Fig. 7.

PITX2c and MYOCD act synergistically to promote VSMC differentiation. a Adenovirus encoding Pitx2c and Myocd were co-transduced into embryonic VSMCs. PITX2c, MYOCD and endogenously expressed SRF were then immunoprecipitated using anti-PITX2, anti-MYOCD and anti-SRF antibodies, respectively. Proteins within immuno-complexes were determined by Western blot using the indicated antibodies. A species-matched IgG served as control. b Schematic diagram of mouse MYOCD domains demonstrating the MYOCD GST fusion proteins analyzed in GST pull-down assays and a summary of MYOCD domains mapped to interact with PITX2c. NTD N-terminal domain, ++ basic domain, Q polyQ domain, SAP, SAF-A/B, Acinus, and PIAS domain, LZ leucine zipper domain, TAD transcriptional activation domain, NT N-terminus, CT C-terminus, N1-4 N-terminal mutants 1–4. c Bacterial expressed full-length MYOCD or/and PITX2c was incubated with SRF-GST fusion protein or with GST control protein. Western blotting was performed to detect the SRF-GST bound MYOCD or/and PITX2c (upper panel). The lower panel indicates the expression of the GST or SRF-GST fusion proteins (arrows). The middle membrane within the dashed lines was cropped for detecting PITX2c. SRF was found to bind to both MYOCD and PITX2c. d Bacterial expressed PITX2c was incubated with NT- or CT-MYOCD-GST fusion protein or control GST protein (arrows). Western blotting was performed to detect the MYOCD-GST bound PITX2c (upper panel) and revealed that PITX2C binds to NT-MYOCD. The middle membrane within the dashed lines was cropped for detecting PITX2c. e Using truncation mutants of NT-MYOCD (N1-4, arrows) and CT-MYOCD GST fusion proteins, GST pull-down assay further showed N1–3 domains of NT-MYOCD specifically binds to PITX2c. f Adenovirus encoding GFP, Pitx2c, or/and Myocd were transduced into embryonic VSMCs and expression of endogenous VSMC-specific gene was determined by Western blot. g The band intensity shown in (f) was measured, normalized to its loading control HSP90 and plotted. N = 4. *P < 0.05, vs. Ad-GFP control group (set to 1, red line); ^#P < 0.05, vs. Ad-Pitx2c or Ad-MYOCD group. Co-expression of MYOCD and Pitx2c in VSMCs synergistically induces the expression of contractile proteins. h WT or mutated CArG Lmod1 gene promoter-luciferase reporters were transfected into MEFs with or without co-expression of Pitx2c and MYOCD expression plasmid. Dual luciferase reporter assays were then performed to determine the combinatorial effects between Pitx2c and MYOCD on Lmod1 gene promoter activity. The promoter activity of the WT reporter at baseline without Pitx2c or MYOCD transfection was set to 1. N = 4–6. *P < 0.05. i Schematic diagram depicting the regulatory mechanisms by which TEAD1 mediates VSMC-specific gene expression during VSMC development. TEAD1 functions upstream of the genetic regulatory hierarchy by directly regulating the critical transcription factors, PITX2c and MYOCD, both of which act not only physically but also synergistically to promote the expression of VSMC-specific genes that are reflective of differentiated VSMCs