[Angiotensin II and aldosterone stimulate alpha1-(I) procollagen mRNA expression in hepatic stellate cells via activation of ERK1/2 and AP-1].
OBJECTIVE
To investigate the signal transduction mechanism underlying the effects of angiotensin II (Ang II) and aldosterone (Aldo) on the signal passageway of active protein-1 (AP-1).


METHODS
In vitro, Hepatic stellate cells (HSCs) of the line HSC-T6 were cultured and treated with Ang II or Aldo, the principal effector molecules of the renin-angiotensin-aldosterone system (RAAS) for 10, 30, 60, 120, and 180 minutes respectively. The protein expression of phospho-P42/44 was detected by Western blotting. In addition, HSC-T6 cells were preincubated for 60 min with U0126, an inhibitor of MAPK/ERK kinase, irbesartan, an AT-1 receptor blocker, N-acetylcysteine (NAC), antioxidant, angiotensin converting enzyme inhibitor (ACEI), or tumor necrosis factor alpha (TNFalpha) prior to exposure to Ang II or Aldo. Then the protein expression of phospho-P42/44 was measured by Western blotting. The DNA biding activity of AP-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of RT-PCR, the mRNA expression of alpha1 (I) procollagen was detected.


RESULTS
The levels of phopho-ERK1/2 protein increased after the treatment of Ang II and Aldo at all time points and both peaked 10 minutes after (both P < 0.01). The levels of phopho-ERK1/2 protein of the irbesartan + Ang II and U0126 + Ang II groups were significantly lower than that of the Ang II group (both P < 0.01). The level of phopho-ERK1/2 protein of the Ang II group was lower than that of the TNFalpha group, however, was especially significantly lower than that of the Ang II + TNFalpha group (P < 0.01). The level of phopho-ERK1/2 protein of the U0126 + Aldo group was significantly lower than that of the Aldo group (P < 0.01). The phopho-ERK1/2 protein level of the NAC + Aldo group was not significantly different from that of the Aldo group (P > 0.05). The phopho-ERK1/2 protein level of the Aldo group was lower than that of the TNFalpha group, however, was especially significantly lower than that of the Aldo + TNFalpha group (P < 0.01). The AP-1 DNA binding protein increased after the treatment of Ang II and peaked 30 min after. U0126, irbesartan, and NAC, as well as ACEUI, significantly inhibited the increased AP-1 DNA binding activity induced by Ang II. The AP-1 DNA binding protein increased after the treatment of Aldo and peaked twice, 30 min and 240 min after. U0126 and NAC significantly and NAC partly inhibited the increased AP-1 DNA binding activity induced by Aldo.


CONCLUSIONS
Stimulation of HSC by Ang II and Aldo results in activation of AP-1 via ERK1/2 pathway leading to up-regulation of AP-1 target gene alpha1 (I) procollagen mRNA expression.
Li
Xu
X
Institute for Digestive Diseases, Nanfang Hospital, Nanfang Medical University, Guangzhou 510515, China.
Meng
Ying
Y
Cai
Shao-xi
SX
Yang
Xi-shan
XS
Zhang
Yi-jun
YJ
Wu
Ping-sheng
PS
D000450:Aldosterone / Q000494:pharmacology*; D000804:Angiotensin II / Q000494:pharmacology*; D002478:Cells, Cultured; D055166:Hepatic Stellate Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D011347:Procollagen / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D011347:Procollagen; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D000804:Angiotensin II; D000450:Aldosterone