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Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucleotides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymography analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of α3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of αv integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor-induced phosphorylation of Erk1,2 and Akt/PkB. The use of a specific pharmacological inhibitor (YM-254890), the depletion of Gα(q/11) by siRNA, or the expression of a constitutively active Gα(q/11) mutant (Q209L) show that activation of Gα(q/11) is responsible for these ATP/ UTP-induced effects. Finally, we report that ATP delays growth factor-induced wound healing of keratinocyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing. [ABSTRACT FROM AUTHOR]

One of the earliest events in epithelial carcinogenesis is the dissolution of tight junctions and cell polarity signals that are essential for normal epithelial barrier function. Here, we report that EFA6B, a guanine nucleotide exchange factor for the Ras superfamily protein Arf6 that helps assemble and stabilize tight junction, is required to maintain apico-basal cell polarity and mesenchymal phenotypes in mammary epithelial cells. In organotypic three-dimensional cell cultures, endogenous levels of EFA6B were critical to determine epithelial-mesenchymal status. EFA6B downregulation correlated with a mesenchymal phenotype and ectopic expression of EFA6B hampered TGFβ-induced epithelial-to-mesenchymal transition (EMT). Transcriptomic and immunohistochemical analyses of human breast tumors revealed that the reduced expression of EFA6B was associated with loss of tight junction components and with increased signatures of EMT, cancer stemness, and poor prognosis. Accordingly, tumors with low levels of EFA6B were enriched in the aggressive triple-negative and claudin-low breast cancer subtypes. Our results identify EFA6B as a novel antagonist in breast cancer and they point to its regulatory and signaling pathways as rational therapeutic targets in aggressive forms of this disease. [ABSTRACT FROM AUTHOR]

12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1761+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis. [ABSTRACT FROM AUTHOR]

A key step of epithelial morphogenesis is the creation of the lumen. Luminogenesis by hollowing proceeds through the fusion of apical vesicles at cell-cell contacts. The small nascent lumens grow through extension, coalescence and enlargement, coordinated with cell division, to give rise to a single central lumen. Here, by using MDCK cells grown in 3D-culture, we show that EFA6A (also known as PSD) participates in luminogenesis. EFA6A recruits α-actinin 1 (ACTN1) through direct binding. In polarized cells, ACTN1 was found to be enriched at the tight junction where it acts as a primary effector of EFA6A for normal luminogenesis. Both proteins are essential for the lumen extension and enlargement, where they mediate their effect by regulating the cortical acto-myosin contractility. Finally, ACTN1 was also found to act as an effector for the isoform EFA6B (also known as PSD4) in the human mammary tumoral MCF7 cell line. EFA6B restored the glandular morphology of this tumoral cell line in an ACTN1-dependent manner. Thus, we identified new regulators of cyst luminogenesis essential for the proper maturation of a newly-formed lumen into a single central lumen., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucleotides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymography analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of alpha3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of alpha(v) integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor-induced phosphorylation of Erk(1,2) and Akt/PkB. The use of a specific pharmacological inhibitor (YM-254890), the depletion of G alpha(q/11) by siRNA, or the expression of a constitutively active G alpha(q/11) mutant (Q209L) show that activation of G alpha(q/11) is responsible for these ATP/UTP-induced effects. Finally, we report that ATP delays growth factor-induced wound healing of keratinocyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.

During neurite elongation, migrating growth cones encounter both permissive and inhibitory substrates, such as laminin and MAG (myelin-associated glycoprotein), respectively. Here, we demonstrated on two neuronal cell lines (PC12 and N1E-115), that laminin and collagen hampered, in a dose-dependent manner, MAG inhibitory activity on several integrin functions, i.e., neurite growth, cell adhesion and cell spreading. Using a function blocking antibody, in PC12 cells, we showed that alpha1beta1 integrin is required in these phenomena. In parallel, we observed that MAG perturbs actin dynamics and lamellipodia formation during early steps of cell spreading. This seemed to be independent of RhoA activation, but dependent of Rac-1 inhibition by MAG. Laminin overrode MAG activity on actin and prevented MAG inhibition NGF-induced Rac1 activation. In conclusion, we evidenced antagonistic signaling between MAG receptors and beta1 integrins, in which Rac-1 may have a central function.

Vascular endothelial growth factor (VEGF) is a potent angiogenic and prognostic factor for many tumors, including those of endocrine-responsive tissues such as the breast and uterus. Recent studies indicate that 17beta-estradiol (E(2)) modulates VEGF expression in breast and uterine cells, involving transcriptional activation through estrogen receptor (ER) alpha. However, molecular mechanisms of VEGF regulation mediated by the two ER subtypes and the potential role of ERbeta in the control of breast cancer angiogenesis have not yet been investigated. In transient transfection assays using the VEGF(-2275/+54) promoter-luciferase construct, E(2) (1 nM) increased transcription activity in MCF-7 cells (either untransfected or cotransfected with ERalpha) and it increased transcription activity in MDA-MB-231 cells cotransfected with ERalpha or ERbeta (1.8- and 2-fold induction, respectively). The positive effect was abolished when MCF-7 cells were treated with pure antiestrogen ICI 182,780 or the agonist/antagonist tamoxifen (1 micro M). To identify response elements involved in this transcriptional regulation, MCF-7 or MDA-MB-231 cells were transfected with several deletion constructs of the VEGF promoter. Deletion of 1.2-2.3 kb upstream to the transcription start in the VEGF promoter abrogated E(2)-dependent transcription in these cells. This region contains an imperfect estrogen-responsive element (ERE), ERE1520, and one activator protein 1 site. Transfection of MCF-7 cells (ERalpha) with the ERE1520-luciferase construct conferred transcriptional activity with 1 nM E(2) (1.9-fold induction). Also, the imperfect ERE formed a complex with ERalpha or ERbeta proteins in gel shift assay using MCF-7 or MDA-MB-231 nuclear extracts. In contrast to ERalpha, ERbeta could transactivate VEGF reporter construct in MDA-MB-231 cells, in the presence of E(2) or tamoxifen, suggesting different transactivational mechanisms between ERalpha and ERbeta in the presence of tamoxifen. Interestingly, E(2) inhibited VEGF transcription in MCF-7 cells transfected with ERbeta or MDA-MB-231 cells cotransfected with ERalpha and ERbeta, suggesting that heterodimerization of ERalpha/ERbeta has the ability to inhibit E(2)-induced VEGF expression in breast cancer cells. These results demonstrate that VEGF is a target gene for ERalpha and ERbeta in breast cancer cells; it remains to be determined whether ERalpha and ERbeta expression in breast biopsies correlates with VEGF expression and vascular density.