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. 2013 Mar 28;2(2):555-86.
doi: 10.3390/biology2020555.

Dynamic Interplay of Smooth Muscle α-Actin Gene-Regulatory Proteins Reflects the Biological Complexity of Myofibroblast Differentiation

Arthur Roger Strauch  1 , Seethalakshmi Hariharan  2
Affiliations

Dynamic Interplay of Smooth Muscle α-Actin Gene-Regulatory Proteins Reflects the Biological Complexity of Myofibroblast Differentiation

Arthur Roger Strauch et al. Biology (Basel). .

Abstract

Myofibroblasts (MFBs) are smooth muscle-like cells that provide contractile force required for tissue repair during wound healing. The leading agonist for MFB differentiation is transforming growth factor β1 (TGFβ1) that induces transcription of genes encoding smooth muscle α-actin (SMαA) and interstitial collagen that are markers for MFB differentiation. TGFβ1 augments activation of Smad transcription factors, pro-survival Akt kinase, and p38 MAP kinase as well as Wingless/int (Wnt) developmental signaling. These actions conspire to activate β-catenin needed for expression of cyclin D, laminin, fibronectin, and metalloproteinases that aid in repairing epithelial cells and their associated basement membranes. Importantly, β-catenin also provides a feed-forward stimulus that amplifies local TGFβ1 autocrine/paracrine signaling causing transition of mesenchymal stromal cells, pericytes, and epithelial cells into contractile MFBs. Complex, mutually interactive mechanisms have evolved that permit several mammalian cell types to activate the SMαA promoter and undergo MFB differentiation. These molecular controls will be reviewed with an emphasis on the dynamic interplay between serum response factor, TGFβ1-activated Smads, Wnt-activated β-catenin, p38/calcium-activated NFAT protein, and the RNA-binding proteins, Purα, Purβ, and YB-1, in governing transcriptional and translational control of the SMαA gene in injury-activated MFBs.

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Figures

Figure 1
Figure 1
The SMαA promoter in quiescent fibroblasts is occupied by the Purβ and YB-1 repressors that occupy opposite strands of the MCAT/THR transcription-activation site. In the presence of TGFβ1, conformational changes occur in the MCAT/THR accompanied by binding of Smad2/3, SRF, and MRTF at cognate sites within and around this site (i.e., Smads at the THR and MRTF/SRF at CArG box B). MRTF normally is sequestered by abundant G-actin monomer that becomes depleted as the SMαA cytoskeleton forms in TGFβ1-activated myofibroblasts (MFBs). The release of MRTF enables its interaction with SRF to enhance SMαA gene transcription. As repressor proteins are removed from the promoter, they bind exon 3 in the nascent SMαA mRNA transcripts to form cytosolic mRNP complexes for transport to polysomes. Association of the mRNA:Purβ:YB1 complex with Pur α in the cytosol may enable coupling with microtubule motor proteins that mediate mRNP intracellular transport known to be under control of Purα in neuronal cells [60]. In theory, reversal of TGFβ1-mediated MFB activation is possible (denoted by the X) upon turnover of Smads and nuclear re-entry of Purβ, YB1, and G-actin monomer after completion of actin cytoskeleton assembly. For simplicity, only Purβ is shown in the nucleus. However, Purα can bind the same DNA site (MCAT-f). Also, the TEF1 and Sp1 trans-activators are omitted from the diagram but are known to bind at the MCAT and downstream SPUR site (not shown), respectively, within the 200 bp SMαA core promoter.
Figure 2
Figure 2
A thermodynamically favorable (ΔG of about −12 kcal/mmol) stem-loop structure can be formed within the MCAT/THR site in the SMαA core promoter. This model depicts formation of single-stranded loops with purine- (DF) and pyrimidine-rich (DR) asymmetry that bind Pur proteins and YB-1, respectively. Our working hypothesis is that Pur and YB-1 repressor proteins minimize SMαA gene transcription in quiescent stromal fibroblasts by disrupting duplex-DNA sites within the MCAT/THR region encompassing the (a) Smad-binding consensus sequence CAGA and/or (b) TEF1-binding site consensus AGGAATG. TGFβ1 activation of the SMαA promoter during MFB differentiation facilitates removal of the Pur and YB-1 repressors transiently exposing their former purine- and pyrimidine-rich binding sites to chemical modification by reagents specific for single-stranded DNA [49]. Nuclear uptake of TGFβ1-regulated Smads helps eliminate Pur and YB-1 repressor binding to the single-stranded loops [13,14] allowing the cruciform to re-fold into duplex B-DNA necessary for binding and transcriptional activation by Smads and TEF1 within the MCAT/THR. Additionally, MRTF/SRF and Sp1 also may bind at nearby re-folded DNA sites in CArG B and SPUR, respectively (not shown). The positions of MCAT/THR mutations that affect SMαA promoter activity are located in sequences depicted by hollow letters.
Figure 3
Figure 3
Substrate-dependent control of SMαA expression in human pulmonary fibroblasts. Cultivation of pulmonary fibroblasts on rigid, plastic (P) substrates in low-serum culture medium sustained baseline expression of SMαA over a 48 h period (upper panel). Treatment with TGFβ1 (5 ng/mL) amplified SMαA expression due to robust signaling provided by accumulation of nuclear stores of phosphorylated Smad 2/3 (lower panel). In contrast, cells maintained on native (C) or denatured type I collagen (dC) substrates showed less baseline SMαA expression during the initial 24 h period and exhibited relatively muted accumulation of phosphorylated Smads 2 and 3 after treatment with TGFβ1 (lower panel). The potentiating effect of rigid-plastic substrate conditions on baseline expression of SMαA correlated with high nuclear levels of SRF trans-activator protein in the apparent absence of TGFβ1-regulated Smad signaling (lower panel). SMαA expression on rigid plastic previously was associated with actin stress fiber assembly in pulmonary fibroblasts [13,92].
Figure 4
Figure 4
YB-1 may function as a porter for mRNA transport in stress-activated MFBs. Tissue injury-associated events including ischemia/reperfusion, thrombosis, inflammation, and biomechanical stress all can trigger MFB differentiation by increasing the levels of active TGFβ1, intracellular calcium, and MAP kinase signaling. These metabolic signals conspire to de-repress the SMαA promoter by removing YB-1 and Pur proteins from the nucleus (for simplicity, only YB-1 is shown in this scheme). With assistance from Purα, displaced YB-1 can function as an mRNA porter to move nascent transcripts from the nucleus to cytosolic polyribosomes where kinases such as Erk1/2 and Akt may phosphorylate YB-1. Phosphorylation of serine 102 in the RNA-binding cold-shock domain could facilitate both unloading of mRNA payload and nuclear re-entry of YB-1 for additional cycles of SMαA mRNA transport or transcriptional suppression depending on whether MFB differentiation is underway or nearing termination. Newly translated G-actin monomers made available by the YB-1 mRNA shuttle are used to polymerize actin stress fibers at focal adhesions needed for directing MFB contractile force in the healing wound.
Figure 5
Figure 5
A diagram summarizing key transcriptional regulatory protein-binding sites in the 3.6 kilobase mammalian SMαA promoter including relative positions of consensus sites in the 5'-flanking and first intron regions. Purα, Purβ, and YB-1 are repressors whereas all other indicated proteins are activators. The exact positions of intron 1-binding sites for SRF and NFATc3 have not yet been identified. TSS refers to transcription-start site. Not drawn to scale.
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