Altered Sensitivity to Single-strand-specific Reagents Associated with the Genomic Vascular Smooth Muscle α-Actin Promoter during Myofibroblast Differentiation

Abstract

Stimulation of quiescent AKR-2B mouse fibroblasts with transforming growth factor ␤1 results in uniform conversion to a myofibroblast-like phenotype as judged by a rapid accumulation of smooth muscle ␣-actin mRNA and protein. Because transcriptional regulation of the smooth muscle ␣-actin gene in these cells might be mediated by single-stranded DNA-binding proteins, we have examined the sensitivity of genomic DNA to chemical reagents with specificity for unpaired bases in a region of the promoter previously implicated in Pur␣, Pur␤, and MSY1 binding in vitro (Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238 -14245). Our data reveal specific differences between purified DNA treated in vitro and nucleoprotein complexes treated in living cells. Although some differences were observed in quiescent cells, treatment with transforming growth factor ␤1 resulted in the development of additional sensitivity within 1 h. This enhancement was most pronounced in bases immediately upstream of an MCAT enhancer element-containing polypurine-polypyrimidine tract. A TATA-proximal element of similar base distribution showed no such hyperreactivities. These results suggest that activation of the endogenous smooth muscle ␣-actin gene during myofibroblast conversion is accompanied by specific structural changes in the promoter that are consistent with a decline in single-stranded DNA repressor protein binding.Although many studies have focused on malignant cells themselves, there is an emerging appreciation of the essential role played by stromal components in regulating tumor growth and progression (1). In the stroma of invasive breast cancer for example, myofibroblasts are the most abundant cell type (2), and their persistence has been linked to stromal modifications believed to facilitate tumor cell invasion and metastasis (3, 4). Myofibroblasts, which exhibit morphological and biochemical features intermediately between fibroblasts and smooth muscle cells, were originally identified in granulation tissue (5) where they appear transiently during wound healing and are thought to provide the contractile force necessary for wound closure (6 -9). Because vascular smooth muscle (VSM) 1 ␣-actin gene expression is generally considered the hallmark of the myofibroblast phenotype (10), elucidation of the molecular mechanisms that control VSM ␣-actin gene expression during fibroblast to myofibroblast conversion will likely provide greater insight into the role of this distinctive cell type in cancer progression and wound healing. Unfortunately, convenient in vitro model systems of myofibroblast cytodifferentiation are lacking due to the manifest heterogeneity of VSM ␣-actin expression in many established fibroblastic cells lines (11).Some of our recent studies have focused on identifying the operative cis-acting elements and DNA-binding proteins that regulate mouse VSM ␣-actin promoter activation and repression in serum-stimulated AKR-2B fibroblasts (12-18), a nontransformed clona...

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

“…As shown above, Egr-1 bound to the SPUR element (positioned between Ϫ59 and Ϫ28 base pairs from the transcriptional start site), whereas regulatory Smads2 and -3 interact exclusively with a THR between positions Ϫ170 and Ϫ150 located ϳ100 base pairs upstream from SPUR (Becker et al, 2000;Subramanian et al, 2004). This eliminated the possibility that Egr-1 and Smads might compete for the same or overlapping DNA binding sites in the promoter.…”

“…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.…”

Nucleoprotein Interactions Governing Cell Type-dependent Repression of the Mouse Smooth Muscle α-Actin Promoter by Single-stranded DNA-binding Proteins Purα and Purβ

“…4, C and D). Relative to A7r5 cells, which exhibit a stable adult VSMC phenotype (46), the VSMP4 reporter predictably showed a higher degree of serum and TGF␤1 inducibility in synchronized AKR-2B cells consistent with transition to a myofibroblast-like phenotype (20). In growth factor-stimulated AKR-2B MEFs, exogenous Pur␣ and Pur␤ were equally effective in attenuating VSMP4 induction (Fig.…”

“…In its double-stranded configuration, the Pur/ Pyr sequence contains a consensus MCAT enhancer motif, which serves as a recognition site for transcription enhancer factor 1 (TEF-1) (18). Importantly, computer modeling of the extended Pur/Pyr tract coupled with ssDNA-selective chemical modification of genomic DNA in cultured fibroblasts indicated that this region has the propensity to assume a partially unpaired configuration in response to transforming growth factor ␤1 (TGF␤1) signaling (20). These findings coupled with results of in vitro DNA binding and cell-based SM␣A promoter mutagenesis studies, led to the development of two alternative models for cryptic MCAT enhancer regulation.…”

Cryptic MCAT Enhancer Regulation in Fibroblasts and Smooth Muscle Cells

“…In this regard, a recent report showing direct protein-protein binding between TEF-1 and SRF (47), leads one to question whether this interaction is subject to regulation by Pur␣, Pur␤, MSY1, or some combination thereof. Furthermore, in light of the changes in DNA structure surrounding the genomic Pu/Py element that accompanies myofibroblast differentiation (48), analysis of nucleoprotein complex assembly/disassembly on the genomic promoter will likely provide further insight into the mechanism of MCAT enhancer regulation by Pur␣, Pur␤, MSY1 vis à vis the proposed models. …”