Abstract 612: The Iroquois Homeobox Protein Irx3 Induces Smooth Muscle Cell Differentiation in Pluripotent P19 Cells
Previous microarray studies conducted in the laboratory identified increased expression of Iroquois homeobox (Irx) genes during VSMC differentiation. We hypothesized that Irx3 is essential for VSMC differentiation in pluripotent cells in part, by regulating downstream target genes critical to this process. We conducted immunohistochemistry studies to determine if Irx3 was expressed in VSMCs in vivo. Our results indicate the presence of Irx3 in mouse brain (positive control) and in the medial layer of mouse brain arterioles when compared to negative control stained sections. Interestingly, Irx3 expression was markedly increased in the neointima of rat balloon-injured carotid arteries after two weeks when compared to control conditions. Likewise, mice treated with AngII (10−7M) for two weeks also exhibited a marked increase in Irx3 expression when compared to IgG controls. To determine if Irx3 could induce VSMC marker gene expression we forced expressed Irx3 in pluripotent P19 cells and examined the expression of the VSMC markers alpha-smooth muscle actin (αSma) and smooth muscle myosin heavy chain (Sm-mhc). Results from quantitative RT-PCR experiments indicate a significant increase in the expression of αSma (2.21-fold; p<0.05) and Sm-mhc (3.02-fold; p<0.05) after 48 hours in cells transfected with Irx3 versus empty vector controls. Likewise, western immunoblot results demonstrated an increase in αSMA expression in Irx3 transfected 10T1/2 cells when compared to vector controls. Myocardin has been previously reported to regulate expression of VSMC marker genes during differentiation. We next examined the effects of Irx3 on the expression levels of myocardin by quantitative RT-PCR. Our results indicate a significant increase in the myocardin expression (1.68-fold; p<0.05) after 48 hours when compared to control conditions. In summary, we herein report the presence and novel function of Irx3 in VSMCs. Our data indicate that forced expression of Irx3 alone is sufficient to increase expression of myocardin and VSMC marker gene expression in pluripotent P19 cells. Thus, these data suggest an essential role for Irx3 during early VSMC differentiation by potentially regulating myocardin and contractile protein gene expression during this process.