The online version of this article (doi:10.1186/1465-9921-15-19) contains supplementary material, which is available to authorized users.
The authors have no competing interests.
MFN Interpreted results of experiments, drafted manuscript, edited and revised manuscript, approved final version of manuscript. AN Performed experiments, analyzed data, interpreted results of experiments, prepared figures, edited and revised manuscript, approved final version of manuscript. MHR Performed experiments and approved final version of manuscript. REP Performed experiments and approved final version of manuscript. SMM Performed experiments, edited and revised manuscript, approved final version of manuscript. IIE Conception and design of research, analyzed data, interpreted results of experiments, edited and revised manuscript, approved final version of manuscript.
Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known.
TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis.
The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis.
TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.
Additional file 1: Supplemental methods and figures. Western-blot-Cells transfected with control siRNA or TRIP-1 siRNA were lysed 48 hours after transfection, and western-blot was performed using antibodies against caldesmon (rabbit monoclonal E89 from Abcam, 1:10,000) and calponin (rabbit monoclonal EP798Y from Abcam, 1:10,000). Real-time PCR-RNA was obtained from cells that had been transfected with control siRNA or TRIP-1 siRNA and treated with TGFβ1 5 ng/ml for 48 hours. Total RNA was isolated using Trizol, DNAse-treated, then processed for First-Strand Synthesis with SuperScript (Life Technologies) and real-time PCR was performed on BioRad MyIQ system using IQ SyberGreen-Supermix from BioRad, Inc. Experiment was performed three times and samples were run in triplicate. Figure Legends: Figure S1. Expression of caldesmon and calponin by western-blot is increased in cells transfected with TRIP-1 siRNA. Figure S2. Real-time PCR analysis of TGF-β -dependent genes A) PAI-1 and B) CTGF in cells with decreased TRIP-1 expression, with or without TGFβ 48 hours treatment. Figure S3. AKT inhibitor II treatment decreases phosphorylation of AKT. Cells in complete media were treated with vehicle (DMSO) or 40 μM AKT inhibitor for 2 hours, then samples were made and SDS PAGE gels run, and Western-Blot performed for P-AKT and Tubulin. (DOC 76 KB)
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- TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation
Michael F Nyp
Mohammad H Rezaiekhaligh
Ricardo E Perez
Sherry M Mabry
Ikechukwu I Ekekezie
- BioMed Central
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