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Podocyte dysfunction plays a crucial role in renal injury and is identified as a key contributor to proteinuria in Fabry disease (FD), primarily impacting glomerular filtration function (GFF). The α3β1 integrins are important for podocyte adhesion to the glomerular basement membrane, and disturbances in these integrins can lead to podocyte injury. Therefore, this study aimed to assess the effects of chloroquine (CQ) on podocytes, as this drug can be used to obtain an in vitro condition analogous to the FD. Murine podocytes were employed in our experiments. The results revealed a dose-dependent reduction in cell viability. CQ at a sub-lethal concentration (1.0 µg/mL) induced lysosomal accumulation significantly ( p < 0.0001). Morphological changes were evident through scanning electron microscopy and immunofluorescence, highlighting alterations in F-actin and nucleus morphology. No significant changes were observed in the gene expression of α3β1 integrins via RT-qPCR. Protein expression of α3 integrin was evaluated with Western Blotting and immunofluorescence, demonstrating its lower detection in podocytes exposed to CQ. Our findings propose a novel in vitro model for exploring secondary Fabry nephropathy, indicating a modulation of α3β1 integrin and morphological alterations in podocytes under the influence of CQ.

Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (∼30-150 nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3β1 and α6β1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 μm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3β1 and α6β1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Integrins are cell surface receptors involved in multiple functions vital for cellular proliferation. Various tumor cells overexpress αβ-integrins, making them ideal biomarkers for diagnostic imaging and tumor-targeted drug delivery. LXY30 is a peptide that can specifically recognize and interact with the integrin α 3 β 1 , a molecule overexpressed in breast, ovarian and colorectal cancer. Hepatitis E virus nanoparticles (HEVNPs) are virus-like particles that have been investigated as drug delivery agents for the targeted delivery of nucleic acids and small proteins. HEVNPs can be a theranostic platform for monitoring and evaluating tumor-targeted therapies if tagged with a suitable diagnostic marker. Herein, we describe the radiolabeling and biological evaluation of integrin α 3 β 1 -targeted HEVNPs. HEVNPs were conjugated with DOTA and radiolabeled with gallium-68 (t 1/2  = 67.7 min), a short-lived positron emitter used in positron emission tomography (PET). The synthesized [ 68 Ga]Ga-DOTA-HEVNPs were used to evaluate the efficacy of conjugated LXY30 peptide to improve HEVNPs binding and internalization to integrin α 3 β 1 expressing human colorectal HCT 116 cells. In vivo tumor accumulation of [ 68 Ga]Ga-DOTA-HEVNP-LXY30 was evaluated in HCT 116 colorectal tumor-bearing mice. [ 68 Ga]Ga-DOTA-HEVNP-LXY30 and non-targeted [ 68 Ga]Ga-DOTA-HEVNP were radiolabeled with radiochemical yields (RCY) of 67.9 ± 3.3% and 73.7 ± 9.8%, respectively. [ 68 Ga]Ga-DOTA-HEVNP-LXY30 exhibited significantly higher internalization in HCT 116 cells than the non-targeted [ 68 Ga]Ga-DOTA-HEVNPs (21.0 ± 0.7% vs. 10.5 ± 0.3% at 3 h, ****P<0.0001). After intravenous administration to mice, accumulation of [ 68 Ga]Ga-DOTA-HEVNP-LXY30 to HCT 116 xenograft tumors was at its highest rate of 0.8 ± 0.4%ID/g at 60 min. [ 68 Ga]Ga-DOTA-HEVNP-LXY30 accumulated mainly in the liver and spleen (39.8 ± 13.0%%ID/g and 24.6 ± 24.1%ID/g, respectively). Despite the low targeting efficiency in vivo, we demonstrated that [ 68 Ga]Ga-DOTA-HEVNP is a promising diagnostic platform for quantitative analysis of HEVNP distribution in vivo. This nanosystem can be utilized in future studies assessing the success of further engineered HEVNP structures with optimized targeting efficiency in vivo., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Endothelial cells engage extracellular matrix and basement membrane components through integrin-mediated adhesion to promote angiogenesis. Angiogenesis involves the sprouting of endothelial cells from pre-existing vessels, their migration into surrounding tissue, the upregulation of angiogenesis-associated genes, and the formation of new endothelial tubes. To determine whether the endothelial laminin-binding integrins, α6β4, and α3β1 contribute to these processes, we employed RNAi technology in organotypic angiogenesis assays, as well in migration assays, in vitro. The endothelial depletion of either α6β4 or α3β1 inhibited endothelial sprouting, indicating that these integrins have non-redundant roles in this process. Interestingly, these phenotypes were accompanied by overlapping and distinct changes in the expression of angiogenesis-associated genes. Lastly, depletion of α6β4, but not α3β1, inhibited migration. Taken together, these results suggest that laminin-binding integrins regulate processes associated with angiogenesis by distinct and overlapping mechanisms.

Junctional adhesion molecule (JAM)-A is a cell adhesion receptor localized at epithelial cell-cell contacts with enrichment at the tight junctions. Its role during cell-cell contact formation and epithelial barrier formation has intensively been studied. In contrast, its role during collective cell migration is largely unexplored. Here, we show that JAM-A regulates collective cell migration of polarized epithelial cells. Depletion of JAM-A in MDCK cells enhances the motility of singly migrating cells but reduces cell motility of cells embedded in a collective by impairing the dynamics of cryptic lamellipodia formation. This activity of JAM-A is observed in cells grown on laminin and collagen-I but not on fibronectin or vitronectin. Accordingly, we find that JAM-A exists in a complex with the laminin- and collagen-I-binding α3β1 integrin. We also find that JAM-A interacts with tetraspanins CD151 and CD9, which both interact with α3β1 integrin and regulate α3β1 integrin activity in different contexts. Mapping experiments indicate that JAM-A associates with α3β1 integrin and tetraspanins CD151 and CD9 through its extracellular domain. Similar to depletion of JAM-A, depletion of either α3β1 integrin or tetraspanins CD151 and CD9 in MDCK cells slows down collective cell migration. Our findings suggest that JAM-A exists with α3β1 integrin and tetraspanins CD151 and CD9 in a functional complex to regulate collective cell migration of polarized epithelial cells., (© 2022. The Author(s).)

Non-muscle-invasive bladder cancer is the most common form of bladder cancer. The main problem in managing bladder tumors is the high recurrence after the transurethral resection of bladder tumors (TURBT). Our study aimed to examine the fate of intravesically applied cancer cells as the implantation of cancer cells after TURBT is thought to be a cause of tumor recurrence. We established an orthotopic mouse bladder tumor model with MB49-GFP cancer cells and traced them during the first three days to define their location and contacts with normal urothelial cells. Data were obtained by Western blot, immunolabeling, and light and electron microscopy. We showed that within the first two hours, applied cancer cells adhered to the traumatized epithelium by cell projections containing α3β1 integrin on their tips. Cancer cells then migrated through the epithelium and on day 3, they reached the basal lamina or even penetrated it. In established bladder tumors, E-cadherin and desmoplakin 1/2 were shown as feasible immunohistochemical markers of tumor margins based on the immunolabeling of various junctional proteins. Altogether, these results for the first time illustrate cancer cell implantation in vivo mimicking cellular events of tumor recurrence in bladder cancer patients.

Background: Tetraspanins CD151, a transmembrane 4 superfamily protein, has been identified participating in the initiation of a variety of cancers. However, the precise function of CD151 in non-small cell lung cancer (NSCLC) remains unclear. Here, we addressed the pro-tumoral role of CD151 in NSCLC by targeting EGFR/ErbB2 which favors tumor proliferation, migration and invasion., Methods: First, the mRNA expression levels of CD151 in NSCLC tissues and cell lines were measured by RT-PCR. Meanwhile, CD151 and its associated proteins were analyzed by western blotting. The expression levels of CD151 in NSCLC samples and its paired adjacent lung tissues were then verified by Immunohistochemistry. The protein interactions are evaluated by co-immunoprecipitation. Flow cytometry was applied to cell cycle analysis. CCK-8, EdU Incorporation, and clonogenic assays were used to analyze cell viability. Wound healing, transwell migration, and matrigel invasion assays were utilized to assess the motility of tumor cells. To investigate the role of CD151 in vivo, lung carcinoma xenograft mouse model was applied., Results: High CD151 expression was identified in NSCLC tissues and cell lines, and its high expression was significantly associated with poor prognosis of NSCLC patients. Further, knockdown of CD151 in vitro inhibited tumor proliferation, migration, and invasion. Besides, inoculation of nude mice with CD151-overexpressing tumor cells exhibited substantial tumor proliferation compared to that in control mice which inoculated with vector-transfected tumor cells. Noteworthy, we found that overexpression of CD151 conferred cell migration and invasion by interacting with integrins. We next sought to demonstrate that CD151 regulated downstream signaling pathways via activation of EGFR/ErbB2 in NSCLC cells. Therefore, we infer that CD151 probably affects the sensitivity of NSCLC in response to anti-cancer drugs., Conclusions: Based on these results, we demonstrated a new mechanism of CD151-mediated tumor progression by targeting EGFR/ErbB2 signaling pathway, by which CD151 promotes NSCLC proliferation, migration, and invasion, which may considered as a potential target of NSCLC treatment.

Integrin α3β1 plays a crucial role in tumor formation in the two-stage chemical carcinogenesis model (DMBA and TPA treatment). However, the mechanisms whereby the expression of α3β1 influences key oncogenic drivers of this established model are not known yet. Using an in vivo mouse model with epidermal deletion of α3β1 and in vitro Matrigel cultures of transformed keratinocytes, we demonstrate the central role of α3β1 in promoting the activation of several protumorigenic signaling pathways during the initiation of DMBA/TPA‒driven tumorigenesis. In transformed keratinocytes, α3β1-mediated focal adhesion kinase/Src activation leads to in vitro growth of spheroids and to strong Akt and STAT 3 activation when the α3β1-binding partner tetraspanin CD151 is present to stabilize cell‒cell adhesion and promote Smad2 phosphorylation. Remarkably, α3β1 and CD151 can support Akt and STAT 3 activity independently of α3β1 ligation by laminin-332 and as such control the essential survival signals required for suprabasal keratin-10 expression during keratinocyte differentiation. These data demonstrate that α3β1 together with CD151 regulate the signaling pathways that control the survival of differentiating keratinocytes and provide a mechanistic understanding of the essential role of α3β1 in early stages of skin cancer development., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of integrin-targeted cancer therapies is hindered by incomplete understanding of integrin function in tumor cells and the tumor microenvironment. Previous studies showed that mice with epidermis-specific deletion of the α3 integrin subunit fail to form skin tumors during two-step chemical tumorigenesis, indicating a protumorigenic role for integrin α3β1. Here, we generated mice with tamoxifen-inducible, epidermis-specific α3 knockout to determine the role of α3β1 in the maintenance of established tumor cells and/or the associated stroma. Genetic ablation of α3 in established skin tumors caused their rapid regression, indicating that α3β1 is essential to maintain tumor growth. Although reduced proliferation and increased apoptosis were observed in α3β1-deficient tumor cells, these changes followed a robust increase in stromal apoptosis. Furthermore, macrophages and fibulin-2 levels were reduced in stroma following α3 deletion from tumor cells. Mass spectrometric analysis of conditioned medium from immortalized keratinocytes showed that α3β1 regulates a substantial fraction of the keratinocyte secretome, including fibulin-2 and macrophage CSF1; RNA in situ hybridization showed that expression of these two genes was reduced in tumor keratinocytes in vivo. Our findings identify α3β1 as a regulator of the keratinocyte secretome and skin tumor microenvironment and as a potential therapeutic target., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Downregulation of integrins α3β1 and α5β1 strongly decreased cell colony formation and in vitro invasion and markedly enhanced anoikis in SK-Mel-147 human melanoma cells. These modifications were accompanied by a marked increase in the levels of active Akt protein kinase, which indicated it played a non-canonical function in the melanoma cells. Pharmacological inhibition of Akt1, an Akt isozyme, in cells depleted of α3β1 or α5β1 restored their invasive activity, while inhibition of the Akt 2 isoform did not cause a visible effect. Similar to our previous results with the α2β1 integrin, this finding suggested that in signaling pathways initiated by α3β1 and α5β1, the Akt1 isoform performs a non-canonical function in regulating invasive phenotype of melanoma cells. In contrast, when the effects of Akt inhibitors on anoikis of the melanoma cells were compared, the Akt2 isoform demonstrated a non-canonical activity in which Akt2 suppression led to a significant attenuation of apoptosis in cells with downregulated α3β1 or α5β1. Our results were the first evidence that, in the same tumor cells, different integrins can control various manifestations of tumor progression through distinct signaling pathways that are both common to various integrins and specific to a particular receptor.

Epidermal-specific deletion of integrin α3β1 almost completely prevents the formation of papillomas during 7,12-Dimethylbenz[ a ]anthracene/12- O -tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage skin carcinogenesis. This dramatic decrease in tumorigenesis was thought to be due to an egress and premature differentiation of α3β1-depleted hair bulge (HB) stem cells (SCs), previously considered to be the cancer cells-of-origin in the DMBA/TPA model. Using a reporter mouse line with inducible deletion of α3β1 in HBs, we show that HB SCs remain confined to their niche regardless of the presence of α3β1 and are largely absent from skin tumors. However, tumor formation was significantly decreased in mice deficient for α3β1 in HB SCs. RNA sequencing of HB SCs isolated from short-term DMBA/TPA-treated skin showed α3β1-dependent expression of the matricellular protein connective tissue growth factor (CCN2), which was confirmed in vitro, where CCN2 promoted colony formation and 3D growth of transformed keratinocytes. Together, these findings show that HBs contribute to skin tumorigenesis in an α3β1-dependent manner and suggest a role of HB SCs in creating a permissive environment for tumor growth through the modulation of CCN2 secretion., (© 2020 Ramovs et al.)

Tetraspanin CD151 has been suggested to regulate cell adhesion through its association with laminin-binding integrins α3β1 and α6β4; however, its precise function in keratinocyte adhesion remains elusive. In this study, we investigated the role of CD151 in the formation and maintenance of laminin-associated adhesions. We show that CD151, through binding to integrin α3β1, plays a critical role in the stabilization of an adhesion structure with a distinct molecular composition of hemidesmosomes with tetraspanin features. These hybrid cell-matrix adhesions, which are formed early during cell adhesion and spreading and at later stages of cell spreading, are present in the central region of the cells. They contain the CD151-α3β1/α6β4 integrin complexes and the cytoskeletal linker protein plectin, but are not anchored to the keratin filaments. In contrast, hemidesmosomes, keratin filament-associated adhesions that contain integrin α6β4, plectin, BP180 (encoded by COL17A1 ) and BP230 (encoded by DST ), do not require CD151 for their formation or maintenance. These findings provide new insights into the dynamic and complex regulation of adhesion structures in keratinocytes and the pathogenic mechanisms underlying skin blistering diseases caused by mutations in the gene for CD151., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

After cutaneous injury, keratinocytes secrete paracrine factors that regulate wound cell functions; dysregulation of this signaling can lead to wound pathologies. Previously, we established that keratinocyte integrin α3β1 promotes wound angiogenesis through paracrine stimulation of endothelial cells. We hypothesize here that α3β1-dependent paracrine signaling from keratinocytes regulates the differentiation state of myofibroblasts. We report that epidermal α3-knockout mice exhibit more wound myofibroblasts and fewer cyclooxygenase 2 (Cox-2)-positive dermal cells than controls. We also found that conditioned medium from α3-expressing mouse keratinocytes (MKα3 + ), but not from α3-null MK cells (MKα3 - ), induces expression of Cox-2 in fibroblasts in a time- and dose-dependent manner and that this induction is mediated by IL-1α. Compared with MKα3 - cells, MKα3 + cells secrete more IL-1α and less IL-1RA, a natural IL-1 receptor antagonist. Treatment with an IL-1α neutralizing antibody, recombinant IL-1RA, or IL-1 receptor-targeting small interfering RNA suppresses MKα3 + conditioned medium-dependent induction of Cox-2 expression in fibroblasts. Finally, active recombinant IL-1α is sufficient to induce Cox-2 in fibroblasts and to inhibit transforming growth factor-β-induced α-SMA expression. Our findings support a role for keratinocyte integrin α3β1 in controlling the secretion of IL-1α, a paracrine factor that regulates the wound myofibroblast phenotype., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Radiation therapy for head and neck cancers results in permanent damage to the saliva producing acinar compartment of the salivary gland. To date, a pure pro-acinar cell line to study underlying mechanisms of acinar cell differentiation in culture has not been described. Here, we report the establishment of a pro-acinar (mSG-PAC1) and ductal (mSG-DUC1) cell line, from the murine submandibular salivary gland (SMG), which recapitulate developmental milestones in differentiation. mSG-DUC1 cells express the ductal markers, keratin-7 and keratin-19, and form lumenized spheroids. mSG-PAC1 cells express the pro-acinar markers SOX10 and aquaporin-5. Using the mSG-PAC1 cell line, we demonstrate that FGF2 regulates specific steps during acinar cell maturation. FGF2 up-regulates aquaporin-5 and the expression of the α3 and α6 subunits of the α3β1 and α6β1 integrins that are known to promote SMG morphogenesis and differentiation. mSG-DUC1 and mSG-PAC1 cells were derived from genetically modified mice, homozygous for floxed alleles of the integrin α3 subunit. Similar to SMGs from α3-null mice, deletion of α3 alleles in mSG-PAC1 cells results in the up-regulation of E-cadherin and the down-regulation of CDC42. Our data indicate that mSG-DUC1 and mSG-PAC1 cells will serve as important tools to gain mechanistic insight into salivary gland morphogenesis and differentiation.

Breast cancer, melanoma and glioblastoma cells undergo cell-mediated aggregation and aggregate coalescence in a transparent 3D Matrigel environment. Cells from normal tissue and non-tumorigenic cell lines do not exhibit these behaviors. Here, 266 monoclonal antibodies (mAbs) demonstrated to interact with a wide variety of membrane, secreted and matrix proteins, have been screened for their capacity to block these tumorigenic cell-specific behaviors in a 3D environment. Remarkably, only six of the 266 tested mAbs exhibited blocking activity, four targeting integrin ß-1, one targeting integrin α-3 and one targeting CD44. Colocalization of integrins ß-1 and α-3 in fixed cells and in live aggregates suggests that the integrin α-3 ß-1 dimer plays a central role in cancer cell aggregation in the 3D environment provided by Matrigel. Our results suggest that blocking by anti-integrin and anti-CD44 mAbs involves interference in cell-cell interactions.

Background: Plasmodium falciparum-infected erythrocytes (IE) sequester in deep vascular beds where their adhesion is mediated by an array of endothelial surface receptors. Because parasite adhesion has been associated with disease, antibodies that block this activity may confer protective immunity. Here, levels of plasma anti-adhesion activity and surface reactivity against freshly collected IEs from malaria-infected children were measured in a Malian birth cohort and related to child age and malaria infection history., Methods: Plasma samples from children enrolled at birth in a longitudinal cohort study of mother-infant pairs in Ouelessebougou, Mali were collected at multiple time points during follow-up visits. Anti-adhesion antibodies (i.e., inhibit IE binding to any of several endothelial receptors) and reactivity with surface IE proteins were measured using a binding inhibition assay and by flow cytometry, respectively., Results: Levels of antibodies that inhibit the binding of children's IE to the receptors ICAM-1, integrin α 3 β 1 and laminin increased with age. The breadth of antibodies that inhibit ICAM-1 and laminin adhesion (defined as the proportion of IE isolates whose binding was reduced by ≥ 50%) also significantly increased with age. The number of malaria infections prior to plasma collection was associated with levels of plasma reactivity to IE surface proteins, but not levels of anti-adhesion activity., Conclusions: Age is associated with increased levels of antibodies that reduce adhesion of children's IE to three of the ten endothelial receptors evaluated here. These results suggest that anti-adhesion antibodies to some but not all endothelial receptors are acquired during the first few years of life.

Aberrant N- glycan sialylation of glycoproteins is closely associated with malignant phenotypes of cancer cells and metastatic potential, which includes cell adhesion, migration, and growth. Recently, phosphatidylinositol 4-kinase IIα (PI4KIIα), which is localized to the trans-Golgi network, was identified as a regulator of Golgi phosphoprotein 3 (GOLPH3) and of vesicle transport in the Golgi apparatus. GOLPH3 is a target of PI4KIIα and helps anchor sialyltransferases and thereby regulates sialylation of cell surface receptors. However, how PI4KIIα-mediated sialyation of cell surface proteins is regulated remains unclear. In this study, using several cell lines, CRISPR/Cas9-based gene knockout and short hairpin RNA-mediated silencing, RT-PCR, lentivirus-mediated overexpression, and immunoblotting methods, we confirmed that PI4KIIα knockdown suppresses the sialylation of N- glycans on the cell surface, in Akt phosphorylation and activation, and integrin α3-mediated cell migration of MDA-MB-231 breast cancer cells. Interestingly, both integrin α3β1 and PI4KIIα co-localized to the trans-Golgi network, where they physically interacted with each other, and PI4KIIα specifically associated with integrin α3 but not α5. Furthermore, overexpression of both integrin α3β1 and PI4KIIα induced hypersialylation. Conversely, integrin α3 knockout significantly inhibited the sialylation of membrane proteins, such as the epidermal growth factor receptor, as well as in total cell lysates. These findings suggest that the malignant phenotype of cancer cells is affected by a sialylation mechanism that is regulated by a complex between PI4KIIα and integrin α3β1., (© 2019 Isaji et al.)

Group A Streptococcus (GAS) is a human-specific pathogen responsible for a wide range of diseases, ranging from superficial to life-threatening invasive infections, including endometritis, and autoimmune sequelae. GAS strains express a vast repertoire of virulence factors that varies depending on the strain genotype, and many adhesin proteins that enable GAS to adhere to host cells are restricted to some genotypes. GAS emm28 is the third most prevalent genotype in invasive infections in France and is associated with gyneco-obstetrical infections. emm28 strains harbor R28, a cell wall-anchored surface protein that has previously been reported to promote adhesion to cervical epithelial cells. Here, using cellular and biochemical approaches, we sought to determine whether R28 supports adhesion also to other cells and to characterize its cognate receptor. We show that through its N-terminal domain, R28 Nt , R28 promotes bacterial adhesion to both endometrial-epithelial and endometrial-stromal cells. R28 Nt was further subdivided into two domains, and we found that both are involved in cell binding. R28 Nt and both subdomains interacted directly with the laminin-binding α3β1, α6β1, and α6β4 integrins; interestingly, these bindings events did not require divalent cations. R28 is the first GAS adhesin reported to bind directly to integrins that are expressed in most epithelial cells. Finally, R28 Nt also promoted binding to keratinocytes and pulmonary epithelial cells, suggesting that it may be involved in supporting the prevalence in invasive infections of the emm28 genotype., (© 2018 Weckel et al.)

Objective: To investigate the regulatory role of fibronectin (FN) in the formation of multicellular aggregate (MCA) in ovarian cancer SKOV3 and OVCAR-3 cells and integrin expression. Methods: The dynamic formation of MCA in SKOV3 and OVCAR-3 was determined using the liquid overlay technique in the presence or absence of FN, anti-FN, RGD peptide, control RGE. The expression of α3β1, α4β1 and α5β1 integrin in monolayer cells, MCA and FN-treated MCA were determined by flow cytometry and quantitative RT-PCR. Results: OVCAR-3 and SKOV3 MCA were formed on the 4th and 8th day and peaked on the 6th and 9th day, respectively. Treatment with different concentrations of FN, LN, type IV collagen and control RGE peptide promoted MCA growth, which was mitigated by anti-FN and RGD peptide. In comparison with monolayer cells, up-regulated α3β1, α4β1 and α5β1 expression were detected in MCA while treatment with FN in both cells. Conclusions: OVCAR-3 and SKOV3 cells had varying dynamic formation of MCA in our experimental system. FN enhanced MCA formation in both cells, which was associated with increased expression of 3β1, α4β1 and α5β1 in the MCA. Therefore, FN and these integrins may be new therapeutic targets for intervention of ovarian cancer metastasis., (Creative Commons Attribution License)

Host receptor usage by Kaposi's sarcoma-associated herpesvirus (KSHV) has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding and then interacts with a complex of EphA2, xCT, and integrins α3β1, αVβ3, and αVβ5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of αV- and β1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2. IMPORTANCE Our data reveal an integrin-independent route of KSHV infection and suggest that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types., (Copyright © 2018 American Society for Microbiology.)

Structural analyses of β2 and β3 integrins have revealed that they generally assume a compact bent conformation in the resting state and undergo a global conformational transition involving extension during upregulation of ligand affinity, collectively called the 'switchblade model'. This hypothesis, however, has not been extensively tested for other classes of integrins. We prepared a set of recombinant integrin ectodomain fragments including αvβ3, α2β1, α3β1, α5β1, α6β1 and α6β4, and used negative-stain electron microscopy to examine their structures under various conditions. In contrast to αvβ3 integrin, which exhibited a severely bent conformation in low-affinity 5 mM Ca 2+ conditions, all β1 integrin heterodimers displayed a mixed population of half-bent to fully extended conformations. Moreover, they did not undergo significant conformational change upon activation by Mn 2+ Integrin α6β4 was even more resistant to conformational regulation, showing a completely extended structure regardless of the buffer conditions. These results suggest that the mechanisms of conformational regulation of integrins are more diverse and complex than previously thought, requiring more experimental scrutiny for each integrin subfamily member., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Proteolytic processing of the laminin-γ2 chain is a hallmark of basement membrane maturation in the skin. Integrin α3β1, a major receptor for epidermal adhesion to laminin-332, is critical for proper basement membrane organization during skin development and wound healing. Previously, we identified a role for α3β1 in promoting the processing of laminin-γ2 in cultured keratinocytes in vitro and in wound epidermis in vivo. In this study we identify the Bmp1 gene, which encodes variants of the mTLD/BMP-1 metalloproteases, as a critical regulator of α3β1-dependent laminin-γ2 processing, thereby expanding the role of this integrin in controlling the secretion by the epidermis of factors that modulate the tissue microenvironment. Because our previous studies identified another epidermal integrin, α9β1, as a suppressive regulator of α3β1-dependent wound angiogenesis, we investigated whether α9β1 has a similar cross-suppressive effect on the ability of α3β1 to promote basement membrane organization. Here, we show that, rather than a cross-suppressive role, α9β1 has an opposing role in basement membrane assembly/maturation through reduced laminin-γ2 processing via mTLD/BMP-1. Although α3β1 promotes this process during wound healing, α9β1 has an inhibitory role, suggesting that regulation of basement membrane assembly requires a complex interplay between these distinct epidermal integrins., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Patients with inflammatory bowel disease often suffer from chronic and relapsing intestinal inflammation that favor the development of colitis associated cancer. An alteration of the epithelial intestinal barrier function observed in IBD is supposed to be a consequence of stress. It has been proposed that corticotrophin-releasing factor receptor (CRF2), one of the two receptors of CRF, the principal neuromediator of stress, acts on cholinergic nerves to induce stress-mediated epithelial barrier dysfunction. Non-neuronal acetylcholine (Ach) and muscarinic receptors (mAchR) also contribute to alterations of epithelial cell functions. In this study, we investigated the mechanisms through which stress and Ach modulate epithelial cell adhesive properties. We show that Ach-induced activation of mAchR in HT-29 cells results in cell dissociation together with changes in cell-matrix contacts, which correlates with the acquisition of invasive potential consistent with a matrix metalloproteinase (MMP) mode of invasion. These processes result from mAchR subsequent stimulation of the cascade of src/Erk and FAK activation. Ach-induced secretion of laminin 332 leads to α3β1 integrin activation and RhoA-dependent reorganization of the actin cytoskeleton. We show that Ach-mediated effects on cell adhesion are blocked by astressin 2b, a CRF2 antagonist, suggesting that Ach action depends partly on CRF2 signaling. This is reinforced by the fact that Ach-mediated activation of mAchR stimulates both the synthesis and the release of CRF2 ligands in HT-29 cells (effects blocked by atropine). In summary, our data provides evidence for a novel intracellular circuit involving mAchR acting on CRF2-signaling that could mediate colonic mucosal barrier dysfunction and exacerbate mucosal inflammation., (Copyright © 2017. Published by Elsevier B.V.)

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Development of wound therapies is hindered by poor understanding of combinatorial integrin function in the epidermis. In this study, we generated mice with epidermis-specific deletion of α3β1, α9β1, or both integrins as well as keratinocyte lines expressing these integrin combinations. Consistent with proangiogenic roles for α3β1, α3-null keratinocytes showed reduced paracrine stimulation of endothelial cell migration and survival, and wounds of epidermis-specific α3 knockout mice displayed impaired angiogenesis. Interestingly, α9β1 in keratinocytes suppressed α3β1-mediated stimulation of endothelial cells, and wounds of epidermis-specific α9 knockout mice displayed delayed vascular normalization and reduced endothelial apoptosis, indicating that α9β1 cross-suppresses α3β1 proangiogenic functions. Moreover, α9β1 inhibited α3β1 signaling downstream of focal adhesion kinase (FAK) autoactivation at the point of Src-mediated phosphorylation of FAK Y861/Y925. Finally, α9β1 cross-suppressed many α3β1-dependent genes, including the gene that encodes MMP-9, which we implicated as a regulator of integrin-dependent cross talk to endothelial cells. Our findings identify a novel physiological context for combinatorial integrin signaling, laying the foundation for therapeutic strategies that manipulate α9β1 and/or α3β1 during wound healing., (© 2017 Longmate et al.)

The transmembrane protein CD82/KAI1 suppresses the metastatic potential of various cancer cell types. Moreover, decrease or loss of CD82 expression is closely associated with malignancy and poor prognosis in many human cancers including prostate cancer. Despite intense scrutiny, the mechanisms underlying the metastasis-suppressing role of CD82 are still not fully understood. Here, we found that a fibronectin matrix induced mesenchymal phenotypes in human prostate cancer cells with no or low CD82 expression levels. However, high CD82 expression rendered prostate cancer cells to have intensified epithelial characteristics upon fibronectin engagement, along with decreased cell motility and invasiveness. The CD82 function of inhibiting fibronectin-induced epithelial-to-mesenchymal transition (EMT) was dependent not only on CD82 interactions with fibronectin-binding α3β1/α5β1 integrins but also on the integrin-mediated intracellular signaling events. Notably, CD82 attenuated the FAK-Src and ILK pathways downstream of the fibronectin-receptor integrins. Immunofluorescence staining of human prostate cancer tissue specimens illustrated a negative association of CD82 with EMT-related gene expression as well as prostate malignancy. Altogether, these results suggest that CD82 suppresses EMT in prostate cancer cells adhered to the fibronectin matrix by repressing adhesion signaling through lateral interactions with the associated α3β1 and α5β1 integrins, leading to reduced cell migration and invasive capacities.

Background/aims: Hepatitis B virus (HBV)-associated glomerulonephritis (HBV-GN) is characterized by a reduced number of podocytes due to apoptosis and shedding from the basement membrane. However, the pathological mechanism of HBV-GN is unclear. We previously showed that hepatitis B virus X protein (HBx) promotes apoptosis in tubular epithelial cells. In this study, we transfected podocytes with HBx and examined the effects on adhesion and apoptosis of these cells., Methods: Podocytes were transfected with pc-DNA3.1 (+)-HBx. One control group was not transfected and another control group was transfected with empty plasmids. Podocyte adhesion was assessed by a fluorescence assay, apoptosis was measured by flow cytometry and fluorescence microscopy, and expression of α3β1 integrin was determined by western blotting and the reverse transcription polymerase chain reaction (RT-PCR). Activity of caspase-8 was measured by a spectrophotometric assay., Results: Relative to controls, podocytes with pc-DNA3.1(+)-HBx had reduced cell adhesion, increased apoptosis, reduced expression of α3β1 integrin, and increased caspase-8 activity. β1 integrin blockage reduced podocyte adhesion, but increased apoptosis and caspase-8 activity. Treatment of transfected podocytes with a caspase-8 inhibitor (Z-IETD-FMK) had no effect on the HBx-mediated integrin downregulation and reduced podocyte adhesion, suggesting that α3β1 integrin downregulaton is sufficient to alter cell adhesion., Conclusions: Our in vitro results indicate that HBx reduced podocyte adhesion and expression of α3β1 integrin, and increased apoptosis. Moreover, HBx-mediated downregulation of α3β1 integrin expression is sufficient to reduce podocyte adhesion. HBx-induced apoptosis of podocytes may contribute to HBV-GN., (© 2017 The Author(s)Published by S. Karger AG, Basel.)

Matrix adhesion via integrins is required for cell survival. Adhesion of epithelial cells to laminin via integrin α3β1 was previously shown to activate at least two independent survival pathways. First, integrin α3β1 is required for autophagy-induced cell survival after growth factor deprivation. Second, integrin α3β1 independently activates two receptor tyrosine kinases, EGFR and Met, in the absence of ligands. EGFR signaling to Erk promotes survival independently of autophagy. To determine how Met promotes cell survival, we inhibited Met kinase activity or blocked its expression with RNA interference. Loss of Met expression, but not inhibition of Met kinase activity, induced apoptosis by reducing integrin α3β1 levels, activating anoikis, and blocking autophagy. Met was specifically required for the assembly of autophagosomes downstream of LC3II processing. Reexpression of wild-type Met, kinase-dead Met, or integrin α3 was sufficient to rescue death upon removal of endogenous Met. Integrin α3β1 coprecipitated and colocalized with Met in cells. The extracellular and transmembrane domain of Met was required to fully rescue cell death and restore integrin α3 expression. Thus Met promotes survival of laminin-adherent cells by maintaining integrin α3β1 via a kinase-independent mechanism., (© 2016 Tesfay, Schulz, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Loss of E-cadherin marks a defect in epithelial integrity and polarity during tissue injury and fibrosis. Whether loss of E-cadherin plays a causal role in fibrosis is uncertain. α3β1 Integrin has been identified to complex with E-cadherin in cell-cell adhesion, but little is known about the details of their cross talk. Herein, E-cadherin gene (Cdh1) was selectively deleted from proximal tubules of murine kidney by Sglt2Cre. Ablation of E-cadherin up-regulated α3β1 integrin at cell-cell adhesion. E-cadherin-deficient proximal tubular epithelial cell displayed enhanced transforming growth factor-β1-induced α-smooth muscle actin (α-SMA) and vimentin expression, which was suppressed by siRNA silencing of α3 integrin, but not β1 integrin. Up-regulation of transforming growth factor-β1-induced α-SMA was mediated by an α3 integrin-dependent increase in integrin-linked kinase (ILK). Src phosphorylation of β-catenin and consequent p-β-catenin-Y654/p-Smad2 transcriptional complex underlies the transcriptional up-regulation of ILK. Kidney fibrosis after unilateral ureteric obstruction or ischemia reperfusion was increased in proximal tubule E-cadherin-deficient mice in comparison to that of E-cadherin intact control mice. The exacerbation of fibrosis was explained by the α3 integrin-dependent increase of ILK, β-catenin nuclear translocation, and α-SMA/proximal tubular-specific Cre double positive staining in proximal tubular epithelial cell. These studies delineate a nonconventional integrin/ILK signaling by α3 integrin-dependent Src/p-β-catenin-Y654/p-Smad2-mediated up-regulation of ILK through which loss of E-cadherin leads to kidney fibrosis., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background. Podocyte injury plays an important role in the onset and progression of diabetic nephropathy (DN). Downregulation of α3β1-integrin expression in podocytes is thought to be associated with podocyte detachment from the glomerular basement membrane, although the mechanisms remain obscure. To determine the mechanism of podocyte detachment, we analyzed the expression levels of α3β1-integrin in podocytes in early and advanced stages of DN. Methods. Surgical specimens from DN patients were examined by in situ hybridization, and the expression levels of α3- and β1-integrin subunits in glomeruli of early (n = 6) and advanced (n = 8) stages were compared with those of normal glomeruli (n = 5). Heat-sensitive mouse podocytes (HSMP) were cultured with TGF-β1 to reproduce the microenvironment of glomeruli of DN, and the expression levels of integrin subunits and the properties of migration and attachment were examined. Results. Podocytes of early-stage DN showed upregulation of α3- and β1-integrin expression while those of advanced stage showed downregulation. Real-time PCR indicated a tendency for upregulation of α3- and β1-integrin in HSMP cultured with TGF-β1. TGF-β1-stimulated HSMP also showed enhanced in vitro migration and attachment on collagen substrate. Conclusions. The results suggested that podocyte detachment during early stage of DN is mediated through upregulation of α3β1-integrin.

Background: The proposed involvement of CD151 in breast cancer (BCa) progression is based on findings from studies in invasive ductal carcinoma (IDC). The IDC and invasive lobular carcinoma (ILC) represent distinct disease entities. Here we evaluated clinical significance of CD151 alone and in association with integrin α3β1 in patients with ILC in context of the data of our recent IDC study., Methods: Expression of CD151 and/or integrin α3β1 was evaluated in ILC samples (N=117) using immunohistochemistry. The findings were analysed in relation to our results from an IDC cohort (N=182) demonstrating a prognostic value of an expression of CD151/integrin α3β1 complex in patients with HER2-negative tumours., Results: Unlike in the IDCs, neither CD151 nor CD151/α3β1 complex showed any correlation with any of the ILC characteristics. Lack of both CD151 and α3β1 was significantly correlated with poor survival (P=0.034) in lymph node-negative ILC N(-) cases. The CD151(-)/α3β1(-) patients had 3.12-fold higher risk of death from BCa in comparison with the rest of the ILC N(-) patients., Conclusions: Biological role of CD151/α3β1 varies between ILC and IDC. Assessment of CD151/α3β1 might help to identify ILC N(-) patients with increased risk of distant metastases.

The present study was performed to investigate the effect of retinoic acid amide (RAA) on the expression of integrin α3β1, rate of cell proliferation and migration in p53-deficient glioma cell line, LN-308. The results revealed promotion of integrin α3 expression, reduction in proliferation and migration in RAA treated cells compared to the control LN-308 glioma cells. Promotion of RAA induced integrin α3β1 expression led to the enhancement in cyclin-dependent kinase nuclear localization and activation of Akt pathway. In addition, RAA treatment inhibited the expression of nuclear factor-κB, Bcl-2 and epidermal growth factor receptor (EGFR). These factors are responsible for promoting the rate of cell proliferation and survival in the carcinoma cells. Thus RAA treatment inhibits rate of LN-308 glioma cell proliferation and migration through increase in integrin α3β1 expression and activation of Akt pathway. Therefore, RAA can be of therapeutic importance for the treatment of glioma.

The α6β4 integrin is known to associate with receptor tyrosine kinases when engaged in epithelial wound healing and in carcinoma invasion and survival. Prior work has shown that HER2 associates with α6β4 integrin and syndecan-1 (Sdc1), in which Sdc1 engages the cytoplasmic domain of the β4 integrin subunit allowing HER2-dependent motility and carcinoma cell survival. In contrast, EGFR associates with Sdc4 and the α6β4 integrin, and EGFR-dependent motility depends on cytoplasmic engagement of β4 integrin with Sdc4. However, how HER2 and EGFR assimilate into a complex with the syndecans and integrin, and why kinase capture is syndecan-specific has remained unknown. In the present study, we demonstrate that HER2 is captured via a site, comprised of amino acids 210-240, in the extracellular domain of human Sdc1, and EGFR is captured via an extracellular site comprised of amino acids 87-131 in human Sdc4. Binding assays using purified recombinant proteins demonstrate that the interaction between the EGFR family members and the syndecans is direct. The α3β1 integrin, which is responsible for the motility of the cells, is captured at these sites as well. Peptides based on the interaction motifs in Sdc1 and Sdc4, called synstatins (SSTN210-240 and SSTN87-131) competitively displace the receptor tyrosine kinase and α3β1 integrin from the syndecan with an IC50 of 100-300 nm. The syndecans remain anchored to the α6β4 integrin via its cytoplasmic domain, but the activation of cell motility is disrupted. These novel SSTN peptides are potential therapeutics for carcinomas that depend on these HER2- and EGFR-coupled mechanisms for their invasion and survival., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9'10), (ii) an Arg to Ala synergy site mutation (FnIII9(R)→(A)10), (iii) a two-Gly (FnIII9(2G)10) insertion, and (iv) a four-Gly (FNIII9(4G)10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9'10 and FnIII9(2G)10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9'10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9'10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glioblastoma, one of the most aggressive forms of brain cancer, is featured by high tumor cell motility and invasiveness, which not only fuel tumor infiltration, but also enable escape from surgical or other clinical interventions. Thus, better understanding of how these malignant traits are controlled will be key to the discovery of novel biomarkers and therapies against this deadly disease. Tetraspanin CD151 and its associated α3β1 integrin have been implicated in facilitating tumor progression across multiple cancer types. How these adhesion molecules are involved in the progression of glioblastoma, however, remains largely unclear. Here, we examined an in-house tissue microarray-based cohort of 96 patient biopsies and TCGA dataset to evaluate the clinical significance of CD151 and α3β1 integrin. Functional and signaling analyses were also conducted to understand how these molecules promote the aggressiveness of glioblastoma at molecular and cellular levels. Results from our analyses showed that CD151 and α3 integrin were significantly elevated in glioblastomas at both protein and mRNA levels, and exhibited strong inverse correlation with patient survival (p < 0.006). These adhesion molecules also formed tight protein complexes and synergized with EGF/EGFR to accelerate tumor cell motility and invasion. Furthermore, disruption of such complexes enhanced the survival of tumor-bearing mice in a xenograft model, and impaired activation of FAK and small GTPases. Also, knockdown- or pharmacological agent-based attenuation of EGFR, FAK or Graf (ARHGAP26)/small GTPase-mediated pathways markedly mitigated the aggressiveness of glioblastoma cells. Collectively, our findings provide clinical, molecular and cellular evidence of CD151-α3β1 integrin complexes as promising prognostic biomarkers and therapeutic targets for glioblastoma.

Background: Connective tissue growth factor (CTGF) plays important roles in normal and pathological conditions. The aim of this study was to investigate the role of CTGF in peritoneal metastasis as well as the underlying mechanism in gastric cancer progression., Methods: CTGF expression levels for wild-type and stable overexpression clones were determined by Western blotting and quantitative polymerase chain reaction (Q-PCR). Univariate and multivariate analyses, immunohistochemistry, and survival probability analyses were performed on gastric cancer patients. The extracellular matrix components involved in CTGF-regulated adhesion were determined. Recombinant CTGF was added to cells or coinoculated with gastric cancer cells into mice to evaluate its therapeutic potential., Results: CTGF overexpression and treatment with the recombinant protein significantly inhibited cell adhesion. In vivo peritoneal metastasis demonstrated that CTGF-stable transfectants markedly decreased the number and size of tumor nodules in the mesentery. Statistical analysis of gastric cancer patient data showed that patients expressing higher CTGF levels had earlier TNM staging and a higher survival probability after the surgery. Integrin α3β1 was the cell adhesion molecule mediating gastric cancer cell adhesion to laminin, and blocking of integrin α3β1 prevented gastric cancer cell adhesion to recombinant CTGF. Coimmunoprecipitation results indicated that CTGF binds to integrin α3. Coinoculation of recombinant CTGF and gastric cancer cell lines in mice showed effective inhibition of peritoneal dissemination., Conclusions: Our results suggested that gastric cancer peritoneal metastasis is mediated through integrin α3β1 binding to laminin, and CTGF effectively blocks the interaction by binding to integrin α3β1, thus demonstrating the therapeutic potential of recombinant CTGF in gastric cancer patients.

The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin-extracellular matrix interactions. The laminin (LM)-binding integrin α3β1 is crucial for this developmental program; however, the LM types and LM/integrin α3β1-dependent signaling pathways are poorly defined. We show that α3 chain-containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin α3β1-dependent collecting duct cell functions. We demonstrate that integrin α3β1-mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin β1 subunit and regulator of integrin α3β1-dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin α3-null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin α3β1-dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways., (© 2015 Yazlovitskaya et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Background: Chinese medicine comprised of all natural herbs is widespread used in the treatment of diabetic nephropathy (DN). Podocyte contributes to the integrity of glomerular filtration barrier whose injury plays an important role in the initiation and progression of DN. Our study aimed to investigate the effect of Qiwei granules on podocyte lesion in diabetic KK-A(y) mice kidney and its underlying mechanism., Methods: Twelve-week-old male KK-A(y) mice were randomly divided in vehicle group and Qiwei granules group, while C57BL/6J mice were used as normal control. The mice were gavage with 1.37 g/kg/day Qiwei granules or water for 10 weeks. We measured water, food intake and body weight (BW) and fasting blood glucose (FBG) every 2 weeks, and urine protein every 4 weeks. At the end of the experiment, all surviving mice were sacrificed. The kidney weight and serum renal parameters were measured, and the renal morphology was observed. To search the underlying mechanism, we examined the podocyte positive marker, slit diaphragm protein expression and some involved cell signal pathway., Results: Qiwei granules treatment significantly improved the metabolic parameters, alleviated the urinary protein, and protected renal function in KK-A(y) mice. In addition, the glomerular injuries and podocyte lesions were mitigated with Qiwei granules treatment. Furthermore, Qiwei granules increased expression of nephrin, CD2AP, and integrin alpha3beta1 in the podocytes of KK-A(y) mice. Qiwei granules improved the phosphoration of Akt and inhibited cleaved caspase-3 protein expression., Conclusion: These finding suggest that Qiwei granules protects the podocyte from the development of DN via improving slit diaphragm (SD) molecules expression and likely activating Akt signaling pathway in KK-A(y) mice.

Integrin α3β1 is highly expressed in both normal and tumorigenic epidermal keratinocytes where it regulates genes that control cellular function and extracellular matrix remodeling during normal and pathological tissue remodeling processes, including wound healing and development of squamous cell carcinoma (SCC). Previous studies identified a role for α3β1 in immortalized and transformed keratinocytes in the regulation of genes that promote tumorigenesis, invasion, and pro-angiogenic crosstalk to endothelial cells. One such gene, matrix metalloproteinase-9 (MMP-9), is induced by α3β1 through a post-transcriptional mechanism of enhanced mRNA stability. In the current study, we sought to investigate the mechanism through which α3β1 controls MMP-9 mRNA stability. First, we utilized a luciferase reporter assay to show that AU-rich elements (AREs) residing within the 3'-untranslated region (3'-UTR) of the MMP-9 mRNA renders the transcript unstable in a manner that is independent of α3β1. Next, we cloned a truncated variant of the MMP-9 mRNA which is generated through usage of an alternative, upstream polyadenylation signal and lacks the 3'-UTR region containing the destabilizing AREs. Using an RNase protection assay to distinguish "long" (full-length 3'-UTR) and "short" (truncated 3'-UTR) MMP-9 mRNA variants, we demonstrated that the shorter, more stable mRNA that lacks 3'-UTR AREs was preferentially generated in α3β1-expressing keratinocytes compared with α3β1-deficient (i.e., α3-null) keratinocytes. Moreover, we determined that α3β1-dependent alternative polyadenylation was acquired by immortalized keratinocytes, as primary neonatal keratinocytes did not display α3β1-dependent differences in the long and short transcripts. Finally, pharmacological inhibition of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway in α3β1-expressing keratinocytes caused a shift towards long variant expression, while Raf-1-mediated activation of ERK in α3-null keratinocytes dramatically enhanced short variant expression, indicating a role for ERK/MAPK signaling in α3β1-mediated selection of the proximal polyadenylation site. These findings identify a novel mode of integrin α3β1-mediated gene regulation through alternative polyadenylation.

Human ovarian cancer is diagnosed in the late, metastatic stages but the underlying mechanisms remain poorly understood. We report a surprising functional link between CD151-α3β1 integrin complexes and the malignancy of serous-type ovarian cancer. Analyses of clinical specimens indicate that CD151 expression is significantly reduced or diminished in 90% of metastatic lesions, while it remains detectable in 58% of primary tumors. These observations suggest a putative tumor-suppressing role of CD151 in ovarian cancer. Indeed, our analyses show that knocking down CD151 or α3 integrin enhances tumor cell proliferation, growth and ascites production in nude mice. These changes are accompanied by impaired cell-cell contacts and aberrant expression of E-cadherin, Mucin 5AC and fibronectin, largely reminiscent of an epithelial to mesenchymal transition (EMT)-like change. Importantly, Slug, a master regulator of EMT, is markedly elevated. Knocking down Slug partially restores CD151-α3β1 integrin complex-dependent suppression of cell proliferation. Moreover, disruption of these adhesion protein complexes is accompanied by a concomitant activation of canonical Wnt signaling, including elevated levels of β-catenin and Axin-2 as well as resistance to the inhibition in β-catenin-dependent transcriptional complexes. Together, our study demonstrates that CD151-α3β1 integrin complexes regulate ovarian tumor growth by repressing Slug-mediated EMT and Wnt signaling.

The laminin-binding integrin α3β1 is highly expressed in epidermal keratinocytes, where it regulates both cell-autonomous and paracrine functions that promote wound healing and skin tumorigenesis. However, the roles for α3β1 in regulating gene expression programs that control the behaviors of immortalized or transformed keratinocytes remain underexplored. In the current study, we used a microarray approach to identify genes that are regulated by α3β1 in immortalized keratinocytes. α3β1-Responsive genes included several genes that are involved in extracellular matrix proteolysis or remodeling, including fibulin-2 and secreted protein acidic and rich in cysteine. However, α3β1-dependent induction of specific target genes was influenced by the genetic lesion that triggered immortalization, as α3β1-dependent fibulin-2 expression occurred in cells immortalized by either SV40 large T antigen or p53-null mutation, whereas α3β1-dependent expression of secreted protein acidic and rich in cysteine occurred only in the former cells. Interestingly, quantitative PCR arrays did not reveal strong patterns of α3β1-dependent gene expression in freshly isolated primary keratinocytes, suggesting that this regulation is acquired during immortalization. p53-null keratinocytes transformed with oncogenic RasV12 retained α3β1-dependent fibulin-2 expression, and RNAi-mediated knockdown of fibulin-2 in these cells reduced invasion, although not their tumorigenic potential. These findings demonstrate a prominent role for α3β1 in immortalized/transformed keratinocytes in regulating fibulin-2 and other genes that promote matrix remodeling and invasion.

Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3β1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor.

The constitutive activation of β-catenin signaling in the mammary basal epithelial cell layer in transgenic K5ΔNβcat mice leads to basal-type tumor development. Integrins of the β1 family and integrin-mediated signaling events have an important role in breast tumor growth and progression. We show here that the deletion of α3β1 integrin, a major laminin receptor, from the basal layer of the mammary epithelium of K5ΔNβcat mice completely prevented the tumorigenesis induced by β-catenin signaling. Moreover, the depletion of α3β1 integrin from a spontaneously transformed mouse mammary basal epithelial cell line (MEC) prevented the cells from forming colonies in soft agar and greatly reduced tumor development in orthotopic grafts. Inhibition of the integrin signaling intermediates Rac1 or PAK1 (P21-activated Kinase 1) in MEC affected tumor cell growth in soft agar, whereas the expression of activated forms of these effectors in α3-depleted cells rescued the capacity of these cells to grow in non-adherent conditions. Similarly, the tumorigenic potential of α3-depleted cells was restored by the expression of activated PAK1, as assessed by orthotopic transplantation assay. In three-dimensional Matrigel culture, MEC survival and proliferation were affected by the depletion of α3β1 integrin, which also significantly decreased the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK). Our data suggest that the activation of signaling cascades downstream from α3β1 and involving the Rac1/PAK1 pathway, MAPK and JNK, promotes prosurvival and proproliferative signals required for the malignant growth of basal mammary epithelial cells, providing further insight into the molecular mechanisms underlying breast cancer initiation and progression.

Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382-393 and α1(IV)531-543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl(393) in α1(IV)382-393 and Hyl(540) and Hyl(543) in α1(IV)531-543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382-393 but right in the middle of α3β1 integrin interaction with α1(IV)531-543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Purpose: To identify a novel biomarker for bladder cancer targeting therapy., Experimental Design: The human bladder cancer cell line T24 cells were used as immunogen to generate mouse monoclonal antibodies. We screened and identified a specific antibody BCMab1 against bladder cancer. We examined BCMab1 antigen expression in the patients with bladder cancer through immunohistochemical staining and investigated the BCMab1 antigen association with clinical severity. We detected the antitumor activity of BCMab1 antibody and investigated its therapeutic efficacy by subcutaneous and orthotopic bladder cancer models., Results: We developed a new monoclonal antibody BCMab1 against bladder cancer that specifically recognized the aberrantly glycosylated Integrin α3β1 epitope on bladder cancer cells. Expression of the BCMab1 antigen was consistent with clinical severity and prognosis of bladder cancer. The glycosyltransferase GALNT1 could contribute to aberrant glycosylation of Integrin α3. The aberrant glycosylation of integrin α3-activated integrin signaling to initiate FAK activation. BCMab1 could block Integrin engagement to inhibit its signaling leading to cell-cycle arrest. In addition, BCMab1 enhanced FcγR-dependent antitumor activity in vivo., Conclusions: BCMab1 antigen is a new biomarker for bladder cancer. BCMab1 antibody exhibited potent antitumor activity against bladder cancer in vivo., (©2014 American Association for Cancer Research.)

Background: Expression of integrin α3β1 is associated with tumor progression, metastasis, and poor prognosis in several cancers, including breast cancer. Moreover, preclinical studies have revealed important pro-tumorigenic and pro-metastatic functions for this integrin, including tumor growth, survival, invasion, and paracrine induction of angiogenesis. Our previously published work in a preclinical breast cancer model showed that integrin α3β1 promotes expression of cyclooxygenase-2 (COX2/PTGS2), a known driver of breast cancer progression. However, the clinical significance of this regulation was unknown. The objective of the current study was to assess the clinical relevance of the relationship between integrin α3β1 and COX2 by testing for their correlated expression among various forms of human breast cancer., Methods: Immunohistochemistry was performed to assess co-expression of α3 and COX2 in specimens of human invasive ductal carcinoma (IDC), either on a commercial tissue microarray (n = 59 samples) or obtained from Albany Medical Center archives (n = 68 samples). Immunostaining intensity for the integrin α3 subunit or COX2 was scored, and Spearman's rank correlation coefficient analysis was performed to assess their co-expression across and within different tumor subtypes or clinicopathologic criteria., Results: Although expression of integrin α3 or COX2 varied among clinical IDC samples, a statistically significant, positive correlation was detected between α3 and COX2 in both tissue microarrays (r(s) = 0.49, p < 0.001, n = 59) and archived samples (r(s) = 0.59, p < 0.0001, n = 68). In both sample sets, this correlation was independent of hormone receptor status, histological grade, or disease stage., Conclusions: COX2 and α3 are correlated in IDC independently of hormone receptor status or other clinicopathologic features, supporting the hypothesis that integrin α3β1 is a determinant of COX2 expression in human breast cancer. These results support the clinical relevance of α3β1-dependent COX2 gene expression that we reported previously in breast cancer cells. The findings also suggest that COX2-positive breast carcinomas of various subtypes might be vulnerable to therapeutic strategies that target α3β1, and that α3 expression might serve as an independent prognostic biomarker.

Extracellular matrix metalloproteinase inducer (EMMPRIN) exhibits overexpression in various cancers and promotes cancer progression and metastasis via the interaction with its associated molecules. The scFv-M6-1B9 intrabody has a potential ability to reduce EMMPRIN cell surface expression. However, the subsequent effect of scFv-M6-1B9 intrabody-mediated EMMPRIN abatement on its related molecules, α3β1-integrin, MCT1, MMP-2 and MMP-9, is undefined. Our results demonstrated that the scFv-M6-1B9 intrabody efficiently decreased α3β1-integrin cell surface expression levels. In addition, intracellular accumulation of MCT1 and lactate were increased. These results lead to suppression of features characteristic for tumor progression, including cell migration, proliferation and invasion, in a colorectal cancer cell line (Caco-2) although there was no difference in MMP expression. Thus, EMMPRIN represents an attractive target molecule for the disruption of cancer proliferation and metastasis. An scFv-M6-1B9 intrabody-based approach could be relevant for cancer gene therapy.

Deficient epidermal adhesion is a hallmark of blistering skin disorders and chronic wounds, implicating integrins as potential therapeutic targets. Integrin α3β1, a major receptor in the epidermis for adhesion to laminin-332 (LN-332), has critical roles in basement membrane (BM) organization during skin development. In the current study we identify a role for α3β1 in promoting stability of nascent epidermal BMs through induction of fibulin-2, a matrix-associated protein that binds LN-332. We demonstrate that mice lacking α3β1 in the epidermis display ruptured BM beneath neo-epidermis of wounds, characterized by extensive blistering. This junctional blistering phenocopies defects reported in newborn α3-null mice, as well as in human patients with α3 gene mutations, indicating that the developmental role of α3β1 in BM organization is recapitulated during wound healing. Mice lacking epidermal α3β1 also have reduced fibulin-2 expression, and fibulin-2-null mice display perinatal skin blisters similar to those in α3β1-deficient mice. Interestingly, α3-null wound epidermis or keratinocytes also show impaired processing of the LN-332 γ2 chain, although this defect was independent of reduced fibulin-2 and did not appear to cause blistering. Our findings indicate a role for integrin α3β1 in BM stability through fibulin-2 induction, both in neonatal skin and in adult wounds.

Integrin α3β1 regulates adhesive interactions of cells with laminins and have a critical role in adhesion-dependent cellular responses. Here, we examined the role of α3β1-integrin in ErbB2-dependent proliferation of breast cancer cells in three-dimensional laminin-rich extracellular matrix (3D lr-ECM). Depletion of α3β1 in ErbB2-overexpressing breast cancer cells suppressed growth and restore cell polarity in 3D lr-ECM. The phenotype of α3β1-depleted cells was reproduced upon depletion of tetraspanin CD151 and mirrored that of the cells treated with Herceptin, an established ErbB2 antagonist. Breast cancer cells expressing the α3β1-CD151 complex have higher steady-state phosphorylation of ErbB2 and show enhanced dimerization of the protein when compared with α3β1-/CD151-depleted cells. Furthermore, Herceptin-dependent dephosphorylation of ErbB2 was only observed in α3β1-CD151-expressing cells. Importantly, the inhibitory activity of Herceptin was more pronounced when cells expressed both α3β1 and CD151. We also found that the level of active RhoA was increased in α3β1- and CD151-depleted cells and that Rho controls dimerization of ErbB2. Expression of α3β1 alone did not have significant prognostic value in patients with invasive ductal carcinoma of the breast. However, expression of α3β1 in combination with CD151 represented a more stringent indicator of poor survival than CD151 alone. Taken together, these results demonstrate that the α3β1-CD151 complex has a critical regulatory role in ErbB2-dependent signalling and thereby may be involved in breast cancer progression.

The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the β1-chain-derived fragment interacts via α3β1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs--key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the α3β1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9.