Initial signaling of the fibronectin receptor (α5β1 integrin) in hepatic stellate cells is independent of tyrosine phosphorylation

Background/Aims: Activation of hepatic stellate cells (HSC) plays an integral role in hepatic fibrosis. HSC activation increases fibronectin (α5β1) receptor expression and interactions between α5β1 and the extracellular matrix increase collagen synthesis. It is unclear how signaling by the α5β1 receptor initiates these changes. We aimed to determine the signaling cascade after α5β1 stimulation in activated HSC. Methods: HSC were isolated from male Sprague–Dawley rats. Activated HSC were exposed to beads coated with fibronectin (ligand for α5β1) or -polylysine (inert control). HSC were stained with FTC-labeled antibodies against classes of signaling molecules. Tyrosine phosphorylation was blocked using genistein or herbimycin A. The fraction of beads with localized immunostaining (indicating accumulation of signaling protein) was determined. Results: The majority of cytoskeletal proteins, Src substrates, Src kinases and members of the ERK and JNK signaling molecule families require actin cytoskeletal organization and tyrosine-kinase-mediated phosphorylation to accumulate. Several proteins (e.g. tensin, FAK) accumulated in the absence of tyrosine phosphorylation. Conclusions: The α5β1 integrin–ligand interaction induces accumulation of cytoskeletal molecules, activating multiple kinase pathways. Initial integrin signaling by α5β1 are associated with cytoskeletal proteins and are independent of tyrosine phosphorylation. We suggest that there may be cytoskeletal changes that may be targeted to diminish HSC activation.