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. 2004 Oct;15(10):4532-43.
doi: 10.1091/mbc.e04-04-0348. Epub 2004 Jul 28.

Induction of vascular smooth muscle alpha-actin gene transcription in transforming growth factor beta1-activated myofibroblasts mediated by dynamic interplay between the Pur repressor proteins and Sp1/Smad coactivators

Sukanya V Subramanian  1 , John A Polikandriotis, Robert J Kelm Jr, Jason J David, Charles G Orosz, Arthur R Strauch
Affiliations

Induction of vascular smooth muscle alpha-actin gene transcription in transforming growth factor beta1-activated myofibroblasts mediated by dynamic interplay between the Pur repressor proteins and Sp1/Smad coactivators

Sukanya V Subramanian et al. Mol Biol Cell. 2004 Oct.

Abstract

The mouse vascular smooth muscle alpha-actin (SMA) gene enhancer is activated in fibroblasts by transforming growth factor beta1 (TGFbeta1), a potent mediator of myofibroblast differentiation and wound healing. The SMA enhancer contains tandem sites for the Sp1 transcriptional activator protein and Puralpha and beta repressor proteins. We have examined dynamic interplay between these divergent proteins to identify checkpoints for possible control of myofibroblast differentiation during chronic inflammatory disease. A novel element in the SMA enhancer named SPUR was responsible for both basal and TGFbeta1-dependent transcriptional activation in fibroblasts and capable of binding Sp1 and Pur proteins. A novel Sp1:Pur:SPUR complex was dissociated when SMA enhancer activity was increased by TGFbeta1 or Smad protein overexpression. Physical association of Pur proteins with Smad2/3 was observed as was binding of Smads to an upstream enhancer region that undergoes DNA duplex unwinding in TGFbeta1-activated myofibroblasts. Purbeta repression of the SMA enhancer could not be relieved by TGFbeta1, whereas repression mediated by Puralpha was partially rescued by TGFbeta1 or overexpression of Smad proteins. Interplay between Pur repressor isoforms and Sp1 and Smad coactivators may regulate SMA enhancer output in TGFbeta1-activated myofibroblasts during episodes of wound repair and tissue remodeling.

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Figures

Figure 1.
Figure 1.
EMSAs depicting interaction of native Sp1, Sp3, and Pur proteins in neonatal mouse ventricle (left and middle) and serum-treated AKR-2B fibroblast (right) extracts with either double-strand SPUR probe (dsSPUR) or a single-strand probe from a different region of the SMA enhancer (ssMCAT) that contains a site for Pur protein binding but not the other proteins. EMSA reactions were processed with antibodies specific for Sp1, Sp3, Purβ, SRF, and TEF1 to size shift DNA:protein complexes containing the cognate transcription factors (size-shifted complexes are within the bracketed area marked with asterisk). The Sp1, Sp3, and Pur proteins all were independently size shifted by their corresponding antibodies but not by unrelated SRF or TEF1 antibodies.
Figure 2.
Figure 2.
(A) DBA of fibroblast nuclear extracts using double- and single-strand SMA enhancer probes. DBA probes are indicated at the top margin. Antibodies used for Western blot analysis of affinity-isolated proteins are noted on the left. ds, double-strand DBA probes; ss, single-strand DBA probes. dsSPUR binds the Pur, Sp1, and Sp3 proteins. (B) DBA of fibroblast nuclear extracts by using mutant SPUR probes. SPUR mutations in double-strand context eliminated Pur binding but had no detrimental effect on binding to single-strand probes. wt, wild-type context; m1, m2, mutant context (Table 1); ns, nonspecific species. Antibodies for DBA Western blots are noted on the left.
Figure 3.
Figure 3.
DBA of nuclear extracts prepared from TGFβ1-activated myofibroblasts. Antibodies used for Western blots are shown at the left of each panel. Over a 6-h exposure to TGFβ1, there was a fourfold decrease in Pur protein binding to the dsSPUR probe.
Figure 4.
Figure 4.
Immunoprecipitation of a Sp1:Pur protein complex from fibroblasts. (A) Sp1 immunoprecipitate isolated from serum-free (control) AKR-2B fibroblast nuclear extracts contained prominent Purα and Purβ bands revealed by Western blot analysis by using a pan-specific anti-Pur antibody. Myofibroblast differentiation during a 6-h exposure to TGFβ1 was accompanied by diminished Sp1:Pur protein interaction. Exposure to TGFβ1 had no detectable effect on Pur protein isoform levels in nuclear extracts (bottom). (B) Pur protein immunoprecipitate isolated from COS7 fibroblasts transfected with empty expression plasmid (-) or equimolar mixtures of plasmids harboring cDNAs encoding Smad 2, 3, and 4 (+) were processed by Western blot by using a Sp1-specific antibody. Transfection with Smad expression plasmids did not effect net expression of Sp1 protein (bottom) but significantly impaired Sp1:Pur protein interaction (top).
Figure 5.
Figure 5.
Pur proteins behave as bimodal mediators of SMA enhancer activity in transfected COS7 fibroblasts. (A) Both Pur proteins were able to suppress Sp1-mediated activation of the SMA enhancer. (B) Smad proteins are robust activators of the SMA enhancer and can neutralize Purα-mediated repression with or without Sp1. (C) Purβ behaved as a dominant negative-type repressor and was unaffected by either Smad or Sp1 protein overexpression. For A to C, the expression plasmids included in individual transfections are noted by (+). (D) Protein overexpression in transfected COS7 fibroblasts was verified by Western blot analysis by using polyclonal antibodies specific for Pur proteins (top); Smad 2, 3, and 4 (middle); or Sp1 (bottom). Lane 1 (all panels), extracts from nontransfected COS7 fibroblasts; lanes 2–4, extracts from COS7 fibroblasts transfected with expression plasmids for Purα alone (lane 2, top), Smad2/3/4 (lane 2, middle), Sp1 (lane 2, bottom), Purβ alone (lane 3, top), or both Purα and Purβ (lane 4, top).
Figure 6.
Figure 6.
Overexpression of Pur proteins in AKR-2B fibroblasts attenuates SMA enhancer and full-length promoter activation by TGFβ1 or Smad proteins. (A) VSMP4 enhancer activation in transfected fibroblasts by TGFβ1, Smad2/3/4, or a combination of both was substantially repressed by Purα overexpression and fully repressed by Purβ. Compare lanes 2, 3, and 4 with lanes 5, 6, and 7 (Purα-mediated repression) or lanes 8, 9, and 10 (Purβ-mediated repression). (B) Smad-mediated activation of the full-length VSMP8 promoter in AKR-2B fibroblasts was substantially repressed by Purα and fully so by Purβ. Compare lane 5 with either lane 6 (Purα-mediated repression) or lane 7 (Purβ-mediated repression). The VSMP8 promoter normally is transcriptionally silent in fibroblasts but activated by TGFβ1 or Smad signaling intermediary proteins (compare lanes 1 and 5). For A and B, the various treatments and/or combinations of expression plasmids used for each transfection are shown at the bottom.
Figure 7.
Figure 7.
Evidence for a Smad 2/3:Pur protein complex in transfected COS7 fibroblasts. Nuclear protein extracts prepared from COS7 fibroblasts overexpressing either Sp1 (lanes 1–4) or Smad 2/3/4 (lanes 5–8) were IPed with antibodies specific for Sp1 or Smads (as noted at the bottom of each panel) and processed for Western blot (WB) using antibodies shown on the left. Both Sp1 (IP:Sp1) and Smad (IP:Flg-Smad) immunoprecipitates contained Purα and Purβ but samples processed with the Smad 7 negative control antibody did not (IP:Smad 7). None of the overexpressed proteins exhibited nonspecific binding to the affinity isolation beads (no IP). Protein overexpression was verified by Western blot analysis of nuclear protein extracts as shown in the bottom two panels. The band denoted “ns” was not studied in detail but may represent a covalently modified Pur protein isoform (unpublished data).
Figure 8.
Figure 8.
SMA enhancer-activating proteins in myofibroblasts exhibit different subcellular localization and enhancer-binding specificity. Left, Smad and Sp1 western blots of nuclear protein extracts prepared from AKR-2B fibroblasts after exposure to TGFβ1 over a 24-h period. Smad 2 and 3 entered the nucleus within 1 h after TGFβ1 treatment, whereas nuclear Sp1 levels were uniformly high. Right, DBA using nuclear extracts from TGFβ1-activated myofibroblasts and various dsDNA probes derived from the SMA enhancer (noted at the top). The Smad and Sp1 enhancer activating proteins bind to different regions of the SMA enhancer.
Figure 9.
Figure 9.
Cardiac remodeling is accompanied by changes in Pur protein isoform content and SMA expression. Protein extracts prepared from neonatal (postpartum day 4, 6, 9, and 13) and adult mouse ventricles as well as ventricles from explanted allogeneic (allograft) heart grafts (30 d posttransplant) were evaluated by Western blot by using Pur- and SMA-specific antibodies. The postnatal developmental period was characterized by dominance of the Purβ isoform and diminishing SMA expression, whereas chronically rejected allografts expressed more Purα and SMA protein compared with nontransplanted native hearts from graft recipients. The band denoted “ns” was not studied in detail but may represent a covalently modified Pur protein isoform (unpublished data).
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References

    1. Armstrong, A.T., Strauch, A.R., Starling, R.C., Sedmak, D.D., and Orosz, C.G. (1997a). Morphometric analysis of neointimal formation in murine cardiac allografts. Transplantation 63, 941-947. - PubMed
    1. Armstrong, A.T., Strauch, A.R., Starling, R.C., Sedmak, D.D., and Orosz, C.G. (1997b). Morphometric analysis of neointimal formation in murine cardiac allografts. II. Rate and location of lesion formation. Transplantation 64, 322-328. - PubMed
    1. Armstrong, A.T., Strauch, A.R., Starling, R.C., Sedmak, D.D., and Orosz, C.G. (1997c). Morphometric analysis of neointimal formation in murine cardiac grafts. III. Dissociation of interstitial fibrosis from neointimal formation. Transplantation 64, 1198-1202. - PubMed
    1. Becker, N.A., Kelm, R.J., Jr., Vrana, J.A., Getz, M.J., and Maher, L.J.I. (2000). Altered sensitivity to single-strand-specific reagents associated with the genomic vascular smooth muscle alpha-actin promoter during myofibroblast differentiation. J. Biol. Chem. 275, 15384-15391. - PubMed
    1. Black, F.M., Packer, S.E., Parker, T.G., Michael, L.H., Roberts, R., Schwartz, R.J., and Schneider, M.D. (1991). The vascular smooth muscle α-actin gene is reactivated during cardiac hypertrophy provoked by load. J. Clin. Investig. 88, 1581-1588. - PMC - PubMed

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