Pressure activates Src-dependent FAK-Akt and ERK1/2 signaling pathways in rat hepatic stellate cells.
OBJECTIVE
Hepatic fibrosis is associated with elevated sinusoidal pressure, which can be transmitted to the hepatic stellate cells (HSCs) in the perisinusoidal space of Disse. Here, we sought to determine the effects of pressure on cellular growth and Src-dependent signaling pathways in the rat HSCs.


METHODS
Cultured rat HSCs were exposed to pressures (0 to 80 mmHg) by using a pressure-inducing apparatus. The proliferation of the cells was determined by a 5-bromo-2'-deoxy-uridine (BrdU) incorporation assay. Reverse transcription-PCR and Western-blot analysis were used to examine the mRNA and protein levels of representative molecules in Src-dependent signaling pathways.


RESULTS
Pressure at 10 to 20 mmHg applied to the HSCs over 1 h upregulated Brdu incorporation and expression of proliferating cell nuclear antigen (PCNA) and type I collagen, while a higher pressure of 40-80 mmHg did not have noticeable effect. The mRNA level of beta (3) integrin was increased by 1-h application of 5 to 20 mmHg. Immunoblot with phospho-specific antibodies demonstrated the phosphorylation of Src (Tyr418), focal adhesion kinase (FAK) (Tyr397), Akt (Ser473) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) (Thr421/Ser424) was increased in response to 10-mmHg pressure. Herbimycin A, an inhibitor of Src phosphorylation, attenuated the pressure-induced HSC proliferation and phosphorylation of above-mentioned signaling molecules.


CONCLUSIONS
Our data demonstrated that pressure alone induced HSC proliferation involving the activation of Src-dependent signaling pathways.
Wu
Han-Jun
HJ
Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
Zhang
Zong-Qi
ZQ
Yu
Bang
B
Liu
Su
S
Qin
Kai-Rong
KR
Zhu
Liang
L
D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D024042:Collagen Type I / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D055166:Hepatic Stellate Cells / Q000201:enzymology* / Q000378:metabolism; D039661:Integrin beta3 / Q000235:genetics / Q000378:metabolism; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D011312:Pressure; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D016227:Benzoquinones; D024042:Collagen Type I; D004791:Enzyme Inhibitors; D039661:Integrin beta3; D047029:Lactams, Macrocyclic; D018809:Proliferating Cell Nuclear Antigen; D017828:Rifabutin; C020754:herbimycin; D051417:Focal Adhesion Kinase 1; C496052:Ptk2 protein, rat; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3