Integrin Subunits Αv Research Articles

Adipokine omentin-1 enhances atherosclerotic plaque stability by binding to macrophage integrin receptor

Abstract Background Omentin-1 is a novel cytokine which is primarily released by epicardial adipose tissues, and the molecule structure analysis revealed that it contained a fibrinogen-like domain. Clinical studies considered that the expression of omentin-1 is tightly associated with the development of cardiovascular diseases. In this research, we sought to investigate the effect of omentin-1 on already-established atherosclerotic lesion in ApoE−/− mouse and find out the cellular receptor of omentin-1 in macrophages. Methods We investigated the effect of omentin-1 on the plaque phenotype by implanting the ALZET minipump (which can continuously inject omentin-1 solution into mice's jugular vein) in western diet-treated ApoE−/− mice. To validate the conjugation of omentin-1 to integrin receptor αvβ3 and αvβ5, we performed immunoprecipitation experiment. Confocal microscopy was used to verify the spatial co-localization of omentin-1 and integrin receptors. Results In vivo studies showed that the administration of omentin-1 increased the collagen content and the average fibrous cap thickness of atherosclerotic plaque in ApoE−/− mice. Omentin-1 mitigated the formation of necrotic cores within the plaque, and reduced the foam cell infiltration concomitantly. Immunohistochemistry analysis of the atherosclerotic lesion from the aorta of mice revealed that the intravenous infusion of omentin-1 can suppress expression of inflammatory cytokines in vivo. Immunoprecipitation experiment and confocal microscopy analysis confirmed the binding of omentin-1 to integrin receptor αvβ3 and αvβ5. To further study the mechanism by which omentin-1 had exerted its protective function, we used human myeloid leukemia mononuclear cell line (THP1) and oxidized low density lipoprotein (ox-LDL) to establish macrophage-derived foam cell model in vitro. Cell studies demonstrated that omentin-1 can attenuated the apoptosis and inflammatory cytokines secretion in ox-LDL-induced macrophages. Besides, omentin-1 can promote the phosphorylation of integrin-relevant signaling pathway in macrophage. After adding the cilengitide (inhibitor of integrin receptor αvβ3 and αvβ5) or intervening the expression of integrin subunit αv, the phenomenon induced by omentin-1 were potently suppressed. Moreover, flowcytometry analysis provided the evidence that omentin-1 had a negative effect on macrophage efferocytosis ability. Conclusions The administration of adipokine omentin-1 can inhibit the necrotic cores formation and pro-inflammatory cytokines expression within the atherosclerotic lesion. The mechanism might be the inhibition of apoptosis and the secretion of pro-inflammatory cytokines in macrophage by binding to integrin receptor αvβ3 and αvβ5. Effect of omentin-1 on AS plaques. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National natural science foundation of China

TGF-β1/WISP1/Integrin-α interaction mediates human chondrocytes dedifferentiation.

To clarify the interaction between TGF-β1 and WISP1, and the effect of Integrin α5/V subunits on the WISP1 caused chondrocyte (CH) dedifferentiated phenotype. The knee joint cartilage from the trauma and osteoarthritis (OA) patients were collected. The patients of trauma group were confirmed to have no OA history. The protein level of WISP1, Integrin-α5/V, and type II/I collagen were analyzed by Western blotting. Besides, we isolated the CHs from the cartilage without OA and treated CHs with exogenic TGF-β1 and WISP1 protein. In addition to this, to regulate the α5 and αV subunits expression of CHs, we silenced two genes by siRNA transfection and upregulated them by exogenic protein supplement. Then, the CHs with different α5 and αV expression were treated with WISP1. To value the chondrogenic gene expression, we determined the type II collagen and SOX9 gene expression by immunofluorescence (IF) and RT-PCR, respectively. Meanwhile, the dedifferentiation markers of CH, type I collagen, and Runx2 expression was also analyzed. Cell proliferation was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The OA cartilage contains a higher level of type I collagen, WISP1, Integrin α5, and Integrin αV, but low type II collagen. The upregulation of TGF-β1 caused the increase of WISP1, as well as the high level of Integrin α5/V, and dedifferentiated gene. Besides, the upregulation of WISP1 also contributed to the TGF-β1 expression and CHs dedifferentiation. Apart from this, the silencing of the α5 subunit of Integrin aggravated the WISP1 induced CHs dedifferentiation, which was reversed by α5 upregulation. However, the αV subunit played an opposite role that mediated the WISP1-induced CHs dedifferentiation. Additionally, the interaction between TGF-β1 and WISP1 promoted the CHs proliferation, which was not affected by the Integrin-α5/V expression. TGF-β1 and WISP1 interact to induce CHs dedifferentiation, which was mainly by the mediation of the Integrin-αV subunit. On the contrary, Integrin-α5 shows a protective effect during the WISP1 caused CHs dedifferentiation.

Structural impact due to PPQEE deletion in multiple cancer associated protein - Integrin αV: An In silico exploration

A heterodimeric receptor subunit, Integrin αV, often complexed with Integrin β3 plays a vital role in cell signaling to regulate angiogenesis and promote cancer progression. The paramount β-turn formed from pentapeptide residues (PPQEE) in the cytoplasmic domain of Integrin αV was previously reported as crucial for cell signaling and its deletion was proved deleterious for protein's cell membrane adhesion and ligand binding properties. This study revealed conformational changes in the Integrin αV subunit upon deletion of PPQEE residues through in silico structural modelling approach followed by analysis of alteration of binding sites. Human Protein Atlas database helped to identify the association of Integrin αV to the unfavourable prognosis of three gastrointestinal cancers: stomach, liver and pancreatic cancers. Molecular modelling and docking techniques were carried out for the necessary complex formations (wild-type and mutant-type). Further comparison was performed for the complexes. The changes in protein's conformation and stability due to PPQEE deletion were observed in both independent subunit and heterodimer. The most noteworthy conformational shift was the disruption of a transmembrane helix into coil, which accounted for protein's impaired cell membrane adhesion, increased solvent accessibility and decreased stability. The deletion also caused a reduction of beta-turn regions, which disrupted ligand binding in the cytoplasmic domain of Integrin αV subunit. This study emphasized on structural basis of how the deletion of PPQEE residues alters stability, ligand binding and signaling activity of Integrin αV subunit highlighting the importance of these residues in maintenance of protein's native structure.

αV integrins in Schwann cells promote attachment to axons, but are dispensable in vivo.

In the developing peripheral nervous system, Schwann cells (SCs) extend their processes to contact, sort, and myelinate axons. The mechanisms that contribute to the interaction between SCs and axons are just beginning to be elucidated. Using a SC-neuron coculture system, we demonstrate that Arg-Gly-Asp (RGD) peptides that inhibit αV -containing integrins delay the extension of SCs elongating on axons. αV integrins in SC localize to sites of contact with axons and are expressed early in development during radial sorting and myelination. Short interfering RNA-mediated knockdown of the αV integrin subunit also delays SC extension along axons in vitro, suggesting that αV -containing integrins participate in axo-glial interactions. However, mice lacking the αV subunit in SCs, alone or in combination with the potentially compensating α5 subunit, or the αV partners β3 or β8 , myelinate normally during development and remyelinate normally after nerve crush, indicating that overlapping or compensatory mechanisms may hide the in vivo role of RGD-binding integrins.

Abstract A41: Inhibition of aberrant YAP and TAZ activity to prevent metastasis formation and growth

Abstract Metastasis causes most cancer-related deaths, and few effective therapies exist for patients with metastatic disease. The development of such therapies requires the identification of proteins essential for the spread and subsequent growth of metastatic cancer cells. Previous work from us and other groups has clearly demonstrated that aberrant YAP or TAZ activity, negatively regulated by the Hippo pathway, promotes cancer development and metastasis. Moreover, many human cancers have abnormally high YAP/TAZ expression or nuclear localization, and this is associated with poor prognosis. We hypothesize that targeting YAP and TAZ or upstream activators will prevent metastasis formation and growth, and we have developed and optimized in vivo model systems to test this. Using these methods, we show that YAP and TAZ are required for metastasis formation in human melanoma and breast cancer cells. An ongoing study using our in vivo metastatic growth assay and inducible YAP/TAZ RNAi is testing if YAP and TAZ are collectively required for metastatic growth after dissemination, and we hope to include the results of this study on our poster. In addition to directly targeting YAP and TAZ, we are also interested in identifying proteins that promote YAP/TAZ activity in metastatic cells, and this could lead to more therapeutic targets. Our recently published work shows that Src signaling can promote YAP/TAZ activity and enhance tumor growth and metastasis. Since integrins promote Src activity and regulate YAP and TAZ, we want to test if there are specific integrins required for YAP/TAZ activity in metastatic cells. Therefore, we used a YAP/TAZ-TEAD transcriptional activity reporter assay to perform an unbiased RNAi screen to identify integrins that regulate YAP/TAZ activity in metastatic melanoma and breast cancer cells. Knockdown of integrin subunits α2, α3, α6, and αV each reduced YAP/TAZ-TEAD transcriptional activity in metastatic breast cancer cells. These results have been confirmed with qPCR for the YAP/TAZ target genes CTGF and CRY61. Interestingly, a different set of integrin subunits appears to be required for YAP/TAZ-TEAD transcriptional activity in metastatic melanoma cells, suggesting the regulation of YAP and TAZ is not the same in different cancer types. Current work aims to use in vivo multiplex metastasis assays, which our lab has developed, to test if knockdown of each integrin either alone, or in combination, prevents metastasis formation and growth. Concurrently, we are focused on elucidating the mechanism by which these integrins enhance YAP/TAZ activity in metastatic cells. Collectively, these studies show the importance of YAP and TAZ and their upstream regulators in metastasis formation and growth and suggest that targeting YAP/TAZ activity may treat metastatic disease. Citation Format: Janine S.A. Warren, Emily Norton, John M. Lamar. Inhibition of aberrant YAP and TAZ activity to prevent metastasis formation and growth [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A41.

Tenascin-C Induces Phenotypic Changes in Fibroblasts to Myofibroblasts with High Contractility through the Integrin αvβ1/Transforming Growth Factor β/SMAD Signaling Axis in Human Breast Cancer

Tenascin-C (TNC) is strongly expressed by fibroblasts and cancer cells in breast cancer. To assess the effects of TNC on stromal formation, we examined phenotypic changes in human mammary fibroblasts treated with TNC. The addition of TNC significantly up-regulated α-smooth muscle actin (α-SMA) and calponin. TNC increased the number of α-SMA- and/or calponin-positive cells with well-developed stress fibers in immunofluorescence, which enhanced contractile ability in collagen gel contraction. The treatment with TNC also significantly up-regulated its own synthesis. Double immunofluorescence of human breast cancer tissues showed α-SMA- and/or calponin-positive myofibroblasts in the TNC-deposited stroma. Among several receptors for TNC, the protein levels of the αv and β1 integrin subunits were significantly increased after the treatment. Immunofluorescence showed the augmented colocalization of αv and β1 at focal adhesions. Immunoprecipitation using an anti-αv antibody revealed a significant increase in coprecipitated β1 with TNC in lysates. The knockdown of αv and β1 suppressed the up-regulation of α-SMA and calponin. The addition of TNC induced the phosphorylation of SMAD2/3, whereas SB-505124 and SIS3 blocked myofibroblast differentiation. Therefore, TNC enhances its own synthesis by forming a positive feedback loop and increases integrin αvβ1 heterodimer levels to activate transforming growth factor-β signaling, which is followed by a change to highly contractile myofibroblasts. TNC may essentially contribute to the stiffer stromal formation characteristic of breast cancer tissues.

Screening of integrins localized on the surface of human epidermal melanocytes.

In vivo, melanocytes occupy three-dimensional (3D) space. Nevertheless, most experiments involving melanocytes are performed in a two-dimensional microenvironment, resulting in difficulty obtaining accurate results. Therefore, it is necessary to construct an artificial in vivo-like 3D microenvironment. Here, as a step towards engineering a precisely defined acellular 3D microenvironment supporting the maintenance of human epidermal melanocytes (HEMs), we examined the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in HEMs. Real-time PCR and fluorescent immunoassay analyses were used to elucidate the expression of integrin α and β subunit genes at the transcriptional and translational levels, respectively. The functionality of the presumed integrin heterodimers was confirmed using attachment and antibody-inhibition assays. Among the genes encoding 12 integrin subunits (α1, α2, α3, α4, α5, α6, α7, αV, β1, β3, β5, and β8) showing significantly higher transcription levels, proteins translated from the integrin α2, α4, α5, β1, β3, and β5 subunit genes were detected on the surface of HEMs. These HEMs showed significantly increased adhesion to collagen I, fibronectin, laminin, and vitronectin, and functional blockade of the integrin α2 subunits significantly inhibited adhesion to collagen I, fibronectin, and laminin. In addition, there was no significant inhibition of the adhesion to fibronectin or vitronectin in HEMs with functional blockade of the integrin α4, α5, or αV subunits. These results indicate that the active integrin α2β1 heterodimer and the inactive integrin α4, α5, αV, β3, and β5 subunits are all localized on the surface of HEMs.

OPN binds alpha V integrin to promote endothelial progenitor cell incorporation into vasculature.

Angiogenesis is fundamental to the expansion of the placental vasculature during pregnancy. Integrins are associated with vascular formation; and osteopontin is a candidate ligand for integrins to promote angiogenesis. Endothelial progenitor cells (EPCs) are released from bone marrow into the blood and incorporate into newly vascularized tissue where they differentiate into mature endothelium. Results of studies in women suggest that EPCs may play an important role in maintaining placental vascular integrity during pregnancy, although little is known about how EPCs are recruited to these tissues. Our goal was to determine the αv integrin mediated effects of osteopontin on EPC adhesion and incorporation into angiogenic vascular networks. EPCs were isolated from 6 h old piglets. RT-PCR revealed that EPCs initially had a monocyte-like phenotype in culture that became more endothelial-like with cell passage. Immunofluorescence microscopy confirmed that the EPCs express platelet endothelial cell adhesion molecule, vascular endothelial cadherin, and von Willebrand factor. When EPCs were cultured on OPN-coated slides, the αv integrin subunit was observed in focal adhesions at the basal surface of EPCs. Silencing of αv integrin reduced EPC binding to OPN and focal adhesion assembly. In vitro siRNA knockdown in EPCs,demonstrated that OPN stimulates EPC incorporation into human umbilical vein endothelial cell (HUVEC) networks via αv-containing integrins. Finally, in situ hybridization and immunohistochemistry localized osteopontin near placental blood vessels. In summary, OPN binds the αv integrin subunit on EPCs to support EPC adhesion and increase EPC incorporation into angiogenic vascular networks.

Stepwise Adipogenesis of Decellularized Cellular Extracellular Matrix Regulates Adipose Tissue-Derived Stem Cell Migration and Differentiation.

Microenvironmental factors can modulate the cellular status of adipose tissue-derived stem cells (ASCs). In response to microenvironmental changes, cells can remodel extracellular matrix (ECM) proteins, which play an important role in regulating cell behaviors. During adipogenic differentiation, ECM components secreted from ASCs remodel dramatically. To evaluate the role of stepwise adipogenesis-induced cellular secretion of ECM on the behavior of ASCs, we cultured ASCs in growth and adipogenic media, and ECM secreted from cells was characterized and decellularized. The ASCs were then reseeded on decellularized ECM (d-ECM) to determine the regulatory effects of ECM on cellular behaviors. During adipogenesis, cell-secreted ECM underwent remodeling characterized by conversion from fibronectin-rich ECM to laminin-rich ECM. The cellular status of ASCs was tested after reseeding on decellularized ECM. When reseeded on growth d-ECM, ASCs exhibited greater migration ability. In contrast, ASCs seeded on adipogenic d-ECM underwent adipogenic differentiation. In addition, integrin subunit αv and integrins α6 and α7 were detected at significantly greater levels in ASCs cultured on growth and adipogenic d-ECM, respectively, suggesting that integrins play an important role in ASC migration and adipogenesis. This study demonstrated that stepwise adipogenesis-induced ECM production plays an important role in ASC migration and differentiation. In addition, this study provided a strategy to achieve precise regulation of stem cell function in adipose tissue engineering.

Integrin αvβ3 enhances replication of porcine epidemic diarrhea virus on Vero E6 and porcine intestinal epithelial cells

The entry mechanism of porcine epidemic diarrhea virus (PEDV) remains unclear, especially the virus receptor. Our previous study revealed a potential correlation between integrin αvβ3 and PEDV infection. In the current study, the effect of overexpression, silencing, antibody inhibition, and co-expression with porcine aminopeptidase N (pAPN) of integrin αvβ3 on PEDV infection was investigated and analyzed in African green monkey Vero E6 cells and porcine intestinal epithelial cells (IECs) using the classical strain CV777 and variant strain HM2017 of PEDV. Integrin αvβ3 significantly enhanced the replication of the classical and variant strains of PEDV in Vero E6 cells and IECs. The integrin αv and β3 subunits were both involved in the enhancement of PEDV infection, the Arg–Gly–Asp peptides targeting integrin αvβ3 significantly inhibited replication of PEDV in Vero E6 cells, and co-expression of integrin αvβ3 with pAPN significantly enhanced replication of PEDV in Vero E6 and BHK-21 cells. These results demonstrate that integrin αvβ3 enhances PEDV replication in Vero E6 cells and IECs. These data provide novel insights into the entry mechanism of PEDV.

MicroRNA-92 Expression in CD133+ Melanoma Stem Cells Regulates Immunosuppression in the Tumor Microenvironment via Integrin-Dependent Activation of TGFβ.

In addition to being refractory to treatment, melanoma cancer stem cells (CSC) are known to suppress host antitumor immunity, the underlying mechanisms of which need further elucidation. In this study, we established a novel role for miR-92 and its associated gene networks in immunosuppression. CSCs were isolated from the B16-F10 murine melanoma cell line based on expression of the putative CSC marker CD133 (Prominin-1). CD133+ cells were functionally distinct from CD133- cells and showed increased proliferation in vitro and enhanced tumorigenesis in vivo. CD133+ CSCs also exhibited a greater capacity to recruit immunosuppressive cell types during tumor formation, including FoxP3+ Tregs, myeloid-derived suppressor cells (MDSC), and M2 macrophages. Using microarray technology, we identified several miRs that were significantly downregulated in CD133+ cells compared with CD133- cells, including miR-92. Decreased expression of miR-92 in CSCs led to higher expression of target molecules integrin αV and α5 subunits, which, in turn, enhanced TGFβ activation, as evidenced by increased phosphorylation of SMAD2. CD133+ cells transfected with miR-92a mimic and injected in vivo showed significantly decreased tumor burden, which was associated with reduced immunosuppressive phenotype intratumorally. Using The Cancer Genome Atlas database of patients with melanoma, we also noted a positive correlation between integrin α5 and TGFβ1 expression levels and an inverse association between miR-92 expression and integrin alpha subunit expression. Collectively, this study suggests that a miR-92-driven signaling axis involving integrin activation of TGFβ in CSCs promotes enhanced tumorigenesis through induction of intratumoral immunosuppression. SIGNIFICANCE: CD133+ cells play an active role in suppressing melanoma antitumor immunity by modulating miR-92, which increases influx of immunosuppressive cells and TGFβ1 expression.

Mast Cell Protease 7 Promotes Angiogenesis by Degradation of Integrin Subunits.

Previous studies from our laboratory have shown that during angiogenesis in vitro, rmMCP-7 (recombinant mouse mast cell protease-7) stimulates endothelial cell spreading and induces their penetration into the matrix. The ability of rmMCP-7 to induce angiogenesis in vivo was assessed in the present study using a directed in vivo angiogenesis assay (DIVAA™). Vessel invasion of the angioreactor was observed in the presence of rmMCP-7 but was not seen in the control. Since integrins are involved in endothelial cell migration, the relationship between rmMCP-7 and integrins during angiogenesis was investigated. Incubation with rmMCP-7 resulted in a reduction in the levels of integrin subunits αv and β1 on SVEC4-10 endothelial cells during angiogenesis in vitro. Furthermore, the degradation of integrin subunits occurs both through the direct action of rmMCP-7 and indirectly via the ubiquitin/proteasome system. Even in the presence of a proteasome inhibitor, incubation of endothelial cells with rmMCP-7 induced cell migration and tube formation as well as the beginning of loop formation. These data indicate that the direct degradation of the integrin subunits by rmMCP-7 is sufficient to initiate angiogenesis. The results demonstrate, for the first time, that mMCP-7 acts in angiogenesis through integrin degradation.

The Streptococcus agalactiae cell wall-anchored protein PbsP mediates adhesion to and invasion of epithelial cells by exploiting the host vitronectin/αv integrin axis.

Binding of microbial pathogens to host vitronectin (Vtn) is a common theme in the pathogenesis of invasive infections. In this study, we characterized the role of Vtn in the invasion of mucosal epithelial cells by Streptococcus agalactiae (i.e. group B streptococcus or GBS), a frequent human pathogen. Moreover, we identified PbsP, a previously described plasminogen-binding protein of GBS, as a dual adhesin that can also interact with human Vtn through its streptococcal surface repeat (SSURE) domains. Deletion of the pbsP gene decreases both bacterial adhesion to Vtn-coated inert surfaces and the ability of GBS to interact with epithelial cells. Bacterial adherence to and invasion of epithelial cells were either inhibited or enhanced by cell pretreatment with, respectively, anti-Vtn antibodies or Vtn, confirming the role of Vtn as a GBS ligand on host cells. Finally, antibodies directed against the integrin αv subunit inhibited Vtn-dependent cell invasion by GBS. Collectively, these results indicate that Vtn acts as a bridge between the SSURE domains of PbsP on the GBS surface andhost integrins to promote bacterial invasion of epithelial cells. Therefore, inhibition of interactions between PbsP and extracellular matrix components could represent a viable strategy to prevent colonization and invasive disease by GBS.

Differential Effects of Integrin αv Knockdown and Cilengitide on Sensitization of Triple-Negative Breast Cancer and Melanoma Cells to Microtubule Poisons.

Low survival rates of patients with metastatic triple-negative breast cancer (TNBC) and melanoma, in which current therapies are ineffective, emphasize the need for new therapeutic approaches. Integrin β1 appears to be a promising target when combined with chemotherapy, but recent data have shown that its inactivation increases metastatic potential owing to the compensatory upregulation of other integrin subunits. Consequently, we analyzed the potential of integrin subunits αv, α3, or α4 as targets for improved therapy in seven TNBC and melanoma cell lines. Experiments performed in an integrin αvβ1-negative melanoma cell line, MDA-MB-435S, showed that knockdown of integrin subunit αv increased sensitivity to microtubule poisons vincristine or paclitaxel and decreased migration and invasion. In the MDA-MB-435S cell line, we also identified a phenomenon in which change in the expression of one integrin subunit changes the expression of other integrins, leading to an unpredictable influence on sensitivity to anticancer drugs and cell migration, referred to as the integrin switching effect. In a panel of six TNBCs and melanoma cell lines, the contribution of integrins αv versus integrins αvβ3/β5 was assessed by the combined action of αv-specific small interfering RNA or αvβ3/β5 inhibitor cilengitide with paclitaxel. Our results suggest that, for TNBC, knockdown of integrin αv in combination with paclitaxel presents a better therapeutic option than a combination of cilengitide with paclitaxel; however, in melanoma, neither of these combinations is advisable because a decreased sensitivity to paclitaxel was observed.

SIN3B promotes integrin αV subunit gene transcription and cell migration of hepatocellular carcinoma.

Paired amphipathic helix protein (SIN3B) is a transcription corepressor for many genes. Here we show a different regulation mechanism of integrin αV gene expression by SIN3B in human hepatocellular carcinoma (HCC). We first observed a close relationship between Integrin αV and SIN3B expressions in HCC patients and tumor cell lines with different metastatic potentials. Overexpression of SIN3B significantly accelerated the cell migration rate of SMMC-7721, but failed when integrin αV expression was silenced. Interestingly, SIN3B stimulated integrin αV subunit promoter activity only in the presence of sulfatide. Importantly, SIN3B was identified in the complex with sulfatide by mass spectrometry. Fat blot assay indicated that SIN3B specifically interacted with sulfatide. Molecular modeling suggested that sulfatide induced the conformational change of SIN3B from compacted α-helices to a relaxed β-sheet in PAH2 domain. The data of immunoprecipitation and ChIP assay indicated that altered SIN3B lost the binding affinity with MAD1 and HDAC2, which reduced the recruitment of HDAC2 on integrin αV gene promoter and prevented the deacetylation of the histone 3. In conclusion, this study demonstrated that SIN3B promoted the transcriptional activation of the integrin αV subunit gene promoter by reducing interaction with HDAC2.

Molecular analysis of biocompatibility of anodized titanium with deposited silver nanodendrites

Titanium (>99.6% purity) and its anodically oxidized modifications, with and without deposited silver nanodendrites regarding its biocompatibility were evaluated. In human gingival fibroblasts and osteoblast cell lines grown on tested samples, the level of expression of genes encoding αV (ITGAV) and β1 (ITGB1) integrin subunits also genes encoding focal adhesion (FAK) and extracellular-signal regulated (ERK) kinases was assessed. For this purpose, the qualitative and quantitative PCR technique was used.The expression of studied genes was dependent on the origin of cell lines and the type of evaluated material. The high expression of PBGD and ITGAV genes in fibroblasts grown on the surface of anodically modified titanium with deposited silver nanodendrites indicates potentially high biocompatibility of these samples for soft tissue cells. The high expression of the ITGB1 and ERK1 genes and the enhanced expression of the FAK gene in osteoblasts cells grown on the tested material was also observed.Summarizing, the nanocrystalline Ti modified with silver deposits showed higher biocompatibility in comparison with the conventional pure Ti samples.

Constitutional absence of epithelial integrin α3 impacts the composition of the cellular microenvironment of ILNEB keratinocytes

Integrin α3β1, a major epidermal adhesion receptor is critical for organization of the basement membrane during development and wound healing. Integrin α3 deficiency leads to interstitial lung disease, nephrotic syndrome and epidermolysis bullosa (ILNEB), an autosomal recessive multiorgan disease characterized by basement membrane abnormalities in skin, lung and kidney. The pathogenetic chains from ITGA3 mutation to tissue abnormalities are still unclear. Although integrin α3 was reported to regulate multiple extracellular proteins, the composition of the extracellular compartment of integrin α3-negative keratinocytes has not been resolved so far. In a comprehensive approach, quantitative proteomics of deposited extracellular matrix, conditioned cultured media as well as of the intracellular compartment of keratinocytes isolated from an ILNEB patient and from normal skin were performed. By mass spectrometry-based proteomics, 167 proteins corresponding to the GO terms “extracellular” and “cell adhesion”, or included in the “human matrisome” were identified in the deposited extracellular matrix, and 217 in the conditioned media of normal human keratinocytes. In the absence of integrin α3, 33% and 26% respectively were dysregulated. Dysregulated proteins were functionally related to integrin α3 or were known interaction partners. The results show that in the absence of integrin α3 ILNEB keratinocytes produce a fibronectin-rich microenvironment and make use of fibronectin-binding integrin subunits αv and α5. The most important results were validated in monolayer and organotypic coculture models. Finally, the in vivo relevance of the most dysregulated components was demonstrated by immunostainings of skin, kidney and lung samples of three ILNEB patients.

PO-160 Downregulation of the oncofetal gene SALL4 in melanoma leads to invasion and metastasis by differential acetylation mediated through binding to HDACs

IntroductionIntegrin signalling regulates proliferation, migration, metastasis and cell death and has been suggested as a possible target in antitumor therapy. The aim of this study was to investigate the potential of different integrin α subunits knockdown in increasing sensitivity of triple-negative breast cancer (TNBC) and melanoma cell lines to cisplatin, microtubule poisons paclitaxel (PTX) and vincristine (VCR), and mitigating metastatic potential.Material and methodsThe knockdown of integrin subunits αv, β3, β5, α3 and α4 was achieved by transfection of siRNA. The sensitivity of cell lines to anticancer drugs was determined using MTT assay. For monitoring cell migration and invasion, migration or matrigel-coated invasion Transwell Cell Culture Inserts were used. Flow cytometry, western blot, and confocal microscopy were done using standard protocols.Results and discussionsWe analysed the proof of concept in melanoma cell line MDA-MB-435S that expresses integrins αvβ3 and αvβ5 but not αvβ1. Following transfection with integrin-specific siRNAs integrin subunit αv knockdown was selected since it increased sensitivity to PTX and VCR, and decreased metastatic potential. We observed an integrin switching effect upon β3, β5 and α4 knockdown that led to differing changes of sensitivity to antitumor drugs and cell migration. Using a panel of TNBC (MDA-MB-231,–468 and −436) and melanoma (RPMI-7951, MeWo and A375) cell lines we showed that knockdown of integrin αv increased sensitivity to PTX and VCR and inhibited metastatic potential in MDA-MB-231, MDA-MB-468, RPMI-7951 and MeWo, while no effect was observed in MDA-MB-436 and A375 cells. To assess the importance of integrins αvβ3/β5 in sensitisation to PTX we exposed all cell lines to combination of αvβ3/β5 inhibitor cilengitide and PTX and observed increased sensitivity to PTX in MDA-MB-231, MDA-MB-436, RPMI-7951 and A375 but surprisingly decreased sensitivity in MeWo and MDA-MB-468 cell lines, implying either involvement of other integrins αv or integrin switching effect. The correlation between loss of phosphorylated focal adhesion kinase pFAK(Y397) and increased sensitivity to PTX was observed in melanoma cell lines, however, sensitisation was not achieved using pFAK(Y397) inhibitor.ConclusionOur results highlight that knocking down integrin αv, rather than inhibiting αvβ3/β5 using cilengitide, could be more effective for increasing sensitivity to microtubule poisons and decreasing metastatic potential in TNBC and melanoma.

PO-156 Differential effect of integrin αV knockdown and cilengitide in sensitisation of triple-negative breast cancer and melanoma cell lines to microtubule poisons

IntroductionIntegrin signalling regulates proliferation, migration, metastasis and cell death and has been suggested as a possible target in antitumor therapy. The aim of this study was to investigate the potential of different integrin α subunits knockdown in increasing sensitivity of triple-negative breast cancer (TNBC) and melanoma cell lines to cisplatin, microtubule poisons paclitaxel (PTX) and vincristine (VCR), and mitigating metastatic potential.Material and methodsThe knockdown of integrin subunits αv, β3, β5, α3 and α4 was achieved by transfection of siRNA. The sensitivity of cell lines to anticancer drugs was determined using MTT assay. For monitoring cell migration and invasion, migration or matrigel-coated invasion Transwell Cell Culture Inserts were used. Flow cytometry, western blot, and confocal microscopy were done using standard protocols.Results and discussionsWe analysed the proof of concept in melanoma cell line MDA-MB-435S that expresses integrins αvβ3 and αvβ5 but not αvβ1. Following transfection with integrin-specific siRNAs integrin subunit αv knockdown was selected since it increased sensitivity to PTX and VCR, and decreased metastatic potential. We observed an integrin switching effect upon β3, β5 and α4 knockdown that led to differing changes of sensitivity to antitumor drugs and cell migration. Using a panel of TNBC (MDA-MB-231,–468 and −436) and melanoma (RPMI-7951, MeWo and A375) cell lines we showed that knockdown of integrin αv increased sensitivity to PTX and VCR and inhibited metastatic potential in MDA-MB-231, MDA-MB-468, RPMI-7951 and MeWo, while no effect was observed in MDA-MB-436 and A375 cells. To assess the importance of integrins αvβ3/β5 in sensitisation to PTX we exposed all cell lines to combination of αvβ3/β5 inhibitor cilengitide and PTX and observed increased sensitivity to PTX in MDA-MB-231, MDA-MB-436, RPMI-7951 and A375 but surprisingly decreased sensitivity in MeWo and MDA-MB-468 cell lines, implying either involvement of other integrins αv or integrin switching effect. The correlation between loss of phosphorylated focal adhesion kinase pFAK(Y397) and increased sensitivity to PTX was observed in melanoma cell lines, however, sensitisation was not achieved using pFAK(Y397) inhibitor.ConclusionOur results highlight that knocking down integrin αv, rather than inhibiting αvβ3/β5 using cilengitide, could be more effective for increasing sensitivity to microtubule poisons and decreasing metastatic potential in TNBC and melanoma.

Identification of integrin heterodimers functioning on the surface of undifferentiated porcine primed embryonic stem cells.

In vitro expansion of undifferentiated porcine primed embryonic stem (ES) cells is facilitated by use of non-cellular niches that mimic three-dimensional (3D) microenvironments enclosing an inner cell mass of porcine blastocysts. Therefore, we investigated the integrin heterodimers on the surface of undifferentiated porcine primed ES cells for the purpose of developing a non-cellular niche to support in vitro maintenance of the self-renewal ability of porcine primed ES cells. Immunocytochemistry and a fluorescence immunoassay were performed to assess integrin α and β subunit levels, and attachment and antibody inhibition assays were used to evaluate the function of integrin heterodimers. The integrin α3 , α5 , α6 , α9 , αV , and β1 subunits, but not the α1 , α2 , α4 , α7 , and α8 subunits, were identified on the surface of undifferentiated porcine primed ES cells. Subsequently, significant increase of their adhesion to fibronectin, tenascin C, and vitronectin were observed and functional blocking of integrin heterodimer α5 β1 , α9 β1 , or αV β1 showed significantly inhibited adhesion to fibronectin, tenascin C, or vitronectin. No integrin α6 β1 heterodimer-mediated adhesion to laminin was detected. These results demonstrate that active α5 β1 , α9 β1 , and αV β1 integrin heterodimers are present on the surface of undifferentiated porcine primed ES cells, together with inactive integrin α3 (presumed) and α6 subunits.