Transforming Growth Factor (beta)3 Regulates the Dynamics of Sertoli Cell Tight Junctions Via the p38 Mitogen-Activated Protein Kinase Pathway

Earlier studies have implicated the significance of transforming growth factor-(beta)3 (TGF(beta)3) in the regulation of Sertoli cell tight junction (TJ) dynamics, possibly via its inhibitory effects on the expression of occludin, claudin-11, and zonula occludens-1 (ZO-1). Yet the mechanism by which TGF(beta)3 regulates the Sertoli cell TJ-permeability barrier is not known. Using techniques of semiquantitative reverse transcription-PCR (RT-PCR), immunoblotting, immunohistochemistry, and inhibitors against different kinases coupled with physiological techniques to assess the Sertoli cell TJ barrier function, it was shown that this TGF(beta)3-induced effect on Sertoli cell TJ dynamics is mediated via the p38 mitogen-activated protein (MAP) kinase pathway. First, the assembly of the Sertoli cell-TJ barrier was shown to be associated with a transient but significant decline in both the TGF(beta)3 production and expression by Sertoli cells. Furthermore, addition of TGF(beta)3 to Sertoli cell cultures during TJ assembly indeed perturbed the TJ barrier with an IC50 at ~9 pM. Second, the TGF(beta)3-induced disruption of the TJ barrier was associated with a transient induction in MEKK2 but not the other upstream signaling molecules that mediate TGF(beta)3 action, such as Smad2, Cdc42, Rac2, and N-Ras, suggesting this effect might be mediated via the p38 MAP kinase pathway. This postulate was confirmed by the observation that TGF(beta)3 also induced the protein level of the activated and phosphorylated form of p38 MAP kinase at the time the TJ barrier was perturbed. Third, and perhaps the most important of all, this TGF(beta)3-mediated inhibitory effect on the TJ barrier and the TGF(beta)3-induced p-p38 MAP kinase production could be blocked by SB202190, a specific p38 MAP kinase inhibitor, but not U0126, a specific MEK1/2 kinase inhibitor. These results thus unequivocally demonstrate that TGF(beta)3 utilizes the p38 MAP kinase pathway to regulate Sertoli cell TJ dynamics.