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1.
J Cell Biochem ; 114(12): 2753-69, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23804301

RESUMO

Myofibroblast differentiation is required for wound healing and accompanied by activation of smooth muscle α-actin (SMαA) gene expression. The stress-response protein, Y-box binding protein-1 (YB-1) binds SMαA mRNA and regulates its translational activity. Activation of SMαA gene expression in human pulmonary myofibroblasts by TGFß1 was associated with formation of denaturation-resistant YB-1 oligomers with selective affinity for a known translation-silencer sequence in SMαA mRNA. We have determined that YB-1 is a substrate for the protein-crosslinking enzyme transglutaminase 2 (TG2) that catalyzes calcium-dependent formation of covalent γ-glutamyl-isopeptide linkages in response to reactive oxygen signaling. TG2 transamidation reactions using intact cells, cell lysates, and recombinant YB-1 revealed covalent crosslinking of the 50 kDa YB-1 polypeptide into protein oligomers that were distributed during SDS-PAGE over a 75-250 kDa size range. In vitro YB-1 transamidation required nanomolar levels of calcium and was enhanced by the presence of SMαA mRNA. In human pulmonary fibroblasts, YB-1 crosslinking was inhibited by (a) anti-oxidant cystamine, (b) the reactive-oxygen antagonist, diphenyleneiodonium, (c) competitive inhibition of TG2 transamidation using the aminyl-surrogate substrate, monodansylcadaverine, and (d) transfection with small-interfering RNA specific for human TG2 mRNA. YB-1 crosslinking was partially reversible as a function of oligomer-substrate availability and TG2 enzyme concentration. Intracellular calcium accumulation and peroxidative stress in injury-activated myofibroblasts may govern SMαA mRNA translational activity during wound healing via TG2-mediated crosslinking of the YB-1 mRNA-binding protein.


Assuntos
Diferenciação Celular/genética , Fator de Crescimento Transformador beta1/metabolismo , Transglutaminases/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Actinas/genética , Cálcio/metabolismo , Proteínas de Ligação ao GTP , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Pulmão/citologia , Pulmão/metabolismo , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Cultura Primária de Células , Biossíntese de Proteínas , Proteína 2 Glutamina gama-Glutamiltransferase , Transdução de Sinais , Transglutaminases/genética
2.
Exp Biol Med (Maywood) ; 237(5): 593-607, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22619371

RESUMO

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.


Assuntos
Transplante de Coração , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Músculo Liso Vascular/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miofibroblastos/metabolismo , Regiões Promotoras Genéticas , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transcrição Gênica , Transplante Heterotópico , Cicatrização , Proteína 1 de Ligação a Y-Box/genética
3.
Am J Physiol Cell Physiol ; 294(3): C702-14, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18344281

RESUMO

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.


Assuntos
Actinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Miócitos Cardíacos/metabolismo , Proteínas Repressoras/metabolismo , Fator de Resposta Sérica/metabolismo , Estresse Fisiológico/metabolismo , Abdome/cirurgia , Actinas/genética , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Fibrose , Rejeição de Enxerto/genética , Rejeição de Enxerto/metabolismo , Transplante de Coração , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/patologia , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Estresse Fisiológico/genética , Estresse Fisiológico/patologia , Estresse Fisiológico/fisiopatologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Transplante Heterotópico , Remodelação Ventricular
4.
Mol Biol Cell ; 15(10): 4532-43, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15282343

RESUMO

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.


Assuntos
Actinas , Proteínas de Ligação a DNA/metabolismo , Músculo Liso Vascular/fisiologia , Fator de Transcrição Sp1/metabolismo , Transativadores/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Células Cultivadas , DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Genes Reporter , Camundongos , Proteínas do Tecido Nervoso , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Smad , Fator de Crescimento Transformador beta1
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