Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts

Abstract

The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including ␣-smooth muscle actin (␣-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/ phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce ␣-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of ␣-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins ␣-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis. INTRODUCTIONA complex histological and architectural structure is a prerequisite for effective lung function. In the lung, specialized structures, the alveoli, increase the surface area of the lung, allowing for efficient gas exchange. The maintenance of these specialized structures is in turn dependent on the underlying connective tissue, comprised principally of fibroblasts and extracellular matrix (ECM; for review, see Gadek et al., 1984), which is essential for the mechanical and structural integrity of the lung. As a response to environmental insults, or as a consequence of local inflammatory processes, structural damage to the lung can occur, resulting in a wound healing response. This response consists of an integrated series of biochemical, immunological, and structural changes that result in the de novo synthesis of a new epithelium, blood vessels, and connective tissue (Razzaque and Taguchi, 2003). The proper repair of connective tissue requires synthesis of new ECM components, such as collagen and fibronectin (Badylak, 2002). In addition, repair of connective tissue requires the proper reconstitution of its support function; that is, an appropriate tensile strength must be recreated. This tensile strength results from the remodeling of the newly formed ECM through a combination of cell locomotion and translocation of the flexible collage...

Contribution of activin receptor–like kinase 5 (transforming growth factor β receptor type I) signaling to the fibrotic phenotype of scleroderma fibroblasts

“…Our results are consistent with the notion that hyperactive TGF␤ receptor signaling is insufficient to explain the complete dcSSc phenotype (4,13,17) and that other pathways also contribute to dcSSc (22). However, these results strongly suggest that TGF␤ receptor antagonists are likely to be of benefit in combating particular, specific phenotypic aspects of dermal fibrosis, namely, the excessive production, adhesion, and contraction of ECM, in patients with early-onset dcSSc.…”

“…Dermal fibroblasts derived from normal subjects and from patients with dcSSc were seeded into a floating 3-dimensional collagen gel matrix and incubated for 24 hours as described in Patients Methods. Under these conditions, the fibroblasts actively mediate remodeling of the collagen gel matrix (22,26,27). The weight of the contracted collagen gel was then measured.…”

“…Experiments were performed essentially as described previously (22). Briefly, 24-well tissue culture plates were precoated with BSA.…”

“…The solidified gel was then detached from the tissue culture plate and incubated for 24 hours in the presence of 0.5% FBS. We then assessed collagen gel contraction by measuring the size and weight of the contracted gel (22).…”

Periostin differentially induces proliferation, contraction and apoptosis of primary Dupuytren's disease and adjacent palmar fascia cells

“…Myofibroblasts have been shown to utilize the PI3 kinase/Akt signaling pathway to avoid apoptosis in abnormal scarring [66][67][68] and periostin signaling has been shown to promote avoidance of apoptosis through this pathway [29,37]. While there was a consistent trend towards a decrease in DD cells apoptosis with periostin treatment, this did not achieve statistical significance in our in vitro collagen culture conditions (DD cells treated with vehicle vs. 0.5 μg/ml recombinant periostin, p=0.075; PF cells treated with vehicle vs. 2.0 μg/ml recombinant periostin, p=0.065).…”