Initial signaling of the fibronectin receptor (alpha5beta1 integrin) in hepatic stellate cells is independent of tyrosine phosphorylation.
OBJECTIVE
Activation of hepatic stellate cells (HSC) plays an integral role in hepatic fibrosis. HSC activation increases fibronectin (alpha(5)beta(1)) receptor expression and interactions between alpha(5)beta(1) and the extracellular matrix increase collagen synthesis. It is unclear how signaling by the alpha(5)beta(1) receptor initiates these changes. We aimed to determine the signaling cascade after alpha(5)beta(1) stimulation in activated HSC.


METHODS
HSC were isolated from male Sprague-Dawley rats. Activated HSC were exposed to beads coated with fibronectin (ligand for alpha(5)beta(1)) or D-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 alpha(5)beta(1) integrin-ligand interaction induces accumulation of cytoskeletal molecules, activating multiple kinase pathways. Initial integrin signaling by alpha(5)beta(1) 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.
Milliano
M T
MT
Division of Gastroenterology and Hepatology, Department of Internal Medicine, University Liver Center, Saint Louis University School of Medicine, 9 South FDT, 3635 Vista Avenue at Grand Boulevard, St. Louis, MO 63110-0250, USA.
Luxon
B A
BA
D000818:Animals; D016227:Benzoquinones; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D019833:Genistein / Q000494:pharmacology; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D047029:Lactams, Macrocyclic; D008024:Ligands; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008863:Microspheres; D010766:Phosphorylation; D011107:Polylysine; D011809:Quinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D039081:Integrin alpha5beta1; D047029:Lactams, Macrocyclic; D008024:Ligands; D011809:Quinones; D017828:Rifabutin; D011107:Polylysine; D014443:Tyrosine; C020754:herbimycin; D019833:Genistein