TGF-β3 and TNFα perturb blood–testis barrier (BTB) dynamics by accelerating the clathrin-mediated endocytosis of integral membrane proteins: A new concept of BTB regulation during spermatogenesis

Abstract

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 stage VIII 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-β3 (TGF-β3) and tumor necrosis factor α (TNFα) 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-β3 (200 ng/testis) to the testis reversibly perturbed 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-β3 (3 ng/ml) and TNFα (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 μM that blocks clathrin-mediated endocytosis was shown to inhibit the TGF-β3-induced protein internalization. This inhibition of TGF-β3-mediated protein endocytosis was further validated by silencing of clathrin. The specific effect of TGF-β3 on protein internalization was confirmed by RNAi using specific TGF-β receptor I (TβR1) siRNA duplexes. When TβR1 was knocked down, the TGF-β3-induced increase in the kinetics of JAM-A and occludin endocytosis was abolished, making them indistinguishable from controls, illustrating the specificity of the TGF-β3 effects on protein endocytosis. In summary, this report demonstrates for the first time that BTB dynamics are regulated by TGF-β3 and TNFα via an enhancement of protein endocytosis at the BTB.

Regulation of blood–testis barrier dynamics by TGF-β3 is a Cdc42-dependent protein trafficking event

“…As Cdc42 switches between active (i.e., GTP-bound) and inactive (i.e., GDP-bound) states, we performed a pull-down assay using GSTp21-binding domain (i.e., PBD) of PAK-1 to precipitate active Cdc42 (Cdc42-GTP). Isolated Sertoli cells were cultured in F12/ DMEM for 4 d to form an intact cell epithelium before being stimulated with 4 ng/mL TGF-β3, at a concentration similar to its level at the BTB microenvironment in vivo (8). It was found that TGF-β3 transiently activated Cdc42 at 5 and 20 min after Sertoli cells were exposed to TGF-β3 ( Fig.…”

“…TGF-β3 is known to disrupt Sertoli cell BTB function by enhancing endocytosis of integral membrane proteins at the BTB (8). To address whether TGF-β3 induces protein internalization via activation of Cdc42, we performed an endocytosis assay using Cdc42-or T17N-expressing Sertoli cells in the presence or absence of TGF-β3 using Sertoli cells cultured for 4 d with an intact epithelium.…”

“…For instance, testosterone maintains the immunological barrier by inducing de novo synthesis of junction proteins (e.g., claudins and occludin) (5-7) as well as transcytosis of proteins from other sites of the Sertoli cells to promote the assembly of "new" TJ fibrils behind a spermatocyte in transit (4). Conversely, cytokines enhance endocytosis of integral membrane proteins above a spermatocyte in transit and target the internalized TJ fibril proteins for intracellular degradation (4,8), thereby destabilizing the "old" BTB ultrastructure to facilitate its transit (2). However, the regulatory component(s) that modulates these events, such as the TGF-β-induced enhancement in protein endocytosis, remains virtually unknown.…”

“…Although studies have shown that an alteration of Cdc42 activity, such as by overexpression of dominant-negative or constitutive-active mutant of Cdc42, induces changes in protein trafficking in cells, the upstream regulators that control Cdc42 activity in this process are unknown (1). Here, we sought to examine whether TGF-β3, when used at a concentration similar to the level at the BTB microenvironment in vivo (8), is an upstream physiological stimulus that activates Cdc42, and if the activation state of Cdc42 is essential for mediating TGF-β3-induced acceleration of protein endocytosis at the BTB. We also examined if Cdc42 determines the extent of endocytosed integral membrane proteins that are recycled and targeted to the cell surface.…”

Association between periodontal status and idiopathic male infertility

“…Similar inflammatory molecules are important in regulating the blood-testis barrier, which affects the function of Sertoli cells. Tumor necrosis factor-alpha, transforming growth factor-β3 (40), and interleukin-1 can disturb blood-testis barrier dynamics (41). Interleukin-6 alters blood-testis barrier function by inhibiting protein degradation (42).…”