Search

In epithelial cancers, the epidermal growth factor receptor (EGFR) and integrin α6β4 are frequently overexpressed and found to synergistically activate intracellular signaling pathways that promote cell proliferation and migration. In cancer cells, the β4 subunit is phosphorylated at tyrosine residues not normally recognized as kinase substrates; however, the function of these phosphotyrosine residues in cancer cells is a subject of much debate. In EGFR-overexpressing carcinoma cells, we found that the Src family kinase (SFK) inhibitor PP2 reduces β4 tyrosine phosphorylation following the activation of EGFR. However, siRNA mediated knockdown of the SFKs Src, Fyn, Yes and Lyn, individually or in combination, did not affect the EGF-induced phosphorylation of β4. Using phospho-peptide affinity chromatography and mass spectrometry, we found that PLCγ1 binds β4 at the phosphorylated residues Y1422/Y1440, but were unable to verify this interaction in A431 carcinoma cells that overexpress the EGFR. Furthermore, using A431 cells devoid of β4 or reconstituted with phenylalanine specific mutants of β4, the activation of several downstream signaling pathways, including PLCγ/PKC, MAPK and PI3K/Akt, were not substantially affected. We conclude that tyrosine-phosphorylated β4 does not enhance EGFR-mediated signaling in EGFR-overexpressing cells, despite the fact that this integrin subunit is highly tyrosine phosphorylated in these cells.

Integrin dimers α3/β1, α6/β1 and α6/β4 are the mammary epithelial cell receptors for laminins, which are major components of the mammary basement membrane. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated using the Cre-Lox approach. During pregnancy, mutant mice displayed decreased luminal progenitor activity and retarded lobulo-alveolar development. Mammary glands appeared functional at the onset of lactation in mutant mice; however, myoepithelial cell morphology was markedly altered, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. Notably, lactation was not sustained in mutant females, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. These results suggest that the p53 pathway is involved in the control of mammary cell proliferation and survival downstream of laminin-binding integrins, and underline an essential role of cell interactions with laminin for lactogenic differentiation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Airway epithelial cells play a significant role in the pathogenesis of asthma. Although the structural and functional defects of airway epithelial cells have been postulated to increase asthma susceptibility and exacerbate asthma severity, the mechanism and implication of these defects remain uncertain. Integrin β4 (ITGB4) is a structural adhesion molecule that is downregulated in the airway epithelium of asthma patients. In this study, we demonstrated that ITGB4 deficiency leads to severe allergy-induced airway inflammation and airway hyper-responsiveness (AHR) in mice. After house dust mite (HDM) challenge, epithelial cell-specific ITGB4-deleted mice showed increased lymphocyte, eosinophil, and neutrophil infiltration into lung compared with that of the wild-type mice. ITGB4 deficiency also resulted in increased expression of the Th2 cytokine IL-4, IL-13, and the Th17 cytokine IL-17A in the lung tissue and in the T cells after HDM challenge. The aggravated inflammation in ITGB4 defect mice was partly caused by enhanced disrupted epithelial barrier integrity after HDM stress, which induced the increased thymic stromal lymphopoietin secretion from airway epithelial cells. This study therefore demonstrates that ITGB4 plays a pivotal role in containing allergen-mediated lung inflammation and airway hyper-responsiveness in allergic asthma., (©2018 Society for Leukocyte Biology.)

MUC5AC is a secretory mucin aberrantly expressed in various cancers. In lung cancer, MUC5AC is overexpressed in both primary and metastatic lesions; however, its functional role is not well understood. The present study was aimed at evaluating mechanistic role of MUC5AC on metastasis of lung cancer cells. Clinically, the overexpression of MUC5AC was observed in lung cancer patient tissues and was associated with poor survival. In addition, the overexpression of Muc5ac was also observed in genetically engineered mouse lung adenocarcinoma tissues (Kras(G12D); Trp53(R172H/+); AdCre) in comparison with normal lung tissues. Our functional studies showed that MUC5AC knockdown resulted in significantly decreased migration in two lung cancer cell lines (A549 and H1437) as compared with scramble cells. Expression of integrins (α5, β1, β3, β4 and β5) was decreased in MUC5AC knockdown cells. As both integrins and MUC5AC have a von Willebrand factor domain, we assessed for possible interaction of MUC5AC and integrins in lung cancer cells. MUC5AC strongly interacted only with integrin β4. The co-localization of MUC5AC and integrin β4 was observed both in A549 lung cancer cells as well as genetically engineered mouse adenocarcinoma tissues. Activated integrins recruit focal adhesion kinase (FAK) that mediates metastatic downstream signaling pathways. Phosphorylation of FAK (Y397) was decreased in MUC5AC knockdown cells. MUC5AC/integrin β4/FAK-mediated lung cancer cell migration was confirmed through experiments utilizing a phosphorylation (Y397)-specific FAK inhibitor. In conclusion, overexpression of MUC5AC is a poor prognostic marker in lung cancer. MUC5AC interacts with integrin β4 that mediates phosphorylation of FAK at Y397 leading to lung cancer cell migration.

Epstein-Barr virus (EBV)-encoded Latent Membrane Protein 2A (LMP2A) is an EBV latency-associated protein regularly expressed in nasopharyngeal carcinoma (NPC). In B cells, LMP2A activity resembles that of a constitutively activated antigen receptor, which recruits the Syk tyrosine kinase to activate a set of downstream signaling pathways. LMP2A also downregulates cellular Syk levels. In the present study, we demonstrate that Syk interacts with the integrin β4 subunit (ITGβ4) of integrin α6β4 in epithelial cells and that concurrent LMP2A expression interferes with this interaction by competitive binding to Syk. We find that both Syk and LMP2A have an effect on ITGβ4 cell surface expression. However, in LMP2A expressing cells, ITGβ4 remains concentrated at the cellular protrusions, an expression pattern characteristic of motile cells, including NPC-derived epithelial cells. This effect of LMP2A on ITGβ4 localization is associated with a greater propensity for migration and invasion in-vitro, and may contribute to the invasive property of LMP2A-expressing NPC.

Following a highly dynamic and complex dialogue between the epithelium and the surrounding microenvironment, the mammary gland develops into a branching structure during puberty, buds during pregnancy, forms intricate polar acini during lactation and, once the babies are weaned, remodels and involutes. At every stage of menstrual and pregnancy cycles, interactions between the cells and the extracellular matrix (ECM) and homotypic and heterotypic cell–cell interactions give rise to the architecture and function of the gland at that junction. These orchestrated programs would not be possible without the important role of the ECM receptors, integrins being the prime examples. The ECM–integrin axis regulates many crucial cellular functions including survival, migration and quiescence; the imbalance in any of these processes could contribute to oncogenesis. In this review we spotlight the involvement of two prominent integrin subunits, β1 and β4 integrins, in cross-talk with tyrosine kinase receptors, and we discuss the roles of these integrin subunits in the biology of normal breast differentiation and as potential prognostic and therapeutic targets in breast cancer.

The development of pulmonary metastasis is the major cause of death in osteosarcoma, and its molecular basis is poorly understood. In this study, we show that beta4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. Furthermore, highly metastatic MNNG-HOS cells have increased levels of beta4 integrin. Suppression of beta4 integrin expression by shRNA and disruption of beta4 integrin function by transfection of dominant-negative beta4 integrin was sufficient to revert this highly metastatic phenotype in the MNNG-HOS model without significantly affecting primary tumor growth. These findings suggest a role for beta4 integrin expression in the metastatic phenotype in human osteosarcoma cells. In addition, we identified a previously uncharacterized interaction between beta4 integrin and ezrin, a membrane-cytoskeletal linker protein that is implicated in the metastatic behavior of osteosarcoma. The beta4 integrin-ezrin interaction appears to be critical for maintenance of beta4 integrin expression. These data begin to integrate ezrin and beta4 integrin expression into a model of action for the mechanism of osteosarcoma metastases.

Flightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in Flii(Tg/+) and Flii(Tg/Tg) mice with their skin being significantly more fragile than Flii(+/-) and WT mice. Flii(+/-) primary keratinocytes showed increased adhesion on laminin and collagen I than WT and Flii(Tg/Tg) primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins alpha3, beta1, alpha6 and beta4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.

Integrin beta4 is a tissue-specific protein, but its role in autophagy of lung adenocarcinoma cells is not clear. In this study, we used microtubule-associated protein 1 light chain 3 processing and acridine orange staining to reveal that knockdown of integrin beta4 by its specific siRNA induced autophagic cell death in A549 lung cancer cells. Next, we investigated the effects of siRNA-mediated downregulation of integrin beta4 on cell death and the level of p53. The proportion of dead cells and level of p53 were significantly increased. Inhibition of autophagy by the inhibitor 3-methyladenine attenuated the cell death induced by integrin beta4 knockdown. To further understand the relationship between p53 and integrin beta4 in autophagic cell death, we inhibited the expression of integrin beta4 by its specific siRNA in p53-mutated H322 lung cancer cells. Knockdown of integrin beta4 could not induce autophagic cell death in H322 cells. The data suggest that integrin beta4 is implicated in and associated with p53 in autophagy of lung cancer cells.

Several different integrins participate in the complex interactions that promote repair of the peripheral nervous system. The role of the integrin alpha6beta4 in peripheral nerve regeneration was investigated in mice by cre-mediated deletion of the Itgb4 (beta4) gene in Schwann cells. After a crush lesion of the sciatic nerve, the recovery of motor, but not that of sensory, nerve function in beta4(-/-) mice was delayed. Immunostaining of neurofilament-200 showed that there also is a significant reduction in the number of newly outgrowing nerve sprouts in beta4(-/-) mice. Morphometric quantitative measurements revealed that fewer axons are myelinated in the nonlesioned beta4(-/-) nerves. After a sciatic nerve crush lesion, beta4(-/-) mice did not only have fewer myelinated axons compared with lesioned wild-type nerve, but their axons also showed a higher g-ratio and a thinner myelin sheath, pointing at reduced myelination. This study revealed that the beta4 protein remains expressed in the early stages of peripheral regeneration, albeit at levels lower than those before the lesion was inflicted, and showed that laminin deposition is not altered in the absence of beta4. These results together demonstrate that integrin alpha6beta4 plays an essential role in axonal regeneration and subsequent myelination.

Although almost two decades of study point to a central role for aberrant ErbB2 activation in breast cancer, many cellular and biochemical mechanisms underlying ErbB2-induced tumor initiation and progression remain to be resolved. A study by Guo et al. published recently in Cell indicates that the signaling function of beta4 integrin actively contributes to the initiation, growth, and invasion of ErbB2-induced mammary tumors in transgenic mice by promoting the activation of c-Jun and STAT3. These observations offer novel mechanistic insight into ErbB2 action and highlight the notion that ErbB2 co-opts the functions of other signaling proteins to elicit tumor progression.

Within each hemidesmosome, alpha6beta4 integrin plays a crucial role in hemidesmosome assembly by binding to laminin-5 in the basement membrane zone of epithelial tissue. Recent analyses have implicated "specificity-determining loops" (SDLs) in the I-like domain of beta integrin in regulating ligand binding. Here, we investigated the function of an SDL-like motif within the extracellular I-like domain of beta4 integrin. We generated point mutations within the SDL of beta4 integrin tagged with green fluorescent protein (GFP-beta4K150A and GFP-beta4Q155L). We also generated a mutation within the I-like domain of the beta4 integrin, lying outside the SDL region (GFP-beta4V284E). We transfected constructs encoding the mutated beta4 integrins and a GFP-conjugated wild type beta4 integrin (GFP-beta4WT) into 804G cells, which assemble hemidesmosomes, and human endothelial cells, which express little endogenous beta4 integrin. In transfected 804G cells, GFP-beta4WT and GFP-beta4V284E colocalize with hemidesmosome proteins, whereas hemidesmosomal components in cells expressing GFP-beta4K150A and GFP-beta4Q155L are aberrantly localized. In endothelial cells, GFP-beta4WT and mutant proteins are co-expressed at the cell surface with alpha6 integrin. When transfected endothelial cells are plated onto laminin-5 matrix, GFP-beta4WT and GFP-beta4V284E localize with laminin-5, whereas GFP-beta4K150A and GFP-beta4Q155L do not. GFP-beta4WT and GFP-beta4V284E expressed in endothelial cells associate with the adaptor protein Shc when the cells are stimulated with laminin-5. However, GFP-beta4K150A and GFP-beta4Q155L fail to associate with Shc even when laminin-5 is present, thus impacting downstream signaling. These results provide evidence that the SDL segment of the beta4 integrin subunit is required for ligand binding and is involved in outside-in signaling.

Helicobacter pylori colonizes the stomach at the interface between the mucus layer and the apical pole of gastric epithelial cells. A number of secreted and shed products from the bacteria, such as proteins and lipopolysaccharide, are likely to have a role in the pathogenesis at the epithelial level. To determine the physiological response of transporting polarized epithelia to released soluble factors from the bacterium, we used the T84 cell line. Monolayers of T84 cells were exposed to soluble extracts from H. pylori. The extracts induced rapid "dome" formation as well as an immediate decrease in transepithelial electrical resistance. Domes are fluid-filled blister-like structures unique to polarized epithelia. Their formation has been linked to sodium-transporting events as well as to diminished adherence of the cells to the substrate. H. pylori-induced dome formation in T84 monolayers was exacerbated by amiloride and inhibited by ouabain. Furthermore, it was associated with changes in the expression of the laminin binding alpha 6 beta 4 integrin and the 67-kDa laminin receptor. Domes formed primarily on laminin-coated filters, rather than on fibronectin or collagen matrices, and their formation was inhibited by preincubating the bacterial extract with soluble laminin. This effect was specific to H. pylori and independent of the urease, vacA, cagA, and Lewis phenotype of the strains. These data indicate that released elements from H. pylori can alter the physiological balance and integrity of the epithelium in the absence of an underlying immune response.

Early metastatic growth occurs at sites of vascular arrest of blood-borne cancer cells and is entirely intravascular. Here we show that lung colonization by B16-F10 cells is licensed by beta(4) integrin adhesion to the mouse lung endothelial Ca(2+)-activated chloride channel protein mCLCA1. In a manner independent of Met, beta(4) integrin-mCLCA1-ligation leads to complexing with and activation of focal adhesion kinase (FAK) and downstream signaling to extracellular signal-regulated kinase (ERK). FAK/ERK signaling is Src-dependent and is interrupted by adhesion blocking antibodies and by dominant-negative (dn)-FAK mutants. Levels of ERK activation in B16-F10 cells transfected with wild-type or mutant FAK are closely associated with rates of proliferation and bromodeoxyuridine (BrdUrd) incorporation of tumor cells grown in mCLCA1-coated dishes, the ability to form tumor cell colonies on CLCA-expressing endothelial cell monolayers, and the extent of pulmonary metastatic growth. Parallel with the transfection rates, B16-F10 cells transfected with dn-FAK mutants and injected intravenously into syngeneic mice generate approximately half the number and size of lung colonies that vector-transfected B16-F10 cells produce. For the first time, beta(4) integrin ligation to its novel CLCA-adhesion partner is shown to be associated with FAK complexing, activation, and signaling to promote early, intravascular, metastatic growth.