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J. Biol. Chem., Vol. 280, Issue 26, 25029-25047, July 1, 2005

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Sertoli-Germ Cell Anchoring Junction Dynamics in the Testis Are Regulated by an Interplay of Lipid and Protein Kinases^*

Michelle K. Y. Siu**, Ching-hang Wong, Will M. Lee¶, and C. Yan Cheng||

From the Center for Biomedical Research, Population Council, New York, New York 10021 and the ^¶Department of Zoology, University of Hong Kong, Hong Kong, China

When Sertoli and germ cells were co-cultured in vitro in serum-free chemically defined medium, functional anchoring junctions such as cell-cell intermediate filament-based desmosome-like junctions and cell-cell actin-based adherens junctions (e.g. ectoplasmic specialization (ES)) were formed within 1-2 days. This event was marked by the induction of several protein kinases such as phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (PKB; also known as Akt), p21-activated kinase-2 (PAK-2), and their downstream effector (ERK) as well as an increase in PKB intrinsic activity. PI3K, phospho (p)-PKB, and PAK were co-localized to the site of apical ES in the seminiferous epithelium of the rat testis in immunohistochemistry studies. Furthermore, PI3K also co-localized with p-PKB to the same site in the epithelium as determined by fluorescence microscopy, consistent with their localization at the ES. These kinases were shown to associate with ES-associated proteins such as 1-integrin, phosphorylated focal adhesion kinase, and c-Src by co-immunoprecipitation, suggesting that the integrin·laminin protein complex at the apical ES likely utilizes these protein kinases as regulatory proteins to modulate Sertoli-germ cell adherens junction dynamics via the ERK signaling pathway. To validate this hypothesis further, an in vivo model using AF-2364 (1-(2,4-dichlorobenzyl)-1H-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 induced by AF-2364 in vivo was also associated with induction of PI3K, p-PKB, PAK-2, and p-ERK as well as a surge in intrinsic PKB activity. Perhaps the most important of all, pretreatment of rats with wortmannin (a PI3K inhibitor) or anti-1-integrin antibody via intratesticular injection indeed delayed AF-2364-induced spermatid loss from the epithelium. In summary, these results illustrate that Sertoli-germ cell anchoring junction dynamics in the testis are regulated, at least in part, via the 1-integrin/PI3K/PKB/ERK signaling pathway.

Received for publication, January 28, 2005 , and in revised form, April 22, 2005.

^* This work was supported in part by NICHD Grants U01 HD045908 and U54 HD029990 (Project 3) from the National Institutes of Health (to C. Y. C.), CONRAD Program Grant CICCR CIG 01-72 (to C. Y. C.), and Hong Kong Research Grant Council Grants HKU 7194/01M and 7413/04M (to W. M. L. and C. Y. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Pathology, Faculty of Medicine, University of Hong Kong, Hong Kong, China.

^** Supported by a postdoctoral fellowship from the University of Hong Kong when this work was conducted in the CYC Laboratory at the Population Council. Also the recipient of the University of Hong Kong Committee for Research and Conference Grant.

^|| To whom correspondence and reprint requests should be addressed: Center for Biomedical Research, Population Council, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8738; Fax: 212-327-8733; E-mail: Y-Cheng{at}popcbr.rockefeller.edu.

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