Sox9 Family Members Negatively Regulate Maturation and Calcification of Chondrocytes through Up-Regulation of Parathyroid Hormone–related Protein

Abstract

Sox9 is a transcription factor that plays an essential role in chondrogenesis and has been proposed to inhibit the late stages of endochondral ossification. However, the molecular mechanisms underlying the regulation of chondrocyte maturation and calcification by Sox9 remain unknown. In this study, we attempted to clarify roles of Sox9 in the late stages of chondrocyte differentiation. We found that overexpression of Sox9 alone or Sox9 together with Sox5 and Sox6 (Sox5/6/9) inhibited the maturation and calcification of murine primary chondrocytes and up-regulated parathyroid hormone-related protein (PTHrP) expression in primary chondrocytes and the mesenchymal cell line C3H10T1/2. Sox5/6/9 stimulated the early stages of chondrocyte proliferation and development. In contrast, Sox5/6/9 inhibited maturation and calcification of chondrocytes in organ culture. The inhibitory effects of Sox5/6/9 were rescued by treating with anti-PTHrP antibody. Moreover, Sox5/6/9 bound to the promoter region of the PTHrP gene and up-regulated PTHrP gene promoter activity. Interestingly, we also found that the Sox9 family members functionally collaborated with Ihh/Gli2 signaling to regulate PTHrP expression and chondrocyte differentiation. Our results provide novel evidence that Sox9 family members mediate endochondral ossification by up-regulating PTHrP expression in association with Ihh/Gli2 signaling.

Potential of Human Fetal Chorionic Stem Cells for the Treatment of Osteogenesis Imperfecta

“…Downstream upregulation of the Sox9 transactivation target Col2a1 [39,40] was also confirmed in transplanted oim compared to oim controls (0.11 -0.01$2 -DCt vs. 0.04 -0.01$2 -DCt respectively, P < 0.05) and of the key cartilage matrix component aggrecan [41,42] (4.8 -0.8$2 -DCt vs. 1.1 -0.4$2 -DCt respectively, P < 0.01), found in proliferating chondrocytes. However, expression of the Sox9 target gene Pthrp, which inhibits chondrocyte maturation [43][44][45], was similar in transplanted and nontransplanted oim (5.2 · 10 -4 -0.7 · 10 -4 $2 -DCt vs. 4.8 · 10 -4 -0.5 · 10 -4 $2 -DCt ). Importantly, Smad3, which inhibits maturation of chondrocytes by mediating TGF-b signaling [46], was downregulated in e-CSC transplanted mice compared with oim controls (1.…”

The transcription factor Znf219 regulates chondrocyte differentiation by assembling a transcription factory with Sox9

“…The recombinant adenoviruses carrying a wild-type of Sox9, Sox5 and Sox6 or a mutant form of Znf219 were constructed by homologous recombination between the expression cosmid cassette (pAxCAwt) and the parental virus genome in 293 cells using an adenovirus construction kit (Takara, Tokyo, Japan), as previously described (Hata et al, 2008;Amano et al, 2009). The viruses showed no proliferative activity owing to a lack of E1A-E1B.…”

Indian Hedgehog Signaling Regulates Transcription and Expression of Collagen Type X via Runx2/Smads Interactions

“…Western Blotting-Preparation for cell lysates and Western blotting was performed as described previously (46). Primary antibodies were used with the following concentrations: rabbit or goat anti-Runx2 antibody (M-70 or S-19, 1:500 dilution, Santa Cruz Biotechnology, Inc.); mouse anti-Runx2 antibody (1:500 dilution, MBL International Corp.); rabbit anti-Col X antibody (1:200 dilution, Abcam); rabbit anti-Mef2c antibody (1:500 dilution, Cell Signaling); mouse anti-␤-actin antibody (1:2000 dilution, Sigma); mouse anti-Myc antibody (9E10, 1:500 dilution, Santa Cruz Biotechnology, Inc.); mouse anti-FLAG antibody (M2, 1:1000 dilution, Sigma); and rabbit antiGli1 antibody (H-300, 1:80 dilution, Santa Cruz Biotechnology, Inc.).…”