Eukaryotic gene transcription requires the coordinated assembly of upstream cis-element binding proteins, intermediary cofactors, and components of the basal transcription machinery into a multicomponent complex competent to initiate transcription. During this process, sequence-specific DNA-binding transcriptional activators and/or repressors play a pivotal role in modulating the cell-type specific expression of genes. While most such proteins bind to double-stranded DNA target sequences, a small but intriguing subclass has been identified that show enhanced affinity and specificity for either the sense or antisense strands of certain cis-regulatory elements required for promoter-specific activation (1-4) or repression (5-9). We have recently cloned and identified two single-stranded DNA (ssDNA)

The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor ␤1 (TGF-␤1) and accompanied by transient activation of the vascular smooth muscle ␣-actin (Sm␣A) gene. Multiple positive-regulatory elements were identified as essential mediators of basal Sm␣A enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-␤1. However, TGF-␤1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the Sm␣A enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate Sm␣A enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the Sm␣A enhancer required for TGF-␤1 amplification. Additionally, the TGF-␤1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for Sm␣A enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the Sm␣A enhancer. The identification of Sp and Smad proteins as essential, independent activators of the Sm␣A enhancer provides new insight into the poorly understood process of myofibroblast differentiation.

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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...