The Role of ZO-2 in Modulating JAM-A and γ-Actin Junctional Recruitment, Apical Membrane and Tight Junction Tension, and Cell Response to Substrate Stiffness and Topography.

This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.

ZO-2 favors Hippo signaling, and its re-expression in the steatotic liver by AMPK restores junctional sealing.

ZO-2 is a peripheral tight junction (TJ) protein whose silencing in renal epithelia induces cell hypertrophy. Here, we found that in ZO-2 KD MDCK cells, in compensatory renal hypertrophy triggered in rats by a unilateral nephrectomy and in liver steatosis of obese Zucker (OZ) rats, ZO-2 silencing is accompanied by the diminished activity of LATS, a kinase of the Hippo pathway, and the nuclear concentration of YAP, the final effector of this signaling route. ZO-2 appears to function as a scaffold for the Hippo pathway as it associates to LATS1. ZO-2 silencing in hypertrophic tissue is due to a diminished abundance of ZO-2 mRNA, and the Sp1 transcription factor is critical for ZO-2 transcription in renal cells. Treatment of OZ rats with metformin, an activator of AMPK that blocks JNK activity, augments ZO-2 and claudin-1 expression in the liver, reduces the paracellular permeability of hepatocytes, and serum bile acid content. Our results suggest that ZO-2 silencing is a common feature of hypertrophy, and that ZO-2 is a positive regulator of the Hippo pathway that regulates cell size. Moreover, our observations highlight the importance of AMPK, JNK, and ZO-2 as therapeutic targets for blood-bile barrier dysfunction.

Zonula occludens 2 and Cell-Cell Contacts Are Required for Normal Nuclear Shape in Epithelia.

MAGUK protein ZO-2 is present at tight junctions (TJs) and nuclei. In MDCK ZO-2 knockdown (KD) cells, nuclei exhibit an irregular shape with lobules and indentations. This condition correlates with an increase in DNA double strand breaks, however cells are not senescent and instead become resistant to UV-induced senescence. The irregular nuclear shape is also observed in isolated cells and in those without TJs, due to the lack of extracellular calcium. The aberrant nuclear shape of ZO-2 KD cells is not accompanied by a reduced expression of lamins A/C and B and lamin B receptors. Instead, it involves a decrease in constitutive and facultative heterochromatin, and microtubule instability that is restored with docetaxel. ZO-2 KD cells over-express SUN-1 that crosses the inner nuclear membrane and connects the nucleoskeleton of lamin A to nesprins, which traverse the outer nuclear membrane. Nesprins-3 and -4 that indirectly bind on their cytoplasmic face to vimentin and microtubules, respectively, are also over-expressed in ZO-2 KD cells, whereas vimentin is depleted. SUN-1 and lamin B1 co-immunoprecipitate with ZO-2, and SUN-1 associates to ZO-2 in a pull-down assay. Our results suggest that ZO-2 forms a complex with SUN-1 and lamin B1 at the inner nuclear membrane, and that ZO-2 and cell-cell contacts are required for a normal nuclear shape.

Methyl parathion causes genetic damage in sperm and disrupts the permeability of the blood-testis barrier by an oxidant mechanism in mice.

Methyl parathion (Me-Pa) is an extremely toxic organophosphorus pesticide still used in developing countries. It has been associated with decreased sperm function and fertility and with oxidative and DNA damage. The blood-testis barrier (BTB) is a structure formed by tight junction (TJ) proteins in Sertoli cells and has a critical role in spermatogenesis. We assessed the effect of repeated doses of Me-Pa (3-12 mg/kg/day for 5 days, i.p.) on sperm quality, lipid oxidation, DNA integrity, and BTB permeability in adult male mice and explored oxidation as a mechanism of toxicity. Me-Pa caused dose-dependent effects on sperm quality, lipoperoxidation, and DNA integrity. Testis histology results showed the disruption of spermatogenesis progression and atrophy of seminiferous tubules. The pesticide opened the BTB, as evidenced by the presence of a biotin tracer in the adluminal compartment of the seminiferous tubules. This effect was not observed after 45 days of exposure when a spermatogenic cycle had completed. The coadministration of the antioxidant α-tocopherol (50 mg/kg/day for 5 days, oral) prevented the effects of Me-Pa on sperm quality, DNA and the BTB, indicating the importance of oxidative stress in the damage generated by Me-Pa. As evidenced by immunochemistry, no changes were found in the localization of the TJ proteins of the BTB, although oxidation (carbonylation) of total proteins in testis homogenates was detected. Our results show that Me-Pa disturbs the BTB and that oxidation is involved in the observed toxic effects on sperm cells.

Syncytiotrophoblast of Placentae from Women with Zika Virus Infection Has Altered Tight Junction Protein Expression and Increased Paracellular Permeability.

The cytotrophoblast of human placenta transitions into an outer multinucleated syncytiotrophoblast (STB) layer that covers chorionic villi which are in contact with maternal blood in the intervillous space. During pregnancy, the Zika virus (ZIKV) poses a serious prenatal threat. STB cells are resistant to ZIKV infections, yet placental cells within the mesenchyme of chorionic villi are targets of ZIKV infection. We seek to determine whether ZIKV can open the paracellular pathway of STB cells. This route is regulated by tight junctions (TJs) which are present in the uppermost portion of the lateral membranes of STB cells. We analyzed the paracellular permeability and expression of E-cadherin, occludin, JAMs -B and -C, claudins -1, -3, -4, -5 and -7, and ZO-1, and ZO-2 in the STB of placentae from ZIKV-infected and non-infected women. In ZIKV-infected placentae, the pattern of expression of TJ proteins was preserved, but the amount of claudin-4 diminished. Placentae from ZIKV-infected women were permeable to ruthenium red, and had chorionic villi with a higher mean diameter and Hofbauer hyperplasia. Finally, ZIKV added to the basolateral surface of a trophoblast cell line reduced the transepithelial electrical resistance. These results suggest that ZIKV can open the paracellular pathway of STB cells.

Activation of the Ca2+ sensing receptor and the PKC/WNK4 downstream signaling cascade induces incorporation of ZO-2 to tight junctions and its separation from 14-3-3.

Zonula occludens-2 (ZO-2) is a tight junction (TJ) cytoplasmic protein, whose localization varies according to cell density and Ca2+ in the media. In cells cultured in low calcium (LC), ZO-2 displays a diffuse cytoplasmic distribution, but activation of the Ca2+ sensing receptor (CaSR) with Gd3+ triggers the appearance of ZO-2 at the cell borders. CaSR downstream signaling involves activation of protein kinase C, which phosphorylates and activates with no lysine kinase-4 that phosphorylates ZO-2 inducing its concentration at TJs. In LC, ZO-2 is protected from degradation by association to 14-3-3 proteins. When monolayers are transferred to normal calcium, the complexes ZO-2/14-3-3ζ and ZO-2/14-3-3σ move to the cell borders and dissociate. The 14-3-3 proteins are then degraded in proteosomes, whereas ZO-2 integrates to TJs. From the plasma membrane residual ZO-2 is endocyted and degradaded in lysosomes. The unique region 2 of ZO-2, and S261 located within a nuclear localization signal, are critical for the interaction with 14-3-3 ζ and σ and for the efficient nuclear importation of ZO-2. These results explain the molecular mechanism through which extracellular Ca2+ triggers the appearance of ZO-2 at TJs in epithelial cells and reveal the novel interaction between ZO-2 and 14-3-3 proteins, which is critical for ZO-2 protection and intracellular traffic.

The organophosphate pesticide methamidophos opens the blood-testis barrier and covalently binds to ZO-2 in mice.

Methamidophos (MET) is an organophosphate (OP) pesticide widely used in agriculture in developing countries. MET causes adverse effects in male reproductive function in humans and experimental animals, but the underlying mechanisms remain largely unknown. We explored the effect of MET on mice testes (5 mg/kg/day/4 days), finding that this pesticide opens the blood-testis barrier and perturbs spermatogenesis, generating the appearance of immature germ cells in the epididymis. In the seminiferous tubules, MET treatment changed the level of expression or modified the stage-specific localization of tight junction (TJ) proteins ZO-1, ZO-2, occludin, and claudin-3. In contrast, claudin-11 was barely altered. MET also modified the shape of claudin-11, and ZO-2 at the cell border, from a zigzag to a more linear pattern. In addition, MET diminished the expression of ZO-2 in spermatids present in seminiferous tubules, induced the phosphorylation of ZO-2 and occludin in testes and reduced the interaction between these proteins assessed by co-immunoprecipitation. MET formed covalent bonds with ZO-2 in serine, tyrosine and lysine residues. The covalent modifications formed on ZO-2 at putative phosphorylation sites might interfere with ZO-2 interaction with regulatory molecules and other TJ proteins. MET bonds formed at ZO-2 ubiquitination sites likely interfere with ZO-2 degradation and TJ sealing, based on results obtained in cultured epithelial cells transfected with ZO-2 mutated at a MET target lysine residue. Our results shed light on MET male reproductive toxicity and are important to improve regulations regarding the use of OP pesticides and to protect the health of agricultural workers.

ZO-2 silencing induces renal hypertrophy through a cell cycle mechanism and the activation of YAP and the mTOR pathway.

Renal compensatory hypertrophy (RCH) restores normal kidney function after disease or loss of kidney tissue and is characterized by an increase in organ size due to cell enlargement and not to cell proliferation. In MDCK renal epithelial cells, silencing of the tight junction protein zona occludens 2 (ZO-2 KD) induces cell hypertrophy by two mechanisms: prolonging the time that cells spend at the G1 phase of the cell cycle due to an increase in cyclin D1 level, and augmenting the rate of protein synthesis. The latter is triggered by the nuclear accumulation and increased transcriptional activity of Yes-associated protein (YAP), the main target of the Hippo pathway, which results in decreased expression of phosphatase and tensin homologue. This in turn increased the level of phosphatidylinositol (3,4,5)-triphosphate, which transactivates the Akt/mammalian target of rapamycin pathway, leading to activation of the kinase S6K1 and increased synthesis of proteins and cell size. In agreement, in a rat model of uninephrectomy, RCH is accompanied by decreased expression of ZO-2 and nuclear expression of YAP. Our results reveal a novel role of ZO-2 as a modulator of cell size.

Heterogeneity between triple negative breast cancer cells due to differential activation of Wnt and PI3K/AKT pathways.

The lack of a successful treatment for triple-negative breast cancer demands the study of the heterogeneity of cells that constitute these tumors. With this aim, two clones from triple negative breast MDA-MB-231 cancer cells were isolated: One with fibroblast-like appearance (F) and another with semi-epithelial (SE) morphology. Cells of the F clone have a higher migration and tumorigenesis capacity than SE cells, suggesting that these cells are in a more advanced stage of epithelial to mesenchymal transformation. In agreement, F cells have a diminished expression of the tight junction proteins claudins 1 and 4, and an increased content of ß-catenin. The latter is due to an augmented activity of the canonical Wnt route and of the EGFR/PI3K/mTORC2/AKT pathway favoring the cytoplasmic accumulation of ß-catenin and its transcriptional activity. In addition, F cells display increased phosphorylation of ß-catenin at Tyr654 by Src. These changes favor in F cells, the over-expression of Snail that promotes EMT. Finally, we observe that both F and SE cells display markers of cancer stem cells, which are more abundant in the F clone.

Anterior and intermediate pituitary tissues express claudin 4 in follicle stellate cells and claudins 2 and 5 in endothelial cells.

Follicle-stellate cells are pituitary non-granular cells that are arranged between secretory cells or organized in follicles with small lumens. Cells from the follicles exhibit the typical phenotype of a transporting epithelium, including apical microvilli with a cilium and tight junctions. Freeze-fracture electron microscopy images show that the tight junctions consist of 5-7 anastomosing strands and that cultured follicle-stellate cells develop a trans-epithelial electrical resistance characteristic of "tight" epithelia. Here, we investigate the molecular composition of the tight junction from follicle stellate cells. We found that the rat anterior pituitary lobe expresses mRNAs for claudins 2, 4 and 5; the proteins of all these claudins are observed in the anterior lobe, whereas the intermediate lobe expresses claudins 2 and 5 and the posterior lobe contains only claudin 5. Follicle-stellate cells, identified by their protein marker S100ß, expresses claudin 4 in the apical membrane, in co-localization with dipeptidyl-peptidase and near acetylated ß-tubulin. Claudin 4 partially co-localizes with E-cadherin, indicating that a fraction of the protein is located in the basolateral domain. Follicle-stellate-enriched cell cultures develop patches of polygonal cells expressing claudin 4 and E-cadherin, encircled by extensive monolayers of fusiform cells. Claudin 2 stains specifically blood vessels, identified by claudin 5 and VE-cadherin labels. Thus, follicles in the anterior pituitary consist of "tight" epithelia that can carry out intense vectorial transport, together with a high cation movement in blood vessels, possibly related to the ion requirements of excitable secretory cells for hormone secretion.

Expression of Eag1 K+ channel and ErbBs in human pituitary adenomas: cytoskeleton arrangement patterns in cultured cells.

Pituitary adenomas can invade surrounded tissue, but the mechanism remains elusive. Ether à go-go-1 (Eag1) potassium channel and epidermal growth factor receptors (ErbB1 and ErbB2) have been associated to invasive phenotypes or poor prognosis in cancer patients. However, cells arrange their cytoskeleton in order to acquire a successful migration pattern. We have studied ErbBs and Eag1 expression, and cytoskeleton arrangements in 11 human pituitary adenomas. Eag1, ErbB1 and ErbB2 expression were studied by immunochemistry in tissue and cultured cells. The cytoskeleton arrangement was analyzed in cultured cells by immunofluorescence. Normal pituitary tissue showed ErbB2 expression and Eag1 only in few cells. However, Eag1 and ErbB2 were expressed in all the tumors analyzed. ErbB1 expression was observed variable and did not show specificity for a tumor characteristic. Cultured cells from micro- and macro-adenomas clinically functional organize their cytoskeleton suggesting a mesenchymal pattern, and a round leucocyte/amoeboid pattern from invasive clinically silent adenoma. Pituitary tumors over-express EGF receptors and the ErbB2 repeated expression suggests is a characteristic of adenomas. Eag 1 was express, in different extent, and could be a therapeutic target. The cytoskeleton arrangements observed suggest that pituitary tumor cells acquire different patterns: mesenchymal, and leucocyte/amoeboid, the last observed in the invasive adenomas. Amoeboid migration pattern has been associated with high invasion capacity.