Endothelin-1 and Transforming Growth Factor-β1 Independently Induce Fibroblast Resistance to Apoptosis via AKT Activation

Abstract

Myofibroblast apoptosis is critical for the normal resolution of wound repair responses, and impaired myofibroblast apoptosis is associated with tissue fibrosis. Lung expression of endothelin (ET)-1, a soluble peptide implicated in fibrogenesis, is increased in murine models of pulmonary fibrosis and in the lungs of humans with pulmonary fibrosis. Mechanistically, ET-1 has been shown to induce fibroblast proliferation, differentiation, contraction, and collagen synthesis. In this study, we examined the role ET-1 in the regulation of lung fibroblast survival and apoptosis. ET-1 rapidly activates the prosurvival phosphatidylinositol 39-OH kinase (PI3K)/AKT signaling pathway in normal and fibrotic human lung fibroblasts. ET-1-induced activation of PI3K/AKT is dependent on p38 mitogenactivated protein kinase (MAPK), but not extracellular signalregulated kinase (ERK) 1/2, JNK, or transforming growth factor (TGF)-b1. Activation of the PI3K/AKT pathway by ET-1 inhibits fibroblast apoptosis, and this inhibition is reversed by blockade of p38 MAPK or PI3K. TGF-b1 has been shown to attenuate myofibroblast apoptosis through the p38 MAPK-dependent secretion of a soluble factor, which activates PI3K/AKT. In this study, we show that, although TGF-b1 induces fibroblast synthesis and secretion of ET-1, TGF-b1 activation of PI3K/AKT is not dependent on ET-1. We conclude that ET-1 and TGF-b1 independently promote fibroblast resistance to apoptosis through signaling pathways involving p38 MAPK and PI3K/AKT. These findings suggest the potential for novel therapies targeting the convergence of prosurvival signaling pathways activated by these two profibrotic mediators.

X-Linked Inhibitor of Apoptosis Regulates Lung Fibroblast Resistance to Fas-Mediated Apoptosis

“…There was no statistically significant difference between the IPF fibroblasts and any of the normal lung fibroblast lines after this combined exposure. Previous studies have shown that cycloheximide alone is insufficient to induce fibroblast apoptosis (11,35), a finding that was confirmed in a subset of experiments for each of the different fibroblast populations studied. Together, these findings are consistent with prior studies showing that normal fibroblasts are relatively resistant to Fas-mediated apoptosis, that IPF lung fibroblasts have increased resistance to Fas-mediated apoptosis compared with normal lung fibroblasts, and that cycloheximide "sensitizes" normal and IPF fibroblasts to undergo robust apoptosis upon Fas activation (8,10,12,(33)(34)(35)(37)(38)(39).…”

“…Whole cell lysates were collected and subjected to SDS-PAGE and Western blotting as previously described (7,11). All Western blots were stripped and reprobed for GAPDH.…”

“…Fibroblasts have an inherent resistance to Fas-mediated apoptosis in vitro, although the response to Fas activation can be enhanced by (1) treatment with the protein translation inhibitor cycloheximide, (2) treatment with the antifibrotic mediator PGE 2 , or (3) treatment with proinflammatory cytokines (10,11,(33)(34)(35)(36). We assessed apoptosis in normal lung fibroblast cell lines (IMR-90 and CCL-210) and patient-derived primary fibroblasts from normal and IPF tissue after exposure to a Fas-activating antibody (Fas-Ab) with and without cycloheximide.…”

“…The resolution phase of normal wound repair coincides with the reduction of fibroblast numbers by apoptosis, but the triggers of apoptosis in normal wound repair and the mechanisms underlying the persistence of fibroblasts during fibrotic wound repair remain poorly understood (5,6). The profibrotic mediators transforming growth factor (TGF)b-1 and endothelin (ET)-1 have been found to promote resistance to apoptotic stimuli in fibroblasts, and the antifibrotic lipid mediator prostaglandin (PG)E 2 has been shown to increase the responsiveness of these cells to apoptotic stimuli, supporting a role for dysregulation of fibroblast apoptosis in the pathogenesis of fibrosis (7)(8)(9)(10)(11)(12).…”

Mechanisms of Lung Fibrosis Resolution

“…First, soluble profibrotic mediators that activate myofibroblasts, such as TGF-b1, also promote resistance to apoptosis. 57 Second, prosurvival protein kinases, including phosphatidylinositol-3 kinases, which are activated by soluble fibrogenic mediators in normal lung fibroblasts, are expressed at higher levels in IPF fibroblastic foci. 73,74 Third, endogenous inhibitors of apoptosis, such as Fas-like IL 1b-converting enzymeeinhibitory protein and X-linked inhibitor of apoptosis, are induced by soluble profibrotic mediators and expressed at increased levels in fibrotic lung fibroblasts.…”