Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ~ 11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALKATI. In ALKATI-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites1. ALKATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALKATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALKATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALKATI, suggesting that patients with ALKATI-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

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The mechanism(s) that regulate and coordinate the events of spermiation and blood-testis barrier (BTB) restructuring in the seminiferous epithelium that occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis are unknown. In this report, fragments derived from the laminin complex composed of laminin ␣3, ␤3, and ␥3 chains (laminin-333) at the apical ectoplasmic specialization (apical ES) were shown to modulate BTB dynamics directly and/or indirectly via hemidesmosome. Experiments were performed using cultured Sertoli cells with functional tight junction (TJ) barrier and the ultrastructural features of the BTB but not apical ES. Recombinant protein fragments of laminin ␤3 and ␥3 chains were shown to reduce the protein levels of occludin and ␤1-integrin dose dependently at the Sertoli-Sertoli and Sertoli-basement membrane interface, respectively, thereby destabilizing the BTB permeability function. These results were corroborated by transient overexpression of laminin fragments in Sertoli cells. To further assess the role of ␤1-integrin in hemidesmosome, knockdown of ␤1-integrin in Sertoli cells by RNAi was found to associate with occludin redistribution at the SertoliSertoli cell interface, wherein occludin moved away from the cell surface and became associated with endosomes, thereby destabilizing the BTB. In short, an apical ES-BTB-hemidesmosome autocrine regulatory axis was identified in testes, coordinating the events of spermiation and BTB restructuring that occur at the opposite ends of the seminiferous epithelium during spermatogenesis.ectoplasmic specialization ͉ hemidesmosome ͉ seminiferous epithelium ͉ Sertoli cells ͉ tight junction D uring spermatogenesis, preleptotene/leptotene spermatocytes at the basal compartment traverse the blood-testis barrier (BTB) at stages ϷVIII-IX of the seminiferous epithelial cycle in adult rat testes, entering the adluminal compartment for further development (1). This event takes place concurrently with spermiation, wherein fully developed spermatids (i.e., spermatozoa) detach from the epithelium at the luminal edge, entering the tubule lumen for their eventual maturation in the epididymis. These morphologic changes, which occur at the opposite ends of the Sertoli cell epithelium, were first described in the 1950s (2). In this report, we provide compelling evidence regarding the mechanism that regulates and coordinates these events. The idea was based on a recent study in which a blockade of the laminin function at the apical ectoplasmic specialization (apical ES) by specific antibodies led to spermatid exfoliation and BTB restructuring (3), even though it was unclear how the anti-laminin ␥3 IgG traversed the BTB to reach the apical ES. Nonetheless, this study shows that a disruption of the apical ES may lead to a transient BTB disruption, illustrating a plausible physiological link between these two ultrastructures.The apical ES is a testis-specific adherens junction (AJ) type that anchors developing spermatids to the Sertoli cell in the ...

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.

Anchoring junctions are cell adhesion apparatus present in all epithelia and endothelia. They are found at the cell-cell interface (adherens junction (AJ) and desmosome) and cell-matrix interface (focal contact and hemidesmosome). In this review, we focus our discussion on AJ in particular the dynamic changes and regulation of this junction type in normal epithelia using testis as a model. There are extensive restructuring of AJ (e.g., ectoplasmic specialization, ES, a testis-specific AJ) at the Sertoli-Sertoli cell interface (basal ES) and Sertoli-elongating spermatid interface (apical ES) during the seminiferous epithelial cycle of spermatogenesis to facilitate the migration of developing germ cells across the seminiferous epithelium. Furthermore, recent findings have shown that ES also confers cell orientation and polarity in the seminiferous epithelium, illustrating that some of the functions initially ascribed to tight junctions (TJ), such as conferring cell polarity, are also part of the inherent properties of the AJ (e.g., apical ES) in the testis. The biology and regulation based on recent studies in the testis are of interest to cell biologists in the field, in particular their regulation, which perhaps is applicable to tumorigenesis.

In mammalian testes, such as rats, the mechanism(s) that regulate blood-testis barrier (BTB) restructuring at stages VIII-IX of the seminiferous epithelial cycle of spermatogenesis to facilitate the transit of preleptotene/leptotene spermatocytes is not known. This is due to the lack of information on the regulatory proteins at the BTB. Herein, focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, is shown to structurally interact with occludin and ZO-1 to form a functional protein complex at the BTB. Its expression at the BTB in the seminiferous epithelium is stage specific, being lowest at stage VIII-IX tubules, analogous to the expression pattern of occludin. Using primary Sertoli cells cultured in vitro with an established tight junction (TJ) permeability barrier that mimics the BTB in vivo, the knockdown of FAK by RNAi led to a transient disruption of the TJ barrier. This was accompanied by a loss of association between occludin and ZO-1, likely the result of reduced occludin phosphorylation at Tyr and Ser residues, but not Thr, which in turn led to a redistribution of occludin at the Sertoli-Sertoli cell interface, moving from cell membrane into cell cytosol, thereby disrupting the BTB. These findings suggest that a similar mechanism is in place in the testis in vivo to regulate BTB restructuring to facilitate the transit of primary spermatocytes. Furthermore, FAK was shown to be a molecular target of cadmium because its knockdown would desensitize Sertoli cells to cadmium-induced TJ barrier disruption. In summary, FAK is a unique regulator of BTB dynamics in the testis.basal ectoplasmic specialization ͉ cell-cell interaction ͉ spermatogenesis ͉ tight junction I n adult rat testes, at stages VIII-IX of the seminiferous epithelial cycle of spermatogenesis, primary preleptotene spermatocytes are in transit at the blood-testis barrier (BTB) while differentiating into leptotene and zygotene spermatocytes (1) so that diplotene spermatocytes can enter meiosis in the adluminal compartment behind the BTB. Although this event has been known to exist for decades (2), the mechanism(s) that regulates BTB dynamics remains largely unknown. In mammals, the BTB is created by adjacent Sertoli cells in the seminiferous epithelium near the basement membrane via coexisting specialized tight junction (TJ), basal ectoplasmic specialization [basal ES, a testis-specific atypical adherens junction (AJ) type], and desmosome-like junction (3, 4). Studies from the past decade have identified several integral membrane protein complexes that constitute the BTB in rodent testes, such as the occludin-ZO-1 complex at the TJ and the N-cadherin-␤-catenin complex at the basal ES (for reviews, see refs. 5 and 6); however, the regulatory proteins that control the Sertoli cell TJ permeability barrier remain unexplored. This information, if known, would be of great importance to investigators in the field. First, the BTB, unlike other blood-tissue barriers such as the blood-brain barrier, is not a static barrier because it must res...

Male infertility caused by exposure to environmental toxicants, such as cadmium, mercury, bisphenol A (BPA) and dioxin, is a global problem, particularly in industrialized countries. Studies in the testis and other organs have illustrated the importance of environmental toxicantinduced oxidative stress in mediating disruption to cell junctions. This, in turn, is regulated by the activation of PI3K/c-Src/FAK and MAPK signaling pathways, with the involvement of polarity proteins, leading to reproductive dysfunction, such as reduced sperm count and semen quality in men. In this review, we discuss how these findings can improve understanding of the modes of action of environmental toxicants in -testicular dysfunction. Thus, specific inhibitors and/or antagonists against signaling molecules in these pathways can possibly reverse and/or block the disruptive effects of toxicant-induced damage. Additional studies comparing high level acute exposure versus low level chronic exposure to environmental toxicants are also needed to elucidate fully the underlying molecular mechanism(s) by which these toxicants disrupt male reproductive function.

The structures of many cell surface adhesion proteins comprise multiple tandem repeats of structurally similar domains. In many cases, the functional significance of this architecture is unknown, and there are several cases in which evidence for individual domain involvement in adhesion has been contradictory. In particular, the extracellular region of the adhesion glycoprotein cadherin consists of five tandemly arranged domains. One proposed mechanism postulated that adhesion involves only trans interactions between the outermost domains. However, subsequent investigations have generated several competing models. Here we describe direct measurements of the distance-dependent interaction potentials between cadherin mutants lacking different domains. By quantifying both the absolute distances at which opposed cadherin fragments bind and the quantized changes in the interaction potentials that result from deletions of individual domains, we demonstrate that two domains participate in homophilic cadherin binding. This finding contrasts with the current view that cadherins bind via a single, unique site on the protein surface. The potentials that result from interactions involving multiple domains generate a novel, modular binding mechanism in which opposed cadherin ectodomains can adhere in any of three antiparallel alignments.