YB-1 Coordinates Vascular Smooth Muscle α-Actin Gene Activation by Transforming Growth Factor β1 and Thrombin during Differentiation of Human Pulmonary Myofibroblasts

Abstract

Profibrotic regulatory mechanisms for tissue repair after traumatic injury have developed under strong evolutionary pressure to rapidly stanch blood loss and close open wounds. We have examined the roles played by two profibrotic mediators, transforming growth factor ␤1 (TGF␤1) and thrombin, in directing expression of the vascular smooth muscle ␣-actin (SM␣A) gene, an important determinant of myofibroblast differentiation and early protein marker for stromal cell response to tissue injury. TGF␤1 is a well known transcriptional activator of the SM␣A gene in myofibroblasts. In contrast, thrombin independently elevates SM␣A expression in human pulmonary myofibroblasts at the posttranscriptional level. A common feature of SM␣A up-regulation mediated by thrombin and TGF␤1 is the involvement of the cold shock domain protein YB-1, a potent repressor of SM␣A gene transcription in human fibroblasts that also binds mRNA and regulates translational efficiency. YB-1 dissociates from SM␣A enhancer DNA in the presence of TGF␤1 or its Smad 2, 3, and 4 coregulatory mediators. Thrombin does not effect SM␣A gene transcription but rather displaces YB-1 from SM␣A exon 3 coding sequences previously shown to be required for mRNA translational silencing. The release of YB-1 from promoter DNA coupled with its ability to bind RNA and shuttle between the nucleus and cytoplasm is suggestive of a regulatory loop for coordinating SM␣A gene output in human pulmonary myofibroblasts at both the transcriptional and translational levels. This loop may help restrict organ-destructive remodeling due to excessive myofibroblast differentiation. INTRODUCTIONFibrosis is a serious complication of chronic cardiopulmonary diseases and postsurgical complication of heart and lung transplant (Pickering and Boughner, 1990;Armstrong et al., 1997;Howell et al., 2002;Chapman, 2004). Although management of acute allograft rejection is accomplished through the use of immunosuppressive agents that can limit immune cell infiltration, the cause of chronic rejection and failure in accepted allografts is poorly understood thus offering no treatment solution short of retransplant. Leading the formation of scar tissue is a specialized stromal cell referred to as the myofibroblast that contains abundant microfilament networks composed of smooth muscle-specific contractile protein isoforms (Tomasek et al., 2002;Hinz and Gabbiani, 2003;Grotendorst et al., 2004). Chronic accumulation of myofibroblasts is associated with excessive extracellular matrix protein biosynthesis, hypercontractility, and organ-destructive remodeling. Vascular smooth muscle ␣-actin (SM␣A) is one of the major contractile proteins expressed by differentiated myofibroblasts (Darby et al., 1990;Ronnov-Jessen and Petersen, 1996;Hinz et al., 2001;Cogan et al., 2002). Normally, SM␣A-enriched myofibroblasts are early, transient participants in stromal wound healing processes. We reason that characterization of the initial molecular events associated with activation of the SM␣A gene in stromal cells would hel...

Transforming Growth Factor β1-mediated Activation of the Smooth Muscle α-Actin Gene in Human Pulmonary Myofibroblasts Is Inhibited by Tumor Necrosis Factor-α via Mitogen-activated Protein Kinase Kinase 1-dependent Induction of the Egr-1 Transcriptional Repressor

“…We previously showed by immunoblot and immunofluorescence microscopy that TGF␤1 substantially increased SM␣A mRNA and protein expression in early passage human pulmonary fibroblasts (Zhang et al, 2005). As shown in Figure 1a, TNF-␣ (10 ng/ml) provided to hPFBs 2 h before treatment with TGF␤1 (5 ng/ml) markedly reduced the accumulation of SM␣A protein relative to the level of expression noted in cells treated with TGF␤1 alone.…”

“…Synthetic oligonucleotide probes used in this report were derived from selected sequences present in the human SM␣A promoter Sun et al, 1995;Kelm et al, 1996;Subramanian et al, 2004;Zhang et al, 2005). Reaction mixtures containing protein extract (100 g of protein) and 3Ј-biotinylated oligonucleotides (100 pmol; Integrated DNA Technologies, Coralville, IA) were incubated in a buffer containing poly(dI-dC), 10 mM Tris, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 0.12 mM PMSF, and 4% glycerol.…”

“…Monolayers of hPFBs maintained at 40 -50% confluence were transfected as described previously (Zhang et al, 2005). Optimized mixtures of the SM␣A promoter: reporter fusion plasmids (VSMP4) with or without mammalian expression plasmids encoding various transcriptional regulatory proteins were transfected using the Mirus (Invitrogen) transfection reagent and protocol provided by the manufacturer.…”

“…Transforming growth factor (TGF) ␤1) is the principle mediator of myofibroblast differentiation in healing wounds where it accumulates in granulation tissue in activated form during leukocyte infiltration and potently stimulates transcription of the type I␣2 collagen subunit (COL1␣2) and SM␣A genes (Becker et al, 2000;Massague and Wotton, 2000;Cogan et al, 2002;Higashi et al, 2003;Grotendorst et al, 2004;Leask and Abraham, 2004;Subramanian et al, 2004;Zhang et al, 2005). Smad proteins 2 and 3 are activated by TGF␤1 receptor engagement and enter the nucleus in which they directly bind SM␣A and COL1␣2 promoter DNA.…”

Increased Expression of Y-Box-Binding Protein-1 in Hind-Limb Muscles During Regeneration from Ischemic Injury in Mice

“…In addition, several genes important for wound healing and cellular proliferation and survival are regulated by YB-1, including collagen alpha1 and alpha2 (Norman et al 2001;Higashi et al 2003), matrix metalloproteinase 2 (Mertens et al 1997), the B-chain isoform of plateletderived growth factor (Stenina et al 2000), vascular endothelial growth factor (Coles et al 2004), granulocyte-macrophage-colony stimulating factor (Coles et al 2000), and Fas death receptor (Lasham et al 2000); moreover, YB-1 can also regulate αSMA and human pulmonary myofibroblasts (Zhang et al 2005). Takahashi et al (2010) reported that YB-1 is increased in the angiogenic endothelial cells of various tumors and is involved in the growth of endothelial cells.…”