Dynamic interplay between serum response factor and Purβ modulates human pulmonary myofibroblast differentiation

Abstract

Smooth muscle α-actin (SMA) enables myofibroblast (MFB) contraction needed for wound healing. The SMA promoter contains CC-(A/T)6-GG boxes (CArGs) that bind serum response factor (SRF). In human pulmonary fibroblasts (hPFBs) the transcriptional repressor, Pur β, interfered with physical binding of SRF binding to CArGs. Collagen inhibited hPFB differentiation into MFBs and dampened response to TGFβ1. There was increased expression of p44 Pur β in collagen-cultivated cells consistent with the observed diminished expression of SMA. While the p67 SRF activator also was detected, a smaller alternatively spliced form that has reduced transcriptional activity was more abundant. Pur β might represent a natural antagonist to SRF and was used in competitive titration studies to examine its ability to displace SRF from CArGs. Pur β gradually displaced native SRF from CArGs when MFB nuclear extract (NE) was pre-mixed with SMA promoter DNA. If Pur β was pre-mixed with DNA before exposure to SRF-rich NE, p67 SRF binding did not change although a SRF size variant was seen. When NE and Pur β were pre-mixed before DNA, SRF binding was nearly fully impaired as if sequestered by Pur β in an off-DNA complex. Analysis of SRF and Pur β interplay during myofibroblast differentiation may reveal aspects of protein structure that could be leveraged to suppress these cells and prevent fibrosis and tissue dysfunction. Supported by HL 085109.