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.

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.

Oxygen sensing by primary cardiac fibroblasts: a key role of p21(Waf1/Cip1/Sdi1).

In mammalian organs under normoxic conditions, O2 concentration ranges from 12% to <0.5%, with O2 approximately 14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to approximately 1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of PO2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the PO2 to which cardiac cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and function of these cells. To test this hypothesis, cardiac fibroblasts (CFs) isolated from adult murine ventricle were cultured in 10% or 21% O2 (hyperoxia relative to the PO2 to which cells are adjusted in vivo) and were compared with those cultured in 3% O2 (mild hypoxia). Compared with cells cultured in 3% O2, cells that were cultured in 10% or 21% O2 demonstrated remarkable reversible G2/M arrest and a phenotype indicative of differentiation to myofibroblasts. These effects were independent of NADPH oxidase function. CFs exposed to high O2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-beta1, (2) lowered telomerase activity, and (3) activation of transforming growth factor-beta1 and p38 mitogen-activated protein kinase. CFs deficient in p21 were resistant to such O2 sensitivity. This study raises the vital broad-based issue of controlling ambient O2 during the culture of primary cells isolated from organs.

Reprogramming of vascular smooth muscle alpha-actin gene expression as an early indicator of dysfunctional remodeling following heart transplant.

OBJECTIVE: Chronic rejection in cardiac allografts depletes vascular smooth muscle (VSM) alpha-actin from the coronary arterial smooth muscle bed while promoting its abnormal accumulation in cardiomyocytes and myofibroblasts. The objective was to determine if the newly discovered TEF1, MSY1, Puralpha and Purbeta VSM alpha-actin transcriptional reprogramming proteins (TRPs) were associated with development of chronic rejection histopathology in accepted murine cardiac allografts. METHODS: A mouse heterotopic cardiac transplant model was employed using H2 locus-mismatched mouse strains (DBA/2 or FVB/N to C57BL/6). Recipients were immunosuppressed to promote long-term allograft acceptance and emergence of chronic rejection. Explanted grafts and isolated heart cells were evaluated for changes in the DNA-binding activity and subcellular distribution of VSM alpha-actin transcriptional regulatory proteins. RESULTS: The DNA-binding activity of all four TRPs was high in the developing mouse ventricle, minimal in adult donor hearts and increased substantially within 30 days after transplantation. Immunohistologic analysis revealed nuclear localization of Purbeta and MSY1 particularly in fibrotic areas of the allograft myocardium demonstrating extravascular accumulation of VSM alpha-actin. Cardiomyocytes isolated from adult, non-transplanted mouse hearts not only exhibited less VSM alpha-actin expression and lower levels of TRPs compared to isolated cardiac fibroblasts or neonatal cardiomyocytes, but also contained a novel size variant of the MSY1 protein. CONCLUSION: Accumulation of TRPs in cardiac allografts, particularly within the fibroblast-enriched myocardial interstitium, was consistent with their potential role in VSM alpha-actin gene reprogramming, fibrosis and dysfunctional remodeling following transplant. These nuclear protein markers could help stage peri-transplant cellular events that precede formation of graft-destructive fibrosis and coronary vasculopathy during chronic rejection.

Y-box binding protein-1 implicated in translational control of fetal myocardial gene expression after cardiac transplant.

Peri-transplant surgical trauma and ischemia/reperfusion injury in accepted murine heterotopic heart grafts has been associated with myofibroblast differentiation, cardiac fibrosis and biomechanical-stress activation of the fetal myocardial smooth muscle α-actin (SMαA) gene. The wound-healing agonists, transforming growth factor ß1 and thrombin, are known to coordinate SMαA mRNA transcription and translation in activated myofibroblasts by altering the subcellular localization and mRNA-binding affinity of the Y-box binding protein-1 (YB-1) cold-shock domain (CSD) protein that governs a variety of cellular responses to metabolic stress. YB-1 accumulated in polyribosome-enriched regions of the sarcoplasm proximal to cardiac intercalated discs in accepted heart grafts. YB-1 binding to a purine-rich motif in exon 3 of SMαA mRNA that regulates translational efficiency increased substantially in perfusion-isolated, rod-shaped adult rat cardiomyocytes during phenotypic de-differentiation in the presence of serum-derived growth factors. Cardiomyocyte de-differentiation was accompanied by the loss of a 60 kDa YB-1 variant that was highly expressed in both adult myocardium and freshly isolated myocytes and replacement with the 50 kDa form of YB-1 (p50) typically expressed in myofibroblasts that demonstrated sequence-specific interaction with SMαA mRNA. Accumulation of p50 YB-1 in reprogrammed, de-differentiated myocytes was associated with a 10-fold increase in SMαA protein expression. Endomyocardial biopsies collected from patients up to 14 years after heart transplant showed variable yet coordinately elevated expression of SMαA and p50 YB-1 protein and demonstrable p50 YB-1:SMαA mRNA interaction. The p60 YB-1 variant in human heart graft samples, but neither mouse p60 nor mouse or human p50, reacted with an antibody specific for the phosphoserine 102 modification in the YB-1 CSD. Modulation of YB-1 subcellular compartmentalization and mRNA-binding activity may be linked with reprogramming of contractile protein gene expression in ventricular cardiomyocytes that could contribute to maladaptive remodeling in accepted, long-term heart grafts.

Serum response factor neutralizes Pur alpha- and Pur beta-mediated repression of the fetal vascular smooth muscle alpha-actin gene in stressed adult cardiomyocytes.

Mouse hearts subjected to repeated transplant surgery and ischemia-reperfusion injury develop substantial interstitial and perivascular fibrosis that was spatially associated with dysfunctional activation of fetal smooth muscle alpha-actin (SM alpha A) gene expression in graft ventricular cardiomyocytes. Compared with cardiac fibroblasts in which nuclear levels of the Sp1 and Smad 2/3 transcriptional-activating proteins increased markedly after transplant injury, the most abundant SM alpha A gene-activating protein in cardiomyocyte nuclei was serum response factor (SRF). Additionally, cardiac intercalated discs in heart grafts contained substantial deposits of Pur alpha, an mRNA-binding protein and known negative modulator of SRF-activated SM alpha A gene transcription. Activation of fetal SM alpha A gene expression in perfusion-isolated adult cardiomyocytes was linked to elevated binding of a novel protein complex consisting of SRF and Pur alpha to a purine-rich DNA element in the SM alpha A promoter called SPUR, previously shown to be required for induction of SM alpha A gene transcription in injury-activated myofibroblasts. Increased SRF binding to SPUR DNA plus one of two nearby CArG box consensus elements was observed in SM alpha A-positive cardiomyocytes in parallel with enhanced Pur alpha:SPUR protein:protein interaction. The data suggest that de novo activation of the normally silent SM alpha A gene in reprogrammed adult cardiomyocytes is linked to elevated interaction of SRF with fetal-specific CArG and injury-activated SPUR elements in the SM alpha A promoter as well as the appearance of novel Pur alpha protein complexes in both the nuclear and cytosolic compartments of these cells.

Impaired wound contraction and delayed myofibroblast differentiation in restraint-stressed mice.

Previous research has shown that psychological stress delays wound closure by >25%. Gene expression of pro-inflammatory cytokines and the maturation of the epithelium were also impaired by stress (Mercado et al.). Wound contraction contributes to the speed of wound closure (Hunt and Hopf). In the current study, wound contraction was decreased by >45% (p<.01) in restraint stressed mice. Fibroblast migration and differentiation into smooth muscle alpha-actin (SmalphaA) -expressing myofibroblasts were delayed in RST mice through day 7 post-wounding. In addition, there was a 25 (p<.01), 48 (p<.01), and 38% (p<.05) decrease in SmalphaA mRNA levels at days 1, 3, and 5 post-wounding in RST mice, respectively. Cytokines that regulate fibroblast migration and differentiation include transforming growth factors-beta1, -beta2, and -beta3 (TGF-betas). Although expression of TGF-beta1 mRNA was downregulated by >25% (p<.01) in RST mice on day 3 post-wounding, no significant differences were detected in active or total TGF-beta1 protein levels. Stress did not alter the expression of TGF-beta2 or -beta3 through day 5 post-wounding. Thus, these data indicate that stress delays wound contraction and myofibroblast differentiation, which are likely independent of expression of TGF-beta1, -beta2, and -beta3.

Vascular smooth muscle alpha-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer.

The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor beta1 (TGF-beta1) and accompanied by transient activation of the vascular smooth muscle alpha-actin (SmalphaA) gene. Multiple positive-regulatory elements were identified as essential mediators of basal SmalphaA 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-beta1. However, TGF-beta1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the SmalphaA 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 SmalphaA enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the SmalphaA enhancer required for TGF-beta1 amplification. Additionally, the TGF-beta1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for SmalphaA 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 SmalphaA enhancer. The identification of Sp and Smad proteins as essential, independent activators of the SmalphaA enhancer provides new insight into the poorly understood process of myofibroblast differentiation.