Fibronectin increases survival of rat hepatic stellate cells--a novel profibrogenic mechanism of fibronectin.
The aims of this study were to determine whether fibronectin increases survival of hepatic stellate cells (HSCs) in starving conditions, and to identify the signal transduction pathways involved in this effect.


METHODS
Primary culture of rat HSCs were plated on fibronectin-uncoated or coated culture wells, and grown in the presence of 0.2% or 20% fetal calf serum. Cell apoptosis was measured by an ELISA procedure. Signal transduction pathways were analyzed by inhibiting major intracellular transduction pathways with appropriated inhibitors and by detecting phosphorylated proteins.


RESULTS
Fibronectin increased survival of serum deprived HSCs. This effect was abrogated by the presence of the RGD peptide, by silencing FAK expression, and by inhibiting PI3K with LY294002 or wortmannin. Growth of HSCs on fibronectin induced integrin alpha5beta1 expression, tyr397, ser473, and ser136 phosphorylation of FAK, Akt, and Bad, respectively, and the binding of phosphorylated Bad to 14-3-3 proteins. Likewise, fibronectin increased Bcl2/Bax ratio and reduced release of mitochondrial cytochrome c into the cytoplasm, formation of apoptosome, and caspase 9 and 3 activity. These effects were avoided by treatment of cells with PI3K inhibitors.


CONCLUSIONS
Fibronectin increases survival of HSCs via a pathway involving integrin alpha5beta1 receptors, FAK, PI3K, Akt and proteins of Bcl2 family.
Rodríguez-Juan
Cristina
C
Department of Gastroenterology, Research Center, University Hospital 12 de Octubre, Madrid, Spain.
de la Torre
Paz
P
García-Ruiz
Inmaculada
I
Díaz-Sanjuán
Teresa
T
Muñoz-Yagüe
Teresa
T
Gómez-Izquierdo
Erica
E
Solís-Muñoz
Pablo
P
Solís-Herruzo
José A
JA
D048948:14-3-3 Proteins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D018929:Cell Culture Techniques; D016923:Cell Death; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D020099:Coated Materials, Biocompatible / Q000378:metabolism; D016895:Culture Media, Serum-Free; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005353:Fibronectins / Q000378:metabolism*; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D039081:Integrin alpha5beta1 / Q000378:metabolism; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D048948:14-3-3 Proteins; D002867:Chromones; D020099:Coated Materials, Biocompatible; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D005353:Fibronectins; D039081:Integrin alpha5beta1; D009025:Morpholines; D009842:Oligopeptides; D000081082:Phosphoinositide-3 Kinase Inhibitors; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C047981:arginyl-glycyl-aspartic acid; D053148:Caspase 3; D053453:Caspase 9