[Effect of TGF-beta1 stimulation on the smad signal transduction pathway of human peritoneal mesothelial cells].
OBJECTIVE
To explore whether the Smad signal transduction pathway in human peritoneal mesothelial cells (HPMCs) influences the process of human peritoneal fibrosis stimulated by TGF-beta1.


METHODS
HPMCs were isolated from normal human omentum and 95% of the primary cultured cells was confirmed to be HPMCs. The third generation of cultured cells was stimulated by 5 ng/ml TGF-beta1. Immunohistochemistry, Western blotting, ELISA, and RT-PCR were employed to investigate the follows: the protein expression of p-Smad2/3 and its migration in HPMCs; the protein and mRNA expressions of SMAD 7 in cells; and the expressions of extracellular fibronectin (FN) protein, intracellular FN mRNA, as well as intracellular collenge-I (COL1) protein and mRNA.


RESULTS
The protein expression of p-Smad2/3 in HPMCs obviously increased 15 min (29% p-Smad2/3-positive cells) after TGF-beta1 stimulation, peaking from 30 min (81% ) to 1 h (84%) and dropping after 2 h (37%); Meanwhile, p-Smad2/3 mainly distributed in cytoplasm at 15 min, concentrated in cell nucleus and peri-nucleus from 30 min to 1 h, and distributed in cytoplasm again at 2 h. The protein expression of SMAD7 in cells obviously increased 24 h after TGF-beta1 stimulation, peaking at 48 h. The mRNA expression of SMAD7 time-dependently increased. The expressions of extracellular FN protein, intracellular FN mRNA, as well as intracellular COL1 protein and mRNA significantly increased and all of them displayed time dependency.


CONCLUSIONS
The SMAD signal transduction pathway of HPMCs can be specifically activated by TGF-beta1 and influence the process of human peritoneal fibrosis. With the stimulation of TGF-beta1, the protein and mRNA expressions of SMAD 7 (an inhibitor of SMAD pathway) significantly increase as a result of feedback.
Zhang
Hao
H
Department of Nephrology, Third Xiangya Hospital, Central South University, Changsha 410013, China. zhanghaoliaoqing@163.com
Liu
Fu-You
FY
Liu
Ying-Hong
YH
D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005355:Fibrosis / Q000209:etiology / Q000473:pathology; D006801:Humans; D010531:Peritoneal Dialysis, Continuous Ambulatory / Q000009:adverse effects; D010537:Peritoneum / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D051785:Smad Proteins / Q000096:biosynthesis* / Q000235:genetics; D051906:Smad7 Protein / Q000096:biosynthesis* / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D012333:RNA, Messenger; C497806:SMAD7 protein, human; D051785:Smad Proteins; D051906:Smad7 Protein; D016212:Transforming Growth Factor beta