Fibrotic disorders are characterized by chronic myofibroblast activation and associated with excessive accumulation of contractile protein smooth muscle α-actin (SMαA), an early marker of myofibroblast differentiation. The SMαA gene is tightly regulated at both the transcriptional and post-transcriptional levels via dynamic interplay of cellular activators and repressors. Transforming growth factor β1 (TGFβ1) is a mediator of myofibroblast differentiation in healing wounds. It activates transcription of the SMαA gene through the binding of Smad transcriptional activators at SMαA enhancer region accompanied by displacement of YB-1 repressor from the promoter. Since unchecked TGFβ1 signaling leads to “endless healing”, which is linked to the pathogenesis of many chronic fibrotic diseases, targeting components of TGFβ1 signaling may be an effective strategy for controlling myofibroblast dysfunction in chronic fibrotic disorders, such as pulmonary fibrosis.
I have examined the ability of pro-inflammatory tumor necrosis factor α (TNFα) to antagonize TGFβ1-mediated human pulmonary myofibroblast differentiation. TNFα abrogated TGFβ1-induced SMαA gene at the level of transcription without affecting phosphorylation of regulatory Smads. Intact MEK-ERK kinase signaling was required for myofibroblast repression by TNFα via induction of the Egr-1 DNA-binding protein, a SMαA transcription repressor. Egr-1 bound to the GC-rich SPUR activation element in the SMαA promoter and potently suppressed Smad3- and TGFβ1-mediated transcription. Reduction in Smad binding to the SMαA promoter in TNFα-treated myofibroblasts was observed with an increase in Egr-1 and YB-1 repressor bindings suggesting that the molecular mechanism underlying repression may involve competitive interplay between Egr-1, YB-1 and Smads.
Moreover I have identified a dual-effect of the coagulation protein thrombin on human pulmonary myofibroblast differentiation. Thrombin translationally activates SMαA gene expression within 5 minutes by removing the translational silencer YB-1 from the exon 3-coding sequence in the SMαA mRNA. When thrombin is applied for a longer period, it inhibits TGFβ1-dependent SMαA activation through the following mechanisms, (1) inducing MEK/ERK-dependent Egr-1 repressor; (2) interfering with the nuclear translocation and DNA binding affinity of Smad activators; (3) promoting nuclear uptake and DNA binding of YB-1 repressor. The dual-function of YB-1 at the DNA and mRNA levels and its nucleocytoplasmic shuttling is suggestive of a regulatory loop for coordinating SMαA gene output at both the transcriptional and translational levels in human pulmonary myofibroblast. This hypothetical regulatory loop may help restrict excessive myofibroblast differentiation.
I conclude that the opposing effects of TNFα and thrombin against TGFβ1 on SMαA expression in activated myofibroblasts may be important to restore tissue homeostasis toward the end of wound healing process. This antagonism could be mediated by a dynamic interaction between YB-1, Egr-1 and Smad proteins through their binding at the SMαA promoter. Understanding these molecular mechanisms may be useful in designing new therapeutic targets to offset destructive tissue remodeling in chronic fibrotic diseases.