The diaphanous-related formins: Dynamic regulators of smooth muscle cell-specific gene transcription

We and others have previously shown that RhoA-dependent actin polymerization stimulates SMC-specific gene transcription by promoting the nuclear accumulation of the myocardin-related transcription factors (MRTF)-A and -B. Very little is known about the downstream RhoA effectors that mediate this response, and the goal of the studies described herein was to define the role of the diaphanous-related formins (DRFs) in regulating smooth muscle cell (SMC) differentiation. The DRFs mDia1 and mDia2 are highly expressed in cultured SMCs and in tissues containing a high smooth muscle component. Activation of mDia1 or mDia2 by RhoA stimulated actin polymerization, MRTF nuclear accumulation, and SMC-specific gene transcription. Interestingly, we found that phosphorylation of the Diaphanous Autoregulatory Domain (DAD) by Rho-kinase also stimulated mDia2 activity and SM-marker gene expression. Knockdown of mDia1/2 using siRNA significantly attenuated expression of numerous SM-marker genes in primary aortic SMCs. While we originally attributed these findings to the regulation of cytoplasmic actin dynamics by the DRFs, recent evidence linking nuclear globular (G)-actin to MRTF nuclear export led us to investigate a possible role for the DRFs in the nucleus. We found that mDia2, but not mDia1 or FHOD1, accumulated in the nucleus following treatment with leptomycin, an inhibitor of Crm-1 dependent nuclear export. Deletion and mutation analyses identified nuclear localization sequences (NLS) in the core formin homology 2 (FH2) domain and extreme N-terminus, and a leucine-rich nuclear export sequence (NES) was identified in the C-terminus of mDia2. Importantly, mDia2 variants that were excluded from the nucleus did not stimulate SMC-specific gene transcription and MRTF-B nuclear accumulation as well as wild-type mDia2. Taken together, these data support a model in which mDia2 activity in the nucleus and cytoplasm depletes cellular G-actin pools resulting in MRTF nuclear accumulation and activation of SMC-specific gene transcription.