Surface-enhanced Raman scattering-based identification of breast cancer progression using extracellular vesicles-derived integrin α6ß4.

Detection of progression is of great importance to breast cancer treatment and can benefit patients. Limited by current detection technologies and biomarkers, early breast cancer progression diagnosis remains challenging. Researchers have found blood extracellular vesicles (EVs)-derived integrin α6ß4 directly facilitate progression in breast cancer, enabling cancer detection. However, EVs size and heterogeneity hinder protein detection, masked by abundant background EVs. Hence, novel tools for efficient detection of EVs with high selectivity and low interference are significantly desired. Here, a new silver-coated gold nanorods SERS probe, termed as Au@Ag@IDA-B/4MSTP, based on DNA aptamer was established for the detection of integrin α6ß4 derived from EVs. Validation of the Au@Ag@IDA-B/4MSTP probes using cell-culture-derived EVs revealed a LOD of 23 particles/µL for EVs detection. This tool was further confirmed to mimic the real state of cancer with subcutaneous tumor model and lung metastasis model in mice. With 10 µL of blood plasma and simple Raman analysis process, the test achieved 85.7 % sensitivity and 83.3 % specificity. Moreover, our method achieves a simplified approach that expedites the detection process. These results demonstrate the good detection performance of Au@Ag@IDA-B/4MSTP probes for EVs integrin α6ß4, and suggest that this non-invasive approach could be a promising tool for early detection of breast cancer progression.

Cerebral arterioles express the laminin subunits α4 and α5 in conjunction with α6ß4 integrin, but strongly downregulate laminin α4 during hypoxia-induced arteriogenic remodeling.

Previous studies have shown that expression of the endothelial laminin receptor α6ß4 integrin in the brain is uniquely restricted to arterioles. As exposure to chronic mild hypoxia (CMH, 8 % O2) stimulates robust angiogenic and arteriogenic remodeling responses in the brain, the goal of this study was to determine how CMH influences cerebrovascular expression of the ß4 integrin as well as its potential ligands, laminin 411 and 511, containing the α4 and α5 laminin subunits respectively, and then define how aging impacts this expression. We observed the following: (i) CMH launched a robust arteriogenic remodeling response both in the young (10 weeks) and aged (20 months) brain, correlating with an increased number of ß4 integrin+ vessels, (ii) while the laminin α4 subunit is expressed evenly across all cerebral blood vessels, laminin α5 was highly expressed preferentially on ß4 integrin+ arterioles, (iii) CMH-induced arteriolar remodeling was associated with strong downregulation of the laminin α4 subunit but no change in the laminin α5 subunit, (iv) in addition to its expression on arterioles, ß4 integrin was also expressed at lower levels on capillaries specifically in white matter (WM) tracts but not in the grey matter (GM), and (v), these observations were consistent in both the brain and spinal cord, and age had no obvious impact. Taken together, our findings suggest that laminin 511 may be a specific ligand for α6ß4 integrin and that dynamic switching of the laminin subunits α4 and α5 might play an instructive role in arteriogenic remodeling. Furthermore, ß4 integrin expression differentiates WM from GM capillaries, highlighting a novel and important difference.

Integrin α6ß4 Confers Doxorubicin Resistance in Cancer Cells by Suppressing Caspase-3-Mediated Apoptosis: Involvement of N-Glycans on ß4 Integrin Subunit.

Drug resistance is a major obstacle to successful cancer treatment. Therefore, it is essential to understand the molecular mechanisms underlying drug resistance to develop successful therapeutic strategies. α6ß4 integrin confers resistance to apoptosis and regulates the survival of cancer cells; however, it remains unclear whether α6ß4 integrin is directly involved in chemoresistance. Here, we show that α6ß4 integrin promotes doxorubicin resistance by decreasing caspase-3-mediated apoptosis. We found that the overexpression of α6ß4 integrin by the ß4 integrin gene rendered MDA-MB435S and Panc-1 cells more resistant to doxorubicin than control cells. The acquired resistance to doxorubicin by α6ß4 integrin expression was abolished by the deletion of the cytoplasmic signal domain in ß4 integrin. Similar results were found in MDA-MB435S and Panc-1 cells when N-glycan-defective ß4 integrin mutants were overexpressed or bisecting GlcNAc residues were increased on ß4 integrin by the co-expression of N-acetylglucosaminyltransferase III with ß4 integrin. The abrogation of α6ß4 integrin-mediated resistance to doxorubicin was accompanied by reduced cell viability and an increased caspase-3 activation. Taken together, our results clearly suggest that α6ß4 integrin signaling plays a key role in the doxorubicin resistance of cancer cells, and N-glycans on ß4 integrin are involved in the regulation of cancer cells.

Mining TCGA and GEO databases for the prediction of poor prognosis in lung adenocarcinoma based on up-regulated expression of TNS4.

To investigate the clinical significance of Tensin4 (TNS4) in human cancers, particularly lung cancer, we mined the Cancer Genome Atlas database for lung adenocarcinoma (TCGA-LUAD) and the Gene Expression Omnibus database to predict poor prognosis based on the up-regulated expression of TNS4 in LUAD. The correlation between the clinical pathologic features of patients and TNS4 gene expression was analyzed using the Wilcoxon signed-rank test. Cox regression analysis was used to evaluate the association of clinicopathologic characteristics with the overall survival (OS) of cancer patients using TCGA data. The relationship between TNS4 expression and cancer patient survival was evaluated with Kaplan-Meier survival curves and meta-analyses. GO and KEGG were also included in the data mining methods. The expression level of TNS4 in LUAD tissue was higher than that in adjacent normal tissue (P < .001). According to the Kaplan-Meier survival curve, LUAD patients with high TNS4 expression had worse prognosis than those with low TNS4 expression (P < .001 for OS; P = .028 for progression-free survival). A positive correlation between TNS4 expression and poor OS was found with both univariate and multivariate analyses. Increased TNS4 expression in LUAD was closely correlated with a higher disease stage (P = .007), positive lymph nodes (P = .005), and larger tumor size (P = .002). Moreover, meta-analysis including seven independent datasets showed LUAD patients with higher TNS4 had poorer OS (combined hazard ratio = 1.27, 95% confidence interval 1.16-1.39). In the high-TNS4 population, regulation of the actin cytoskeleton, extracellular matrix receptor interactions, and focal adhesion were differentially enriched. Integrin α6ß4 and laminin-5 genes were also associated with TNS4. TNS4 expression may be a potential biomarker for predicting poor survival in LUAD. Moreover, the correlation between TNS4 and integrin α6ß4 may be attributed to the role of TNS4 in LUAD.

Integrin alpha 6 is upregulated and drives hepatocellular carcinoma progression through integrin α6ß4 complex.

Integrin α6 (ITGA6) forms integrin receptors with either integrin ß1 (ITGB1) or integrin ß4 (ITGB4). How it functions to regulate hepatocellular carcinoma (HCC) progression is not well-elucidated. We found that ITGA6 RNA and protein expression levels are significantly elevated in human HCC tissues in comparison with paired adjacent nontumor tissues by RNA sequencing, RT-qPCR, Western blotting and immunofluorescence staining. Stable knockdown of ITGA6 with different ITGA6 shRNA expression lentivectors significantly inhibited proliferation, migration and anchorage-independent growth of HCC cell lines in vitro, and xenograft tumor growth in vivo. The inhibition of anchorage-dependent and -independent growth of HCC cell lines was also confirmed with anti-ITGA6 antibody. ITGA6 knockdown was shown to induce cell-cycle arrest at G0/G1 phase. Immunoprecipitation assay revealed apparent interaction of ITGA6 with ITGB4, but not ITGB1. Expression studies showed that ITGA6 positively regulates the expression of ITGB4 with no or negative regulation of ITGB1 expression. Finally, while high levels of ITGA6 and ITGB4 together were associated with significantly worse survival of HCC patients in TCGA data set, the association was not significant for high levels of ITGA6 and ITGB1. In conclusion, ITGA6 is upregulated in HCC tumors and has a malignant promoting role in HCC cells through integrin α6ß4 complex. Thus, integrin α6ß4 may be a therapeutic target for treating patients with HCC.

Laminin-integrin a6b4 interaction activates notch signaling to facilitate bladder cancer development.

BACKGROUND: Laminins are high-molecular weight (400 ~ 900 kDa) proteins in extracellular matrix, which serve as major component of the basal lamina, and play a crucial role in promoting tumor cell migration. This study aimed at characterizing the role of laminin in promoting cancer development, and elucidating the mechanism of tumor progression driven by laminin-Notch signaling in bladder cancer. METHODS: 2D collagen/laminin culture system was established and CCK-8/transwell assay was conducted to evaluate the proliferation/migration ability of Biu-87 and MB49 cells cultured on 2D gels. Activation of integrins-Notch1 signaling was determined by western blotting. Orthotopic bladder cancer mice model was established to assess the therapeutic effects of Notch inhibitor. RESULTS: Our study demonstrated that extracellular laminin can trigger tumor cell proliferation/migration through integrin α6ß4/Notch1 signaling in bladder cancer. Inhibition of Telomere repeat-binding factor 3 (TRB3)/Jagged Canonical Notch Ligand 1 (JAG1) signaling suppressed Notch signals activation induced by laminin-integrin axis. In MB49 orthotopic bladder cancer mice model, Notch inhibitor SAHM1 efficiently improved tumor suppressive effects of chemotherapy and prolonged survival time of tumor-bearing mice. CONCLUSION: In conclusion, we show that, in bladder cancer, extracellular laminin induced the activation of Notch pathway through integrin α6ß4/TRB3/JAG3, and disclosed a novel role of laminin in bladder cancer cells proliferation or migration.

KRAS Mutants Upregulate Integrin ß4 to Promote Invasion and Metastasis in Colorectal Cancer.

KRAS mutation in colorectal cancer is associated with aggressive tumor behavior through increased invasiveness and higher rates of lung metastases, but the biological mechanisms behind these features are not fully understood. In this study, we show that KRAS-mutant colorectal cancer upregulates integrin α6ß4 through ERK/MEK signaling. Knocking-out integrin ß4 (ITGB4) specifically depleted the expression of integrin α6ß4 and this resulted in a reduction in the invasion and migration ability of the cancer cells. We also observed a reduction in the number and area of lung metastatic foci in mice that were injected with ITGB4 knockout KRAS-mutant colorectal cancer cells compared with the mice injected with ITGB4 wild-type KRAS-mutant colorectal cancer cells, while no difference was observed in liver metastases. Inhibiting integrin α6ß4 in KRAS-mutant colorectal cancer could be a potential therapeutic target to diminish the KRAS-invasive phenotype and associated pulmonary metastasis rate. IMPLICATIONS: Knocking-out ITGB4, which is overexpressed in KRAS-mutant colorectal cancer and promotes tumor aggressiveness, diminishes local invasiveness and rates of pulmonary metastasis.

Contribution of Endothelial Laminin-Binding Integrins to Cellular Processes Associated with Angiogenesis.

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, α6ß4, and α3ß1 contribute to these processes, we employed RNAi technology in organotypic angiogenesis assays, as well in migration assays, in vitro. The endothelial depletion of either α6ß4 or α3ß1 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 α6ß4, but not α3ß1, inhibited migration. Taken together, these results suggest that laminin-binding integrins regulate processes associated with angiogenesis by distinct and overlapping mechanisms.

Exosome-derived ENO1 regulates integrin α6ß4 expression and promotes hepatocellular carcinoma growth and metastasis.

Alpha-enolase (ENO1) has been found to be dysregulated in several human malignancies, including hepatocellular carcinoma (HCC). Although the role of ENO1 as a glycolytic enzyme in HCC cells has been well characterized, little is known about the other roles of ENO1, especially exosome-derived ENO1, in regulating HCC progression. Here, we demonstrated that ENO1 is frequently upregulated in HCC cells or tissues, with even higher expression in highly metastatic HCC cells or metastatic tissues as well as in exosomes derived from highly metastatic sources. Moreover, ENO1 expression is associated with the tumor-node-metastasis (TNM) stage, differentiation grade and poor prognosis in HCC patients. Surprisingly, ENO1 can be transferred between HCC cells via exosome-mediated crosstalk, exhibiting an effect similar to that of ENO1 overexpression in HCC cells, which promoted the growth and metastasis of HCC cells with low ENO1 expression by upregulating integrin α6ß4 expression and activating the FAK/Src-p38MAPK pathway. In summary, our data suggest that exosome-derived ENO1 is essential to promoting HCC growth, metastasis, and further patient deterioration. The findings from this study implicate a novel biomarker for the clinical evaluation of HCC progression, especially the prediction of HCC metastatic risk.

Comparative interactomics analysis reveals potential regulators of α6ß4 distribution in keratinocytes.

The integrin α6ß4 and cytoskeletal adaptor plectin are essential components of type I and type II hemidesmosomes (HDs). We recently identified an alternative type II HD adhesion complex that also contains CD151 and the integrin α3ß1. Here, we have taken a BioID proximity labeling approach to define the proximity protein environment for α6ß4 in keratinocytes. We identified 37 proteins that interacted with both α6 and ß4, while 20 and 78 proteins specifically interacted with the α6 and ß4 subunits, respectively. Many of the proximity interactors of α6ß4 are components of focal adhesions (FAs) and the cortical microtubule stabilizing complex (CMSC). Though the close association of CMSCs with α6ß4 in HDs was confirmed by immunofluorescence analysis, CMSCs have no role in the assembly of HDs. Analysis of the ß4 interactome in the presence or absence of CD151 revealed that they are strikingly similar; only 11 different interactors were identified. One of these was the integrin α3ß1, which interacted with α6ß4 more strongly in the presence of CD151 than in its absence. These findings indicate that CD151 does not significantly contribute to the interactome of α6ß4, but suggest a role of CD151 in linking α3ß1 and α6ß4 together in tetraspanin adhesion structures.

Tetraspanin CD151 and integrin α3ß1 contribute to the stabilization of integrin α6ß4-containing cell-matrix adhesions.

Tetraspanin CD151 has been suggested to regulate cell adhesion through its association with laminin-binding integrins α3ß1 and α6ß4; 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 α3ß1, 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-α3ß1/α6ß4 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 α6ß4, 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.

A DNA Nanodevice Simultaneously Activating the EGFR and Integrin for Enhancing Cytoskeletal Activity and Cancer Cell Treatment.

Cell-surface receptors (e.g., EGFR and integrin) and their interactions play determining roles in signal transduction and cytoskeletal activation, which affect cell attachment/detachment, invasion, motility, metastasis (intracellular), and cell-cell signaling. For instance, the interactions between the EGFR and integrin (α6ß4) may cause increased mechanical force and shear stress via enhanced cytoskeleton activation. Here, we design a DNA nanodevice (DNA-ND) that can simultaneously target the EGFR and integrin receptors on the caveolae. The piconewton (pN) forces in response to the EGFR-integrin coactivation can be sensed upon the unfolding of the DNA hairpin structure on the side arm of the device via changes of the fluorescence and plasmonic signals. We find that simultaneous activation of EGFR-integrin receptors causes enhanced signal transduction, contractions of the cells, and initiation of the biochemical pathways, thus resulting in a change of the cell division and endocytosis/exocytosis processes that affect the cell proliferation/apoptosis. The DNA-ND further enables us to visualize the cointernalization and degradation of the receptors by lysosomes, providing a novel approach toward bioimaging and mechano-pharmacology.

Screening of Hydrogels for Human Pluripotent Stem Cell-Derived Neural Cells: Hyaluronan-Polyvinyl Alcohol-Collagen-Based Interpenetrating Polymer Network Provides an Improved Hydrogel Scaffold.

There is a clear need for novel in vitro models, especially for neuronal applications. Development of in vitro models is a multiparameter task consisting of cell-, biomaterial-, and environment-related parameters. Here, three different human origin neuronal cell sources are studied and cultured in various hydrogel 3D scaffolds. For the efficient evaluation of complex results, an indexing method for data is developed and used in principal component analysis (PCA). It is found that no single hydrogel is superior to other hydrogels, and collagen I (Col1) and hyaluronan-poly(vinyl alcohol) (HA1-PVA) gels are combined into an interpenetrating network (IPN) hydrogel. The IPN gel combines cell supportiveness of the collagen gel and stability of the HA1-PVA gel. Moreover, cell adhesion is studied in particular and it is found that adhesion of neurons differs from that observed for fibroblasts. In conclusion, the HA1-PVA-col1 hydrogel is a suitable scaffold for neuronal cells and supports adhesion formation in 3D.

Integrin α6ß4 Recognition of a Linear Motif of Bullous Pemphigoid Antigen BP230 Controls Its Recruitment to Hemidesmosomes.

Mechanical stability of epithelia requires firm attachment to the basement membrane via hemidesmosomes. Dysfunction of hemidesmosomal proteins causes severe skin-blistering diseases. Two plakins, plectin and BP230 (BPAG1e), link the integrin α6ß4 to intermediate filaments in epidermal hemidesmosomes. Here, we show that a linear sequence within the isoform-specific N-terminal region of BP230 binds to the third and fourth FnIII domains of ß4. The crystal structure of the complex and mutagenesis analysis revealed that BP230 binds between the two domains of ß4. BP230 induces closing of the two FnIII domains that are locked in place by an interdomain ionic clasp required for binding. Disruption of BP230-ß4 binding prevents recruitment of BP230 to hemidesmosomes in human keratinocytes, revealing a key role of this interaction for hemidesmosome assembly. Phosphomimetic substitutions in ß4 and BP230 destabilize the complex. Thus, our study provides insights into the architecture of hemidesmosomes and potential mechanisms of regulation.

Integrin α6ß4E variant is associated with actin and CD9 structures and modifies the biophysical properties of cell-cell and cell-extracellular matrix interactions.

Integrin α6ß4 is an essential, dynamic adhesion receptor for laminin 332 found on epithelial cells, required for formation of strong cell-extracellular matrix (ECM) adhesion and induced migration, and coordinated by regions of the ß4C cytoplasmic domain. ß4E, a unique splice variant of ß4 expressed in normal tissue, contains a cytoplasmic domain of 231 amino acids with a unique sequence of 114 amino acids instead of ß4C's canonical 1089 amino acids. We determined the distribution of α6ß4E within normal human glandular epithelium and its regulation and effect on cellular biophysical properties. Canonical α6ß4C expressed in all basal cells, as expected, while α6ß4E expressed within a subset of luminal cells. α6ß4E expression was induced by three-dimensional culture conditions, activated Src, was reversible, and was stabilized by bortezomib, a proteasome inhibitor. α6ß4C expressed in all cells during induced migration, whereas α6ß4E was restricted to a subset of cells with increased kinetics of cell-cell and cell-ECM resistance properties. Interestingly, α6ß4E presented in "ringlike" patterns measuring ∼1.75 × 0.72 microns and containing actin and CD9 at cell-ECM locations. In contrast, α6ß4C expressed only within hemidesmosome-like structures containing BP180. Integrin α6ß4E is an inducible adhesion isoform in normal epithelial cells that can alter biophysical properties of cell-cell and cell-ECM interactions.

Integrin α6ß4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues.

Cellular senescence refers to an irreversible growth arrest that is triggered by various intrinsic and extrinsic stresses. Many recent studies have demonstrated that cellular senescence plays a crucial role in the regression of tumors exposed to ionizing radiation (IR), but the underlying mechanism remains unknown. Here we show that the activation of integrin ß4 is essential for IR-induced cellular senescence. IR treatment results in the phosphorylation of integrin ß4 at tyrosine residue 1510, leading to activation of the integrin α6ß4-Src-AKT signaling pathway. We further reveal that the IR-induced phosphorylation of integrin ß4 is regulated by the cholesterol content and membrane fluidity. We also find that IR-induced p53-caspase signaling is independent of integrin α6ß4-Src-AKT signaling. Finally, we show that siRNA- or inhibitor-mediated blockade of integrin α6ß4-Src-AKT signaling switches the post-irradiation fate from senescence to apoptosis, under p53 activated condition, in both cancer cells and tumor tissues of xenograft mice. On the basis of our finding that, integrin α6ß4 is specifically activated and acts primarily to induce premature senescence in irradiated cancer cells, we propose that this integrin may be a valuable target and biomarker for radiotherapy.

The laminin binding α3 and α6 integrins cooperate to promote epithelial cell adhesion and growth.

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 α3ß1, α6ß1 and α6ß4 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 α3ß1 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.

The N-terminal domain of the R28 protein promotes emm28 group A Streptococcus adhesion to host cells via direct binding to three integrins.

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, R28Nt, R28 promotes bacterial adhesion to both endometrial-epithelial and endometrial-stromal cells. R28Nt was further subdivided into two domains, and we found that both are involved in cell binding. R28Nt and both subdomains interacted directly with the laminin-binding α3ß1, α6ß1, and α6ß4 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, R28Nt 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.

Enteric Species F Human Adenoviruses use Laminin-Binding Integrins as Co-Receptors for Infection of Ht-29 Cells.

The enteric species F human adenovirus types 40 and 41 (HAdV-40 and -41) are the third most common cause of infantile gastroenteritis in the world. Knowledge about HAdV-40 and -41 cellular infection is assumed to be fundamentally different from that of other HAdVs since HAdV-40 and -41 penton bases lack the αV-integrin-interacting RGD motif. This motif is used by other HAdVs mainly for internalization and endosomal escape. We hypothesised that the penton bases of HAdV-40 and -41 interact with integrins independently of the RGD motif. HAdV-41 transduction of a library of rodent cells expressing specific human integrin subunits pointed to the use of laminin-binding α2-, α3- and α6-containing integrins as well as other integrins as candidate co-receptors. Specific laminins prevented internalisation and infection, and recombinant, soluble HAdV-41 penton base proteins prevented infection of human intestinal HT-29 cells. Surface plasmon resonance analysis demonstrated that HAdV-40 and -41 penton base proteins bind to α6-containing integrins with an affinity similar to that of previously characterised penton base:integrin interactions. With these results, we propose that laminin-binding integrins are co-receptors for HAdV-40 and -41.

Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa.

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin ß3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin ß3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin ß3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin ß3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6ß4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.