Transforming growth factor-ß1 impairs CFTR-mediated anion secretion across cultured porcine vas deferens epithelial monolayer via the p38 MAPK pathway.

ABSTRACT

The goal of this study was to determine whether transforming growth factor-ß1 (TGF-ß1) affects epithelial cells lining the vas deferens, an organ that is universally affected in cystic fibrosis male patients. In PVD9902 cells, which are derived from porcine vas deferens epithelium, TGF-ß1 exposure significantly reduced short-circuit current (Isc) stimulated by forskolin or a cell membrane-permeant cAMP analog, 8-pCPT-cAMP, suggesting that TGF-ß1 affects targets of the cAMP signaling pathway. Electrophysiological results indicated that TGF-ß1 reduces the magnitude of current inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) channel blockers. Real-time RT-PCR revealed that TGF-ß1 downregulates the abundance of mRNA coding for CFTR, while biotinylation and Western blot showed that TGF-ß1 reduces both total CFTR and apical cell surface CFTR abundance. These results suggest that TGF-ß1 causes a reduction in CFTR expression, which limits CFTR-mediated anion secretion. TGF-ß1-associated attenuation of anion secretion was abrogated by SB431542, a TGF-ß1 receptor I inhibitor. Signaling pathway studies showed that the effect of TGF-ß1 on Isc was reduced by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). TGF-ß1 exposure also increased the amount of phospho-p38 MAPK substantially. In addition, anisomycin, a p38 MAPK activator, mimicked the effect of TGF-ß1, which further suggests that TGF-ß1 affects PVD9902 cells through a p38 MAPK pathway. These observations suggest that TGF-ß1, via TGF-ß1 receptor I and p38 MAPK signaling, reduces CFTR expression to impair CFTR-mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations and ultimately would compromise male fertility.