A TGF-beta control element required for SM alpha -actin expression in vivo also partially mediates GKLF-dependent transcriptional repression.

We previously demonstrated that a conserved TGF-beta control element (TCE) within the 5 region of the smooth muscle cell (SMC) differentiation marker gene SM alpha-actin could mediate both transcriptional activation and repression in cultured SMCs through interaction with members of the zinc finger Kruppel like transcription factor (KLF) family. The aims of the present studies were to: 1) determine the role of the SM alpha-actin TCE in vivo through mutagenesis studies in transgenic mice; and 2) further characterize the possible role and mechanisms by which the TCE binding factor GKLF/KLF4 induces repression of SMC marker genes in various SMC model systems in vitro. Our results showed that the TCE was required for SM alpha-actin promoter activity in transgenic mice in vivo. Results of transient transfection studies showed that GKLF-induced repression of a SM alpha-actin promoter/luciferase reporter gene partially depended on the TCE. Furthermore, a GKLF over-expressing adenovirus inhibited whereas GKLF morpholino anti-sense oligos increased expression of endogenous SMC marker genes. Results of chromatin immuno-precipitation assays showed GKLF binding to TCE containing regions of various SMC marker gene promoters within intact chromatin. Finally, results of co-transfection studies showed that over-expression of IKLF/KLF5 reversed GKLF dependent repression thus supporting a model of reciprocal activation-repression of SMC gene expression by different members of the KLF gene family.