The Purα/Purβ Single‐Strand DNA‐Binding Proteins Attenuate Smooth‐Muscle Actin Gene Transactivation in Myofibroblasts

Abstract

Expression of smooth muscle alpha-actin (SMαA) is essential for myofibroblast-mediated wound contraction following tissue injury. The Pur α/β and YB-1 transcriptional repressors govern the DNA-binding activity of serum response factor (SRF) and phosphorylated Smad3 (pSmad3) transcriptional activators during induction of SMαA gene expression in human pulmonary myofibroblasts. In quiescent fibroblasts, Pur α exhibited a novel function in enhancing stability of pre-existing SRF complexes with SMαA core promoter DNA, whereas Pur β was more effective in disrupting SRF-DNA interaction. Pur proteins were less efficient competitors of pre-existing, core-promoter complexes containing both SRF and pSmad3 in nuclear extracts from TGFβ1-activated myofibroblasts. TGFβ1 signaling dissociated a SRF/Pur protein complex with concurrent formation of a transient pSmad3/MRTF-A/Pur β complex during early phase myofibroblast differentiation. Pur β was replaced by Pur α in the pSmad3/MRTF-A complex in mature myofibroblasts. Combining all three repressors potently inhibited SRF and pSmad3 binding to promoter DNA in quiescent fibroblasts and TGFβ1-activated myofibroblasts, respectively. The results point to dynamic interplay between transcriptional activators and repressors in regulating SMαA gene output during myofibroblast differentiation. Therapeutic targeting of nucleoprotein complexes regulating the SMαA promoter may prevent excessive myofibroblast accumulation associated with chronic cardiopulmonary fibrosis and dysfunctional tissue remodeling.

Structural and functional analysis of single‐nucleotide polymorphic variants of purine‐rich element‐binding protein B

“…3,5,9 Possible therapeutic strategies to limit destructive tissue fibrosis include the targeting of molecular regulators that promote sustained connective tissue fibroblast to myofibroblast differentiation. 16,20,21 A number of recent reports have shed light on the structural features of Purβ that account for its functional activity as a ssDNA-binding protein and repressor of Acta2 gene expression. 17 An alternative approach is to target the regulatory factors that control Acta2 expression since smooth muscle α-actin contributes to myofibroblast contractility and is the classic marker of the myofibroblast phenotype.…”

“…AKR-2B MEFs and HEK293 cells were used as a source of mouse and human nuclear proteins. 20 Although the ELISA format using nuclear extract as a source of protein-binding partners does not allow the ready discrimination of direct versus indirect interactions, the apparent inability of Purβ P223L to efficiently bind Smad proteins may explain why this variant is partially deficient in Acta2 repressor activity. Increased interaction of the P223L variant with MSY1/YB1 may facilitate more efficient removal of the variant from the nucleus owing to the coordinated transport of Purβ-MSY1/YB1 complexes by Acta2 mRNA.…”

“…For example, the Smad activator proteins have been reported to induce Acta2 expression by binding to CAGA elements following the exposure of fibroblasts to transforming growth factor β1 (TGF-β1). 16,20,21 Purα and Purβ proteins may also mediate gene repression by binding directly to other transcription factors associated with positive and negative regulation of Acta2 including TEF1, Smad3, and YB1. 16,18,19 Conversely, repression of Acta2 in fibroblasts appears to be governed by the purinerich element-binding proteins A and B (Purα and Purβ) and Y-box binding protein 1 (YB1), which bind in a strand-specific manner to DNA sequences that overlap the G/C-rich and MCAT elements.…”

“…17,18 Specificity proteins 1 and 3 (Sp1, Sp3), serum response factor (SRF), and transcription enhancer factor 1 (TEF1) are also components of a multiprotein Acta2 activation complex formed by their interaction with G/C-rich, CArG, and muscle-CAT (MCAT) recogni-tion motifs, respectively. 16,20 Insights into the specific molecular mechanism by which Purα and Purβ repress Acta2 have been provided by studies that have uncovered the unique structural features of each protein. 16,20,21 Purα and Purβ proteins may also mediate gene repression by binding directly to other transcription factors associated with positive and negative regulation of Acta2 including TEF1, Smad3, and YB1.…”

Circulating miRNAs as Putative Biomarkers of Exercise Adaptation in Endurance Horses

“…In addition, up-regulation of myomiRs, as shown in our experiment, leads to an anti-hypertrophic signal (Soci et al, 2016 ) throughout regulation of PURB . PURB (CNC 9, Table 6 , Figure 3 ) acts on the myosin genes and is considered a repressor of slow muscle contractions (Soci et al, 2016 ) that acts by controlling vascular smooth muscle alpha-actin gene transcription and repressing myoblasts (Hariharan et al, 2014 ).…”