the regulation of intracellular pH and cell volume homeostasis. Mutations of these genes result in genetic diseases with abnormal bone deformation and body size, indicating that ClCs may have a role in chondrogenesis. In the present study, we isolated chicken mandibular mesenchymal cells (CMMC) from Hamburg-Hamilton (HH) stage 26 chick embryos and induced chondrocyte maturation by using ascorbic acid and ß-glycerophosphate (AA-BGP). We also determined the effect of the chloride channel inhibitor NPPB [5-nitro-2-(3-phenylpropylamino) benzoic acid] on regulation of growth, differentiation, and gene expression in these cells using MTT and real-time PCR assays. We found that CLCN1 and CLCN3-7 mRNA were expressed in CMMC and NPPB reduced expression of CLCN3, CLCN5, and CLCN7 mRNA in these cells. At the same time, NPPB inhibited the growth of the CMMC, but had no effect on the mRNA level of cyclin D1 and cyclin E (P>0.05) with/without AA-BGP treatment. AA-BGP increased markers for early chondrocyte differentiation including type II collagen, aggrecan (P<0.01) and Sox9 (P<0.05), whilst had no effect on the late chondrocyte differentiation marker type X collagen. NPPB antagonized AA-BGP-induced expression of type II collagen and aggrecan (P<0.05). Furthermore, NPPB downregulated type X collagen (P<0.05) with/without AA-BGP treatment. We conclude that abundant chloride channel genes in CMMC play important roles in regulating chondrocyte proliferation and differentiation. Type X collagen might function as a target of chloride channel inhibitors during the differentiation process.

Regulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases by basic fibroblast growth factor and dexamethasone in periodontal ligament cells.

BACKGROUND AND OBJECTIVES: In this study, we investigated the effect of basic fibroblast growth factor (bFGF) and dexamethasone (Dex) on mRNA expressions of collagen (COL) type I, III and X, matrix metalloproteinases (MMP)-1, -2, -3 and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2, and also on mineralization and morphology of periodontal ligament (PDL) cells. MATERIAL AND METHODS: Periodontal ligament cells were obtained from premolar teeth extracted for orthodontic reasons. Periodontal ligament cells were cultured with Dulbecco's modified Eagle's medium containing: (1) 5% fetal bovine serum (FBS); (2) 5% FBS + ascorbic acid (AA, 50 microg/mL); (3) 5% FBS + Dex (10(-7) m) + AA; (4) 5% FBS + bFGF (10 ng/mL) + AA; or (5) 5% FBS + Dex (10(-7) m) + bFGF + AA. Cells within each group were evaluated for gene expression profile using semi-quantitative reverse transcriptase-polymerase chain reaction for COL I, III and X, MMP-1, -2, -3 and -9 and TIMP-1 and -2 on days 14 and 21 and for biomineralization by von Kossa stain in vitro on day 21. Images of PDL cells were examined using a phase contrast microscope. RESULTS: Basic fibroblast growth factor stimulated MMP-1, MMP-3 and MMP-9 mRNA expressions and inhibited TIMP-2 mRNA expression. Treatment of cells with Dex + bFGF led to downregulation of MMP-1, MMP-3 and MMP-9 transcripts. Whilst AA alone and Dex alone induced biomineralization of PDL cells, bFGF blocked the mineralization activity of the cells. In the Dex + bFGF group, more mineral nodules were noted when compared to AA alone and Dex alone groups. CONCLUSION: The addition of Dex to culture reversed bFGF-mediated inhibition of mineralization. Use of combined bFGF and Dex to regulate PDL cell function may be a good therapeutic option to obtain periodontal regeneration.

Retinoids directly activate the collagen X promoter in prehypertrophic chondrocytes through a distal retinoic acid response element.

Retinoids are essential for the terminal differentiation of chondrocytes during endochondral bone formation. This maturation process is characterized by increased cell size, expression of a unique extracellular matrix protein, collagen X, and eventually by mineralization of the matrix. Retinoids stimulate chondrocyte maturation in cultured cells and experimental animals, as well as in clinical studies of synthetic retinoids; furthermore, retinoid antagonists prevent chondrocyte maturation in vivo. However, the mechanisms by which retinoids regulate this process are poorly understood. We and others showed previously that retinoic acid (RA) stimulates expression of genes encoding bone morphogenetic proteins (BMPs), suggesting that retinoid effects on chondrocyte maturation may be indirect. However, we now show that RA also directly stimulates transcription of the collagen X gene promoter. We have identified three RA response element (RARE) half-sites in the promoter, located 2,600 nucleotides upstream from the transcription start site. These three half-sites function as two overlapping RAREs that share the middle half-site. Ablation of the middle half-site destroys both elements, abolishing RA receptor (RAR) binding and drastically decreasing RA stimulation of transcription. Ablation of each of the other two half-sites destroys only one RARE, resulting in an intermediate level of RAR binding and transcriptional stimulation. These results, together with our previously published data, indicate that retinoids stimulate collagen X transcription both directly, through activation of RARs, and indirectly, through increased BMP production.

Effects of retinoic acid on the differentiation of chondrogenic progenitor cells, ATDC5.

Chondrocyte differentiation is a fundamental process during endochondral ossification. Retinoic acid (RA) has been shown to regulate this process, however, the mechanisms underlying RA regulation of chondrogenesis are not clearly understood. Chondroprogenitor cells, ATDC5 have been shown to be a useful in vitro model for examining the multiple step differentiation of chondrocytes. The present study investigated the mechanisms underlying RA regulation of chondrogenesis using ATDC5 cell culture. In this study, we show that RA suppresses the cell growth, cartilage nodule formation, accumulation of proteoglycan, alkaline phosphatase (ALPase) activity and mineralization and that RA dose dependently upregulates the levels of type X collagen and matrix metalloproteinase-13 (MMP-13) mRNA which are marker proteins of hypertrophic chondrocytes, in ATDC5 cells. The addition of protein synthesis inhibitor, cycloheximide (CHX), partially inhibits the induction of type X collagen and MMP-13 mRNA by RA. In this system, RA upregulates the mRNA level of Runx2/Cbfa1 (type II), a positive regulator for mineralization, and downregulates the mRNA of Indian hedgehog (Ihh), parathyroid hormone related protein (PTHrP), negative regulators for terminal differentiation. However, RA downregulates ALPase, bone gla protein (BGP) mRNAs and mineralization. These data indicate that RA stimulates cartilage differentiation, however, cell condensation and cartilage nodule formation may be candidates of primary importance in the terminal differentiation of chondrocytes.

PGE2 inhibits chondrocyte differentiation through PKA and PKC signaling.

Prostaglandins are ubiquitous metabolites of arachidonic acid, and cyclooxygenase inhibitors prevent their production and secretion. Animals with loss of cyclooxygenase-2 function have reduced reparative bone formation, but the role of prostaglandins during endochondral bone formation is not defined. The role of PGE2 as a regulator of chondrocyte differentiation in chick growth plate chondrocytes (GPCs) was examined. While PGE2, PGD2, PGF2alpha, and PGJ2 all inhibited colX expression, approximately 80% at 10(-6) M, PGE2 was the most potent activator of cAMP response element (CRE)-mediated transcription. PGE2 dose-dependently inhibited the expression of the differentiation-related genes, colX, VEGF, MMP-13, and alkaline phosphatase gene, and enzyme activity with significant effects at concentrations as low as 10(-10) M. PGE2 induced cyclic AMP response element binding protein (CREB) phosphorylation and increased c-Fos protein levels by 5 min, and activated transcription at CRE-Luc, AP-1-Luc, and c-Fos promoter constructs. The protein kinase A (PKA) inhibitor, H-89, completely blocked PGE2-mediated induction of CRE-Luc and c-Fos promoter-Luc promoters, and partially inhibited induction of AP-1-Luc, while the protein kinase C (PKC) inhibitor Go-6976 partially inhibited all three promoters, demonstrating substantial cross-talk between these signaling pathways. PGE2 inhibition of colX gene expression was dependent upon both PKA and PKC signaling. These observations demonstrate potent prostaglandin regulatory effects on chondrocyte maturation and show a role for both PKA and PKC signaling in PGE2 regulatory events.

Parathyroid hormone-related peptide (PTHrP) inhibits Runx2 expression through the PKA signaling pathway.

The bone-related transcription factor Runx2 (Cbfa1) has been extensively shown to regulate osteoblast differentiation and function. Recent studies demonstrate that Runx2 is also a positive regulator of chondrocyte maturation and vascular invasion in cartilage. Runx2 activity can be modulated in several ways, including direct stimulation of gene expression, post-translational modification, and protein-protein interactions. We have previously reported cooperative effects between BMP and RA downstream signaling involving Smad proteins and Runx2. Furthermore, our previous studies showed that PTHrP inhibits chondrocyte maturation primarily through CREB and AP-1 signaling pathways. In the present study, we investigated the effect of PTHrP on Runx2 expression in chick upper sternal chondrocytes (USCs). We further determined the signaling pathways through which PTHrP regulates Runx2 transcription. Our results show that PTHrP inhibits Runx2 expression at both the mRNA and protein levels concomitant with a PTHrP-mediated suppression of the phenotypic marker of hypertrophy, type X collagen. We further determined potential signaling pathways through which PTHrP inhibits Runx2 expression using protein kinase inhibitors, H89 (PKA inhibitor): Go-6976 (PKC inhibitor): SB203850 (p38 MAPK inhibitor), and U0126 (MEK inhibitor). We show that pretreatment with PKA and, to a lesser extent, PKC inhibitors significantly blocked PTHrP suppression of Runx2, while p38 MAPK and MEK inhibitors had no significant effect. Furthermore, PTHrP suppression of Runx2 mRNA was partially blocked in USCs infected with RCAS-A-CREB, a dominant negative reagent that abrogates CREB activity. Overall, our results demonstrate that PTHrP downregulates Runx2 expression primarily through the PKA signaling pathway.

Action of estradiol on epiphyseal growth plate chondrocytes.

Estrogen plays an important role in the human growth plate by accelerating growth and promoting epiphyseal fusion in both sexes. Nevertheless, the precise mechanisms responsible for these effects are poorly understood. In the present study, we examined the role of 17beta-estradiol (E2) on cell proliferation and viability, type X collagen synthesis, alkaline phosphatase activity, and matrix calcification in primary cultures of resting, proliferating, and prehypertrophic chondrocytes derived from explants of the bovine fetal epiphyseal growth plate. Growth plate chondrocytes were isolated and separated into maturationally distinct subpopulations, which were cultured for 7-21 days to high density in either (1) serum-free medium, (2) 1 nM thyroid hormone (T3), (3) E2 concentrations ranging from 10(-13) M to 10(-7) M, or (4) a combination of T3 and E2. To compare E2 effects in both sexes, chondrocytes were harvested from 8 fetuses of both sexes. After hormone treatment, cell cultures were analyzed for cell number and viability, collagen type X, alkaline phosphatase (ALP), and matrix calcification. Neither DNA content nor cell viability were affected by the duration or type of hormone treatment. By itself, E2 stimulated maturation of all subpopulations only in pharmacologic doses (10(-7) M). Physiologic E2 concentrations were no different than negative controls treated with ITS (insulin, transferrin, and selenite). Regardless of E2 concentrations, the addition of E2 to 1 nM T3 did not appreciably affect the response to T3 alone, which stimulates maturation of the phenotype. All effects were comparable in both male and female chondrocytes, in all cell subpopulations (maturation stages) and fetuses of varying gestational age. These findings indicate that at physiologic concentrations, the effects of E2 on fetal bovine growth plate chondrocyte appear to be indirect and independent of T3, suggesting that, in vivo, E2 acts in concert with other factors or hormones to induce fusion of the growth plate.

Recombinant human bone morphogenetic protein-2 promotes Indian hedgehog-mediated osteo-chondrogenic differentiation of a human chondrocytic cell line in vivo and in vitro.

We examined osteo-chondrogenic differentiation of a human chondrocytic cell line (USAC) by rhBMP-2 in vivo and in vitro. USAC was established from a transplanted tumor to athymic mouse derived from an osteogenic sarcoma of the mandible. USAC usually shows chondrocytic phenotypes in vivo and in vitro. rhBMP-2 up-regulated not only the mRNA expression of types II and X collagen, but also the mRNA expression of osteocalcin and Cbfa1 in USAC cells in vitro. In vivo experimental cartilaginous tissue formation was prominent in the chamber with rhBMP-2 when compared with the chamber without rhBMP-2. USAC cells implanted with rhBMP-2 often formed osteoid-like tissues surrounded by osteoblastic cells positive for type I collagen. rhBMP up-regulated Ihh, and the expression of Ihh was well correlated with osteo-chondrogenic cell differentiation. These results suggest that rhBMP-2 promotes chondrogenesis and also induces osteogenic differentiation of USAC cells in vivo and in vitro through up-regulation of Ihh.

CREB Cooperates with BMP-stimulated Smad signaling to enhance transcription of the Smad6 promoter.

Growth plate chondrocytes integrate a multitude of growth factor signals during maturation. PTHrP inhibits maturation through stimulation of PKA/CREB signaling while the bone morphogenetic proteins (BMPs) stimulate maturation through Smad mediated signaling. In this manuscript, we show that interactions between CREB and the BMP associated Smads are promoter specific, and demonstrate for the first time the requirement of CREB signaling for Smad mediated activation of a BMP responsive region of the Smad6 promoter. The 28 base pairs (bp) BMP responsive element of the Smad6 promoter contains an 11 bp Smad binding region and an adjacent 17 bp region in which we characterize a putative CRE site. PKA/CREB gain of function enhanced BMP stimulation of this reporter, while loss of CREB function diminished transcriptional activity. In contrast, ATF-2 and AP-1 transcription factors had minimal effects. Electrophoretic mobility shift assay (EMSA) confirmed CREB binding to the Smad6 promoter element. Mutations eliminating binding resulted in loss of transcriptional activity, while mutations that maintained CREB binding had continued reporter activation by CREB and BMP-2. The Smad6 gene was similarly regulated by CREB. Dominant negative CREB reduced BMP-2 stimulated Smad6 gene transcription by 50%, but markedly increased BMP-2 mediated stimulation of colX and Ihh expression. In contrast, PTHrP which activates CREB signaling, blocked the stimulatory effect of BMP-2 on colX and Ihh, but minimally inhibited the stimulatory effect of BMP on Smad6. These findings are the first to demonstrate a cooperative association between CREB and BMP regulated Smads in cells from vertebrates and demonstrate that promoter-specific rather than generalized interactions between PKA/CREB and BMP signaling regulate gene expression in chondrocytes.