Sertoli-germ cell adherens junction dynamics in the testis are regulated by RhoB GTPase via the ROCK/LIMK signaling pathway (HTML)
Lui,Wing-Yee; Lee,William M.; Cheng,Chuen-yan
Biology of Reproduction 68(6): 2189-2206
Publication date: 2003
During spermatogenesis, cell-cell actin-based adherens junctions(AJs), such as ectoplasmic specializations (ESs), between Sertoliand germ cells undergo extensive restructuring in the seminiferousepithelium to facilitate germ cell movement across the epithelium.Although the mechanism(s) that regulates AJ dynamics in thetestis is virtually unknown, Rho GTPases have been implicatedin the regulation of these events in other epithelia. Studieshave shown that the in vitro assembly of the Sertoli-germ cellAJs but not of the Sertoli cell tight junctions (TJs) is associatedwith a transient but significant induction of RhoB. Immunohistochemistryhas shown that the localization of RhoB in the seminiferousepithelium is stage specific, being lowest in stages VII-VIIIprior to spermiation, and displays cell-specific associationduring the epithelial cycle. Throughout the cycle, RhoB waslocalized near the site of basal and apical ESs but was restrictedto the periphery of the nuclei in elongating (but not elongated)spermatids, spermatocytes, and Sertoli cells. However, RhoBwas not detected near the site of apical ESs at stages VII-VIII.Furthermore, disruption of AJs in Sertoli-germ cell cocultureseither by hypotonic treatment or by treatment with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide(AF-2364) also induced RhoB expression. When adult rats weretreated with AF-2364 to perturb Sertoli-germ cell AJs in vivo,a 4-fold induction in RhoB in the testis, but not in kidneyand brain, was detected within 1 h, at least 1-4 daysbefore germ cell loss from the epithelium could be detectedby histological analysis. The signaling pathway(s) by whichAF-2364 perturbed the Sertoli-germ cell AJs apparently beganwith an initial activation of integrin, which in turn activatedRhoB, ROCK1, (ho-assiated protein inase 1, also called ROKß), LIMK1 (LIM kinase 1, also called in-11 sl-1 ec3 kinase 1), and cofilin but not p140mDia and profilin via phosphorylation. Immunoprecipitationand immunoblots revealed that the induction of LIMK1 was mediatedvia an increase in its phospho-Ser but not phospho-Tyr content.Furthermore, Y-27632 ([(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide,2HCl]), a specific ROCK inhibitor, could effectively delay theAF-2364-induced germ cell loss from the seminiferous epitheliumin vivo, illustrating that the integrin/RhoB/ROCK/LIMK pathwayindeed plays a crucial role in the regulation of Sertoli-germcell AJ dynamics. The fact that the RhoB pathway in the kidneyand brain was not activated suggests that AF-2364 exerts itseffects primarily at the testis-specific ES multiprotein complexstructures between Sertoli cells and spermatids. In summary,this report illustrates that Sertoli germ cell AJ dynamics areregulated, at least in part, via the integrin/ROCK/LIMK/cofilinsignaling pathway.