PPARgamma2 expression in growth plate chondrocytes is regulated by p38 and GSK-3.

Although peroxisome proliferator activated receptor (PPAR)gamma remains a critical regulator of preadipocyte differentiation, new roles have been discovered in inflammation, bone morphogenesis, endothelial function, cancer, longevity and atherosclerosis. Despite the demonstration of PPARgamma expression in chondrocytes, its role and the pathways affecting its expression and activity in chondrocytes remain largely unknown. We investigated the effects of PPARgamma activation on chondrocyte differentiation and its participation in chondrocyte lipid metabolism. PPARgamma2 expression is highly regulated during chondrocyte differentiation in vivo and in vitro PPARgamma activation with troglitazone resulted in increased Indian hedgehog expression and reduced collagen X expression, confirming previously described roles in the inhibition of differentiation. However, the major effect of PPARgamma2 in chondrocytes appears to be on lipid metabolism. During differentiation chondrocytes increase expression of the lipid-associated metabolizing protein, Lpl, which is accompanied by increased gene expression of PPARgamma. PPARgamma expression is suppressed by p38 activity, but requires GSK-3 activity. Furthermore, Lpl expression is regulated by p38 and GSK-3 signalling. This is the first study demonstrating a relationship between PPARgamma2 expression and chondrocyte lipid metabolism and its regulation by p38 and GSK-3 signalling.

Human stanniocalcin-1 or -2 expressed in mice reduces bone size and severely inhibits cranial intramembranous bone growth.

Stanniocalcin-1 (STC1) and -2 (STC2) are highly related, secreted, homodimeric glycoproteins that are significantly upregulated by different forms of stress including high phosphate levels. Transgenic mice that constitutively express either human STC1 or STC2 exhibit intra-uterine growth restriction and permanent post-natal growth retardation. STC1 is expressed in chondrocytic and osteoblastic cells during murine development and can enhance differentiation of calvarial cells in culture. Therefore, there is mounting evidence that stanniocalcins (STCs) modulate bone development in vivo. To further define the effects of stanniocalcins on skeletal development, we performed a series of measurements on components of the axial, appendicular, and cranial skeleton in transgenic and wildtype mice. We show that skeletal growth is retarded and that the intramembranous bones of the cranium exhibit a particularly severe delay in suture closure. The posterior frontal suture remains patent throughout the lifetime of human STC1 and STC2 transgenic mice. We did not observe significant effects on chondrogenesis: however, calvarial cells exhibited reduced viability, proliferation and delayed differentiation, indicating that developing osteoblasts are particularly sensitive to the levels of STCs. Given the evidence linking STC1 to cellular phosphate homeostasis, we assessed the expression of a variety of phosphate regulators in transgenic and wildtype calvarial cells and found significantly lower levels of Mepe, Dmp1, Sfrp4 in transgenic cells without a change in Pit1 or Pit2. Collectively these data support a direct regulatory role for STCs in osteoblasts and suggest that overexposure to these factors inhibits normal skeletal development without significant changes in patterning.

C-type natriuretic peptide regulates endochondral bone growth through p38 MAP kinase-dependent and -independent pathways.

BACKGROUND: C-type natriuretic peptide (CNP) has recently been identified as an important anabolic regulator of endochondral bone growth, but the molecular mechanisms mediating its effects are not completely understood. RESULTS: We demonstrate in a tibia organ culture system that pharmacological inhibition of p38 blocks the anabolic effects of CNP. We further show that CNP stimulates endochondral bone growth largely through expansion of the hypertrophic zone of the growth plate, while delaying mineralization. Both effects are reversed by p38 inhibition. We also performed Affymetrix microarray analyses on micro-dissected tibiae to identify CNP target genes. These studies confirmed that hypertrophic chondrocytes are the main targets of CNP signaling in the growth plate, since many more genes were regulated by CNP in this zone than in the others. While CNP receptors are expressed at similar levels in all three zones, cGMP-dependent kinases I and II, important transducers of CNP signaling, are expressed at much higher levels in hypertrophic cells than in other areas of the tibia, providing a potential explanation for the spatial distribution of CNP effects. In addition, our data show that CNP induces the expression of NPR3, a decoy receptor for natriuretic peptides, suggesting the existence of a feedback loop to limit CNP signaling. Finally, detailed analyses of our microarray data showed that CNP regulates numerous genes involved in BMP signaling and cell adhesion. CONCLUSION: Our data identify novel target genes of CNP and demonstrate that the p38 pathway is a novel, essential mediator of CNP effects on endochondral bone growth, with potential implications for understanding and treatment of numerous skeletal diseases.

p38 MAP kinase signaling is necessary for rat chondrosarcoma cell proliferation.

Chondrosarcomas represent the second most frequent class of primary skeletal malignancies. This tumor type is highly resistant to radiation therapy and currently available chemotherapies, thereby limiting treatment choice to surgical resection. Identifying the mechanisms responsible for chondrosarcoma cell proliferation is therefore crucial for the development of new treatment strategies. Here, we demonstrate a significant reduction in rat chondrosarcoma cell proliferation following treatment with pharmacological inhibitors (SB202190 and PD169316) of p38 mitogen-activated protein (MAP) kinases. In an attempt to dissect possible mechanisms, we investigated the effect of p38 inhibition on promoter activity of cell-cycle genes. Surprisingly, p38 inhibition resulted in upregulation of the activities of all three D-type cyclin promoters. In addition, p38 inhibitors induced increased transcription of the cell-cycle inhibitor p21(waf1/cip1). As expected, promoter activity of the cyclin A gene, which lies downstream of D-type cyclins and p21 in cell-cycle progression, was strongly reduced by p38 inhibitors. These effects were independent of a cyclic AMP response element and conferred by the proximal 150 nucleotides of the cyclin A promoter. Decreased transcription was accompanied by greatly reduced cyclin A protein levels upon p38 inhibition. These observations indicate complex regulation of chondrosarcoma cell-cycle progression by p38 signaling, and suggest that components of p38 MAP kinase pathways may be effective targets in the treatment of these tumors.

p38 MAP kinase signalling is required for hypertrophic chondrocyte differentiation.

Longitudinal growth of endochondral bones is accomplished through the co-ordinated proliferation and hypertrophic differentiation of growth plate chondrocytes. The molecular mechanisms and signalling cascades controlling these processes are not well understood. To analyse the expression and roles of p38 mitogen-activated protein kinases in this process, we have established a micromass system for the reproducible hypertrophic differentiation of mouse mesenchymal limb bud cells. Our results show that all four mammalian p38 kinase genes are expressed during the chondrogenic programme, as well as their upstream regulators MKK3 (mitogen-activated protein kinase kinase 3) and MKK6. Treatment of micromass cultures with pharmacological inhibitors of p38 results in a marked delay in hypertrophic differentiation in micromass cultures, indicating a requirement for p38 signalling in chondrocyte differentiation. Inhibition of p38 kinase activity leads to reduced and delayed induction of alkaline phosphatase activity and matrix mineralization. In addition, p38 inhibition causes reduced expression of hypertrophic marker genes such as collagen X, matrix metalloproteinase 13 and bone sialoprotein. The function of p38 in hypertrophic differentiation appears to be mediated, at least in part, by the transcription factor myocyte enhancer factor 2C. In summary, we have demonstrated a novel requirement for p38 signalling in hypertrophic differentiation of chondrocytes and identified myocyte enhancer factor 2C as an important regulator of chondrocyte gene expression.

Genome-wide analyses of gene expression during mouse endochondral ossification.

BACKGROUND: Endochondral ossification is a complex process involving a series of events that are initiated by the establishment of a chondrogenic template and culminate in its replacement through the coordinated activity of osteoblasts, osteoclasts and endothelial cells. Comprehensive analyses of in vivo gene expression profiles during these processes are essential to obtain a complete understanding of the regulatory mechanisms involved. METHODOLOGY/PRINCIPAL FINDINGS: To address these issues, we completed a microarray screen of three zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs both confirmed reported data and provided novel insights into endochondral ossification. Parallel comparisons of the microdissected tibiae data set with our previously completed micromass culture screen further corroborated the suitability of micromass cultures for modeling gene expression in chondrocyte development. The micromass culture system demonstrated striking similarities to the in vivo microdissected tibiae screen; however, the micromass system was unable to accurately distinguish gene expression differences in the hypertrophic and mineralized zones of the tibia. CONCLUSIONS/SIGNIFICANCE: These studies allow us to better understand gene expression patterns in the growth plate and endochondral bones and provide an important technical resource for comparison of gene expression in diseased or experimentally-manipulated cartilages. Ultimately, this work will help to define the genomic context in which genes are expressed in long bones and to understand physiological and pathological ossification.

Inhibition of p38 MAPK signaling in chondrocyte cultures results in enhanced osteogenic differentiation of perichondral cells.

Chondrocytes and osteoblasts originate from the same progenitor cell; however, both are characterized by distinct gene expression profiles once they are differentiated. Signals from differentiating chondrocytes, such as Indian hedgehog (Ihh), regulate the differentiation of osteoblast precursor cells. The MAPK pathways play important roles in controlling the differentiation of both chondrocytes and osteoblasts, with the p38 pathway being particularly relevant in skeletal cells. In the present study, we investigated the effects of p38 inhibition on osteoblastic marker gene expression in chondrocyte cultures. Using high-density micromass cultures of mesenchymal cells as well as chondrocytes that had differentiated in vivo and were maintained in short-term monolayer culture, we demonstrate elevated Runx2, Osterix and Osteocalcin transcript levels in chondrocyte cultures upon inhibition of p38 activity with the pharmacological inhibitor PD169316. Osteocalcin immunolocalization was restricted to perichondral/periosteal cells in micromass cultures, suggesting that inhibition of p38 results in increased periosteal osteogenesis. Coinciding with increased expression of these genes, we observed elevated levels of transcripts for Ihh and its target gene, Ptch, in response to p38 inhibition. Addition of recombinant hedgehog protein mimicked some effects of p38 inhibitors. We therefore suggest that p38 signaling regulates chondrocyte-perichondral cell communication during skeletal development, partially through increased Ihh signaling.

CCN2 is necessary for adhesive responses to transforming growth factor-beta1 in embryonic fibroblasts.

CCN2 is induced by transforming growth factor-beta (TGFbeta) in fibroblasts and is overexpressed in connective tissue disease. CCN2 has been proposed to be a downstream mediator of TGFbeta action in fibroblasts; however, the role of CCN2 in regulating this process unclear. By using embryonic fibroblasts isolated from ccn2-/- mice, we showed that CCN2 is required for a subset of responses to TGFbeta. Affymetrix genome-wide expression profiling revealed that 942 transcripts were induced by TGFbeta greater than 2-fold in ccn2+/+ fibroblasts, of which 345 were not induced in ccn2-/- fibroblasts, including pro-adhesive and matrix remodeling genes. Whereas TGFbeta properly induced a generic Smad3-responsive promoter in ccn2-/- fibroblasts, TGFbeta-induced activation of focal adhesion kinase (FAK) and Akt was reduced in ccn2-/- fibroblasts. Emphasizing the importance of FAK and Akt activation in CCN2-dependent transcriptional responses to TGFbeta in fibroblasts, CCN2-dependent transcripts were not induced by TGFbeta in fak-/- fibroblasts and were reduced by wortmannin in wild-type fibroblasts. Akt1 overexpression in ccn2-/- fibroblasts rescued the TGFbeta-induced transcription of CCN2-dependent mRNA. Finally, induction of TGFbeta-induced fibroblast adhesion to fibronectin and type I collagen was significantly diminished in ccn2-/- fibroblasts. Thus in embryonic fibroblasts, CCN2 is a necessary cofactor required for TGFbeta to activate the adhesive FAK/Akt/phosphatidylinositol 3-kinase cascade, FAK/Akt-dependent genes, and adhesion to matrix.

MAP kinases in chondrocyte differentiation.

The majority of the vertebrate skeleton develops through the process of endochondral ossification and involves successive steps of chondrogenesis, chondrocyte proliferation, and hypertrophic chondrocyte differentiation. Interruption of this program through gene mutations and hormonal or environmental factors contributes to numerous diseases, including growth disorders and chondrodysplasias. While a large number of growth factors and hormones have been implicated in the regulation of chondrocyte biology, relatively little is known about the intracellular signaling pathways involved. Recent data provide novel insights into the mechanisms governing acquisition of new phenotypes within the chondrogenic program and suggest multiple pivotal roles for members of the mitogen-activated protein kinase family and their downstream targets in cartilage development. These data are summarized and discussed here.

RhoA/ROCK signaling suppresses hypertrophic chondrocyte differentiation.

Coordinated proliferation and differentiation of growth plate chondrocytes is required for normal growth and development of the endochondral skeleton, but little is known about the intracellular signal transduction pathways regulating these processes. We have investigated the roles of the GTPase RhoA and its effector kinases ROCK1/2 in hypertrophic chondrocyte differentiation. RhoA, ROCK1, and ROCK2 are expressed throughout chondrogenic differentiation. RhoA overexpression in chondrogenic ATDC5 cells results in increased proliferation and a marked delay of hypertrophic differentiation, as shown by decreased induction of alkaline phosphatase activity, mineralization, and expression of the hypertrophic markers collagen X, bone sialoprotein, and matrix metalloproteinase 13. These effects are accompanied by activation of cyclin D1 transcription and repression of the collagen X promoter by RhoA. In contrast, inhibition of Rho/ROCK signaling by the pharmacological inhibitor Y27632 inhibits chondrocyte proliferation and accelerates hypertrophic differentiation. Dominant-negative RhoA also inhibits induction of the cyclin D1 promoter by parathyroid hormone-related peptide. Finally, Y27632 treatment partially rescues the effects of RhoA overexpression. In summary, we identify the RhoA/ROCK signaling pathway as a novel and important regulator of chondrocyte proliferation and differentiation.

Immunophenotyping of macrophages in human pulmonary tuberculosis and sarcoidosis.

Classic studies of tuberculosis (TB) revealed morphologic evidence of considerable heterogeneity of macrophages (MØs), but the functional significance of this heterogeneity remains unknown. We have used newly available specific antibodies for selected membrane and secretory molecules to examine the phenotype of MØs in situ in a range of South African patients with TB, compared with sarcoidosis. Patients were human immunodeficiency virus-negative adults and children, and the examined biopsy specimens included lung and lymph nodes. Mature pulmonary MØs (alveolar, interstitial, epithelioid and multinucleated giant cells) selectively expressed scavenger receptor type A and a novel carboxypeptidase-like antigen called carboxypeptidase-related vitellogenin-like MØ molecule (CPVL). CPVL did not display enhanced expression in sarcoidosis, vs. TB patients, as observed with angiotensin-converting enzyme (ACE), a related molecule. Immunocytochemical studies with surfactant proteins (SP)-A and -D showed that type II alveolar cells expressed these collectins, as did MØs, possibly after binding of secreted proteins. Studies with an antibody specific for the C-terminus of fractalkine, a tethered CX3C chemokine, confirmed synthesis of this molecule by bronchiolar epithelial cells and occasional endothelial cells. These studies provide new marker antigens and extend previous studies on MØ differentiation, activation and local interactions in chronic human granulomatous inflammation in the lung.