Connexin 43 and plakophilin-2 as a protein complex that regulates blood-testis barrier dynamics

Doxorubicin (DOX) is an anthracycline derivative antibiotic that still frequently used in the treatment of solid tumors and hematological malignancies. The clinical use of DOX is largely restricted due to acute and chronic renal, cardiac, hematological, and testicular toxicities. Previous studies have indicated that oxidative stress, lipid peroxidation, and apoptosis in germ cells are the main factors in DOX-induced testicular toxicity, but the entire molecular mechanisms that responsible for DOX-induced testicular damage are not yet fully understood. Fluvastatin is a cholesterol-lowering agent that acts by inhibiting hydroxylmethyl glutaryl coenzyme A, the key enzyme for cholesterol biosynthesis. In addition to its cholesterol-lowering effect, fluvastatin showed an antioxidant effect by cleaning hydroxyl and superoxide radicals and this drug could have a protective effect by acting on the mammalian target of rapamycin (mTOR) signal pathway in testicular damage caused by obesity. This study aimed to investigate the possible protective and therapeutic effects of fluvastatin on the DOX-induced testicular toxicity model by histochemical, immunohistochemical, biochemical, and real-time polymerase chain reaction analyses. The present study indicates that fluvastatin may have a protective and therapeutic effect by removing reactive oxygen species and by regulating the mTOR, connexin 43, and matrix metalloproteinase 9 protein and messenger ribonucleic acid expressions, which play an important role in regulating the blood–testis barrier. On the other hand, the use of fluvastatin as a protective/prophylactic agent was found to be more effective than the use of this drug for treatment. In light of this information, fluvastatin may be a candidate agent that can be used to prevent testicular toxicity observed in men receiving DOX treatment.

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Blood Testis Barrier and Somatic Cells Impairment in a Series of 35 Adult Klinefelter Syndrome Patients

International Journal of Molecular Sciences ◽

10.3390/ijms20225717 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5717 ◽

Cited By ~ 7

Author(s):

Giudice ◽

Vermeulen ◽

Wyns

Keyword(s):

Germ Cell ◽

Connexin 43 ◽

Klinefelter Syndrome ◽

Cell Loss ◽

Testicular Tissue ◽

Seminiferous Tubules ◽

Morphological Alterations ◽

Normal Spermatogenesis ◽

Underlying Mechanisms ◽

Blood Testis Barrier

Klinefelter Syndrome (KS) is the most common genetic cause of infertility in men. Degeneration of the testicular tissue starts in utero and accelerates at puberty with hyalinisation of seminiferous tubules, spermatogonia apoptosis and germ cell maturation arrest. Therefore, fertility preservation in young KS boys has been proposed, although this measure is still debated due to insufficient knowledge of the pathophysiology of the disease. To better understand the underlying mechanisms of testicular failure and germ cell loss, we analysed functional and morphological alterations in the somatic compartment of KS testis, i.e., Sertoli cells, including the blood–testis barrier (BTB) and Leydig cells (LC). We compared three populations: 35 KS 47,XXY non-mosaic patients, 28 Sertoli-cell-only (SCO) syndrome patients and 9 patients with normal spermatogenesis. In KS patients the expression of BTB proteins connexin-43 and claudin-11 assessed with a semi-quantitative scoring system appeared significantly reduced with a disorganised pattern. A significant reduction in seminiferous tubules expressing androgen receptors (AR) was observed in KS compared to normal spermatogenesis controls. INSL3 expression, a marker of LC maturation, was also significantly reduced in KS compared to patients with normal spermatogenesis or SCO. Hence, the somatic compartment impairment in KS could be involved in degeneration of seminiferous tubules.

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The Scribble/Lgl/Dlg Polarity Protein Complex Is a Regulator of Blood-Testis Barrier Dynamics and Spermatid Polarity during Spermatogenesis

Endocrinology ◽

10.1210/en.2012-1670 ◽

2012 ◽

Vol 153 (12) ◽

pp. 6041-6053 ◽

Cited By ~ 33

Author(s):

Wenhui Su ◽

Elissa W. P. Wong ◽

Dolores D. Mruk ◽

C. Yan Cheng

Keyword(s):

Protein Complex ◽

Blood Testis Barrier

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An Occludin-Focal Adhesion Kinase Protein Complex at the Blood-Testis Barrier: A Study Using the Cadmium Model

Endocrinology ◽

10.1210/en.2008-1741 ◽

2009 ◽

Vol 150 (7) ◽

pp. 3336-3344 ◽

Cited By ~ 76

Author(s):

Erica R. Siu ◽

Elissa W. P. Wong ◽

Dolores D. Mruk ◽

K. L. Sze ◽

Catarina S. Porto ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Sertoli Cell ◽

Focal Adhesion ◽

Adhesion Molecule ◽

Protein Complex ◽

Permeability Barrier ◽

Ectoplasmic Specialization ◽

Blood Testis Barrier

Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII–IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl2 to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics.

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Uropathogenic Escherichia coli Infection Compromises the Blood-Testis Barrier by Disturbing mTORC1-mTORC2 Balance

Frontiers in Immunology ◽

10.3389/fimmu.2021.582858 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yongning Lu ◽

Miao Liu ◽

Nicholas J. Tursi ◽

Bin Yan ◽

Xiang Cao ◽

...

Keyword(s):

Signaling Pathway ◽

Connexin 43 ◽

Mtor Signaling ◽

Cell Junctions ◽

Cell Junction ◽

Adjacent Cell ◽

Mtor Signaling Pathway ◽

Short Term Treatment ◽

Blood Testis Barrier

The structural and functional destruction of the blood-testis barrier (BTB) following uropathogenic E. coli (UPEC) infection may be a critical component of the pathologic progress of orchitis. Recent findings indicate that the mammalian target of the rapamycin (mTOR)-signaling pathway is implicated in the regulation of BTB assembly and restructuring. To explore the mechanisms underlying BTB damage induced by UPEC infection, we analyzed BTB integrity and the involvement of the mTOR-signaling pathway using in vivo and in vitro UPEC-infection models. We initially confirmed that soluble virulent factors secreted from UPEC trigger a stress response in Sertoli cells and disturb adjacent cell junctions via down-regulation of junctional proteins, including occludin, zonula occludens-1 (ZO-1), F-actin, connexin-43 (CX-43), β-catenin, and N-cadherin. The BTB was ultimately disrupted in UPEC-infected rat testes, and blood samples from UPEC-induced orchitis in these animals were positive for anti-sperm antibodies. Furthermore, we herein also demonstrated that mTOR complex 1 (mTORC1) over-activation and mTORC2 suppression contributed to the disturbance in the balance between BTB “opening” and “closing.” More importantly, rapamycin (a specific mTORC1 inhibitor) significantly restored the expression of cell-junction proteins and exerted a protective effect on the BTB during UPEC infection. We further confirmed that short-term treatment with rapamycin did not aggravate spermatogenic degeneration in infected rats. Collectively, this study showed an association between abnormal activation of the mTOR-signaling pathway and BTB impairment during UPEC-induced orchitis, which may provide new insights into a potential treatment strategy for testicular infection.

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Disruption of Ca 2+ i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2–Deficient Mice

Circulation ◽

10.1161/circulationaha.119.039710 ◽

2019 ◽

Vol 140 (12) ◽

pp. 1015-1030 ◽

Cited By ~ 21

Author(s):

Joon-Chul Kim ◽

Marta Pérez-Hernández ◽

Francisco J. Alvarado ◽

Svetlana R. Maurya ◽

Jerome Montnach ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Sarcoplasmic Reticulum ◽

Right Ventricular ◽

Membrane Permeability ◽

Connexin 43 ◽

Left Ventricular ◽

Plakophilin 2 ◽

Kinase C ◽

Multiple Imaging

Background: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. Methods: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Results: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca 2+ transients, increased Ca 2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca 2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca 2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca 2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca 2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca 2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca 2+ , increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca 2+ ] i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Conclusions: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca 2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca 2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca 2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

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Connexin 43 reboots meiosis and reseals blood‐testis barrier following toxicant‐mediated aspermatogenesis and barrier disruption

The FASEB Journal ◽

10.1096/fj.15-276527 ◽

2015 ◽

Vol 30 (4) ◽

pp. 1436-1452 ◽

Cited By ~ 25

Author(s):

Nan Li ◽

Dolores D. Mruk ◽

Ka‐Wai Mok ◽

Michelle W. M. Li ◽

Chris K. C. Wong ◽

...

Keyword(s):

Connexin 43 ◽

Barrier Disruption ◽

Blood Testis Barrier

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Perfluorooctanesulfonate (PFOS) Perturbs Male Rat Sertoli Cell Blood-Testis Barrier Function by Affecting F-Actin Organization via p-FAK-Tyr407: An in Vitro Study

Endocrinology ◽

10.1210/en.2013-1657 ◽

2014 ◽

Vol 155 (1) ◽

pp. 249-262 ◽

Cited By ~ 64

Author(s):

Hin-Ting Wan ◽

Dolores D. Mruk ◽

Chris K. C. Wong ◽

C. Yan Cheng

Keyword(s):

Tight Junction ◽

Sertoli Cell ◽

Sertoli Cells ◽

Connexin 43 ◽

Male Rat ◽

Environmental Toxicants ◽

Adhesion Proteins ◽

Actin Organization ◽

Blood Testis Barrier

Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10–20 μM (∼5–10 μg/mL) was found to be more potent than bisphenol A (100 μM) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr407 was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr407 was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr407 and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB.

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