Regulation of cell junction dynamics by cytokines in the testis: a molecular and biochemical perspective

Abstract

Studies in the past decade in the field have demonstrated the significance of cytokines in regulating epithelial and endothelial cell junctions including tight and anchoring junctions in multiple organs including the testis. There are mounting evidences in recent years that cytokines play a crucial role in the restructuring of junctions at the Sertoli-Sertoli and Sertoli-germ cell interface in the seminiferous epithelium during spermatogenesis. These earlier studies, however, were focused on the effects of cytokines in maintaining the steady-state protein levels of integral membrane proteins at the sites of the blood-testis barrier (BTB) and anchoring junctions at the Sertoli-Sertoli and Sertoli-germ cell interface, such as basal and apical ectoplasmic specialization, respectively. The molecular pathway(s) and/or mechanism(s) underlying these effects remained virtually unexplored until very recently. Herein, we summarize and provide some discussions on studies that focused on the role of cytokines in regulating junction restructuring events in epithelia from a molecular and biochemical perspective. Specifically, we use the adult rat or mouse testis as a model to highlight the significance of transcriptional and translational regulation. Specific areas of research that require further attentions are also highlighted.

Fig. 1. Ultrastructural features of the major junction types in the adult rat testis

(A) This is the cross-section of a stage V seminiferous tubule from adult rat testes, showing the intimate relationship between the seminiferous epithelium, composed of Sertoli and developing germ cells (spermatogonia, spermatocytes, round spermatids, and elongating spermatids), resting on the tunica propria. The seminiferous epithelium is physically divided into the basal and adluminal compartment by the blood-testis barrier. SC, Sertoli cell Sg, spermatogonium Ps, pachytene spermatocyte RS, round spermatid Es, elongating spermatid. (B) This is an enlarged image in the seminiferous epithelium showing an early elongating spermatid (both the condensed nucleus and the early acrosome, Ac, are visible) attached to Sertoli cells via the apical ectoplasmic specialization (ES) (a testis-specific adherens junction type) characterized by the presence of actin filament bundles (white arrowheads) sandwiched between the endoplasmic reticulum (ER) and the Sertoli cell plasma membrane. The two apposing white arrowheads illustrate the relative location of the two apposing Sertoli and germ cell plasma membranes. The ultrastructural features of apical ES (namely actin filament bundles sandwiched between ER and plasma membrane) are limited to the Sertoli cell side. In contrast, the basal ES at the blood-testis barrier (BTB) (the boxed area in C illustrates the typical features of the BTB in adult rat testes) (C) found between two adjacent Sertoli cells (white arrowheads represent the two apposing Sertoli cell plasma membranes) near the basement membrane displaying these ultrastructural ES features, namely, actin filament bundles sandwiched between ER and plasma membrane of the Sertoli cell, are present on both sides of the two Sertoli cells, which also co-exist with tight junctions (TJ, see black arrowheads with white tails). Also clearly visible at the BTB is the desmosome-like junctions (Des) typified by the presence of electron-dense substances but without the basal ES. The tunica propria shown in A was also magnified in C which is composed of two acellular zones (namely the basement membrane, see white asterisks, and the collagen fibril network, c) and two cellular zones (namely the peritubular myoid cell layer, and the lymphatic vessel underneath). Bar in A, B and C is 10 μm, 0.2 μm, and 1 μm, respectively.

Fig. 2. A schematic drawing showing different junction types and their relative location in the seminiferous epithelium of adult rat testes

The intimate relationship between developing germ and Sertoli cells and the relative location of different junction types in the epithelium at the Sertoli-Sertoli and Sertoli-germ cell interface are shown (see left panel). Differentiating preleptotene spermatocytes must traverse the BTB at stage VIII of the epithelial cycle, and the BTB has also physically divided the seminiferous epithelium into the basal and adluminal compartment. The molecular architecture of apical ES (middle right panel) and tight junction at the BTB (bottom right panel) in the seminiferous epithelium are also illustrated.

Fig. 3. A schematic drawing illustrating the dynamic regulation of integral membrane proteins through ubiquitination and endocytosis

At steady-state, junction proteins are organized themselves at cell-cell contact sites. Upon certain stimulation, target proteins will be recognized and underwent ubiquitination (top panel) or endocytosis (bottom panel) for degradation. For ubiquitination, E3 ligase first recognizes and binds to the target protein. Meanwhile, E1 activates ubiquitin and activated ubiquitin is then conjugated to the target protein with the help of E2, resulting in the formation of polyubiquitin tail onto the target protein. The polyubiquitinated protein may either be degraded by 26S proteasome or undergo deubiquitination by deubiquitinating enzymes (DUBs). The action of DUBs helps cleaving the poly-ubiquitin chain from the target protein, resulting in rescuing the protein from degradation. Protein can then be recycled back to the membrane surface and facilitate the reassembly of junctions. For endocytosis, junction proteins are internalized via various endocytic structures including clathrin-coated vesicle, caveolin-coated vesicle and actin-coated vacuolae. Internalized proteins are delivered into early endosomes. They are then delivered to either late endosomes in which targets will be degraded by lysosomes or recycling endosomes for channeling back to the cell membrane.

Fig. 4. A schematic drawing illustrating the differential interaction of TGF-β3 and its receptor with different adaptors in Sertoli cells can lead to the activation of different signaling pathways, which selectively perturbs the BTB and/or anchoring junction dynamics

The interactions of TAB1 and CD2AP with the TGF-β3/TGF-β-receptor complex can activate both the p38 or the ERK signaling pathways, which, in turn, causes the disruption of both BTB and AJs (right panel). When the TGF-β3/TGF-β-receptor complex associates only with CD2AP, but not TAB1, this selectively activates only the ERK signaling pathway, resulting in AJ disruption without compromising the BTB (left panel). Without such selective interaction between the TGF-β3/receptor complex and any of the adaptors, both the BTB and anchoring junction remains intact (middle panel). Keys to the symbols in this figure can be found in Fig. 2.