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Regulation of blood-testis barrier dynamics by TGF-beta3 is a Cdc42-dependent protein trafficking event.

ABSTRACT: In the testis, the blood-testis barrier (BTB) is constituted by specialized junctions between adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. Although the BTB is one of the tightest blood-tissue barriers in the mammalian body, it undergoes extensive restructuring at stage VIII of the seminiferous epithelial cycle to facilitate the transit of preleptotene spermatocytes. Thus, meiosis and postmeiotic germ cell development take place in the seminiferous epithelium behind the BTB. Cytokines (e.g., TGF-beta3) are known to regulate BTB dynamics by enhancing the endocytosis of integral membrane proteins and their intracellular degradation. This thus reduces the levels of proteins above the spermatocytes in transit at the BTB, causing its disruption after testosterone-induced new tight junction (TJ) fibrils are formed behind these cells. By using Sertoli cells cultured in vitro with an established TJ permeability barrier that mimicked the BTB in vivo, Cdc42 was shown to be a crucial regulator that mediated the TGF-beta3-induced BTB disruption. TGF-beta3 was shown to activate Cdc42 to its active GTP-bound form. However, an inactivation of Cdc42 by overexpressing its dominant-negative mutant T17N in Sertoli cell epithelium was shown to block the TGF-beta3-induced acceleration in protein endocytosis. Consequently, this prevented the disruption of Sertoli cell TJ permeability barrier and redistribution of TJ proteins (e.g., CAR and ZO-1) from the cell-cell interface to cell cytosol caused by TGF-beta3. In summary, Cdc42 is a crucial regulatory component in the TGF-beta3-mediated cascade of events that leads to the disruption of the TJ fibrils above the preleptotene spermatocytes to facilitate their transit.

SUBMITTER: Wong EW

PROVIDER: S-EPMC2895131 | biostudies-other | 2010 Jun

REPOSITORIES: biostudies-other

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Regulation of blood-testis barrier dynamics by TGF-beta3 is a Cdc42-dependent protein trafficking event.

Wong Elissa W P EW Mruk Dolores D DD Lee Will M WM Cheng C Yan CY

Proceedings of the National Academy of Sciences of the United States of America 20100607 25

In the testis, the blood-testis barrier (BTB) is constituted by specialized junctions between adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. Although the BTB is one of the tightest blood-tissue barriers in the mammalian body, it undergoes extensive restructuring at stage VIII of the seminiferous epithelial cycle to facilitate the transit of preleptotene spermatocytes. Thus, meiosis and postmeiotic germ cell development take place in the seminiferous epithelium ...[more]

PMID: 20534521

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Project description:In adult mammals such as rats, the blood-testis barrier (BTB) conferred by adjacent Sertoli cells in the seminiferous epithelium segregates post-meiotic germ cell development from the systemic circulation and is one of the tightest blood-tissue barriers. Yet it must "open" transiently at stages VIII to IX of the epithelial cycle to accommodate the migration of preleptotene/leptotene spermatocytes. While this is a vital event of spermatogenesis, the mechanism(s) that regulates BTB dynamics is virtually unknown. Recent studies have suggested that transforming growth factor-beta3 (TGF-beta3) and tumor necrosis factor alpha (TNFalpha) secreted by Sertoli and germ cells into the microenvironment of the BTB are capable of inducing reversible BTB disruption in vivo, apparently by reducing the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB via the p38 mitogen activated protein (MAP) kinase signaling pathway. In this study, local administration of TGF-beta3 (200 ng/testis) to the testis was shown to reversibly perturb the BTB integrity in vivo. We next sought to delineate the mechanism by which these cytokines maintain the steady-state level of integral membrane proteins: occludin, junctional adhesion molecule-A (JAM-A) and N-cadherin at the BTB. Primary Sertoli cells cultured in vitro were shown to establish intact tight junctions and functional BTB within two days when assessed by transepithelial electrical resistance (TER) measurement across the cell epithelium. Sertoli cell integral membrane protein internalization at the BTB was assessed by biotinylation of cell surface proteins, to be followed by tracking the endocytosed/biotinylated proteins by using specific antibodies. Both TGF-beta3 (3 ng/ml) and TNFalpha (10 ng/ml) were shown to significantly accelerate the kinetics of internalization of JAM-A, N-cadherin, and occludin versus controls. Treatment of cells with phenylarsine oxide (PAO) at 10 microM that blocks clathrin-mediated endocytosis was shown to inhibit the TGF-beta3-induced protein internalization. This inhibition of TGF-beta3-mediated protein endocytosis was further validated by silencing of clathrin. The specific effect of TGF-beta3 on protein internalization was further confirmed by RNAi using specific TGF-beta receptor I (TbetaR1) siRNA duplexes. When TbetaR1 was knocked down, the TGF-beta3-induced increase in the kinetics of JAM-A and occludin endocytosis was abolished, making them indistinguishable from controls, illustrating the specificity of the TGF-beta3 effects on protein endocytosis. In summary, this report demonstrates for the first time that BTB dynamics are regulated by TGF-beta3 and TNFalpha via an enhancement of protein endocytosis at the BTB.

Project description:The intricate interaction between protein endocytosis, transcytosis, recycling and endosome- or ubiquitin-mediated protein degradation determines the junction integrity in epithelial cells including Sertoli cells at the blood-testis barrier (BTB). Studies have shown that androgens and cytokines (e.g., TGF-?3) that are known to promote and disrupt BTB integrity, respectively, accelerate protein endocytosis at the BTB. We hypothesized that testosterone-induced endocytosed proteins are transcytosed and recycled back to the Sertoli cell surface, whereas cytokine-induced endocytosed proteins are degraded so that androgens and cytokines have opposing effects on BTB integrity. Herein, we report that both testosterone and TGF-?3 induced the steady-state level of clathrin, an endocytic vesicle protein. Testosterone and TGF-?3 also induced the association between internalized occludin (a BTB integral membrane protein) and clathrin, as well as early endosome antigen-1 (EEA-1). Interestingly, testosterone, but not TGF-?3, also induced the levels of proteins that regulate protein transcytosis (e.g., caveolin-1) and recycling (e.g., Rab11), and their association with internalized occludin and N-cadherin from the cell surface. In contrast, TGF-?3, but not testosterone, induced the level of ubiquitin-conjugating enzyme E2 J1 (Ube2j1), a protein crucial to the intracellular protein degradation pathway, and its association with internalized occludin. Based on these findings and recent reports in the field, we hypothesize that the concerted effects of testosterone and TGF-?3 likely facilitate the transit of preleptotene spermatocytes at the BTB while maintaining the immunological barrier in that testosterone induces the assembly of "new" tight junction (TJ)-fibrils below migrating spermatocytes via protein transcytosis and recycling before cytokines induce the disassembly of "old" TJ-fibrils above spermatocytes via endocytic vesicle-mediated degradation of internalized proteins. This thus provides a unique mechanism in the testis to facilitate the transit of preleptotene spermatocytes, many of which are connected in "clones" via cytoplasmic bridges, at the BTB while maintaining the immunological barrier during stage VIII of the seminiferous epithelial cycle of spermatogenesis.

Project description:The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the "opening" and "closing" of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing "old" BTB above preleptotene spermatocytes in transit in "clones" at the BTB, but also contribute to the assembly of "new" BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the transit of spermatocytes, can be the "gateway" to making the BTB so vulnerable to toxicants and/or chemicals, causing male reproductive dysfunction.

Dataset Information

Regulation of blood-testis barrier dynamics by TGF-beta3 is a Cdc42-dependent protein trafficking event.

Publications

Regulation of blood-testis barrier dynamics by TGF-beta3 is a Cdc42-dependent protein trafficking event.

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