Neuronal Wiskott-Aldrich syndrome protein (N-WASP) is critical for formation of α-smooth muscle actin filaments during myofibroblast differentiation

Abstract

Myofibroblasts are implicated in pathological stromal responses associated with lung fibrosis. One prominent phenotypic marker of fully differentiated myofibroblasts is the polymerized, thick cytoplasmic filaments containing newly synthesized α-smooth muscle actin (α-SMA). These α-SMA-containing cytoplasmic filaments are important for myofibroblast contractility during tissue remodeling. However, the molecular mechanisms regulating the formation and maturation of α-SMA-containing filaments have not been defined. This study demonstrates a critical role for neuronal Wiskott-Aldrich syndrome protein (N-WASP) in regulating the formation of α-SMA-containing cytoplasmic filaments during myofibroblast differentiation and in myofibroblast contractility. Focal adhesion kinase (FAK) is activated by transforming growth factor-β1 (TGF-β1) and is required for phosphorylation of tyrosine residue 256 (Y256) of N-WASP. Phosphorylation of Y256 of N-WASP is essential for TGF-β1-induced formation of α-SMA-containing cytoplasmic filaments in primary human lung fibroblasts. In addition, we demonstrate that actin-related protein (Arp) 2/3 complex is downstream of N-WASP and mediates the maturation of α-SMA-containing cytoplasmic filaments. Together, this study supports a critical role of N-WASP in integrating FAK and Arp2/3 signaling to mediate formation of α-SMA-containing cytoplasmic filaments during myofibroblast differentiation and maturation.

Therapeutic Targeting of Src Kinase in Myofibroblast Differentiation and Pulmonary Fibrosis

“…FAK has been identified as a key signaling protein in myofibroblast differentiation, as FAK activation is required for TGF-b1-induced myofibroblast differentiation Ding et al, 2008;Grove et al, 2014). FAK promotes the formation of a-SMA-containing fibers through neuronal Wiskott-Aldrich syndrome protein (Cai et al, 2012). FAK inhibition also reduced bleomycin-induced lung fibrosis in mice (Lagares et al, 2012;Ding et al, 2013).…”

“…Equivalent micrograms of whole-cell lysates were electrophoresed on SDS-PAGE, transferred to an Immobilon-P membrane (Millipore), probed with indicated antibodies, and developed with the electrochemiluminescence system (Pharmacia Biotech, Piscataway, NJ). Densitometry analysis of band density was performed as described elsewhere (Cai et al, 2012). The expression of glyceraldehyde 3-phosphate dehydrogenase protein was used as a loading control.…”

“…Collagen-gel contraction assays were performed as described previously elsewhere (Cai et al, 2012). Briefly, collagen gels were cast in six-well plates using type I collagen/Dulbecco's modified Eagle's medium solution composed of bovine skin collagen type I (Invitrogen, Palo Alto, CA).…”

Neuronal Wiskott‐Aldrich syndrome protein regulates TGF‐β1‐mediated lung vascular permeability

“…RMVECs or L2 alveolar epithelial cells were transfected with lentiviral vectors as previously described (33), and the knockdown effects were confirmed by Western blot analysis (for protein level) and by quantitative RT-PCR (for mRNA level) as previously described (34,35). The protein concentration of cell lysates was determined by using a bicinchoninic acid kit (Pierce, Rockford, IL, USA) per the manufacturer's instructions.…”

“…Quantitative RT-PCR was performed as previously described (33). In brief, total RNA was extracted by using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.…”