Fig. 7

ATF6 associates with Runx2 in chondrogenesis and ATF6 enhances Runx2-mediated chondrocyte hypertrophy. a ATF6 binds to Runx2 inhypertrophic chondrocyte differentiation of C3H10T1/2 cells treated with BMP2 5 days. Cells lysates prepared from micromass culture of C3H10T1/2 cells treated with BMP2 5 days were collected and were incubated with either control IgG (lane 3) or Runx2 antibodies (lane 2), followed by protein A-agarose. The immunoprecipitated (IP) protein complex and cell extracts (lane 1; serving as a positive control) were examined by Western blotting with anti-ATF6 antibody. The arrow is protein ATF6. b ATF6 binds to Runx2 in hypertrophic chondrocyte differentiation of C3H10T1/2 cells treated with BMP2 7 days. Cell lysates prepared from micromass culture of C3H10T1/2 cells treated with BMP2 7 days were collected and analyzed, respectively, as described in a. The arrow is protein ATF6. c ATF6 enhanced RUNX2-dependent Col X expression in C3H10T1/2 cells. Transcript levels of Col X were detected by real-time PCR analysis of RNA isolated from C3H10T1/2 cells infected with either Ad-GFP (control), Ad-RUNX2 (MOI = 50), or Ad-ATF6 + Ad-RUNX2, as indicated. *p < 0.05. d ATF6 enhanced RUNX2-dependent MMP13 expression in C3H10T1/2 cells. Transcript levels of MMP13 were detected by real-time PCR analysis of RNA isolated from C3H10T1/2 cells infected with Ad-GFP(control), Ad-RUNX2 (MOI = 50), or Ad-ATF6 + Ad-RUNX2, as indicated. RNA was extracted as described in a, and MMP13 mRNA was detected by real-time PCR. *p < 0.05. e siRUNX2 decreases BMP2-induced Col X expression stimulated by ATF6 in C3H10T1/2 cells. Transcript levels of Col X were detected by real-time PCR analysis of RNA isolated from micromass culture of C3H10T1/2 cells infected with either BMP2, BMP2 + Ad-ATF6(MOI = 50), or BMP2 + Ad-ATF6+ siRUNX2(MOI = 50), as indicated. *p < 0.05,**p < 0.01. f siRUNX2 decreases BMP2-induced MMP13 expression stimulated by ATF6 in C3H10T1/2 cells. Transcript levels of MMP13 were detected by real-time PCR analysis of RNA isolated from micromass culture of C3H10T1/2 cells infected with either BMP2, BMP2 + Ad-ATF6, or BMP2 + Ad-ATF6 + siRUNX2, as indicated. *p < 0.05,**p < 0.01. Error bars, SD