Myofibroblast Differentiation by Transforming Growth Factor-ॆ1 Is Dependent on Cell Adhesion and Integrin Signaling via Focal Adhesion Kinase

Abstract

Myofibroblast differentiation and activation by transforming growth factor-beta1 (TGF-beta1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-beta1 and adhesion-dependent signals. TGF-beta1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-beta receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-beta1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-beta1-induced alpha-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of alpha-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-beta1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-beta1/Smad signaling.

Combinatorial activation of FAK and AKT by transforming growth factor-β1 confers an anoikis-resistant phenotype to myofibroblasts

“…Our studies in human lung fibroblasts have demonstrated that early and rapid activation of p38 MAPK by TGF-β1 is essential for the production of an autocrine growth factor that mediates activation of the PI3K-AKT pathway [8]. Additionally, our prior studies have demonstrated delayed, but sustained, activation of FAK that is required for stable induction of myofibroblast differentiation by TGF-β1 [12]. A specific requirement for SMAD3-dependent signalling in the activation of FAK and AKT, potential crosstalk between SMAD-dependent and -independent signalling, or their role (s) in the anoikis-resistance of myofibroblasts have not been previously reported.…”

“…Our previous studies showed that AKT activation by TGF-β1 in human lung fibroblasts is dependent on early p38-MAPK [8]. Additionally, FAK activation by TGF-β1 is mediated via a transcription-dependent process that is associated with upregulation of fibronectin and its integrin receptor subunits [12]. Crosstalk between these two pro-survival pathways and the specific role(s) of SMAD3, a key regulator of TGF-β1 pro-fibrotic signalling [24], in the delayed activation of FAK and AKT have not been defined.…”

“…TGF-β1 signalling in fibroblasts involves early receptor(s)-mediated SMAD-dependent andindependent pathways with more delayed activation of the nonreceptor protein kinases, FAK and AKT [8,12]. Our previous studies showed that AKT activation by TGF-β1 in human lung fibroblasts is dependent on early p38-MAPK [8].…”

“…We have previously shown that myofibroblast differentiation by TGF-β1 is dependent on adhesion-mediated signalling through focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase that is upregulated by TGF-β1 [12]. FAK is also an important regulator of cell survival signalling [13].…”

Role for ephrinB2 in postnatal lung alveolar development and elastic matrix integrity

“…In those mice, alveolarization is delayed, but they finally form normal lungs (Sonja Mund and J.C.S., unpublished results). The alterations in cellular number and phenotype seen in older mutant lungs could in principle be a secondary consequence of the ECM alterations present at P1 (e.g., McGowan and Torday, 1997;Neptune et al, 2003;Thannickal et al, 2003); or could reflect an independent requirement for ephrinB2 function.…”