Objective: To prove whether the SMAD signal transduction pathway in human peritoneal mesothelial cells (HPMCs) influenced the process of human peritoneal fibrosis stimulated by TGF-β1.
Methods: HPMCs were isolated from normal human omentum and the third generation cells were stimulated by 5 ng/ml TGF-β1. Immunohistochemistry, Western blotting, ELISA and RT-PCR were employed to investigate the protein expression of p-Smad2/3 and the protein and mRNA expressions of SMAD 7, fibronectin (FN) and collagen-I (COL1).
Results: The protein expression of p-Smad2/3 in HPMCs was remarkably increased 15 min (29% p-Smad2/3-positive cells) after TGF-β1 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 SMAD 7 in cells was obviously increased 24 h after TGF-β1 stimulation, peaking at 48 h. The mRNA expression of SMAD7 was time-dependently increased. The expressions of extracellular FN protein, intracellular FN mRNA, as well as intracellular COL1 protein and mRNA were significantly increased and all of them displayed time dependency.
Conclusions: The SMAD signal transduction pathway of HPMCs can be specifically activated by TGF-β1 and influence the process of human peritoneal fibrosis. The protein and mRNA expression of SMAD 7 (an inhibitor of SMAD pathway) are significantly increased as a result of feedback.
Keywords: Smad2/3; Smad7; TGF-β1; extracellular matrix; mesothelial cells.