Fibroblast-specific integrin-alpha V differentially regulates type 17 and type 2 driven inflammation and fibrosis.

Fibroproliferative diseases affect a significant proportion of the world's population. Despite this, core mechanisms driving organ fibrosis of diverse etiologies remain ill defined. Recent studies suggest that integrin-alpha V serves as a master driver of fibrosis in multiple organs. Although diverse mechanisms contribute to the progression of fibrosis, TGF-ß and IL-13 have emerged as central mediators of fibrosis during type 1/type 17, and type 2 polarized inflammatory responses, respectively. To investigate if integrin-alpha V interactions or signaling is critical to the development of type 2 fibrosis, we analyzed fibroblast-specific integrin-alpha V knockout mice in three type 2-driven inflammatory disease models. While we confirmed a role for integrin-alpha V in type 17-associated fibrosis, integrin-alpha V was not critical to the development of type 2-driven fibrosis. Additionally, our studies support a novel mechanism through which fibroblasts, via integrin-alpha V expression, are capable of regulating immune polarization. We show that when integrin-alpha V is deleted on fibroblasts, initiation of type 17 inflammation is inhibited leading to a deregulation of type 2 inflammation. This mechanism is most evident in a model of severe asthma, which is characterized by a mixed type 2/type 17 inflammatory response. Together, these findings suggest dual targeting of integrin-alpha V and type 2 pathways may be needed to ameliorate fibrosis and prevent rebound of opposing pro-fibrotic and inflammatory mechanisms. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Periostin promotes migration and invasion of renal cell carcinoma through the integrin/focal adhesion kinase/c-Jun N-terminal kinase pathway.

Periostin (POSTN) is an extracellular matrix protein which is overexpressed in a variety of cancers and has been related to tumorigenesis of renal cell carcinoma. However, the involvement of POSTN in renal cell carcinoma migration, invasion, and their underlying mechanisms has not been established. In this study, renal cell carcinoma cell lines stably overexpressing POSTN were established using a lentiviral vector, and the effects of POSTN on renal cell carcinoma cell migration and invasion were investigated. POSTN overexpression increased the migration and invasion capabilities of renal cell carcinoma cell lines as well as activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. Integrin αvß3 and αvß5 antibodies inhibited POSTN overexpression or recombinant POSTN-induced focal adhesion kinase activation, cell migration, and invasion. Furthermore, lentivirus-mediated focal adhesion kinase knockdown and c-Jun N-terminal kinase inhibitor reduced POSTN-enhanced phosphorylation of c-Jun N-terminal kinase, matrix metalloproteinase-9 and matrix metalloproteinase-2 expressions, cell migration, and invasion. Our research thus indicates that POSTN promotes renal cell carcinoma cell migration and invasion through interaction with integrins αvß3 and αvß5 and subsequent activation of the focal adhesion kinase/c-Jun N-terminal kinase pathway. These results suggest that POSTN plays a critical role in renal cell carcinoma metastasis and may represent a potential target for novel therapeutic approaches against renal cell carcinoma.

Clustering of integrin α5 at the lateral membrane restores epithelial polarity in invasive colorectal cancer cells.

Apicobasolateral polarity is a fundamental property of epithelial cells, and its loss is a hallmark of cancer. Integrin-mediated contact with the extracellular matrix defines the basal surface, setting in motion E-cadherin-mediated cell-cell contact, which establishes apicobasolateral polarity. Role(s) for lateral integrins in this polarization process and the consequences of their disruption are incompletely understood. We show that addition of an integrin ß1-activating monoclonal antibody, P4G11, to invasive colorectal cancer cells in three-dimensional type 1 collagen reverts the invasive phenotype and restores apicobasolateral polarity. P4G11 induces clustering of integrin α5ß1 at lateral, intercellular surfaces. This leads to deposition and polymerization of fibronectin and recruitment of paxillin to sites of lateral integrin α5ß1 clustering and is followed by tight junction formation, as determined by ZO-1 localization. Inducible elimination of integrin α5 abrogates the epithelial-organizing effects of P4G11. In addition, polymerization of fibronectin is required for the effects of P4G11, and addition of polymerized superfibronectin is sufficient to induce tight junction formation and apicobasolateral polarization. In the normal human colon, we show that integrin α5 localizes to the lateral membrane of terminally differentiated colonocytes and that integrin α5 staining may be reduced in colorectal cancer. Thus we propose a novel role for integrin α5ß1 in regulating epithelial morphogenesis.

Mice deficient in endothelial α5 integrin are profoundly resistant to experimental ischemic stroke.

Stroke is a disease in dire need of better therapies. We have previously shown that a fragment of the extracellular matrix proteoglycan, perlecan, has beneficial effects following cerebral ischemia via the α5ß1 integrin receptor. We now report that endothelial cell selective α5 integrin deficient mice (α5 KO) are profoundly resistant to ischemic infarct after transient middle cerebral artery occlusion. Specifically, α5 KOs had little to no infarct 2-3 days post-stroke, whereas controls had an increase in mean infarct volume over the same time period as expected. Functional outcome is also improved in the α5 KOs compared with controls. Importantly, no differences in cerebrovascular anatomy or collateral blood flow were noted that could account for this difference in ischemic injury. Rather, we demonstrate that α5 KOs have increased blood-brain barrier integrity (increased expression of claudin-5, and absent brain parenchymal IgG extravasation) after stroke compared with controls, which could explain their resistance to ischemic injury. Additionally, inhibition of α5 integrin in vitro leads to decreased permeability of brain endothelial cells following oxygen-glucose deprivation. Together, these findings indicate endothelial cell α5 integrin plays an important role in stroke outcome and blood-brain barrier integrity, suggesting that α5 integrin could be a novel therapeutic target for stroke.

Alpha 5 Integrin Mediates Osteoarthritic Changes in Mouse Knee Joints.

Osteoarthritis (OA) is one of most common skeletal disorders and can affect synovial joints such as knee and ankle joints. α5 integrin, a major fibronectin receptor, is expressed in articular cartilage and has been demonstrated to play roles in synovial joint development and in the regulation of chondrocyte survival and matrix degradation in articular cartilage. We hypothesized that α5 integrin signaling is involved in pathogenesis of OA. To test this, we generated compound mice that conditionally ablate α5 integrin in the synovial joints using the Gdf5Cre system. The compound mice were born normally and had an overall appearance similar to the control mice. However, when the mutant mice received the OA surgery, they showed stronger resistance to osteoarthritic changes than the control. Specifically the mutant knee joints presented lower levels of cartilage matrix and structure loss and synovial changes and showed stronger biomechanical properties than the control knee joints. These findings indicate that α5 integrin may not be essential for synovial joint development but play a causative role in induction of osteoarthritic changes.

TM4SF5 suppression disturbs integrin α5-related signalling and muscle development in zebrafish.

TM4SF5 (transmembrane 4 L six family member 5) is involved in EMT (epithelial-mesenchymal transition) for liver fibrosis and cancer metastasis; however, the function(s) of TM4SF5 during embryogenesis remains unknown. In the present study the effects of TM4SF5 on embryogenesis of zebrafish were investigated. tm4sf5 mRNA was expressed in the posterior somites during somitogenesis and in whole myotome 1 dpf (day post-fertilization). tm4sf5 suppression impaired development of the trunk with aberrant morphology of muscle fibres and altered expression of integrin α5. The arrangement and adhesion of muscle cells were abnormally disorganized in tm4sf5 morphants with reduced muscle fibre masses, where integrin α5-related signalling molecules, including fibronectin, FAK (focal adhesion kinase), vinculin and actin were aberrantly localized, compared with those in control fish. Aberrant muscle developments in tm4sf5 morphants were recovered by additional tm4sf5 or integrin α5 mRNA injection. Such a role for TM4SF5 was observed in the differentiation of C2C12 mouse myoblast cells to multinuclear muscle cells. Taken together, the results show that TM4SF5 controls muscle differentiation via co-operation with integrin α5-related signalling.

The role of integrin-α5 in the proliferation and odontogenic differentiation of human dental pulp stem cells.

INTRODUCTION: It has been reported that integrin-α5 (ITGA5) activity is related to cell proliferation, differentiation, migration, and organ development. However, the involvement of ITGA5 in the biological functions of human dental pulp stem cells (hDPSCs) has not been explored. The aim of this study was to investigate the role of ITGA5 in the proliferation and odontogenic differentiation of hDPSCs. METHODS: We knocked down ITGA5 in hDPSCs using lentivirus-mediated ITGA5 short hairpin RNA (shRNA). Changes in the proliferation in hDPSCs infected with lentiviruses expressing ITGA5-specific shRNA or negative control shRNA were examined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-ethynyl-2'-deoxyuridine labeling. Both ITGA5 knockdown cells and shMock cells were cultured in mineralization medium for 3 weeks, and the differentiation of cells was detected with alizarin red S staining. The expression of odontogenic differentiation-related molecular markers was assessed using real-time polymerase chain reaction and Western blot assays. RESULTS: The knockdown of ITGA5 decreased the proliferation capacity of hDPSCs. ITGA5 shRNA promoted odontogenic differentiation of hDPSCs with the enhanced formation of mineralized nodules. It also up-regulated the messenger RNA expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. CONCLUSIONS: These findings suggest that ITGA5 plays an important role in maintaining hDPSCs in a proliferative state. The inhibition of ITGA5 signaling promotes the odontogenic differentiation of hDPSCs.

Remanent cell traction force in renal vascular smooth muscle cells induced by integrin-mediated mechanotransduction.

It was previously demonstrated in isolated renal vascular smooth muscle cells (VSMCs) that integrin-mediated mechanotransduction triggers intracellular Ca(2+) mobilization, which is the hallmark of myogenic response in VSMCs. To test directly whether integrin-mediated mechanotransduction results in the myogenic response-like behavior in renal VSMCs, cell traction force microscopy was used to monitor cell traction force when the cells were pulled with fibronectin-coated or low density lipoprotein (LDL)-coated paramagnetic beads. LDL-coated beads were used as a control for nonintegrin-mediated mechanotransduction. Pulling with LDL-coated beads increased the cell traction force by 61 ± 12% (9 cells), which returned to the prepull level after the pulling process was terminated. Pulling with noncoated beads had a minimal increase in the cell traction force (12 ± 9%, 8 cells). Pulling with fibronectin-coated beads increased the cell traction force by 56 ± 20% (7 cells). However, the cell traction force was still elevated by 23 ± 14% after the pulling process was terminated. This behavior is analogous to the changes of vascular resistance in pressure-induced myogenic response, in which vascular resistance remains elevated after myogenic constriction. Fibronectin is a native ligand for α(5)ß(1)-integrins in VSMCs. Similar remanent cell traction force was found when cells were pulled with beads coated with ß(1)-integrin antibody (Ha2/5). Activation of ß(1)-integrin with soluble antibody also triggered variations of cell traction force and Ca(2+) mobilization, which were abolished by the Src inhibitor. In conclusion, mechanical force transduced by α(5)ß(1)-integrins triggered a myogenic response-like behavior in isolated renal VSMCs.

An angiogenic role for the α5ß1 integrin in promoting endothelial cell proliferation during cerebral hypoxia.

Fibronectin is a critical regulator of vascular modelling, both in development and in the adult. In the hypoxic adult central nervous system (CNS), fibronectin is induced on angiogenic vessels, and endothelial cells show strong induction of the two fibronectin receptors α5ß1 and αvß3 integrins. In a previous study, we found that the αvß3 integrin is dispensable for hypoxic-induced cerebral angiogenesis, but a role for the endothelial α5ß1 integrin was suggested. To directly investigate the role of endothelial α5 integrin in cerebral angiogenesis, wild-type mice and mice lacking α5 integrin expression in endothelial cells (α5-EC-KO) were subject to hypoxia (8% O(2)) for 0, 2, 4, 7 or 14 days. Quantification of cerebral vessel density and endothelial-specific proteins claudin-5 and Glut-1 revealed that α5-EC-KO mice displayed an attenuated angiogenic response, which correlated with delayed endothelial proliferation. α5-EC-KO mice showed no defect in the ability to organize a cerebrovascular fibronectin matrix, and no compensatory increase in vascular αvß3 integrin expression. Consistent with these findings, primary α5KO brain endothelial cells (BEC) in culture exhibited delayed growth and proliferation. Taken together, these studies demonstrate an important angiogenic role for the α5ß1 integrin in promoting BEC proliferation in response to cerebral hypoxia.

Integrin-α5 coordinates assembly of posterior cranial placodes in zebrafish and enhances Fgf-dependent regulation of otic/epibranchial cells.

Vertebrate sensory organs develop in part from cranial placodes, a series of ectodermal thickenings that coalesce from a common domain of preplacodal ectoderm. Mechanisms coordinating morphogenesis and differentiation of discrete placodes are still poorly understood. We have investigated whether placodal assembly in zebrafish requires Integrin- α5 (itga5), an extracellular matrix receptor initially expressed throughout the preplacodal ectoderm. Morpholino knockdown of itga5 had no detectable effect on anterior placodes (pituitary, nasal and lens), but posterior placodes developed abnormally, resulting in disorganization of trigeminal and epibranchial ganglia and reduction of the otic vesicle. Cell motion analysis in GFP-transgenic embryos showed that cell migration in itga5 morphants was highly erratic and unfocused, impairing convergence and blocking successive recruitment of new cells into these placodes. Further studies revealed genetic interactions between itga5 and Fgf signaling. First, itga5 morphants showed changes in gene expression mimicking modest reduction in Fgf signaling. Second, itga5 morphants showed elevated apoptosis in the otic/epibranchial domain, which was rescued by misexpression of Fgf8. Third, knockdown of the Fgf effector erm had no effect by itself but strongly enhanced defects in itga5 morphants. Finally, proper regulation of itga5 requires dlx3b/4b and pax8, which are themselves regulated by Fgf. These findings support a model in which itga5 coordinates cell migration into posterior placodes and augments Fgf signaling required for patterning of these tissues and cell survival in otic/epibranchial placodes.

Integrin subunits alpha5 and alpha6 regulate cell cycle by modulating the chk1 and Rb/E2F pathways to affect breast cancer metastasis.

BACKGROUND: Although integrins have been implicated in the progression of breast cancer, the exact mechanism whereby they exert this regulation is clearly not understood. To understand the role of integrins in breast cancer, we examined the expression levels of several integrins in mouse breast cancer cell lines by flow cytometry and the data were validated by Western and RT-PCR analysis. The importance of integrins in cell migration and cell invasion was examined by in vitro assays. Further the effect of integrins on metastasis was investigated by in vivo experimental metastasis assays using mouse models. RESULTS: Integrin α5 subunit is highly expressed in the nonmetastatic cell line 67NR and is significantly low in the highly invasive cell line 4T1. In contrast, expression levels of integrin α6 subunit are high in 4T1 cells and low in 67NR cells. In vitro data indicated that overexpression of α5 subunit and knockdown of α6 integrin subunit inhibited cell proliferation, migration, and invasion. Our in vivo findings indicated that overexpression of integrin α5 subunit and knockdown of α6 subunit decreased the pulmonary metastasis property of 4T1 cells. Our data also indicated that overexpression of alpha 5 integrin subunit and suppression of alpha6 integrin subunit inhibited cells entering into S phase by up-regulating p27, which results in downregulation of cyclinE/CDK2 complexes, This suggests that these integrins regulate cell growth through their effects on cell-cycle-regulated proteins. We also found that modulation of these integrins upregulates E2F, which may induce the expression of chk1 to regulate cdc25A/cyclin E/CDK2/Rb in a feedback loop mechanism. CONCLUSION: This study indicates that Integrin α5 subunit functions as a potential metastasis suppressor, while α6 subunit functions as a metastasis promoter. The modulation of integrins reduces cdc25 A, another possible mechanism for downregulation of CDK2. Taken together we demonstrate a link between integrins and the chk1-cdc25-cyclin E/CDK2-Rb pathway.

Combinatorial and distinct roles of α5 and α4 integrins in stress erythropoiesis in mice.

To delineate the role of specific members of ß1 integrins in stress erythropoiesis in the adult, we compared the response to phenylhydrazine stress in 3 genetically deficient models. The survival of ß1-conditionally deficient mice after phenylhydrazine is severely compromised because of their inability to mount a successful life saving splenic erythroid response, a phenotype reproduced in ß1(Δ/Δ) reconstituted animals. The response of bone marrow to phenylhydrazine-induced stress was, unlike that of spleen, appropriate in terms of progenitor cell expansion and mobilization to peripheral blood although late differentiation defects qualitatively similar to those in spleen were present in bone marrow. In contrast to ß1-deficient mice, α4(Δ/Δ) mice showed only a kinetic delay in recovery and similar to ß1(Δ/Δ), terminal maturation defects in both bone marrow and spleen, which were not present in VCAM-1(Δ/Δ) mice. Convergence of information from these comparative studies lends new insight to the distinct in vivo roles of α4 and α5 integrins in erythroid stress, suggesting that the presence of mainly α5ß1 integrin in all hematopoietic progenitor cells interacting with splenic microenvironmental ligands/cells is instrumental for their survival and accumulation during hemolytic stress, whereas presence of α4 or of both α5 and α4, is important for completion of terminal maturation steps.

Endothelial alpha5 and alphav integrins cooperate in remodeling of the vasculature during development.

Integrin cell adhesion receptors and fibronectin, one of their extracellular matrix ligands, have been demonstrated to be important for angiogenesis using functional perturbation studies and complete knockout mouse models. Here, we report on the roles of the alpha5 and alphav integrins, which are the major endothelial fibronectin receptors, in developmental angiogenesis. We generated an integrin alpha5-floxed mouse line and ablated alpha5 integrin in endothelial cells. Unexpectedly, endothelial-specific knockout of integrin alpha5 has no obvious effect on developmental angiogenesis. We provide evidence for genetic interaction between mutations in integrin alpha5 and alphav and for overlapping functions and compensation between these integrins and perhaps others. Nonetheless, in embryos lacking both alpha5 and alphav integrins in their endothelial cells, initial vasculogenesis and angiogenesis proceed normally, at least up to E11.5, including the formation of apparently normal embryonic vasculature and development of the branchial arches. However, in the absence of endothelial alpha5 and alphav integrins, but not of either alone, there are extensive defects in remodeling of the great vessels and heart resulting in death at ~E14.5. We also found that fibronectin assembly is somewhat affected in integrin alpha5 knockout endothelial cells and markedly reduced in integrin alpha5/alphav double-knockout endothelial cell lines. Therefore, neither alpha5 nor alphav integrins are required in endothelial cells for initial vasculogenesis and angiogenesis, although they are required for remodeling of the heart and great vessels. These integrins on other cells, and/or other integrins on endothelial cells, might contribute to fibronectin assembly and vascular development.

Interleukin-8 (CXCL8) stimulates trophoblast cell migration and invasion by increasing levels of matrix metalloproteinase (MMP)2 and MMP9 and integrins alpha5 and beta1.

Interleukin-8 (IL8/CXCL8) is present in decidua and trophoblast, which also expresses the IL8 receptors, CXCR1 and CXCR2. IL8 was shown to stimulate trophoblast migration. Matrix metalloproteinase (MMP)2, MMP9, and integrins alpha(5)beta(1) and alpha(1)beta(1) were found to play important roles in trophoblast invasion. We hypothesized that IL8 would increase this cell migration and invasion by HTR-8/SVneo cells through the activity of MMPs and integrins. Isolated first trimester of pregnancy cytotrophoblast (CT) and HTR-8/SVneo cell line were used. Migration was studied by monolayer wounding test, and invasion by Matrigel invasion test. The effects of IL8 on MMPs and integrin subunit expression were determined in HTR-8/SVneo cells by gelatin zymography and western blot respectively. The results that were obtained showed that exogenous IL8 stimulated HTR-8/SVneo cell migration and invasion. MMP2 and MMP9 levels were stimulated to 182% (P<0.01) and 134% (P<0.01) respectively. Integrin alpha(5) expression was increased to 119% (P<0.05) and integrin beta(1) expression to 173% (P<0.001) of the control values. The data that were obtained show for the first time the sensitivity of the HTR-8/SVneo cells, in addition to isolated first trimester CT, to IL8. Exogenous IL8/CXCL8 increased trophoblast cell migration and invasion, which may be partly attributable to stimulation of MMP2 and MMP9 levels and an increase in integrins. HTR-8/SVneo cell viability and proliferation were also increased.

TMPRSS4 induces invasion and epithelial-mesenchymal transition through upregulation of integrin alpha5 and its signaling pathways.

TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed on the cell surface in pancreatic, thyroid and other cancer tissues, although its oncogenic significance and molecular mechanisms are unknown. Previously, we have shown that TMPRSS4 promotes invasion, migration and metastasis of human tumor cells by facilitating an epithelial-mesenchymal transition (EMT). In this study, we explored the molecular basis underlying TMPRSS4-mediated effects. We show that multiple downstream signaling pathways, including focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), Akt, Src and Rac1, are activated by TMPRSS4 expression and that FAK signaling and ERK activation are required for TMPRSS4-induced invasiveness and EMT, including cadherin switch. Inhibition of PI3K or Src reduced invasiveness and actin rearrangement mediated by TMPRSS4 without restoring E-cadherin expression. Downregulation of E-cadherin was required for TMPRSS4-mediated effects but was not sufficient to induce EMT and invasion. TMPRSS4 induced integrin alpha5 expression and its signal transduction, leading to invasiveness and EMT accompanied by downregulation of E-cadherin. Functional blocking confirmed that integrin alpha5beta1 is a critical signaling molecule that is sufficient to induce TMPRSS4-mediated effects. Immunohistochemical analysis showed that TMPRSS4 expression was significantly higher in human colorectal cancer tissues from advanced stages than in that of early stage. Furthermore, upregulation of TMPRSS4 was correlated with enhanced integrin alpha5 expression. These observations implicate integrin alpha5 upregulation as a molecular mechanism by which TMPRSS4 induces invasion and contributes to cancer progression.

Monocyte CD49e and 110-120 kDa fibronectin fragments: HIV prognostic indicators independent of viral load and CD4 T-cell counts.

OBJECTIVE: To investigate the prognostic impact of chronic inflammation associated with HIV infections. Previously, we had observed that proteases, released in the course of HIV infections, cause 110-120 kDa fibronectin fragments (FNf) to appear in the blood of many patients. In vitro, at concentrations within the range found in patients' plasma, FNf stimulate monocytes to release proteolytic enzymes that remove CD49e from the cell surface and produce cytokines that suppress proliferation of activated T cells when stimulated by agents that crosslink their antigen receptors. DESIGN: A long-term observational study of patients whose plasma FNf and monocyte CD49e had been measured at 90-day intervals for 1.4 + or - 0.5 years. METHODS: Plasma FNf was measured by a quantitative western blot assay and monocyte CD49e expression by flow cytometry. Patients were monitored clinically for up to 5 years after enrollment. RESULTS: All-cause mortality was significantly higher in patients who had at least 5 microg/ml FNf in more than 50% of plasma samples and/or persistent depletion of monocyte CD49e. Persistence of FNf and depletion of monocyte CD49e were not associated with changes in viral load or CD4 T-cell counts. CONCLUSION: Persistently reduced expression of blood monocyte CD49e and/or the persistent presence of FNf in plasma are adverse prognostic markers in HIV-infected patients.

alpha5-integrin is crucial for L1CAM-mediated chemoresistance in pancreatic adenocarcinoma.

We recently showed that the adhesion molecule L1CAM (CD171) is overexpressed in pancreatic adenocarcinoma (PDAC) essentially contributing to chemoresistance of PDAC cells. In search of the mechanisms of this effect we now identified alpha5-integrin as the L1CAM ligand being essential for L1CAM-mediated chemoresistance of these highly malignant tumor cells. Thus, blockade or knock-down of alpha5-integrin in the L1CAM expressing PDAC cell lines PT45-P1res, Colo357 and Panc1 increased anti-cancer drug sensitivity. In line with the previously reported NO-dependent caspase inhibition resulting from L1CAM induced iNOS expression, the loss of chemoresistance upon alpha5-integrin inhibition was preceded by decreased iNOS expression and enhanced caspase-3/-7 activation. Accordingly, the loss of anti-cancer drug protection by alpha5-integrin inhibition could be overcome by administration of the NO-donor SNAP. Moreover, the gain of chemoresistance of parental PT45-P1 cells when transfected with L1CAM was abrogated by alpha5-integrin inhibition, whereas transfection of PT45-P1 cells with an integrin binding-deficient L1CAM mutant (L1mutRGE) did neither induce chemoresistance or iNOS expression nor conferred sensitivity to alpha5-integrin inhibition as seen upon transfection with wild-type L1CAM. Thus, mutational loss of the integrin binding site in the L1CAM molecule or the blockade of alpha5-integrin abolished the induction of iNOS expression and chemoresistance by L1CAM, indicating that both a functional L1CAM and alpha5-integrin are indispensable of L1CAM-induced drug resistance in PDAC cells.

Hypoxia-inducible factor 1alpha mediates anoikis resistance via suppression of alpha5 integrin.

The transcription factor hypoxia-inducible factor 1 (HIF-1) alpha is abundantly expressed in the majority of human carcinomas and their metastases. HIF-1alpha controls central metastasis-associated pathways such as glycolysis, angiogenesis, and invasion. Functional inhibition of HIF-1alpha leads to impaired metastasis formation in murine tumor models. However, the precise molecular mechanisms underlying the metastasis-promoting role of HIF-1alpha have not been fully characterized. The ability of transformed epithelial cells to initiate the metastatic cascade relies on their ability to escape anoikis, a default program of apoptosis induction following loss of integrin anchoring to the extracellular matrix. Therefore, we addressed the function of HIF-1alpha in anoikis resistance and anchorage-independent growth. Inhibition of HIF-1alpha via RNA interference resulted in up-regulation of alpha5 integrin on the cell surface of human gastric cancer cells, whereas other integrins remained unaffected. Integrin alpha5 induction occurred at the level of transcription and was dependent on elevated intracellular superoxide in HIF-1alpha-knockdown cells. HIF-1alpha-deficient cells displayed significantly increased anoikis susceptibility due to up-regulated alpha5 integrin. Finally, colony formation in soft agar was shown to be dependent on HIF-1alpha as HIF-1alpha-deficient cells displayed a 70% reduction in anchorage-independent proliferation. Results obtained by RNA interference could be entirely confirmed by application of the pharmacologic HIF-1alpha-inhibitor 2-methoxyestradiol. Hence, our data argue for a pivotal role for HIF-1alpha in anoikis control via suppression of alpha5 integrin. HIF-1alpha-inhibiting drugs might therefore offer an innovative strategy for antimetastatic cancer therapy.

Etk/BMX, a Btk family tyrosine kinase, and Mal contribute to the cross-talk between MyD88 and FAK pathways.

MyD88 and focal adhesion kinase (FAK) are key adaptors involved in signaling downstream of TLR2, TLR4, and integrin alpha5beta1, linking pathogen-associated molecule detection to the initiation of proinflammatory response. The MyD88 and integrin pathways are interlinked, but the mechanism of this cross-talk is not yet understood. In this study we addressed the involvement of Etk, which belongs to the Tec family of tyrosine kinases, in the cross-talk between the integrin/FAK and the MyD88 pathways in fibroblast-like synoviocytes (FLS) and in IL-6 synthesis. Using small interfering RNA blockade, we report that Etk plays a major role in LPS- and protein I/II (a model activator of FAK)-dependent IL-6 release by activated FLS. Etk is associated with MyD88, FAK, and Mal as shown by coimmunoprecipitation. Interestingly, knockdown of Mal appreciably inhibited IL-6 synthesis in response to LPS and protein I/II. Our results also indicate that LPS and protein I/II induced phosphorylation of Etk and Mal in rheumatoid arthritis FLS via a FAK-dependent pathway. In conclusion, our data provide support that, in FLS, Etk and Mal are implicated in the cross-talk between FAK and MyD88 and that their being brought into play is clearly dependent on FAK.

Antibody-mediated blockade of integrin alpha v beta 6 inhibits tumor progression in vivo by a transforming growth factor-beta-regulated mechanism.

The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.