Sertoli-germ cell anchoring junction dynamics in the testis are regulated by an interplay of lipid and protein kinases
Siu,Michelle K.Y.; Wong,Ching-hang; Lee,Will M.; Cheng,Chuen-yan
Journal of Biological Chemistry 280(26): 25029-25047
Publication date: 2005
When Sertoli and germ cells were cocultured in vitro in serum-free chemically defined media, functional anchoring junctions, such as cell-cell intermediate filament based desmosome-like junctions and cell-cell actin-based adherens junctions (AJ) (e.g., ectoplasmic specialization, ES), are formed within 1-2 days. This event was marked by an induction of several protein kinases, such as PI 3-kinase (phosphatidylinositol 3-kinase), phosphorylated protein kinase B (p-PKB, PKB is also known as Akt), PAK-2 (p21-activated kinase-2), and their downstream effector, ERK as well as an increase in PKB intrinsic activity. Studies by immunohistochemistry indeed co-localized PI 3-kinase, p-PKB and PAK to the site of apical ES in the seminiferous epithelium of the rat testis. Furthermore, PI 3-kinase was also co-localized with p-PKB to the same site in the epithelium by fluorescent microscopy consistent with their localization at the ES. These kinases were also shown to associate with ES-associated protein complexes, such as b1 integrin, p-FAK and c-Src, by co-immunoprecipitation, suggesting that the integrin/laminin protein complex at the apical ES is likely utilized these protein kinases as regulatory proteins to modulate Sertoli-germ cell AJ dynamics via the ERK signaling pathway. To further validate this hypothesis, an in vivo model using AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to perturb Sertoli-germ cell anchoring junction function, inducing germ cell loss from the epithelium in adult rats, was used in conjunction with specific inhibitors. Interestingly, the event of germ cell loss from the epithelium induced by AF-2364 in vivo was also associated with an induction of PI 3-kinase, p-PKB, PAK-2, and p-ERK; as well as a surge in intrinsic PAK activity when spermatids began to dislodge from the epithelium. Perhaps the most important of all, it was shown that pre-treatment of rats with wortmannin (a PI 3-kinase inhibitor) or an anti-b1-integrin antibody via intratesticular injection indeed delayed the AF-2364-induced spermatid loss from the epithelium versus rats treated with AF-2364 or IgG alone. In summary, these results thus illustrate that Sertoli-germ cell anchoring junction dynamics in the rat testis are regulated, at least in part, via the b1 integrin-PI 3-kinase-PKB-ERK signaling pathway.