Phenotypic modulation of human urinary tract stroma-derived fibroblasts by transforming growth factor beta3.

ABSTRACT

OBJECTIVES: Animal models have described critical roles for transforming growth factor beta (TGFbeta) isoforms in modulating urinary tract stroma phenotype. TGFbeta3 is of particular interest because it may regulate TGFbeta1 and TGFbeta2 expression, but its modulatory affect has not been so well characterized in human cells. In this study, we aim to determine whether TGFbeta3 treatment induced differentiation of human urinary tract stroma-derived fibroblasts to a smooth muscle-like phenotype. METHODS: We established cultures of human urinary tract stroma-derived fibroblasts and studied the effects of TGFbeta3 treatment using proliferation assays, cell cycle analysis, immunocytochemistry, and Western blotting for expression of differentiation marker and downstream regulators, and fura-2 fluorescence to study the effects on intracellular calcium. RESULTS: TGFbeta3 treatment induced proliferation that peaked at 72 hours, followed by enhanced expression of alpha-smooth muscle actin (alpha-SMA) with a maximal 3.4-fold increase at 168 hours. TGFbeta3 treatment decreased resting [Ca(2+)](i) by 70% and caused a 95% decrease in stimulated internal Ca(2+) release regulated by the sarcoplasmic/endoplasmic calcium-ATPase pump. These effects were associated with upregulation of nuclear activator of T cells -1 (NFAT), a known regulator of cell differentiation. CONCLUSIONS: TGFbeta3 treatment causes a time-specific response in the presence of serum, whereby fibroblasts initially proliferate and subsequently differentiate to a smooth muscle-like phenotype. This sequence was associated with stabilization of [Ca(2+)](i) stores, suggesting a role in the induction of hyperplasia and reduction of contractility; phenomena associated with a number of urinary tract pathologies.