Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.
Bottom Line: Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood.Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B.Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction.
Affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298.Show MeSH
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Mentions: SRF is a multifunctional transcription factor that is known to regulate the expression not only of cytoskeletal proteins but also of so-called “early response” genes such as c-Fos, JunD, and Erg-1 (Soh et al., 1999; Lee et al., 2010b). Therefore we asked whether increased SRF expression after γ-CYA depletion resulted in ubiquitous activation of different SRF targets. Quantitative RT-PCR analysis showed that, in contrast to contractile proteins, mRNA levels of early-response genes were significantly decreased by γ-CYA knockdown (Supplemental Figure S3). This result indicates a specific shift of SRF activity toward transcriptional up-regulation of myogenic markers. Similar transcriptional reprogramming observed in previous studies was associated with a family of specific SRF coactivators that includes myocardin and myocardin-related transcriptional factor A (MRTF-A) and MRTF-B, expressed in muscle and nonmuscle cells, respectively (Posern et al., 2002; Miralles et al., 2003; Wang et al., 2004). Therefore we next investigated whether MRTF is involved in γ-CYA–dependent EMyT. MRTF is known to dual localize in the nucleus and the cytoplasm, and it has to be translocated into the nucleus in order to stimulate gene expression (Miralles et al., 2003; Pawlowski et al., 2010). According to our immunofluorescence labeling and confocal microscopy analysis, loss of γ-CYA triggered a considerable translocation of endogenous MRTF-A into the nucleus, as manifested by an approximately twofold increase in its nuclear/cytoplasmic ratio of the MRTF signal (Figure 4, A and B). Similar nuclear translocation was observed for exogenously expressed, Flag-tagged MRTF-A, as well as for endogenous or exogenous MRTF-B (Supplemental Figure S4). Furthermore, dual MRTF-A–γ-CYA or MRTF-B–γ-CYA knockdown significantly attenuated induction of all EMyT markers as compared with γ-CYA–only depleted cells (Figure 4, C and D). Of interest, depletion of an individual MRTF isoform (either MRTF-A or MRTF-B) almost completely suppressed induction of several contractile proteins despite the presence of the other MRTF isoform. This most likely indicates that both isoforms cooperate in expressional up-regulation of contractile proteins and that a certain threshold concentration of MRTF is required in order to stimulate the EMyT.
Affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298.