The transcriptional activation by SRY-type high mobility group box 9 (SOX9) and the transforming growth factor ␤ (TGF-␤) signals are necessary for chondrogenic differentiation. We have previously shown that CREB-binding protein (CBP/p300) act as an important SOX9 co-activator during chondrogenesis. In the present study, we investigated the relationship between TGF-␤-dependent Smad2/3 signaling pathways and the SOX9-CBP/p300 transcriptional complex at the early stage of chondrogenesis. Overexpressed Smad3 strongly induced the primary chondrogenesis of human mesenchymal stem cells. In addition, Smad3 enhanced the transcriptional activity of SOX9 and the expression of ␣1(II) collagen gene (COL2A1), and small interference RNA against Smad3 (si-Smad3) inhibited them. We observed that Smad2/3 associated with Sox9 in a TGF-␤-dependent manner and formed the transcriptional complexes with SOX9 on the enhancer region of COL2A1. Interestingly, the association between Sox9 and CBP/p300 was increased by Smad3 overexpression and was suppressed by si-Smad3. Our findings indicate that Smad3 has a stronger potential to stimulate the SOX9-dependent transcriptional activity by modulating the interaction between SOX9 and CBP/ p300, rather than Smad2. This study suggests that the Smad3 pathway presents a key role for the SOX9-dependent transcriptional activation in primary chondrogenesis.

Objective. To examine whether depsipeptide (FK228), a histone deacetylase (HDA) inhibitor, has inhibitory effects on the proliferation of synovial fibroblasts from rheumatoid arthritis (RA) patients, and to examine the effects of systemic administration of FK228 in an animal model of arthritis.Methods. Autoantibody-mediated arthritis (AMA) was induced in 19 male DBA/1 mice (6-7 weeks old); 10 of them were treated by intravenous administration of FK228 (2.5 mg/kg), and 9 were used as controls. The effects of FK228 were examined by radiographic, histologic, and immunohistochemical analyses and arthritis scores. RA synovial fibroblasts (RASFs) were obtained at the time of joint replacement surgery. In vitro effects of FK228 on cell proliferation were assessed by MTT assay. Cell morphology was examined by light and transmission electron microscopy. The effects on the expression of the cell cycle regulators p16INK4a and p21 WAF1/Cip1 were examined by real-time polymerase chain reaction and Western blot analysis. The acetylation status of the promoter regions of p16INK4a and p21 WAF1/Cip1 were determined by chromatin immunoprecipitation assay.Results. A single intravenous injection of FK228 (2.5 mg/ml) successfully inhibited joint swelling, synovial inflammation, and subsequent bone and cartilage destruction in mice with AMA. FK228 treatment induced histone hyperacetylation in the synovial cells and decreased the levels of tumor necrosis factor ␣ and interleukin-1␤ in the synovial tissues of mice with AMA. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia, with excessive inflammatory cell infiltration in the joints, leading to erosion of the articular cartilage and marginal bone, with subsequent joint destruction (1). Despite an explosion of information over the last 2 decades, a detailed understanding of the mechanisms of synovial hyperplasia and inflammation is lacking. Recent reports have implicated rapid proliferation of synovial cells, overexpression of inflammatory genes, and impairment of apoptosis, which can allow the persistence of abnormal cells (2-4), in the disease process.Cyclin-dependent kinases (CDKs) are essential Dr.

Chromatin structure is a fundamental component of gene regulation, expression, and cellular differentiation. We have previously reported that the multifunctional coactivator p300 is a member of the Sox9 (Sry-type high mobility group box 9)-related transcriptional apparatus and activates Sox9-dependent transcription during chondrogenesis. However, the mechanism of synergy between Sox9 and p300 in chromatin-mediated transcription has not been elucidated. In the present study we investigated the activity of Sox9 and p300 on chromatinized templates in vitro. Recombinant Sox9 was shown to be associated with several transcriptional cofactors including p300. In vitro transcription assays revealed that p300 potentiated Sox9-dependent transcription on chromatinized DNA and, importantly, was associated with hyperacetylated histones. Consistent with these results, the histone deacetylase inhibitor trichostatin A stimulated the expression of Sox9-regulated cartilage matrix genes and induced histone acetylation around the enhancer region of the collagen ␣1 (II) gene in chondrocytes. These findings suggest that Sox9 interacts with chromatin and activates transcription via regulation of chromatin modification.Chondrocyte differentiation from mesenchymal stem cells (MSCs) 2 to hypertrophic chondrocytes is a dynamic process in endochondral bone formation. The sequential differentiation and maturation steps of chondrocytes are regulated by several transcription factors such as Sox5/6/9 and runt-related gene Runx2 (1-3). Sox9 (Sry-type high mobility group box 9) plays an essential role in establishing the condensation of prechondrogenic mesenchymal cells and initiating chondrocyte differentiation (1, 2). Mutations that inactivate SOX9 cause the skeletal malformation syndrome campomelic dysplasia (4, 5). Expression of the ␣1 chain of type II collagen (Col2a1), a major component of the cartilage extracellular matrix, is regulated by Sox9 through the Sox9-binding site on the Col2a1 enhancer region (6) However, Sox9-dependent transcriptional regulation in chondrogenesis seems to require other additional cofactors (7). We have previously demonstrated that p300/ CREB-binding protein (CBP), peroxisome proliferator-activated receptor-␥ (PPAR-␥) coactivator-1␣ (PGC-1␣), and Smad3 associate with Sox9 and enhance the transcriptional activity of Sox9 during chondrogenesis (8 -10). However, little is known about the mechanism of Sox9-driven activation.The eukaryotic genome is packaged into chromatin in which DNA wraps around the surface of a histone octamer (two sets of histone H2A, H2B, H3, and H4) and forms the nucleosome-repeated structure (11-13). The chromatin structure controls gene expression and other fundamental cellular processes (11-13) by inhibiting the access of regulatory factors to DNA. These inhibitory effects can be reversed by the activity of chromatin remodeling factors.In vitro chromatin assembly and transcription assays have been very useful in elucidating the effects of chromatin structure on gene expression. Several prot...

RUNX-2 might have a role as a key downstream mediator of p38's ability to regulate mechanical stress-induced MMP-13 and ADAMTS-5 expression.

Chondrogenesis is a multistep pathway in which multipotential mesenchymal stem cells (MSC) differentiate into chondrocytes. The transcription factor Sox9 (SRY-related high mobility group-Box gene 9) regulates chondrocyte differentiation and cartilagespecific expression of genes, such as Col2a1 (collagen type II ␣1). However, Sox9 expression is detected not only in chondrogenic tissue but also in nonchondrogenic tissues, suggesting the existence of a molecular partner(s) required for Sox9 to control chondrogenesis and chondrogenic gene expression. Here, we report identification of peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣) as a coactivator for Sox9 during chondrogenesis. Expression of PGC-1␣ is induced at chondrogenesis sites during mouse embryonic limb development and during chondrogenesis in human MSC cultures. PGC-1␣ directly interacts with Sox9 and promotes Sox9-dependent transcriptional activity, suggesting that PGC-1␣ acts as a transcriptional coactivator for Sox9. Consistent with this finding, PGC-1␣ disruption in MSC by small interfering RNA inhibits Col2a1 expression during chondrogenesis. Furthermore, overexpression of both PGC-1␣ and Sox9 induced expression of chondrogenic genes, including Col2a1, followed by chondrogenesis in the MSC and developing chick limb. Together, our results suggest a transcriptional mechanism for chondrogenesis that is coordinated by PGC-1␣.cartilage ͉ mesenchymal stem cell ͉ peroxisome proliferator-activated receptor ␥ ͉ coactivator 1␣ ͉ limb development