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The aim of the study was to analyze the effect of polymorphic variants previously associated with arterial hypertension (AH) in Genome Wide Association Studies (GWASs) in/next to genes and locuses CYP7A1 and PLEKHA7 on the development of coronary artery disease (CAD) in Bulgarian patients. A hundred and nine consecutive patients with angiographically documented CAD were studied. The genotyping was done with 7900 HT Fast Real-Time PCR (Applied Biosystems) with TaqMan® method. The control group consisted of 192 healthy population controls, selected from the bio- bank of the Molecular Medicine Center. SPSS and PLINK were used for the statistical analysis with level of significance < 0.05 and confidence interval 95%. The mean age of the studied patients was 63.71 ± 9.35 years; 35 (35%) females. Previous myocardial infarction (MI) had 38(38%); one-vessel – 39 (39%); two-vessel – 28 (28%); three-vessel disease – 34 (34%); 43 (43%) were with diabetes mellitus; 92 (92%) – with arterial hypertension (AH); 77 (77%) – with dyslipidemia; 42 (42%) were smokers; 25 (25%) were obese. We did not find any significant association between CAD and poly- morphism rs11191548 near CYP17A1 and only a tendency for genotype of rs381815 in PLEKHA7 (p = 0.06; OR 0.64; CI 0.40-1.02 for CAD) under dominant model. This is of practical importance both for studying the genetic aspects of CAD in the future and for enlargement of the current database.

Angle-closure glaucoma diagnosis verification is based on biometric indicators and computer tomography data of the anterior segment of the eye. However, not every patient with a narrow angle and a shellow anterior chamber develops an acute attack. The review summarizes the literature data on risk factors for an acute glaucoma attack and lists additional parameters to be considered when examining patients with angle-closure glaucoma to identify a predisposition to an acute glaucoma attack.

The extracellular matrix (ECM) plays crucial roles in tissue homeostasis. Abnormalities in ECM composition are associated with pathological conditions, such as fibrosis and cancer. These ECM alterations are sensed by the epithelium and can influence its behavior through crosstalk with other mechanosensitive complexes, including the adherens junctions (AJs). We have previously shown that the AJs, through their component PLEKHA7, recruit the RNAi machinery to regulate miRNA levels and function. We have particularly shown that the junctional localization of RNAi components is critical for their function. Here, we investigated whether different ECM substrates can influence the junctional localization of RNAi complexes. To do this, we plated colon epithelial Caco2 cells on four key ECM substrates found in the colon under normal or pathogenic conditions, namely laminin, fibronectin, collagen I, and collagen IV, and we examined the subcellular distribution of PLEKHA7, and of the key RNAi components AGO2 and DROSHA. Fibronectin and collagen I negatively impacted the junctional localization of PLEKHA7, AGO2, and DROSHA when compared to laminin. Furthermore, fibronectin, collagen I, and collagen IV disrupted interactions of AGO2 and DROSHA with their essential partners GW182 and DGCR8, respectively, both at AJs and throughout the cell. Combinations of all substrates with fibronectin also negatively impacted junctional localization of PLEKHA7 and AGO2. Additionally, collagen I triggered accumulation of DROSHA at tri-cellular junctions, while both collagen I and collagen IV resulted in DROSHA accumulation at basal areas of cell–cell contact. Altogether, fibronectin and collagens I and IV, which are elevated in the stroma of fibrotic and cancerous tissues, altered localization patterns and disrupted complex formation of PLEKHA7 and RNAi components. Combined with our prior studies showing that apical junctional localization of the PLEKHA7-RNAi complex is critical for regulating tumor-suppressing miRNAs, this work points to a yet unstudied mechanism that could contribute to epithelial cell transformation. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is the most lethal leukemia with an extremely poor prognosis and high relapse rates. In leukemogenesis, adhesion abnormalities can readily guide an imbalance between hematopoietic progenitor cells and bone marrow stromal cells, altering the normal hematopoietic bone marrow microenvironment into leukemic transformation that enhances leukemic proliferation. Here, we have firstly studied the PLEKHA7 expression in leukemic cells to assess their growth capability affected by the restoration of PLEKHA7 in the cells. The efficacy of PLEKHA7-loaded cRGD-mediated PEGylated cationic lipid nanoparticles for efficient PLEKHA7 delivery in leukemic cells as well as the effect of PLEKHA7 on the regulated induction of AML behavior and growth alterations were investigated. PLEKHA7 re-expression diminished colony-forming ability and reinforced the incidence of growth retardation without apoptosis in AML cell lines. PLEKHA7 regulated the restoration of cell surface adhesion and integrity during normal homeostasis. Our findings revealed that PLEKHA7 functions as a behavior and growth modulator in AML. To our knowledge, the role of PLEKHA7 in AML had not been studied previously and our data could be exploited for further mechanistic studies and insights into altering human AML behavior and growth.

Plekha7 is a key adherens junction component involved in numerous functions in mammalian cells. Plekha7 is the most studied member of the PLEKHA protein family, which includes eight members with diverse functions. However, the evolutionary history of Plekha7 remains unexplored. Here, we outline the phylogeny and identify the origins of this gene and its paralogs. We show that Plekha7, together with Plekha4, Plekha5, and Plekha6, belong to a subfamily that we name PLEKHA4/5/6/7. This subfamily is distinct from the other Plekha proteins, which form two additional separate subfamilies, namely PLEKHA1/2 and PLEKHA3/8. Sequence, phylogenetic, exon-intron organization, and syntenic analyses reveal that the PLEKHA4/5/6/7 subfamily is represented by a single gene in invertebrates, which remained single in the last common ancestor of all chordates and underwent gene duplications distinctly in jawless and jawed vertebrates. In the latter species, a first round of gene duplications gave rise to the Plekha4/7 and Plekha5/6 pairs and a second round to the four extant members of the subfamily. These observations are consistent with the 1R/2R hypothesis of vertebrate genome evolution. Plekha7 and Plekha5 also exist in two copies in ray-finned fishes, due to the Teleostei-specific whole genome duplication. Similarities between the vertebrate Plekha4/5/6/7 members and non-chordate sequences are restricted to their N-terminal PH domains, whereas similarities across the remaining protein molecule are only sporadically found among few invertebrate species and are limited to the coiled-coil and extreme C-terminal ends. The vertebrate Plekha4/5/6/7 proteins contain extensive intrinsically disordered domains, which are topologically and structurally conserved in all chordates, but not in non-chordate invertebrates. In summary, our study sheds light on the origins and evolution of Plekha7 and the PLEKHA4/5/6/7 subfamily and unveils new critical information suitable for future functional studies of this still understudied group of proteins.

PLEKHA5, PLEKHA6, and PLEKHA7 (WW-PLEKHAs) are members of the PLEKHA family of proteins that interact with PDZD11 through their tandem WW domains. WW-PLEKHAs contribute to the trafficking and retention of transmembrane proteins, including nectins, Tspan33, and the copper pump ATP7A, at cell-cell junctions and lateral membranes. However, the structural basis for the distinct subcellular localizations of PLEKHA5, PLEKHA6, and PLEKHA7 is not clear. Here we expressed mutant and chimeric proteins of WW-PLEKHAs in cultured cells to clarify the role of their structural domains in their localization. We found that the WW-mediated interaction between PLEKHA5 and PDZD11 is required for their respective association with cytoplasmic microtubules. The PH domain of PLEKHA5 is required for its localization along the lateral plasma membrane and promotes the lateral localization of PLEKHA7 in a chimeric molecule. Although the PH domain of PLEKHA7 is not required for its localization at the adherens junctions (AJ), it promotes a AJ localization of chimeric proteins. The C-terminal region of PLEKHA6 and PLEKHA7 and the coiled-coil region of PLEKHA7 promote their localization at AJ of epithelial cells. These observations indicate that the localizations of WW-PLEKHAs at specific subcellular sites, where they recruit PDZD11, are the result of multiple cooperative protein-lipid and protein-protein interactions and provide a rational basis for the identification of additional proteins involved in trafficking and sorting of WW-PLEKHAs.

Abstract Background The disruption of E-cadherin-mediated adhesion is considered an important driver of tumor progression. Nevertheless, numerous studies have demonstrated that E-cadherin promotes growth- or invasion-related signaling, contrary to the prevailing notion. During tumor progression, epithelial ovarian cancer (EOC) maintains E-cadherin expression and can positively affect EOC cell growth by contributing to PI3K/AKT activation. In polarized epithelia PLEKHA7, a regulator of the zonula adherens integrity, impinges E-cadherin functionality, but its role in EOCs has been never studied. Methods Ex-vivo EOC cells and cell lines were used to study E-cadherin contribution to growth and EGFR activation. The expression of the proteins involved was assessed by real time RT-PCR, immunohistochemistry and western blotting. Cells growth and drug susceptibility was monitored in different 3-dimensional (3D) systems. Recombinant lentivirus-mediated gene expression, western blotting, immunoprecipitation and confocal microscopy were applied to investigate the biological impact of PLEKHA7 on E-cadherin behaviour. The clinical impact of PLEKHA7 was determined in publicly available datasets. Results We show that E-cadherin expression contributes to growth of EOC cells and forms a complex with EGFR thus positively affecting ligand-dependent EGFR/CDK5 signaling. Accordingly, 3D cultures of E-cadherin-expressing EOC cells are sensitive to the CDK5 inhibitor roscovitine combined with cisplatin. We determined that PLEKHA7 overexpression reduces the formation of E-cadherin-EGFR complex, EGFR activation and cell tumorigenicity. Clinically, PLEKHA7 mRNA is statistically decreased in high grade EOCs respect to low malignant potential and low grade EOCs and correlates with better EOC patient outcome. Conclusions These data represent a significant step towards untangling the role of E-cadherin in EOCs by assessing its positive effects on EGFR/CDK5 signaling and its contribution to cell growth. Hence, the inhibition of this signaling using a CDK5 inhibitor exerts a synergistic effect with cisplatin prompting on the design of new therapeutic strategies to inhibit growth of EOC cells. We assessed for the first time in EOC cells that PLEKHA7 induces changes in the asset of E-cadherin-containing cell-cell contacts thus inhibiting E-cadherin/EGFR crosstalk and leading to a less aggressive tumor phenotype. Accordingly, PLEKHA7 levels are lower in high grade EOC patient tumors and EOC patients with better outcomes display higher PLEKHA7 levels.

ALK-rearranged renal cell carcinoma is a provisional entity in the 2016 WHO Classification of Tumors of the Urinary System and Male Genital Organs. The reported fusion partners included VCL, TPM3, EML4, STRN, and HOOK1. Herein, we present a peculiar renal cell carcinoma morphologically resembling metanephric adenoma and harboring a novel PLEKHA7-ALK fusion. Microscopically, the tumor is composed of bland epithelial cells with scant to moderate amount of amphophilic cytoplasm, round and uniform nuclei, delicate chromatin, and inconspicuous nucleoli, arranged in tightly packed small acini and angulated tubules. Papillary formation, intraluminal glomeruloid tufts, microcysts, and solid nests were focally observed. Psammomatous calcifications were evident. The tumor cells were diffusely reactive for CK7, AMACR, PAX8, and ALK, while non-reactive for WT1, BRAF V600E, CD57, carbonic anhydrase IX, TFE3, and cathepsin K. Fluorescence in situ hybridization showed breaking apart of ALK. A novel PLEKHA7exon18-ALKexon20 fusion was detected using ArcherDX FusionPlex next-generation sequencing panel and was further confirmed with reverse-transcriptase PCR. Our case demonstrates that in contrast to prior cases showing high-grade tumor cells, ALK-rearranged renal cell carcinoma may also present as a low-grade renal tumor mimicking metanephric adenoma. Immunohistochemistry and molecular testing are helpful to identify this tumor, which may be eligible for ALK inhibitor-targeted therapy. [ABSTRACT FROM AUTHOR]

In the majority of cases coronary heart disease is a polygenic disease. The search of all genetic polymorphisms that can be correlated with elevated risk is still under way. Their frequencies are specific for the various populations. Thus, to discover their local frequency is of practical significance for the precise definition of the cardio-vascular status. On the other hand, the development of the options for genetic treatment necessitate the knowledge of all culprit polymorphic variants and their local distribution. This is a review of the clinical point of view on some genetic aspects of the cardio-vascular diseases. The discussion is focused on polymorphic variants of the genes CYP17A1 and PLEKHA7. They have been the aim of study, conducted in a group of patients with coronary heart disease. As a national study for a potential link between these two genes and coronary heart disease is lacking at the moment, a thorough search of papers in referred journals and international studies was done in order to make the current review. [ABSTRACT FROM AUTHOR]

Cadherins are homophilic adhesion molecules with important functions in cell-cell adhesion, tissue morphogenesis, and cancer. In epithelial cells, E-cadherin accumulates at areas of cell-cell contact, coalesces into macromolecular complexes to form the adherens junctions (AJs), and associates via accessory partners with a subcortical ring of actin to form the apical zonula adherens (ZA). As a master regulator of the epithelial phenotype, E-cadherin is essential for the overall maintenance and homeostasis of polarized epithelial monolayers. Its expression is regulated by a host of genetic and epigenetic mechanisms related to cancer, and its function is modulated by mechanical forces at the junctions, by direct binding and phosphorylation of accessory proteins collectively termed catenins, by endocytosis, recycling and degradation, as well as, by multiple signaling pathways and developmental processes, like the epithelial to mesenchymal transition (EMT). Nuclear signaling mediated by the cadherin associated proteins β-catenin and p120 promotes growth, migration and pluripotency. Receptor tyrosine kinase, PI3K/AKT, Rho GTPase, and HIPPO signaling, are all regulated by E-cadherin mediated cell-cell adhesion. Finally, the recruitment of the microprocessor complex to the ZA by PLEKHA7, and the subsequent regulation of a small subset of miRNAs provide an additional mechanism by which the state of epithelial cell-cell adhesion affects translation of target genes to maintain the homeostasis of polarized epithelial monolayers. Collectively, the data indicate that loss of E-cadherin function, especially at the ZA, is a common and crucial step in cancer progression. [ABSTRACT FROM AUTHOR]

PLEKHA7 is a recently characterized component of the cytoplasmic region of epithelial adherens junctions (AJ). It comprises two WW domains, a pleckstrin-homology domain, and proline-rich and coiled-coil domains. PLEKHA7 interacts with cytoplasmic components of the AJ (p120-catenin, paracingulin, afadin), stabilizes the E-cadherin complex by linking it to the minus ends of noncentrosomal microtubules, and stabilizes junctional nectins through the newly identified interactor PDZD11. Similarly to afadin, and unlike E-cadherin and p120-catenin, the localization of PLEKHA7 at AJ is strictly zonular (in the zonula adhaerens subdomain of AJ), and does not extend along the basolateral contacts. Genome-wide association studies and experiments on animal and cellular models show that although PLEKHA7 is not required for organism viability, it is implicated in cardiovascular physiology, hypertension, primary angle closure glaucoma, susceptibility to staphylococcal α-toxin, and epithelial morphogenesis and growth. Thus, PLEKHA7 is a cytoskeletal adaptor protein important for AJ organization, and at the center of junction-associated signaling pathways which fine-tune important pathophysiological processes. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin.We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7-/- mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections. [ABSTRACT FROM AUTHOR]

Background Single nucleotide polymorphisms ( SNPs) rs11024102 in PLEKHA 7 and rs3753841 in COL 11 A 1 were identified to be associated with primary angle closure glaucoma ( PACG) by a recent large genome-wide association study. This present study is to evaluate the association of PLEKHA 7 rs11024102 and COL 11 A 1 rs3753841 with PACG. Design A systematic review and meta-analysis. Participants A total of 25 271 subjects (4895 PACG patients and 20 376 controls) in different ethnicities were tested for PLEKHA 7 rs11024102 and COL 11 A 1 rs3753841. Methods A comprehensive literature search was conducted on studies published up to July 2014. Summary odds ratios ( ORs) and 95% confidence intervals were analysed. Publication bias of the included articles was evaluated using funnel plots and Egger's test. Main Outcome Measures OR for the effects of PLEKHA 7 rs11024102 and COL 11 A 1 rs3753841 on PACG risk. Results Four eligible articles were included in this study for meta-analysis. The overall result showed that SNPs rs11024102 and rs3753841 were statistically associated with PACG ( P < 0.001) in fixed-effects model. Stratified analyses showed that the association of PLEKHA 7 rs11024102 and COL 11 A 1 rs3753841 with PACG was statistically significant in Asian population (including South Indian cohort) ( P < 0.001). In Caucasian population, significant association of COL 11 A 1 rs3753841 with PACG was detected ( P = 0.004), but PLEKHA 7 rs11024102 did not show any association with PACG ( P = 0.140). Conclusions This meta-analysis suggests that PLEKHA 7 rs11024102 is associated with PACG in Asian population and COL 11 A 1 rs3753841 has a genetic association with the development of PACG both in Caucasian and Asian populations. [ABSTRACT FROM AUTHOR]

Summary: Molecular differentiation between invasive lobular carcinomas (ILCs) and invasive ductal carcinomas (IDCs) of the breast has not been well defined. We investigated gene expression differences between ILCs and IDCs and their correlation with variations in invasiveness and tumor growth. Total RNA was isolated from 30 frozen tumor samples: 10 from ILCs and 20 from IDCs. Gene expression was investigated using the Affymetrix GeneChip Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA). Data and validation were performed by reverse transcriptase polymerase chain reaction and immunohistochemistry. Gene expression differences between ILCs and IDCs were found in 140 genes. Overall, ILCs showed up-regulation of genes related with cell migration, lipid and fatty acid metabolism, and some transcription factors and showed down-regulation of cell adhesion, actin cytoskeleton, cell proliferation, and energetic metabolism of the tumor cells. Our reverse transcriptase polymerase chain reaction results showed that PLEKHA and TMSB10 expression discriminated ILCs from luminal A IDCs, whereas PLEKHA7, TMSB10, PRDX4, and SERPINB5 discriminated ILCs from luminal B IDCs. At the protein level, Plekha7 was overexpressed in ILCs but not in normal tissue or low-grade IDCs. Moreover, Plekha7 overexpression had an inverse relation with E-cadherin expression. The gene expression profile in ILCs and IDCs differs in several signaling pathways. Our findings suggest that overexpression of PLEKHA7 is common in ILCs and could be a molecular marker to differentiate ILCs from IDCs. [ABSTRACT FROM AUTHOR]

Cingulin, paracingulin, and PLEKHA7 are proteins localized in the cytoplasmic region of the apical junctional complex of vertebrate epithelial cells. Cingulin has been detected at tight junctions (TJs), whereas paracingulin has been detected at both TJs and adherens junctions (AJs) and PLEKHA7 has been detected at AJs. One function of cingulin and paracingulin is to regulate the activity of Rho family GTPases at junctions through their direct interaction with guanidine exchange factors of RhoA and Rac1. Cingulin also contributes to the regulation of transcription of several genes in different types of cultured cells, in part through its ability to modulate RhoA activity. PLEKHA7, together with paracingulin, is part of a protein complex that links E-cadherin to the microtubule cytoskeleton at AJs. In this paper, we review the current knowledge about these proteins, including their discovery, the characterization of their expression, localization, structure, molecular interactions, and their roles in different developmental and disease model systems. [ABSTRACT FROM AUTHOR]

The plasma membrane calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adapter protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here, we test this hypothesis using cultured cell model systems. We show using immunofluorescence microscopy and a surface biotinylation assay that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct epithelial cells results in increased accumulation of endogenous PMCA at lateral cell-cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, coexpression of PDZD11 reduces membrane accumulation of overexpressed PMCA4x/b, and analysis of cytosolic calcium transients shows that PDZD11 counteracts calcium extrusion activity of overexpressed PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mouse kidney collecting duct) cells increases the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7-PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Plekha7 (Pleckstrin homology [PH] domain containing, family A member 7) regulates the assembly of proteins of the cytoplasmic apical zonula adherens junction (AJ), thus ensuring cell-cell adhesion and tight-junction barrier integrity. Little is known of Plekha7 function in cancer. In colorectal cancer (CRC) Plekha7 expression is elevated compared to adjacent normal tissue levels, increasing with clinical stage. Plekha7 was present at plasma membrane AJ with wild-type KRas (wt-KRas) but was dispersed in cells expressing mutant KRas (mut-KRas). Fluorescence lifetime imaging microscopy (FLIM) indicated a direct Plekha7 interaction with wt-KRas but scantily with mut-KRas. Inhibiting Plekha7 specifically decreased mut-KRas cell signaling, proliferation, attachment, migration, and retarded mut-KRAS CRC tumor growth. Binding of diC8-phosphoinositides (PI) to the PH domain of Plekha7 was relatively low affinity. This may be because a D175 amino acid residue plays a "sentry" role preventing PI(3,4)P 2 and PI(3,4,5)P 3 binding. Molecular or pharmacological inhibition of the Plekha7 PH domain prevented the growth of mut-KRas but not wt-KRas cells. Taken together the studies suggest that Plekha7, in addition to maintaining AJ structure plays a role in mut-KRas signaling and phenotype through interaction of its PH domain with membrane mut-KRas, but not wt-KRas, to increase the efficiency of mut-KRas downstream signaling. [ABSTRACT FROM AUTHOR]

Inflammatory breast cancer is a highly aggressive form of breast cancer that forms clusters of tumor emboli in dermal lymphatics and readily metastasizes. These cancers express high levels of E-cadherin, the major mediator of adherens junctions, which enhances formation of tumor emboli. Previous studies suggest that E-cadherin promotes cancer when the balance between apical and basolateral cadherin complexes is disrupted. Here, we used immunohistochemistry of inflammatory breast cancer patient samples and analysis of cell lines to determine the expression of PLEKHA7, an apical adherens junction protein. We used viral transduction to re-express PLEKHA7 in inflammatory breast cancer cells and examined their aggressiveness in 2D and 3D cultures and in vivo. We determined that PLEKHA7 was deregulated in inflammatory breast cancer, demonstrating improper localization or lost expression in most patient samples and very low expression in cell lines. Re-expressing PLEKHA7 suppressed proliferation, anchorage independent growth, spheroid viability, and tumor growth in vivo. The data indicate that PLEKHA7 is frequently deregulated and acts to suppress inflammatory breast cancer. The data also promote the need for future inquiry into the imbalance between apical and basolateral cadherin complexes as driving forces in inflammatory breast cancer. [ABSTRACT FROM AUTHOR]

Tight and adherens junctions are specialized sites of cell-cell interaction in epithelia and endothelia, and are involved in barrier, adhesion, and signaling functions. These functions are orchestrated by a highly organized meshwork of macromolecules in the membrane and cytoplasmic compartments. In this review, we discuss the structural organization and functions of the major cytoplasmic scaffolding and adaptor proteins of vertebrate apical junctions (ZO proteins, afadin, PLEKHA7, cingulin, paracingulin, polarity complex proteins, and a few others), focusing on their interactions with cytoskeletal and signaling proteins. Furthermore, we discuss recent results highlighting how mechanical tension, protein-protein interactions and post-translational modifications regulate the conformation and function of scaffolding proteins, and how spontaneous phase separation into biomolecular condensates contributes to apical junction assembly. Using a sequence-based algorithm, a large fraction of cytoplasmic proteins of apical junctions are predicted to be phase separating proteins (PSPs), suggesting that formation of biomolecular condensates is a general mechanism to organize cell-cell contacts by clustering proteins. • Scaffolding proteins localize at the cytoplasmic plaque of tight junction and zonula adhaerens. • Scaffolding proteins cluster transmembrane and cytoplasmic ligands to modulate their function. • Their interaction with the actomyosin cytoskeleton is crucial for assembly and mechanoregulation. • A large fraction of scaffolding and associated proteins are predicted to undergo phase separation. [ABSTRACT FROM AUTHOR]

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function. [ABSTRACT FROM AUTHOR]

The membrane pore-forming α-toxin is an important virulence factor of Staphylococcus aureus. Target cells can remove pores from their surface, but recent work shows that α-toxin may undermine this self-defense by clinging to epithelial cell junctions. The findings could lead to the development of novel remedies against S. aureus infections. [ABSTRACT FROM AUTHOR]

Pleckstrin homology domain-containing A7 (PLEKHA7) is a cytoplasmic protein at adherens junctions that has been implicated in hypertension, glaucoma, and responses to Staphylococcus aureus α-toxin. Complex formation between PLEKHA7, PDZ domain-containing 11 (PDZD11), tetraspanin 33, and the α-toxin receptor ADAM metallopeptidase domain 10 (ADAM10) promotes junctional clustering of ADAM10 and α-toxin-mediated pore formation. However, how the N-terminal region of PDZD11 interacts with the N-terminal tandem WW domains of PLEKHA7 and how this interaction promotes tetraspanin 33 binding to the WW1 domain is unclear. Here, we used site-directed mutagenesis, glutathione S -transferase pulldown experiments, immunofluorescence, molecular modeling, and docking experiments to characterize the mechanisms driving these interactions. We found that Asp-30 of WW1 and His-75 of WW2 interact through a hydrogen bond and, together with Thr-35 of WW1, form a binding pocket that accommodates a polyproline stretch within the N-terminal PDZD11 region. By strengthening the interactions of the ternary complex, the WW2 domain stabilized the WW1 domain and cooperatively promoted the interaction with PDZD11. Modeling results indicated that, in turn, PDZD11 binding induces a conformational rearrangement, which strengthens the ternary complex, and contributes to enlarging a "hydrophobic hot spot" region on the WW1 domain. The last two lipophilic residues of tetraspanin 33, Trp-283 and Tyr-282, were required for its interaction with PLEKHA7. Docking of the tetraspanin 33 C terminus revealed that it fits into the hydrophobic hot spot region of the accessible surface of WW1. We conclude that communication between the two tandem WW domains of PLEKHA7 and the PLEKHA7-PDZD11 interaction modulate the ligand-binding properties of PLEKHA7., Competing Interests: Conflict of interest—The authors declare that they have no conflict of interest with the contents of this article., (© 2020 Rouaud et al.)

Summary We previously identified PLEKHA7 and other junctional proteins as host factors mediating death by S. aureus α-toxin, but the mechanism through which junctions promote toxicity was unclear. Using cell biological and biochemical methods, we now show that ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Instead, disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. These results uncover a dock-and-lock molecular mechanism to target ADAM10 to junctions and provide a paradigm for how junctions regulate transmembrane receptors through their clustering. Graphical Abstract Highlights • The α-toxin receptor ADAM10 is clustered at junctions by the PLEKHA7-PDZD11 complex • Tspan33 docks ADAM10 to junctions by binding to the WW domain of PLEKHA7 • ADAM10 junctional clustering promotes the efficient formation of stable toxin pores • Loss of PLEKHA7-PDZD11 leads to toxin pore removal by endocytosis and cell survival Shah et al. clarify the mechanism through which adherens junctions render cells more susceptible to death by S. aureus α-toxin. PLEKHA7 and associated proteins create an Achilles heel by clustering the toxin receptor ADAM10 and toxin pores at junctions. Unclustered toxin pores are unstable and endocytosed, promoting cell survival. [ABSTRACT FROM AUTHOR]

Background: The disruption of E-cadherin-mediated adhesion is considered an important driver of tumor progression. Nevertheless, numerous studies have demonstrated that E-cadherin promotes growth- or invasion-related signaling, contrary to the prevailing notion. During tumor progression, epithelial ovarian cancer (EOC) maintains E-cadherin expression and can positively affect EOC cell growth by contributing to PI3K/AKT activation. In polarized epithelia PLEKHA7, a regulator of the zonula adherens integrity, impinges E-cadherin functionality, but its role in EOCs has been never studied. Methods: Ex-vivo EOC cells and cell lines were used to study E-cadherin contribution to growth and EGFR activation. The expression of the proteins involved was assessed by real time RT-PCR, immunohistochemistry and western blotting. Cells growth and drug susceptibility was monitored in different 3-dimensional (3D) systems. Recombinant lentivirus-mediated gene expression, western blotting, immunoprecipitation and confocal microscopy were applied to investigate the biological impact of PLEKHA7 on E-cadherin behaviour. The clinical impact of PLEKHA7 was determined in publicly available datasets. Results: We show that E-cadherin expression contributes to growth of EOC cells and forms a complex with EGFR thus positively affecting ligand-dependent EGFR/CDK5 signaling. Accordingly, 3D cultures of E-cadherin-expressing EOC cells are sensitive to the CDK5 inhibitor roscovitine combined with cisplatin. We determined that PLEKHA7 overexpression reduces the formation of E-cadherin-EGFR complex, EGFR activation and cell tumorigenicity. Clinically, PLEKHA7 mRNA is statistically decreased in high grade EOCs respect to low malignant potential and low grade EOCs and correlates with better EOC patient outcome. Conclusions: These data represent a significant step towards untangling the role of E-cadherin in EOCs by assessing its positive effects on EGFR/CDK5 signaling and its contribution to cell growth. Hence, the inhibition of this signaling using a CDK5 inhibitor exerts a synergistic effect with cisplatin prompting on the design of new therapeutic strategies to inhibit growth of EOC cells. We assessed for the first time in EOC cells that PLEKHA7 induces changes in the asset of E-cadherin-containing cell-cell contacts thus inhibiting E-cadherin/EGFR crosstalk and leading to a less aggressive tumor phenotype. Accordingly, PLEKHA7 levels are lower in high grade EOC patient tumors and EOC patients with better outcomes display higher PLEKHA7 levels. [ABSTRACT FROM AUTHOR]

The cytoskeleton is crucially important for the assembly of cell-cell junctions and the homeostatic regulation of their functions. Junctional proteins act, in turn, as anchors for cytoskeletal filaments, and as regulators of cytoskeletal dynamics and signalling proteins. The cross-talk between junctions and the cytoskeleton is critical for the morphogenesis and physiology of epithelial and other tissues, but is not completely understood. Microtubules are implicated in the delivery of junctional proteins to cell-cell contact sites, in the differentiation and spatial organization of the cytoplasm, and in the stabilization of the barrier and adhesive functions of junctions. Here we focus on the relationships between microtubules and junctions of vertebrate epithelial cells. We highlight recent discoveries on the molecular underpinnings of microtubule-junction interactions, and report new data about the interaction of cingulin and paracingulin with microtubules. We also propose a possible new role of junctions as "molecular sinks" for microtubule-associated signalling proteins. [ABSTRACT FROM AUTHOR]

Summary Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such. [ABSTRACT FROM AUTHOR]

PLEKHA7 is a junctional protein implicated in stabilization of the cadherin protein complex, hypertension, cardiac contractility, glaucoma, microRNA processing, and susceptibility to bacterial toxins. To gain insight into the molecular basis for the functions of PLEKHA7, we looked for new PLEKHA7 interactors. Here, we report the identification of PDZ domain-containing protein 11 (PDZD11) as a new interactor of PLEKHA7 by yeast two-hybrid screening and by mass spectrometry analysis of PLEKHA7 immunoprecipitates. We show that PDZD11 (17 kDa) is expressed in epithelial and endothelial cells, where it forms a complex with PLEKHA7, as determined by co-immunoprecipitation analysis. The N-terminal Trp-Trp (WW) domain of PLEKHA7 interacts directly with the N-terminal 44 amino acids of PDZD11, as shown by GST-pulldown assays. Immunofluorescence analysis shows that PDZD11 is localized at adherens junctions in a PLEKHA7-dependent manner, because its junctional localization is abolished by knock-out of PLEKHA7, and is rescued by re-expression of exogenous PLEKHA7. The junctional recruitment of nectin-1 and nectin-3 and their protein levels are decreased via proteasome-mediated degradation in epithelial cells where either PDZD11 or PLEKHA7 have been knocked-out. PDZD11 forms a complex with nectin-1 and nectin-3, and its PDZ domain interacts directly with the PDZ-binding motif of nectin-1. PDZD11 is required for the efficient assembly of apical junctions of epithelial cells at early time points in the calcium-switch model. These results show that the PLEKHA7-PDZD11 complex stabilizes nectins to promote efficient early junction assembly and uncover a new molecular mechanism through which PLEKHA7 recruits PDZ-binding membrane proteins to epithelial adherens junctions., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The transition of sessile epithelial cells to a migratory, mesenchymal phenotype is essential for metazoan development and tissue repair, but this program is exploited by tumor cells in order to escape the confines of the primary organ site, evade immunosurveillance, and resist chemo-radiation. In addition, epithelial-to-mesenchymal transition (EMT) confers stem-like properties that increase efficiency of colonization of distant organs. This review evaluates the role of cell-cell junctions in suppressing EMT and maintaining a quiescent epithelium. We discuss the conflicting data on junctional signaling in cancer and recent developments that resolve some of these conflicts. We focus on evidence from breast cancer, but include other organ sites where appropriate. Current and potential strategies for inhibition of EMT are discussed.

Purpose: To investigate the association of PLEKHA7 and COL11A1 with primary angle closure glaucoma, as well as acute and chronic subphenotype, in a Han Chinese population., Methods: A total of 984 cases, including 606 primary angle closure glaucoma (PACG) and 378 primary angle closure (PAC), and 922 normal controls were recruited. Twelve single nucleotide polymorphisms (SNPs) (rs1676486, rs3753841, rs12138977, rs2126642, rs2622848, rs216489, rs1027617, rs366590, rs11024060, rs6486330, rs11024097, and rs11024102) in the PLEKHA7 gene and COL11A12 gene were genotyped. Distributions of allele frequencies were compared between cases and controls as well as in patient subgroups with or without acute attacks., Results: Four of the 12 SNPs, including rs1676486 (P = 0.0060) and rs12138977 (P = 0.028) in COL11A1, as well as rs216489 (P = 0.0074) and rs11024102 (P = 0.038) in PLEKHA7, were found to have a statistically significant association with PAC/PACG. In the subgroup analysis, 6 out of 12 SNPs (rs1676486, rs3753841, rs12138977, rs216489, rs11024060, and rs11024102) showed statistically significant differences between acute PAC/PACG cases and controls. However, none of them showed statistically significant differences between chronic PAC/PACG cases and controls., Conclusions: Our study suggests that rs1676486 and rs12138977 in COL11A1 as well as rs216489 and rs11024102 in PLEKHA7 are associated with an increased risk of PAC/PACG in the Han Chinese population, supporting prior reports of the association of COL11A1 and PLEKH7 with angle closure glaucoma. Both COL11A1 and PLEKHA7 were shown to confer significant risk for acute PAC/PACG. Further work is necessary to confirm the importance of COL11A1 and PLEKHA7 in the pathogenesis of glaucoma., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

PLEKHA7 is a recently identified protein of the epithelial zonula adhaerens (ZA), and is part of a protein complex that stabilizes the ZA, by linking it to microtubules. Since the ZA is important in the assembly and disassembly of tight junctions (TJ), we asked whether PLEKHA7 is involved in modulating epithelial TJ barrier function. We generated clonal MDCK cell lines in which one of four different constructs of PLEKHA7 was inducibly expressed. All constructs were localized at junctions, but constructs lacking the C-terminal region were also distributed diffusely in the cytoplasm. Inducible expression of PLEKHA7 constructs did not affect the expression and localization of TJ proteins, the steady-state value of transepithelial resistance (TER), the development of TER during the calcium switch, and the flux of large molecules across confluent monolayers. In contrast, expression of three out of four constructs resulted both in enhanced recruitment of E-cadherin and associated proteins at the apical ZA and at lateral puncta adherentia (PA), a decreased TER at 18 h after assembly at normal calcium, and an attenuation in the fall in TER after extracellular calcium removal. This latter effect was inhibited when cells were treated with nocodazole. Immunoprecipitation analysis showed that PLEKHA7 forms a complex with the cytoplasmic TJ proteins ZO-1 and cingulin, and this association does not depend on the integrity of microtubules. These results suggest that PLEKHA7 modulates the dynamics of assembly and disassembly of the TJ barrier, through E-cadherin protein complex- and microtubule-dependent mechanisms.

Adherens junction (AJ) is a specialized cell-cell junction structure that plays a role in mechanically connecting adjacent cells to resist strong contractile forces and to maintain tissue structure, particularly in the epithelium. AJ is mainly comprised of cell adhesion molecules cadherin and nectin and their associating cytoplasmic proteins including β-catenin, α-catenin, p120(ctn), and afadin. Our series of studies have revealed that nectin first forms cell-cell adhesion and then recruits cadherin to form AJ. The recruitment of cadherin by nectin is mediated by the binding of α-catenin and p120(ctn) to afadin. Recent studies showed that PLEKHA7 binds to p120(ctn), which is associated with E-cadherin, and maintains the integrity of AJ in epithelial cells. In this study, we showed that PLEKHA7 bound to afadin in addition to p120(ctn) and was recruited to the nectin-3α-based cell-cell adhesion site in a manner dependent on afadin, but not on p120(ctn). The binding of PLEKHA7 to afadin was required for the proper formation of AJ, but not for the formation of tight junction, in EpH4 mouse mammary gland epithelial cells. These results indicate that PLEKHA7 plays a cooperative role with nectin and afadin in the proper formation of AJ in epithelial cells.