miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability

Background The brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer’s disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined. Methods Using a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored. Results Our results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ß-catenin signaling, a key pathway required for the BBB maintenance. Conclusion For the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier’s function.

Reciprocal EGFR signaling in the anchor cell ensures precise inter-organ connection during Caenorhabditis elegans vulval morphogenesis

ABSTRACT During Caenorhabditis elegans vulval development, the uterine anchor cell (AC) first secretes an epidermal growth factor (EGF) to specify the vulval cell fates and then invades the underlying vulval epithelium. By doing so, the AC establishes direct contact with the invaginating primary vulF cells and attaches the developing uterus to the vulva. The signals involved and the exact sequence of events joining these two organs are not fully understood. Using a conditional let-23 EGF receptor (EGFR) allele along with novel microfluidic short- and long-term imaging methods, we discovered a specific function of the EGFR in the AC during vulval lumen morphogenesis. Tissue-specific inactivation of let-23 in the AC resulted in imprecise alignment of the AC with the primary vulval cells, delayed AC invasion and disorganized adherens junctions at the contact site forming between the AC and the dorsal vulF toroid. We propose that EGFR signaling, activated by a reciprocal EGF cue from the primary vulval cells, positions the AC at the vulval midline, guides it during invasion and assembles a cytoskeletal scaffold organizing the adherens junctions that connect the developing uterus to the dorsal vulF toroid. Thus, EGFR signaling in the AC ensures the precise alignment of the two developing organs.

Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

The roles of distinct Ca2+ signaling mediated by Piezo and inositol triphosphate receptor (IP3R) in the remodeling of E-cadherin during cell dissemination

Given the role of E-cadherin (E-cad) in holding epithelial cells together, the inverse relationship between E-cad levels and cell invasion has been perceived as a principle underlying the invasiveness of tumor cells. In contrast, our study employing the Drosophila model of cell dissemination demonstrates that E-cad is necessary for the invasiveness of RasV12-transformed cells in vivo. Drosophila E-cad/β-catenin disassembles at adherens junctions and assembles at invasive protrusions—the actin- and cortactin-rich invadopodia-like protrusions associated with breach of the extracellular matrix (ECM)—during cell dissemination. Loss of E-cad attenuates dissemination of RasV12-transformed cells by impairing their ability to compromise the ECM. Strikingly, the remodeling of E-cad/β-catenin subcellular distribution is controlled by two discrete intracellular calcium signaling pathways: Ca2+ release from endoplasmic reticulum via the inositol triphosphate receptor (IP3R) disassembles E-cad at adherens junctions while Ca2+ entry via the mechanosensitive channel Piezo assembles E-cad at invasive protrusions. Thus, our study provides molecular insights into the unconventional role of E-cad in cell invasion during cell dissemination in vivo and describes the discrete roles of intracellular calcium signaling in the remodeling of E-cad/β-catenin subcellular localization.

Anti-Malignant Effect of Tensile Loading to Adherens Junctions in Cutaneous Squamous Cell Carcinoma Cells

Actomyosin contractility regulates various cellular processes including proliferation and differentiation while dysregulation of actomyosin activity contributes to cancer development and progression. Previously, we have reported that actomyosin-generated tension at adherens junctions is required for cell density-dependent inhibition of proliferation of normal skin keratinocytes. However, it remains unclear how actomyosin contractility affects the hyperproliferation ability of cutaneous squamous cell carcinoma (cSCC) cells. In this study, we find that actomyosin activity is impaired in cSCC cells both in vitro and in vivo. External application of tensile loads to adherens junctions by sustained mechanical stretch attenuates the proliferation of cSCC cells, which depends on intact adherens junctions. Forced activation of actomyosin of cSCC cells also inhibits their proliferation in a cell-cell contact-dependent manner. Furthermore, the cell cycle arrest induced by tensile loading to adherens junctions is accompanied by epidermal differentiation in cSCC cells. Our results show that the degree of malignant properties of cSCC cells can be reduced by applying tensile loads to adherens junctions, which implies that the mechanical status of adherens junctions may serve as a novel therapeutic target for cSCC.

Effect of p18 on Endothelial Barrier Function by Mediating Vascular Endothelial Rab11a-VE-cadherin Recycling

Endothelial barrier integrity requires recycling of VE-cadherin to adherens junctions. Both p18 and Rab11a play significant roles in VE-cadherin recycling. However, the underlying mechanism and the role of p18 in activating Rab11a have yet to be elucidated. Performing in vitro and in vivo experiments, we showed that p18 protein bound to VE-cadherin before Rab11a through its VE-cadherin-binding domain (aa 1–39). Transendothelial resistance showed that overexpression of p18 promoted the circulation of VE-cadherin to adherens junctions and the recovery of the endothelial barrier. Silencing of p18 caused endothelial barrier dysfunction and prevented Rab11a-positive recycling endosome accumulation in the perinuclear recycling compartments. Furthermore, p18 knockdown in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide and cecal ligation puncture. This study showed that p18 regulated the pulmonary endothelial barrier function in vitro and in vivo by regulating the binding of Rab11a to VE-cadherin and the activation of Rab11a.

Membrane architecture and adherens junctions contribute to strong Notch pathway activation

ABSTRACT The Notch pathway mediates cell-to-cell communication in a variety of tissues, developmental stages and organisms. Pathway activation relies on the interaction between transmembrane ligands and receptors on adjacent cells. As such, pathway activity could be influenced by the size, composition or dynamics of contacts between membranes. The initiation of Notch signalling in the Drosophila embryo occurs during cellularization, when lateral cell membranes and adherens junctions are first being deposited, allowing us to investigate the importance of membrane architecture and specific junctional domains for signalling. By measuring Notch-dependent transcription in live embryos, we established that it initiates while lateral membranes are growing and that signalling onset correlates with a specific phase in their formation. However, the length of the lateral membranes per se was not limiting. Rather, the adherens junctions, which assemble concurrently with membrane deposition, contributed to the high levels of signalling required for transcription, as indicated by the consequences of α-Catenin depletion. Together, these results demonstrate that the establishment of lateral membrane contacts can be limiting for Notch trans-activation and suggest that adherens junctions play an important role in modulating Notch activity.

VE-Cadherin and Vesicles Differentially Regulate Lymphatic Vascular Permeability to Solutes of Various Sizes

Lymphatic vascular permeability prevents lymph leakage that is associated with lymphedema, lymphatic malformations, obesity, and inflammation. However, the molecular control of lymphatic permeability remains poorly understood. Recent studies have suggested that adherens junctions and vesicle transport may be involved in regulating lymphatic vessel permeability. To determine the contribution of each transport pathway, we utilized an ex vivo permeability assay to directly measure the solute flux of various molecular weight solutes across a range of pressures in intact murine collecting lymphatic vessels. Pharmacological and biological tools were used to probe the relative contributions of vesicles and junction proteins in the lymphatic vasculature. We show that the permeability of collecting lymphatic vessels is inversely related to the solute molecular weight. Further, our data reveal that vesicles selectively transport BSA, as an inhibitor of vesicle formation significantly decreased the permeability to BSA (∼60% decrease, n = 8, P = 0.02), but not to 3 kDa dextran (n = 7, P = 0.41), α-lactalbumin (n = 5, P = 0.26) or 70 kDa dextran (n = 8, P = 0.13). In contrast, disruption of VE-cadherin binding with a function blocking antibody significantly increased lymphatic vessel permeability to both 3 kDa dextran (5.7-fold increase, n = 5, P < 0.0001) and BSA (5.8-fold increase, n = 5, P < 0.0001). Thus, in the lymphatic vasculature, adherens junctions did not exhibit selectivity for any of the solutes tested here, whereas vesicles specifically transport BSA. Overall, the findings suggest that disease states that disrupt VE-cadherin localization or expression will cause significant leakage of solutes and fluid from the lymphatic vasculature.