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(113) THE ADHERENS JUNCTION DYNAMICS IN THE TESTIS ARE REGULATED BY RhoB GUANOSINE TRIPHOSPHATASE VIA THE ROCK/LIMK SIGNALING PATHWAY. Lui, Wing Yee1,2, Lee, Will M2, Cheng, C Yan1, 1 Population Council, New York, NY2 Department of Zoology, Hong Kong, China ABSTRACT- During spermatogenesis, the cell-cell actin-based adherens junctions (AJ), such as ectoplasmic specializations, in the seiminiferous epithelium undergo extensive restructuring to facilitate the movement of developing germ cells across the epithelium. Yet, the mechanism(s) that regulates AJ dynamics is virtually unknown. Other studies have implicated Rho GTPases in the regulation of AJ dynamics via their effects on actin organization in the cytoskeletal network and the redistribution of cadherins at the site of AJs. As such, we sought to investigate the role of Rho GTPases in AJ dynamics in the testis. Using RT-PCR and immunoblottings, Sertoli and germ cells were found to express RhoB GTPase and the RhoB-associated downstream signaling molecules, such as LIMK. Moreover, in vitro assembly of Sertoli-germ cell AJs, but not Sertoli cell tight junctions (TJs), was associated with a transient RhoB induction. Furthermore, disruption of AJs in Sertoli-germ cell cocultures by hypotonic treatment also induced a transient surge in RhoB expression. Also, addition of 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to Sertoli-germ cell cocultures that perturbed Sertoli-germ AJs also induced RhoB expression. To expand these observations to the in vivo level, adult rats (~300 gm b.w.) received a single dose of AF-2364 at 300 mg/kg b.w. by gavage, which was known to induce germ cells loss from the seminiferous epithelium by perturbing AJs, RhoB was induced by ~4-fold within 1 hr. This is long before germ cell loss from the epithelium became visible histologically, which is at least ~4 days after treatment. Studies by immunoblottings and immunohistochemistry to investigate the signaling pathway(s) by which AF-2364 utilizes to perturb AJs have shown that the AF-2364-induced germ cells loss initially activated integrin KEY WORDS: Testis, Adherens junction dynamics, RhoB GTPase, Signal transduction |
2. This in turn activated RhoB, ROCK1, LIMK1 and cofilin, but not p140mDia and profilin. Furthermore, immunoprecipitation studies demonstrated not only the ROCK1 and LIMK1 proteins were stimulated during AF-2364-induced AJ disruption, the phospho-Ser, but not phospho-Tyr, content of LIMK1 was also enhanced. Summary: these results illustrate unequivocally that the AJ dynamics in the testis are regulated, at least in part, via the integrin/RhoB/ROCK/LIMK signaling pathway.