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The role and mechanisms of DEP exposure on thyroid injury are not yet clear. This study explores thyroid damage induced by in vivo DEP exposure using a mouse model. This study has observed alterations in thyroid follicular architecture, including rupture, colloid overflow, and the formation of voids. Additionally, there was a significant decrease in the expression levels of proteins involved in thyroid hormone synthesis, such as thyroid peroxidase and thyroglobulin, their trend of change is consistent with the damage to the thyroid structure. Serum levels of triiodothyronine and tetraiodothyronine were raise. However, the decrease in TSH expression suggests that the function of the HPT axis is unaffected. To delve deeper into the intrinsic mechanisms of thyroid injury, we performed KEGG pathway enrichment analysis, which revealed notable alterations in the cell adhesion signaling pathway. Our immunofluorescence results show that DEP exposure impairs thyroid adhesion, and integrin α3β1 plays an important role. CD151 binds to α3β1, promoting multimolecular complex formation and activating adhesion-dependent small GTPases. Our in vitro model has confirmed the pivotal role of integrin α3β1 in thyroid cell adhesion, which may be mediated by the CD151/α3β1/Rac1 pathway. In summary, exposure to DEP disrupts the structure and function of the thyroid, a process that likely involves the regulation of cell adhesion through the CD151/α3β1/Rac1 pathway, leading to glandular damage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The emergence of lysosome-targeting chimeras (LYTACs), which represents a promising strategy for membrane protein degradation based on lysosomal pathways, has attracted much attention in disease intervention and treatment. However, the expression level of commonly used lysosome-targeting receptors (LTRs) varies in different cell lines, thus limiting the broad applications of LYTACs. To overcome this difficulty, we herein report the development of integrin α3β1 (ITGA3B1)-facilitated bispecific aptamer chimeras (ITGBACs) as a platform for the degradation of membrane proteins. ITGBACs consist of two aptamers, one targeting ITGA3B1 and another binding to the membrane-associated protein of interest (POI), effectively transporting the POI into lysosomes for degradation. Our findings demonstrate that ITGBACs effectively eliminate pathological membrane proteins, such as CD71 and PTK7, inducing significant cell-cycle arrest and apoptosis and markedly inhibiting tumor growth in tumor-bearing mice models. Therefore, this work provides a novel and versatile membrane protein degradation platform, offering a promising targeted therapy based on tumor-specific LTRs.

Abstract: Background: The recruitment of neutrophils to sites of localized injury or infection is initiated by changes on the surface of endothelial cells located in proximity to tissue damage. Inflammatory mediators, such as TNF-α, increase surface expression of adhesive ligands and receptors on the endothelial surface to which neutrophils tether and adhere. Neutrophils then transit through the activated endothelium to reach sites of tissue injury with little lasting vascular injury. However, in cases of sepsis, the interaction of endothelial cells with highly activated neutrophils can cause damage vascular damage. The identification of molecules that are essential for neutrophil diapedesis may reveal targets of therapeutic opportunity for preservation of endothelial function in the presence of critical illness. We tested the hypothesis that inhibition of neutrophil β1 integrin very late antigen-3 (VLA-3; α3β1) and/or inhibition of the tetraspanin (TM4) family member CD151 would protect against neutrophil-mediated loss of endothelial function. Methods: Blood was obtained from septic patients or healthy donors. Neutrophils were purified, and aliquots were treated with/without proinflammatory molecules. Confluent human umbilical vascular endothelial cells were activated with TNF-α. Electric cell impedance sensing was used to determine monolayer resistance over time after the addition of neutrophils that were treated with blocking antibodies against VLA-3 and/or CD151 or isotype controls. Groups (depending on relevancy) were analyzed by Mann-Whitney U test, Wilcoxon test, or repeated-measures one-way ANOVA. Results: Neutrophils from septic patients and neutrophils activated ex vivo reduced endothelial monolayer resistance to a greater extent than neutrophils from healthy donors. Antibody blockade of VLA-3 and/or CD151 significantly reduced activation-associated endothelial damage. Similar findings were demonstrated on fibronectin, collagen I, collagen IV, and laminin, suggesting that neutrophil surface VLA-3 and CD151 are responsible for endothelial damage regardless of substrata and are likely to be operative in all bodily tissues. Conclusion: This report identifies VLA-3 and CD151 on the activated human neutrophil, which are responsible for damage to endothelial function. Targeting these molecules in vivo may demonstrate preservation of organ function during critical illness., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Shock Society.)

Integrin receptors for the extracellular matrix activate intracellular signaling pathways that are critical for tissue development, homeostasis, and regeneration/repair, and their loss or dysregulation contributes to many developmental defects and tissue pathologies. This review will focus on tissue remodeling roles for integrin α3β1, a receptor for laminins found in the basement membranes (BMs) that underlie epithelial cell layers. As a paradigm, we will discuss literature that supports a role for α3β1 in promoting ability of epidermal keratinocytes to modify their tissue microenvironment during skin development, wound healing, or tumorigenesis. Preclinical and clinical studies have shown that this role depends largely on ability of α3β1 to govern the keratinocyte's repertoire of secreted proteins, or the "secretome," including 1 ) matrix proteins and proteases involved in matrix remodeling and 2 ) paracrine-acting growth factors/cytokines that stimulate other cells with important tissue remodeling functions (e.g., endothelial cells, fibroblasts, inflammatory cells). Moreover, α3β1 signaling controls gene expression that helps epithelial cells carry out these functions, including genes that encode secreted matrix proteins, proteases, growth factors, or cytokines. We will review what is known about α3β1-dependent gene regulation through both transcription and posttranscriptional mRNA stability. Regarding the latter, we will discuss examples of α3β1-dependent alternative splicing (AS) or alternative polyadenylation (APA) that prevents inclusion of cis -acting mRNA sequences that would otherwise target the transcript for degradation via nonsense-mediated decay or destabilizing AU-rich elements (AREs) in the 3'-untranslated region (3'-UTR). Finally, we will discuss prospects and anticipated challenges of exploiting α3β1 as a clinical target for the treatment of cancer or wound healing.

The development of wound therapy targeting integrins is hampered by inadequate understanding of integrin function in cutaneous wound healing and the wound microenvironment. Following cutaneous injury, keratinocytes migrate to restore the skin barrier, and macrophages aid in debris clearance. Thus, both keratinocytes and macrophages are critical to the coordination of tissue repair. Keratinocyte integrins have been shown to participate in this coordinated effort by regulating secreted factors, some of which crosstalk to distinct cells in the wound microenvironment. Epidermal integrin α3β1 is a receptor for laminin-332 in the cutaneous basement membrane. Here we show that wounds deficient in epidermal α3β1 express less epidermal-derived macrophage colony-stimulating factor 1 (CSF-1), the primary macrophage-stimulating growth factor. α3β1-deficient wounds also have fewer wound-proximal macrophages, suggesting that keratinocyte α3β1 may stimulate wound macrophages through the regulation of CSF-1. Indeed, using a set of immortalized keratinocytes, we demonstrate that keratinocyte-derived CSF-1 supports macrophage growth, and that α3β1 regulates Csf1 expression through Src-dependent stimulation of Yes-associated protein (YAP)-Transcriptional enhanced associate domain (TEAD)-mediated transcription. Consistently, α3β1-deficient wounds in vivo display a substantially reduced number of keratinocytes with YAP-positive nuclei. Overall, our current findings identify a novel role for epidermal integrin α3β1 in regulating the cutaneous wound microenvironment by mediating paracrine crosstalk from keratinocytes to wound macrophages, implicating α3β1 as a potential target of wound therapy., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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).)

Using a model of the human SK-Mel-147 melanoma cell line, it was shown that blocking the expression of integrin α3β1 by transduction of cells with α3-specific shRNA did not affect their proliferation, but sharply increased the proportion of SA-β-Gal-positive cells, a phenotypic feature of cell senescence. These findings were accompanied by a significant increase in the activity of the Akt and mTOR protein kinases and the expression of p53 and p21 oncosupressors. Pharmacological inhibition of mTORC1 reduced the number SA-β-Gal-positive cells in the SK-Mel-147 cell population depleted of α3β1. Based on our recent data on a non-canonical function of Akt isomers in the regulation of SK-Mel-147 cell senescence caused by deficiency of α2β1 receptor, we investigated the role of Akt isomers in senescence induced by the α3β1 knockdown. It appeared that in the cell population with downregulated α3β1, inhibition of Akt1 reduced the number SA-β-Gal positive cells to the level of control cell population, while inhibition of Akt2 had no visible effect. Our results demonstrate that the laminin-specific integrin α3β1, like the collagen-specific receptor α2β1, is involved in tumor cell protection from senescence, and senescence induced by α3β1 depletion, like that caused by α2β1 deficiency, is based on a signaling mechanism employing a non-canonical function of the Akt1 isoform.

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.

Pancreatic cancer is highly malignant and has a five-year survival rate of 5% due to an early lymph node, nerve, and vascular metastasis. Integrin α 3 β 1 (also called very late antigen-3, VLA-3) is overexpressed in many tumors and plays a vital role in tumor formation, recurrence, and metastasis. In this study, we developed a 68 Ga-radiolabeled peptide tracer targeting the α 3 unit of VLA-3 and evaluated its potential application in positron emission computed tomography (PET) imaging of pancreatic cancer. NOTA-CK11 was prepared by solid-phase synthesis and successfully radiolabeled with 68 Ga with greater than 99% radiochemical purity and a specific activity of 37 ± 5 MBq/nmol ( n = 5). The expression level of integrin α 3 in three human pancreatic cancer cells was evaluated with the order of SW1990, BXPC-3, and PANC-1 from high to low, while the expression level of integrin β 1 was relatively close. When SW1990 cells with the highest expression level of VLA-3 were stained with FITC-CK11, strong fluorescence was observed by flow cytometry and under a laser confocal microscope. However, no significant fluorescence was observed in the blocking group when treated with excessive CK11. 68 Ga-NOTA-CK11 showed significant radioactivity accumulation in SW1990 cells and was blocked by CK11 successfully. Subsequent small-animal PET imaging and biodistribution studies in mice bearing SW1990 xenografts confirmed its high tumor uptake with a good tumor-to-blood ratio and tumor-to-muscle ratio (2.45 ± 0.31 and 3.65 ± 0.33, respectively) at 1 h post injection of the probe. In summary, we successfully developed a peptide-based imaging agent, 68 Ga-NOTA-CK11, that showed a strong binding affinity with VLA-3 and good target specificity for SW1990 cells and xenografted pancreatic tumor, rending it a promising radiotracer for PET imaging of VLA-3 expression in pancreatic cancer.

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.)

Docetaxel (DTX) widely used for treating nonsmall cell lung cancer (NSCLC) patients is associated with dose-limiting side effects, especially neurotoxicity and myelosuppression. Here, we have developed cyclic cNGQGEQc peptide-directed polymersomal docetaxel (cNGQ-PS-DTX) as a targeted and multifunctional formulation for NSCLC. cNGQ-PS-DTX carrying 8.1 wt % DTX had a size of 93 nm, neutral surface charge, high stability, and glutathione-triggered DTX release behavior. Cytotoxicity studies demonstrated a clearly better antitumor activity of cNGQ-PS-DTX in α 3 β 1 integrin overexpressing A549 human lung cancer cells than free DTX and nontargeted PS-DTX. cNGQ-PS-DTX showed a remarkably high tolerability (over 8 times better than free DTX) and slow elimination in mice. Importantly, cNGQ-PS-DTX exhibited greatly improved tumor accumulation and higher suppression of subcutaneous and orthotopic A549 xenografts as compared to PS-DTX and free DTX controls. α 3 β 1 integrin-targeting polymersomal docetaxel emerges as an advanced nanotherapeutic for NSCLC treatment.

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.

Integrins, the major receptors for cell-extracellular matrix (ECM) interactions, regulate multiple cell biological processes including adhesion, migration, proliferation and growth factor-dependent signaling. The principal laminin (LM) binding integrins α3β1, α6β1 and α6β4 are usually co-expressed in cells and bind to multiple laminins with different affinities making it difficult to define their specific function. In this study, we generated kidney epithelial collecting duct (CD) cells that lack both the α3 and α6 integrin subunits. This deletion impaired cell adhesion and migration to LM-332 and LM-511 more than deleting α3 or α6 alone. Cell adhesion mediated by both α3β1 and α6 integrins was PI3K independent, but required K63-linked polyubiquitination of Akt by the ubiquitin-modifying enzyme TRAF6. Moreover, we provide evidence that glial-derived neurotrophic factor (GDNF) and fibroblast growth factor 10 (FGF10)- mediated cell signaling, spreading and proliferation were severely compromised in double integrin α3/α6- but not single α3- or α6-null CD cells. Interestingly, these growth factor-dependent cell functions required both PI3K- and TRAF6-dependent Akt activation. These data suggest that expression of the integrin α3 or α6 subunit is sufficient to mediate GDNF- and FGF10-dependent spreading, proliferation and signaling on LM-511. Thus, our study shows that α3 and α6 containing integrins promote distinct functions and signaling by CD cells on laminin substrata., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.)

In this study, we established a long-term three-dimensional (3D) culture system by using integrin ligand modified alginate hydrogels to encapsulate and differentiate neural progenitor cells (NPCs) toward oligodendrocyte (OL) lineage cells. The porosity of the hydrogel was optimized by varying the alginate concentrations and then characterized by scanning electronic microscopy (SEM). The surface plasmon resonance (SPR) test was used to confirm the ligand-integrin interactions indicating adherence between the NPC surfaces and the hydrogels. Following encapsulation in the hydrogels, both mouse and human NPC sphere cultures could be maintained up to 90 days. Mouse NPC spheres were differentiated into viable neurons, astrocytes and mature OLs by day 60 in all groups whereas human NPC spheres were differentiated into neurons and later into GFAP positive astrocytes and O4 positive pre-OL within 90 days. The species difference in the timeline of OL development between mouse and human was reflected in this system. The ligand LXY30 interacting with the α3β1 integrin receptor was more effective in promoting the differentiation of hNPCs to OL lineage cells compared with the ligand LXW64 interacting with the αvβ3 integrin receptor, hyaluronic acid interacting with CD44 receptor or without any ligand. This study is the first to differentiate O4 + pre-OLs from hNPCs in a LXY30-α3β1 (integrin-ligand) modified alginate 3D hydrogel culture. This 3D platform could serve as a valuable tool in disease modeling, drug discovery, and NPC transplantation.

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.)

Cancer cells from a primary tumor can disseminate to other tissues, remaining dormant and clinically undetectable for many years. Little is known about the cues that cause these dormant cells to awaken, resume proliferating, and develop into metastases. Studying mouse models, we found that sustained lung inflammation caused by tobacco smoke exposure or nasal instillation of lipopolysaccharide converted disseminated, dormant cancer cells to aggressively growing metastases. Sustained inflammation induced the formation of neutrophil extracellular traps (NETs), and these were required for awakening dormant cancer. Mechanistic analysis revealed that two NET-associated proteases, neutrophil elastase and matrix metalloproteinase 9, sequentially cleaved laminin. The proteolytically remodeled laminin induced proliferation of dormant cancer cells by activating integrin α3β1 signaling. Antibodies against NET-remodeled laminin prevented awakening of dormant cells. Therapies aimed at preventing dormant cell awakening could potentially prolong the survival of cancer patients., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.)

The host injury involved in multiorgan system failure during severe inflammation is mediated, in part, by massive infiltration and sequestration of hyperactive neutrophils in the visceral organ. A recombinant form of human activated protein C (rhAPC) has shown cytoprotective and anti-inflammatory functions in some clinical and animal studies, but the direct mechanism is not fully understood. Recently, we reported that, during endotoxemia and severe polymicrobial peritonitis, integrin VLA-3 (CD49c/CD29) is specifically upregulated on hyperinflammatory neutrophils and that targeting the VLA-3 high neutrophil subpopulation improved survival in mice. In this article, we report that rhAPC binds to human neutrophils via integrin VLA-3 (CD49c/CD29) with a higher affinity compared with other Arg-Gly-Asp binding integrins. Similarly, there is preferential binding of activated protein C (PC) to Gr1 high CD11b high VLA-3 high cells isolated from the bone marrow of septic mice. Furthermore, specific binding of rhAPC to human neutrophils via VLA-3 was inhibited by an antagonistic peptide (LXY2). In addition, genetically modified mutant activated PC, with a high affinity for VLA-3, shows significantly improved binding to neutrophils compared with wild-type activated PC and significantly reduced neutrophil infiltration into the lungs of septic mice. These data indicate that variants of activated PC have a stronger affinity for integrin VLA-3, which reveals novel therapeutic possibilities., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Parkinson's disease (PD) is a neurodegenerative disorder in which the loss of dopaminergic neurons in the midbrain (mDA neurons) causes progressive loss of motor control and function. Using embryonic and mDA neurons, midbrain tissue from mice, and differentiated human neural stem cells, we investigated the mechanisms controlling the survival of mDA neurons. We found that the extracellular matrix protein laminin-511 (LM511) promoted the survival and differentiation of mDA neurons. LM511 bound to integrin α 3 β 1 and activated the transcriptional cofactor YAP. LM511-YAP signaling enhanced cell survival by inducing the expression of the microRNA miR-130a, which suppressed the synthesis of the cell death-associated protein PTEN. In addition, LM511-YAP signaling increased the expression of transcription factors critical for mDA identity, such as LMX1A and PITX3, and prevented the loss of mDA neurons in response to oxidative stress, a finding that warrants further investigation to assess therapeutic potential for PD patients. We propose that by enhancing LM511-YAP signaling, it may be possible to prevent mDA neuron degeneration in PD or enhance the survival of mDA neurons in cell replacement therapies., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Although the effects of long-term experimental dysthyroidism on long-term potentiation (LTP) and long-term depression (LTD) have been documented, the relationship between LTP/LTD and acute administration of L-thyroxine (T4) has not been described. Here, we investigated the effects of intra-hippocampal administration of T4 on synaptic plasticity in the dentate gyrus of the hippocampal formation. After a 15-minute baseline recording, LTP and LTD were induced by application of high- and low-frequency stimulation protocols, respectively. Infusions of saline or T4 and tetraiodothyroacetic acid (tetrac), a T4 analog that inhibits binding of iodothyronines to the integrin αvβ3 receptor, either alone or together, were made during the stimulation protocols. The averages of the excitatory postsynaptic potential (EPSP) slopes and population spike (PS) amplitudes, between 55 to 60 minutes, were used as a measure of the LTP/LTD magnitude and were analyzed by two-way univariate ANOVA with T4 and tetrac as between-subjects factors. The input-output curves of the infusion groups were comparable to each other, as shown by the non significant interaction observed between stimulus intensity and infused drug. The magnitude of the LTP in T4-infused rats was significantly lower as compared to saline-infused rats. Both the PS amplitude and the EPSP slope were depressed more markedly with T4 infusion than with saline, tetrac, and T4 + tetrac infusion. Data of this study provide in vivo evidence that T4 can promote LTD over LTP via the integrin αvβ3 receptor, and that the effect of endogenous T4 on this receptor can be suppressed by tetrac in the hippocampus. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

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.

Integrin α3β1 has been shown to be a novel candidate target for the imaging and specific therapy of non-small-cell lung cancer. We have previously reported on a peptide containing a novel motif of NGXG that specifically binds to the integrin α3 receptor on lung cancer cells using a one-bead one-peptide combinatorial library. In this study, we developed the peptide cNGEGQQc-based therapeutic agent labeling with radionuclide iodine-131 (I) and evaluated its characteristics including stability, biodistribution, antitumor activity, and safety. The results showed that I-cNGEGQQc was stable in serum. Furthermore, the biodistribution of I-cNGEGQQc was determined in normal mice and rabbits. In-vivo biodistribution studies showed that radiolabeled peptide in the kidney was significantly higher than that in other organs. Nude mice bearing lung cancer cell xenografts (H1975 and L78) were used as an in-vivo model for tumor-inhibition efficacy studies with I-cNGEGQQc. The tumor growth decreased significantly in mice receiving I-labeled peptide compared with the controls and the effect of I-labeled peptide can be blocked by unlabeled cNGEGQQc. Safety studies showed that I-cNGEGQQc was relatively safe for animals without significant toxicity. Our data suggest that I-cNGEGQQc has potential as a targeted radiotherapeutic agent for non-small-cell lung cancer.

Dermatopontin (DPT) is a matricellular protein with cardinal roles in cutaneous wound healing. The protein is also reported to be altered in various anomalies including cancer. The present study is aimed to unravel the role of DPT in angiogenesis which is imperative in many physiological and pathological processes. DPT's capabilities on promoting angiogenesis were assessed using various in vitro and ex vivo systems. The results indicated that DPT enhances cell motility and induces lamellipodia formation in endothelial cells. Additionally, we noticed that DPT stimulates tube formation in endothelial cells when plated on a matrigel substrate. However, it was observed that DPT had no effect on the proliferation of endothelial cells even at higher concentrations and prolonged treatment periods. Additional experiments on CAM and aortic arch assays apparently depicted that DPT promotes neovascularisation and tube sprouting, thus unraveling its prominent role in angiogenesis. Further, PCR analysis revealed that endothelial cells are devoid of DPT expression, but when exogenously supplied, modulates the expression of transforming growth factor β1 and integrin α3β1 which are reported to have crucial roles in endothelial cell behaviour during angiogenesis. In conclusion, DPT possess vital pro-angiogenic properties and thus retains promising therapeutic values in managing chronic wounds and cancer., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

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.

Integrins play an important role in cell adhesion by linking the cytoskeleton of cells to components in the extracellular matrix. In this capacity, integrins cooperate with different cell surface receptors, including growth factor receptors and G-protein coupled receptors, to regulate intracellular signaling pathways that control cell polarization, spreading, migration, survival, and gene expression. A distinct subfamily of molecules in the integrin family of adhesion receptors is formed by receptors that mediate cell adhesion to laminins, major components of the basement membrane that lie under clusters of cells or surround them, separating them from other cells and/or adjacent connective tissue. During the past decades, many studies have provided evidence for a role of laminin-binding integrins in tumorigenesis, and both tumor-promoting and suppressive activities have been identified. In this review we discuss the dual role of the laminin-binding integrins α3β1 and α6β4 in tumor development and progression, and examine the factors and mechanisms involved in these opposing effects., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.)

Epithelial cells, both normal and precancerous, stably anchor to basement membranes, whereas malignant tumors pass through them to achieve metastasis. Of basement membrane components, laminin-511 (α5, β1, γ1; LM-511) has been found to be a major isoform in many adult basement membranes. Several studies have shown that LM-511 promotes not only cell adhesion but also tumor cell migration. Thus, LM-511 can be viewed like two distinct molecules in normal vs. tumor cells; tumor cells seem to be able to alter their response (adhesive vs. migratory) to LM-511. In this study we examined the effects of biologically active molecules on A549 lung adenocarcinoma cell adhesion to LM-511. Of them, phorbol 12-myristate 13-acetate (PMA) induced transition to a rounded cell shape and significantly promoted cell migration on LM-511. The attachment of PMA-treated A549 cells to LM-511 was weaker than that of control cells. PMA-stimulated signaling pathway reduced the binding of integrin α3β1 to LM-511. Cell migration assays using inhibitors for signal transduction and cytoskeletal organization showed that suppression of cell adhesion via the rho-associated protein kinase (ROCK) pathway promoted tumor cell migration on LM-511. Our results suggest that the ROCK pathway is involved in the transition from static to migratory cell behaviors on LM-511., (Copyright © 2016 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.

Purpose of Review: Neutrophils have traditionally been viewed in the context of acute infection and inflammation forming the first line of defense against invading pathogens. Neutrophil trafficking to the site of inflammation requires adhesion and transmigration through blood vessels, which is orchestrated by adhesion molecules, such as β2 and β1-integrins, chemokines, and cytokines. The review focuses on recent advances in understanding the regulators of neutrophil recruitment during inflammation in both acute and chronic settings., Recent Findings: Recent findings suggest that besides the established pathways of selectin or chemokine-mediated integrin activation, signaling by distinct Toll-like receptors (TLRs) (especially TLR2, TLR4, and TLR5) can activate integrin-dependent neutrophil adhesion. Moreover, the integrin α3β1 has been vitally implicated as a new player in neutrophil recruitment and TLR-mediated responses in septic inflammation. Furthermore, several endogenous inhibitory mechanisms of leukocyte recruitment have been identified, including the secreted molecules Del-1, PTX3, and GDF-15, which block distinct steps of the leukocyte adhesion cascade, as well as novel regulatory signaling pathways, involving the protein kinase AKT1 and IFN-λ2/IL-28A., Summary: The leukocyte adhesion cascade is a tightly regulated process, subjected to both positive and negative regulators. Dysregulation of this process and hence neutrophil recruitment can lead to the development of inflammatory and autoimmune diseases.

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.