RESUMO
Phenotypic heterogeneity poses a significant hurdle for cancer treatment but is under-characterized in the context of tumor invasion. Amidst the range of phenotypic heterogeneity across solid tumor types, collectively invading cells and single cells have been extensively characterized as independent modes of invasion, but their intercellular interactions have rarely been explored. Here, we isolated collectively invading cells and single cells from the heterogeneous 4T1 cell line and observed extensive transcriptional and epigenetic diversity across these subpopulations. By integrating these datasets, we identified laminin-332 as a protein complex exclusively secreted by collectively invading cells. Live-cell imaging revealed that laminin-332 derived from collectively invading cells increased the velocity and directionality of single cells. Despite collectively invading and single cells having similar expression of the integrin α6ß4 dimer, single cells demonstrated higher Rac1 activation upon laminin-332 binding to integrin α6ß4. This mechanism suggests a novel commensal relationship between collectively invading and single cells, wherein collectively invading cells promote the invasive potential of single cells through a laminin-332/Rac1 axis.
Assuntos
Laminina , Proteínas rac1 de Ligação ao GTP , Humanos , Movimento Celular , Integrina alfa6beta4/genética , Calinina , Laminina/genética , Laminina/metabolismo , Neoplasias/genética , Simbiose , Animais , Camundongos , Linhagem Celular Tumoral , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Integrina alfa6beta4 , Neoplasias , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Integrina beta4/genética , Transdução de Sinais , Apoptose/fisiologiaRESUMO
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.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Regulação Neoplásica da Expressão Gênica , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/patologia , Prognóstico , Tensinas/genética , Tensinas/metabolismoRESUMO
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.
Assuntos
Carcinoma Hepatocelular , Integrina alfa6 , Integrina alfa6beta4 , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Integrina beta4/genética , Integrina beta4/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologiaRESUMO
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.
Assuntos
Neoplasias Colorretais , Integrina beta4 , Neoplasias Pulmonares , Animais , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Integrina beta4/genética , Integrina beta4/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos , Invasividade Neoplásica/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6ß4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin ß4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6ß4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proliferação de Células , Hemidesmossomos/metabolismo , Humanos , Integrina alfa6beta4/genética , Laminina/metabolismo , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
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.
Assuntos
Integrina alfa6beta4 , Laminina , Adesão Celular/fisiologia , Células Endoteliais/metabolismo , Integrina alfa3beta1/genética , Integrina alfa3beta1/metabolismo , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Laminina/metabolismoRESUMO
Integrin α6ß4 binds plectin to associate with vimentin; however, the biological function remains unclear. Here, we utilized various integrin ß4 mutants and CRISPR-Cas9 editing to investigate this association. Upon laminin binding, integrin α6ß4 distinctly distributed peripherally as well as centrally, proximal to the nucleus. Upon fibronectin addition, integrin α6ß4 was centrally recruited to large focal adhesions (FAs) and enhanced Fak (also known as PTK2) phosphorylation. Integrin ß4 plectin-binding mutants or genetic deletion of plectin inhibited ß4 recruitment to FAs and integrin α6ß4-enhanced cell spreading, migration and three-dimensional invasive growth. Loss of the ß4 signaling domain (but retaining plectin binding) blocked migration and invasiveness but not cell spreading, recruitment to FAs or colony growth. Immunostaining revealed that integrin α6ß4 redistributed vimentin perinuclearly, where it colocalized with plectin and FAs. Depletion of vimentin completely blocked integrin ß4-enhanced invasive growth, Fak phosphorylation and proliferation in three dimensions but not two dimensions. In summary, we demonstrate the essential roles of plectin and vimentin in promoting an invasive phenotype downstream of integrin α6ß4. This article has an associated First Person interview with the first author of the paper.
Assuntos
Integrina alfa6beta4 , Plectina , Adesão Celular , Humanos , Integrina alfa6beta4/genética , Integrina beta4/genética , Filamentos Intermediários , Plectina/genética , Vimentina/genéticaRESUMO
Hemidesmosomes (HDs) are specialized multiprotein complexes that connect the keratin cytoskeleton of epithelial cells to the extracellular matrix (ECM). In the skin, these complexes provide stable adhesion of basal keratinocytes to the underlying basement membrane. Integrin α6ß4 is a receptor for laminins and plays a vital role in mediating cell adhesion by initiating the assembly of HDs. In addition, α6ß4 has been implicated in signal transduction events that regulate diverse cellular processes, including proliferation and survival. In this Review, we detail the role of α6ß4 in HD assembly and beyond, and we discuss the molecular mechanisms that regulate its function.
Assuntos
Hemidesmossomos , Integrina alfa6beta4 , Adesão Celular , Integrina alfa6beta4/genética , Queratinócitos , Transdução de SinaisRESUMO
Hemidesmosomes are specialized cell-matrix adhesion structures that are associated with the keratin cytoskeleton. Although the adhesion function of hemidesmosomes has been extensively studied, their role in mechanosignaling and transduction remains largely unexplored. Here, we show that keratinocytes lacking hemidesmosomal integrin α6ß4 exhibit increased focal adhesion formation, cell spreading, and traction-force generation. Moreover, disruption of the interaction between α6ß4 and intermediate filaments or laminin-332 results in similar phenotypical changes. We further demonstrate that integrin α6ß4 regulates the activity of the mechanosensitive transcriptional regulator YAP through inhibition of Rho-ROCK-MLC- and FAK-PI3K-dependent signaling pathways. Additionally, increased tension caused by impaired hemidesmosome assembly leads to a redistribution of integrin αVß5 from clathrin lattices to focal adhesions. Our results reveal a novel role for hemidesmosomes as regulators of cellular mechanical forces and establish the existence of a mechanical coupling between adhesion complexes.
Assuntos
Hemidesmossomos/genética , Integrina alfa6beta4/genética , Queratinas/genética , Mecanotransdução Celular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Moléculas de Adesão Celular/genética , Movimento Celular/genética , Junções Célula-Matriz/genética , Junções Célula-Matriz/metabolismo , Células Cultivadas , Citoesqueleto/genética , Adesões Focais/genética , Adesões Focais/metabolismo , Humanos , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas de Sinalização YAP , Quinases Associadas a rho/genética , CalininaRESUMO
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.
Assuntos
Distonina/química , Hemidesmossomos/metabolismo , Integrina alfa6beta4/química , Penfigoide Bolhoso/metabolismo , Domínios Proteicos , Sequência de Aminoácidos , Membrana Basal/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Distonina/genética , Distonina/metabolismo , Hemidesmossomos/genética , Humanos , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Modelos Moleculares , Mutagênese , Penfigoide Bolhoso/genética , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
The α6ß4 integrin heterodimer is an essential component of hemidesmosomes (HDs) and HD-related structures, which adhere epithelial cells to the underlying extracellular matrix. In this study, we focused on the importance of the α6 integrin 3' untranslated region (UTR) in α6ß4 integrin localization. To do so, A549â¯cells (a type II lung alveolar cell line) and immortalized human epidermal keratinocytes (iHEK) were infected with adenovirus encoding the entire α6 integrin protein with or without portions of its 3'UTR. In infected A549â¯cells, we detected α6ß4 integrin heterodimers containing the product of the adenovirus, regardless of whether the α6 integrin 3'UTR was present. However, only those α6 integrin proteins whose messages contained bases 4770-5633 of the α6 integrin 3'UTR were targeted to matrix adhesion sites. Moreover, overexpression of the full length α6 integrin 3'UTR, minus the coding sequence, in A549â¯cells disrupts the localization of endogenous α6ß4 integrin heterodimers. Following infection of iHEKs with the same adenovirus, the induced α6 integrin protein localizes to HDs regardless of whether its message possessed a 3'UTR. In sharp contrast, in α6 integrin depleted iHEKs, restoring α6 integrin expression using the coding sequence alone via adenoviral transduction resulted in α6 integrin preferentially forming α6ß1 rather than α6ß4 integrin heterodimers. α6ß4 integrin was only observed in knocked down cells following infection of adenovirus encoding the α6 integrin coding sequence with its 3'UTR. In summary, our data indicate that the α6 integrin 3'UTR is a key regulator of α6ß4 integrin heterodimer assembly and incorporation at sites of cell-matrix adhesion.
Assuntos
Regiões 3' não Traduzidas , Integrina alfa6/análise , Integrina alfa6beta4/genética , Células A549 , Linhagem Celular , Humanos , Integrina alfa6/genética , Queratinócitos/metabolismo , Multimerização Proteica , Estabilidade Proteica , Regulação para CimaRESUMO
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 â¼1.75 × 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.
Assuntos
Adesão Celular/fisiologia , Movimento Celular/fisiologia , Integrina alfa6beta4/fisiologia , Actinas/metabolismo , Actinas/fisiologia , Linhagem Celular Tumoral , Desmossomos/metabolismo , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Hemidesmossomos/metabolismo , Humanos , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Integrinas/metabolismo , Laminina/metabolismo , Isoformas de Proteínas , Tetraspanina 29/metabolismo , Tetraspanina 29/fisiologiaRESUMO
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.
Assuntos
Moléculas de Adesão Celular/metabolismo , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Integrina alfa3/metabolismo , Integrina alfa6/metabolismo , Laminina/metabolismo , Transdução de Sinais , Animais , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/química , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Matriz Extracelular/química , Matriz Extracelular/efeitos dos fármacos , Fator 10 de Crescimento de Fibroblastos/farmacologia , Deleção de Genes , Regulação da Expressão Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Humanos , Integrina alfa3/genética , Integrina alfa3beta1/genética , Integrina alfa3beta1/metabolismo , Integrina alfa6/genética , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Laminina/química , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , CalininaRESUMO
Podocyte injury is an early pathological change characteristic of various glomerular diseases, and apoptosis and F-actin cytoskeletal disruption are typical features of podocyte injury. In this study, we found that adriamycin (ADR) treatment resulted in typical podocyte injury and repressed plectin expression. Restoring plectin expression protected against ADR-induced podocyte injury whereas siRNA-mediated plectin silencing produced similar effects as ADR-induced podocyte injury, suggesting that plectin plays a key role in preventing podocyte injury. Further analysis showed that plectin repression induced significant integrin α6ß4, focal adhesion kinase (FAK) and p38 MAPK phosphorylation. Mutating Y1494, a key tyrosine residue in the integrin ß4 subunit, blocked FAK and p38 phosphorylation, thereby alleviating podocyte injury. Inhibitor studies demonstrated that FAK Y397 phosphorylation promoted p38 activation, resulting in podocyte apoptosis and F-actin cytoskeletal disruption. In vivo studies showed that administration of ADR to rats resulted in significantly increased 24-hour urine protein levels along with decreased plectin expression and activated integrin α6ß4, FAK, and p38. Taken together, these findings indicated that plectin protects podocytes from ADR-induced apoptosis and F-actin cytoskeletal disruption by inhibiting integrin α6ß4/FAK/p38 pathway activation and that plectin may be a therapeutic target for podocyte injury-related glomerular diseases.
Assuntos
Quinase 1 de Adesão Focal/genética , Rim/metabolismo , Plectina/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Citoesqueleto de Actina/genética , Actinas/genética , Animais , Apoptose/efeitos dos fármacos , Doxorrubicina/toxicidade , Humanos , Integrina alfa6beta4/genética , Rim/lesões , Rim/patologia , Glomérulos Renais/lesões , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Fosforilação , Podócitos/metabolismo , Ratos , Transdução de Sinais/genéticaRESUMO
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 â¼95% 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.
Assuntos
Moléculas de Adesão Celular , Códon sem Sentido , Epidermólise Bolhosa Juncional , Gentamicinas/farmacologia , Queratinócitos/metabolismo , Mutagênese/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/metabolismo , Epidermólise Bolhosa Juncional/patologia , Células HEK293 , Humanos , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Queratinócitos/patologia , CalininaRESUMO
Structural analyses of ß2 and ß3 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 αvß3, α2ß1, α3ß1, α5ß1, α6ß1 and α6ß4, and used negative-stain electron microscopy to examine their structures under various conditions. In contrast to αvß3 integrin, which exhibited a severely bent conformation in low-affinity 5â mM Ca2+ conditions, all ß1 integrin heterodimers displayed a mixed population of half-bent to fully extended conformations. Moreover, they did not undergo significant conformational change upon activation by Mn2+ Integrin α6ß4 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.
Assuntos
Integrina alfa6beta4/química , Integrina beta1/química , Integrina beta4/química , Cálcio/química , Cálcio/metabolismo , Linhagem Celular , Humanos , Integrina alfa3beta1/química , Integrina alfa3beta1/genética , Integrina alfa3beta1/metabolismo , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Integrina beta4/genética , Integrina beta4/metabolismo , Ligantes , Microscopia Eletrônica , Conformação Proteica , Domínios ProteicosRESUMO
Increases in lipid peroxidation can cause ferroptosis, a form of cell death triggered by inhibition of glutathione peroxidase 4 (GPX4), which catalyzes the reduction of lipid peroxides and is a target of ferroptosis inducers, such as erastin. The α6ß4 integrin protects adherent epithelial and carcinoma cells from ferroptosis induced by erastin. In addition, extracellular matrix (ECM) detachment is a physiologic trigger of ferroptosis, which is evaded by α6ß4. The mechanism that enables α6ß4 to evade ferroptosis involves its ability to protect changes in membrane lipids that are proferroptotic. Specifically, α6ß4-mediated activation of Src and STAT3 suppresses expression of ACSL4, an enzyme that enriches membranes with long polyunsaturated fatty acids and is required for ferroptosis. Adherent cells lacking α6ß4 require an inducer, such as erastin, to undergo ferroptosis because they sustain GPX4 expression, despite their increase in ACSL4. In contrast, ECM detachment of cells lacking α6ß4 is sufficient to trigger ferroptosis because GPX4 is suppressed. This causal link between α6ß4 and ferroptosis has implications for cancer biology and therapy.
Assuntos
Coenzima A Ligases/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Glutationa Peroxidase/genética , Integrina alfa6beta4/genética , Quinases da Família src/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Coenzima A Ligases/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Feminino , Glutationa Peroxidase/antagonistas & inibidores , Glutationa Peroxidase/metabolismo , Humanos , Integrina alfa6beta4/metabolismo , Ferro/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Piperazinas/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound α and ß subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin α3ß1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of α6 containing integrins (α6ß1 and α6ß4), the other major laminin receptors, is unknown. Patients with mutations in α6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the α6 or ß4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin α6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that α6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa.
Assuntos
Membrana Basal/metabolismo , Integrina alfa6beta1/genética , Integrina alfa6beta4/genética , Túbulos Renais Coletores/metabolismo , Laminina/genética , Obstrução Ureteral/metabolismo , Animais , Apoptose , Membrana Basal/patologia , Adesão Celular , Movimento Celular , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose , Regulação da Expressão Gênica , Humanos , Integrina alfa6beta1/deficiência , Integrina alfa6beta4/deficiência , Túbulos Renais Coletores/patologia , Laminina/metabolismo , Camundongos , Camundongos Knockout , Ligação Proteica , Transdução de Sinais , Ureter/cirurgia , Obstrução Ureteral/patologia , Obstrução Ureteral/cirurgiaRESUMO
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 α3ß1 and α6ß4 in tumor development and progression, and examine the factors and mechanisms involved in these opposing effects.