Figure 5: Wwp2 mediates formation of transcriptional complex with Sox9 and Med25 and regulates Sox9 transcriptional activity.

(a) Luciferase reporter assay using the p89/4×48 Col2a1 reporter and Col11a2 reporter plasmid in C3H10T1/2 cells. Wwp2-CA includes a mutation in the HECT domain of Wwp2. (mean ± s.d.; n=5). (b, c) Knockdown of Wwp2 (b) or Med25 (c) using siRNAs led to the downregulation of Col2a1 transcription in C3H10T1/2 cells detected by qRT–PCR. (mean ± s.d.; *P<0.05, **P<0.01, ***P<0.001; n=4). (d) Downregulation of Col2a1 induced by knockdown of Wwp2 or Med25 in C3H10T1/2 cells was not restored by exogenous Sox9 overexpression. (mean ± s.d.; Dunnett's test, **P<0.01; ***P<0.001; n=5). (e) EMSA using nuclear protein from C3H10T1/2 cells that were co-transfected with Flag-tagged Sox9, myc-tagged Wwp2 and HA-tagged Med25. The 48-bp Col2a1 enhancer oligonucleotide probe was used. Nuc-protein, nuclear protein. (f) ChIP assay detected recruitment of Sox9, Wwp2 and Med25 to the chondrocyte-specific enhancer region of the Col2a1 gene. Cell lysate extracted from C3H10T1/2 cells that were co-transfected with Flag-tagged Sox9, myc-tagged Wwp2 and HA-tagged Med25 was immunoprecipitated with antibodies for each tag. (g) Downregulation of Wwp2 led to the attenuation of the physical binding between Sox9 and Med25. The Wwp2 siRNA was initially transfected into ATDC5 cells and HA-tagged Med25 was additionally transfected 24 h after transfection with Wwp2 siRNA. Nuclear protein was immunoprecipitated with an anti-Sox9 antibody, followed by immunoblotting with an anti-HA antibody. (h) Model describing the mechanism underlying the physical and functional interaction between Sox9, Wwp2 and Med25. Sox9 regulates the transcription of Wwp2 and mediates the nuclear translocation of Wwp2, resulting in the formation of a transcriptional complex, including RNA polymerase II and Sox 5/6, which regulates the expression of the Col2a1 gene.