RESUMO
Expression of smooth muscle alpha-actin (SMαA) is essential for myofibroblast-mediated wound contraction following tissue injury. The Pur α/ß and YB-1 transcriptional repressors govern the DNA-binding activity of serum response factor (SRF) and phosphorylated Smad3 (pSmad3) transcriptional activators during induction of SMαA gene expression in human pulmonary myofibroblasts. In quiescent fibroblasts, Pur α exhibited a novel function in enhancing stability of pre-existing SRF complexes with SMαA core promoter DNA, whereas Pur ß was more effective in disrupting SRF-DNA interaction. Pur proteins were less efficient competitors of pre-existing, core-promoter complexes containing both SRF and pSmad3 in nuclear extracts from TGFß1-activated myofibroblasts. TGFß1 signaling dissociated a SRF/Pur protein complex with concurrent formation of a transient pSmad3/MRTF-A/Pur ß complex during early phase myofibroblast differentiation. Pur ß was replaced by Pur α in the pSmad3/MRTF-A complex in mature myofibroblasts. Combining all three repressors potently inhibited SRF and pSmad3 binding to promoter DNA in quiescent fibroblasts and TGFß1-activated myofibroblasts, respectively. The results point to dynamic interplay between transcriptional activators and repressors in regulating SMαA gene output during myofibroblast differentiation. Therapeutic targeting of nucleoprotein complexes regulating the SMαA promoter may prevent excessive myofibroblast accumulation associated with chronic cardiopulmonary fibrosis and dysfunctional tissue remodeling.
Assuntos
Actinas/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Pulmão/metabolismo , Miofibroblastos/metabolismo , Fibrose Pulmonar/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Actinas/genética , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/genética , Fibrose , Humanos , Pulmão/patologia , Miofibroblastos/patologia , Proteínas de Fusão Oncogênica/metabolismo , Fosforilação , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Interferência de RNA , Elemento de Resposta Sérica , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Fatores de Tempo , Transativadores , Fatores de Transcrição/genética , Transfecção , Fator de Crescimento Transformador beta1/metabolismo , Regulação para CimaRESUMO
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/genéticaRESUMO
Myofibroblasts (MFBs) are smooth muscle-like cells that provide contractile force required for tissue repair during wound healing. The leading agonist for MFB differentiation is transforming growth factor ß1 (TGFß1) that induces transcription of genes encoding smooth muscle α-actin (SMαA) and interstitial collagen that are markers for MFB differentiation. TGFß1 augments activation of Smad transcription factors, pro-survival Akt kinase, and p38 MAP kinase as well as Wingless/int (Wnt) developmental signaling. These actions conspire to activate ß-catenin needed for expression of cyclin D, laminin, fibronectin, and metalloproteinases that aid in repairing epithelial cells and their associated basement membranes. Importantly, ß-catenin also provides a feed-forward stimulus that amplifies local TGFß1 autocrine/paracrine signaling causing transition of mesenchymal stromal cells, pericytes, and epithelial cells into contractile MFBs. Complex, mutually interactive mechanisms have evolved that permit several mammalian cell types to activate the SMαA promoter and undergo MFB differentiation. These molecular controls will be reviewed with an emphasis on the dynamic interplay between serum response factor, TGFß1-activated Smads, Wnt-activated ß-catenin, p38/calcium-activated NFAT protein, and the RNA-binding proteins, Purα, Purß, and YB-1, in governing transcriptional and translational control of the SMαA gene in injury-activated MFBs.
