RESUMO
Integrin-mediated laminin adhesions mediate epithelial cell anchorage to basement membranes and are critical regulators of epithelial cell polarity. Integrins assemble large multiprotein complexes that link to the cytoskeleton and convey signals into the cells. Comprehensive proteomic analyses of actin network-linked focal adhesions (FA) have been performed, but the molecular composition of intermediate filament-linked hemidesmosomes (HD) remains incompletely characterized. Here we have used proximity-dependent biotin identification (BioID) technology to label and characterize the interactome of epithelia-specific ß4-integrin that, as α6ß4-heterodimer, forms the core of HDs. The analysis identified â¼150 proteins that were specifically labeled by BirA-tagged integrin-ß4. In addition to known HDs proteins, the interactome revealed proteins that may indirectly link integrin-ß4 to actin-connected protein complexes, such as FAs and dystrophin/dystroglycan complexes. The specificity of the screening approach was validated by confirming the HD localization of two candidate ß4-interacting proteins, utrophin (UTRN) and ELKS/Rab6-interacting/CAST family member 1 (ERC1). Interestingly, although establishment of functional HDs depends on the formation of α6ß4-heterodimers, the assembly of ß4-interactome was not strictly dependent on α6-integrin expression. Our survey to the HD interactome sets a precedent for future studies and provides novel insight into the mechanisms of HD assembly and function of the ß4-integrin.
Assuntos
Integrina beta4/química , Integrina beta4/metabolismo , Proteômica/métodos , Animais , Biotinilação , Cromatografia Líquida , Cães , Células Madin Darby de Rim Canino , Mapas de Interação de Proteínas , Multimerização Proteica , Espectrometria de Massas em TandemRESUMO
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
12-Lipoxygenase (12-LOX) metabolizes arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, or 12(S)-HETE, a proinflammatory bioactive lipid implicated in tumor angiogenesis, growth, and metastasis. The mechanisms underlying 12-LOX-mediated signaling in cancer progression are still ill-defined. In the present study we demonstrate that 12-LOX phosphorylation and subsequent enzymatic activity occurs after integrin ß4 stimulation and Src kinase recruitment to the integrin subunit. Inhibition of Src activity by PP2 or Src dominant-negative mutants reduced 12-LOX tyrosine phosphorylation and 12(S)-HETE production in response to integrin ß4 stimulation in A431 cells. The pertinent Src-targeted residues for 12-LOX activity were mapped to Y19 and Y614, where 12-LOX mutants Y19F and Y614F showed 70% less enzymatic activity. Furthermore, we have shown that the 12-LOX activity modulated by these residues impacts migration. To our knowledge, this is the first report that c-Src kinase activity is required for ß4-integrin-mediated phosphorylation of 12-LOX.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Araquidonato 12-Lipoxigenase/metabolismo , Movimento Celular , Integrina beta4/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Integrina beta4/químicaRESUMO
Integrin α6ß4 is a major component of hemidesmosomes that mediate the stable anchorage of epithelial cells to the underlying basement membrane. Integrin α6ß4 has also been implicated in cell proliferation and migration and in carcinoma progression. The third and fourth fibronectin type III domains (FnIII-3,4) of integrin ß4 mediate binding to the hemidesmosomal proteins BPAG1e and BPAG2, and participate in signalling. Here, it is demonstrated that X-ray crystallography, small-angle X-ray scattering and double electron-electron resonance (DEER) complement each other to solve the structure of the FnIII-3,4 region. The crystal structures of the individual FnIII-3 and FnIII-4 domains were solved and the relative arrangement of the FnIII domains was elucidated by combining DEER with site-directed spin labelling. Multiple structures of the interdomain linker were modelled by Monte Carlo methods complying with DEER constraints, and the final structures were selected against experimental scattering data. FnIII-3,4 has a compact and cambered flat structure with an evolutionary conserved surface that is likely to correspond to a protein-interaction site. Finally, this hybrid method is of general application for the study of other macromolecules and complexes.
Assuntos
Fibronectinas/química , Integrina beta4/química , Sequência de Aminoácidos , Cristalografia por Raios X , Fibronectinas/metabolismo , Humanos , Integrina beta4/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios XRESUMO
BACKGROUND: Integrins and enzymes of the eicosanoid pathway are both well-established contributors to cancer. However, this is the first report of the interdependence of the two signaling systems. In a screen for proteins that interacted with, and thereby potentially regulated, the human platelet-type 12-lipoxygenase (12-LOX, ALOX12), we identified the integrin ß4 (ITGB4). METHODS: Using a cultured mammalian cell model, we have demonstrated that ITGB4 stimulation leads to recruitment of 12-LOX from the cytosol to the membrane where it physically interacts with the integrin to become enzymatically active to produce 12(S)-HETE, a known bioactive lipid metabolite that regulates numerous cancer phenotypes. RESULTS: The net effect of the interaction was the prevention of cell death in response to starvation. Additionally, regulation of ß4-mediated, EGF-stimulated invasion was shown to be dependent on 12-LOX, and downstream Erk signaling in response to ITGB4 activation also required 12-LOX. CONCLUSIONS: This is the first report of an enzyme of the eicosanoid pathway being recruited to and regulated by activated ß4 integrin. Integrin ß4 has recently been shown to induce expansion of prostate tumor progenitors and there is a strong correlation between stage/grade of prostate cancer and 12-LOX expression. The 12-LOX enzymatic product, 12(S)-HETE, regulates angiogenesis and cell migration in many cancer types. Therefore, disruption of integrin ß4-12LOX interaction could reduce the pro-inflammatory oncogenic activity of 12-LOX. This report on the consequences of 12-LOX and ITGB4 interaction sets a precedent for the linkage of integrin and eicosanoid biology through direct protein-protein association.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Eicosanoides/metabolismo , Integrina beta4/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células CHO , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cromatografia Líquida , Cricetinae , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Integrina beta4/química , Inibidores de Lipoxigenase/farmacologia , Espectrometria de Massas , Invasividade Neoplásica , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacosRESUMO
Targeted drugs are less toxic than traditional chemotherapeutic therapies; however, the proportion of patients that benefit from these drugs is often smaller. A marker that confidently predicts patient response to a specific therapy would allow an individual therapy selection most likely to benefit the patient. Here, we used quantitative mass spectrometry to globally profile the basal phosphoproteome of a panel of non-small cell lung cancer cell lines. The effect of the kinase inhibitor dasatinib on cellular growth was tested against the same panel. From the phosphoproteome profiles, we identified 58 phosphorylation sites, which consistently differ between sensitive and resistant cell lines. Many of the corresponding proteins are involved in cell adhesion and cytoskeleton organization. We showed that a signature of only 12 phosphorylation sites is sufficient to accurately predict dasatinib sensitivity. Four of the phosphorylation sites belong to integrin ß4, a protein that mediates cell-matrix or cell-cell adhesion. The signature was validated in cross-validation and label switch experiments and in six independently profiled breast cancer cell lines. The study supports that the phosphorylation of integrin ß4, as well as eight further proteins comprising the signature, are candidate biomarkers for predicting response to dasatinib in solid tumors. Furthermore, our results show that identifying predictive phosphorylation signatures from global, quantitative phosphoproteomic data is possible and can open a new path to discovering molecular markers for response prediction.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Fosfoproteínas/análise , Pirimidinas/farmacologia , Tiazóis/farmacologia , Biomarcadores Tumorais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dasatinibe , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Humanos , Integrina beta4/química , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Espectrometria de Massas , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteoma/análiseRESUMO
Integrin ß4 and its Y-1494 phosphorylation play an important role in cell signaling. We found a small molecule, ethyl1-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3-(4-chlorophenyl)-1H-pyrazole-5-carboxylate (ECPC), that could elevate the levels of KIT ligand (KITLG), interleukin 8 (IL-8), prostaglandin-endoperoxide synthase 2 (PTGS2) and activating transcription factor 3 (ATF3) and promote apoptosis in vascular endothelial cells (VECs) through integrin ß4. We investigated the underlying mechanism of integrin ß4 participating in this process. ECPC treatment increased the phosphorylation of Y-1494 in the integrin ß4 cytoplasmic domain via a well-known receptor tyrosine kinase, fibroblast growth factor receptor 1 (FGFR1), and integrin ß4 translocated from the cytoplasm to nucleus. With suppression of Y-1494 phosphorylation by FGF-2 or siRNA of FGFR1, ECPC failed to promote integrin ß4 nuclear translocation and could not increase the expression of KITLG, IL-8, PTGS2 or ATF3. Y-1494 phosphorylation and nuclear translocation of integrin ß4 may be important during ECPC-induced apoptosis in VECs.
Assuntos
Apoptose , Núcleo Celular/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Integrina beta4/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Motivos de Aminoácidos , Apoptose/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Integrina beta4/química , Integrina beta4/genética , Fosforilação/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Bibliotecas de Moléculas Pequenas/síntese químicaRESUMO
Migration of keratinocytes requires a regulated and dynamic turnover of hemidesmosomes (HDs). We and others have previously identified three serine residues on the integrin ß4 cytoplasmic domain that play a critical role in the regulation of HD disassembly. In this study we show that only two of these residues (Ser-1356 and Ser-1364) are phosphorylated in keratinocytes after stimulation with either PMA or EGF. Furthermore, in direct contrast to previous studies performed in vitro, we found that the PMA- and EGF-stimulated phosphorylation of ß4 is not mediated by PKC, but by ERK1/2 and its downstream effector kinase p90RSK1/2. EGF-stimulated phosphorylation of ß4 increased keratinocyte migration, and reduced the number of stable HDs. Furthermore, mutation of the two serines in ß4 to phospho-mimicking aspartic acid decreased its interaction with the cytoskeletal linker protein plectin, as well as the strength of α6ß4-mediated adhesion to laminin-332. During mitotic cell rounding, when the overall cell-substrate area is decreased and the number of HDs is reduced, ß4 was only phosphorylated on Ser-1356 by a distinct, yet unidentified, kinase. Collectively, these data demonstrate an important role of ß4 phosphorylation on residues Ser-1356 and Ser-1364 in the formation and/or stability of HDs.
Assuntos
Regulação para Baixo , Fator de Crescimento Epidérmico/metabolismo , Hemidesmossomos/metabolismo , Integrina beta4/metabolismo , Sistema de Sinalização das MAP Quinases , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Ciclo Celular , Linhagem Celular , Chlorocebus aethiops , Hemidesmossomos/enzimologia , Integrina beta4/química , Integrina beta4/genética , Queratinócitos/citologia , Queratinócitos/enzimologia , Queratinócitos/metabolismo , Camundongos , Dados de Sequência Molecular , FosforilaçãoRESUMO
Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the basement membrane. The binding of alpha6beta4 to plectin plays a central role in their assembly. We have defined three regions on beta4 that together harbor all the serine and threonine phosphorylation sites and show that three serines (S1356, S1360, and S1364), previously implicated in HD regulation, prevent the interaction of beta4 with the plectin actin-binding domain when phosphorylated. We have also established that epidermal growth factor receptor activation, which is known to function upstream of HD disassembly, results in the phosphorylation of only one or more of these three residues and the partial disassembly of HDs in keratinocytes. Additionally, we show that S1360 and S1364 of beta4 are the only residues phosphorylated by PKC and PKA in cells, respectively. Taken together, our studies indicate that multiple kinases act in concert to breakdown the structural integrity of HDs in keratinocytes, which is primarily achieved through the phosphorylation of S1356, S1360, and S1364 on the beta4 subunit.
Assuntos
Receptores ErbB/metabolismo , Hemidesmossomos/metabolismo , Integrina beta4/metabolismo , Fosfosserina/metabolismo , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Hemidesmossomos/efeitos dos fármacos , Humanos , Integrina beta4/química , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Plectina/química , Plectina/metabolismo , Proteína Quinase C/metabolismo , Estrutura Terciária de ProteínaRESUMO
The integrin alpha6beta4 is a receptor for laminins and provides stable adhesion of epithelial cells to the basement membranes. In addition, alpha6beta4 is important for keratinocyte migration during wound healing and favours the invasion of carcinomas into surrounding tissue. The cytoplasmic domain of the beta4 subunit is responsible for most of the intracellular interactions of the integrin; it contains four fibronectin type III domains and a Calx-beta motif. The crystal structure of the Calx-beta domain of beta4 was determined to 1.48 A resolution. The structure does not contain cations and biophysical data support the supposition that the Calx-beta domain of beta4 does not bind calcium. Comparison of the Calx-beta domain of beta4 with the calcium-binding domains of Na(+)/Ca(2+)-exchanger 1 reveals that in beta4 Arg1003 occupies a position equivalent to that of the calcium ions in the Na(+)/Ca(2+)-exchanger. By combining mutagenesis and thermally induced unfolding, it is shown that Arg1003 contributes to the stability of the Calx-beta domain. The structure of the Calx-beta domain is discussed in the context of the function and intracellular interactions of the integrin beta4 subunit and a putative functional site is proposed.
Assuntos
Integrina beta4/química , Proteínas Mutantes/química , Arginina/química , Arginina/metabolismo , Cálcio , Cátions , Adesão Celular , Cristalização , Cristalografia por Raios X , Humanos , Integrina beta4/genética , Integrina beta4/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Trocador de Sódio e Cálcio/química , Relação Estrutura-AtividadeRESUMO
Endogenous DC electric fields (EF) are present during embryogenesis and are generated in vivo upon wounding, providing guidance cues for directional cell migration (galvanotaxis) required in these processes. To understand the role of beta (beta)4 integrin in directional migration, the migratory paths of either primary human keratinocytes (NHK), beta4 integrin-null human keratinocytes (beta4-), or those in which beta4 integrin was reexpressed (beta4+), were tracked during exposure to EFs of physiological magnitude (100 mV/mm). Although the expression of beta4 integrin had no effect on the rate of cell movement, it was essential for directional (cathodal) migration in the absence of epidermal growth factor (EGF). The addition of EGF potentiated the directional response, suggesting that at least two distinct but synergistic signaling pathways coordinate galvanotaxis. Expression of either a ligand binding-defective beta4 (beta4+AD) or beta4 with a truncated cytoplasmic tail (beta4+CT) resulted in loss of directionality in the absence of EGF, whereas inhibition of Rac1 blinded the cells to the EF even in the presence of EGF. In summary, both the beta4 integrin ligand-binding and cytoplasmic domains together with EGF were required for the synergistic activation of a Rac-dependent signaling pathway that was essential for keratinocyte directional migration in response to a galvanotactic stimulus.
Assuntos
Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Condutividade Elétrica , Fator de Crescimento Epidérmico/farmacologia , Integrina beta4/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células Cultivadas , Citoplasma/efeitos dos fármacos , Adesões Focais/efeitos dos fármacos , Humanos , Integrina beta4/química , Queratinócitos/efeitos dos fármacos , Laminina/metabolismo , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Pseudópodes/efeitos dos fármacosRESUMO
Aging is characterized by a progressive loss of physiological integrity, leading to impaired organ function and, ultimately, increased vulnerability to death. Many complex diseases are related to aging, including asthma. In the lung, the airway epithelium serves as the first barrier to prevent the access of inspired external stimuli and dictates the initial stress responses. Notably, in the airway mucosa of asthma patients, an increase in senescent airway epithelial cells has been detected. Although it has been speculated that the senescence of airway epithelial cells could increase asthma susceptibility and aggravate asthma severity, the role of cell senescence in the development of asthma remains unclear. Integrin ß4 (ITGB4) is a structural adhesion molecule with complex physiological functions that is downregulated in airway epithelial cells of asthma patients. This study demonstrates that the expression of ITGB4 in airway epithelial cells is downregulated significantly under oxidative stress or upon inflammatory stimulation. Moreover, we show that ITGB4 deficiency induces the senescence of airway epithelial cells through the activation of the p53 pathway both in vitro and in vivo. Together, our results demonstrate that airway epithelial senescence induced by ITGB4 deficiency after oxidative stress or inflammatory stimulation may be involved in the pathogenesis of asthma. Understanding the contribution of ITGB4 deficiency to the senescence of airway epithelial cells in asthma patients may provide new therapeutic approaches for the treatment of asthma.
Assuntos
Asma/etiologia , Senescência Celular , Células Epiteliais/patologia , Integrina beta4/metabolismo , Mucosa Respiratória/patologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Asma/patologia , Proliferação de Células , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , Integrina beta4/química , Integrina beta4/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Respiratória/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin stress fiber mediated nuclear pore opening, however the protein mediators of YAP translocation remain unclear. Here, we show that YAP co-localizes with F-actin during activating conditions, such as sparse plating and culturing on stiff 2D substrates. To identify proteins mediating YAP translocation, we performed co-immunoprecipitation followed by mass spectrometry (co-IP/MS) for proteins that differentially associated with YAP under activating conditions. Interestingly, YAP preferentially associates with ß1 integrin under activating conditions, and ß4 integrin under inactivating conditions. In activating conditions, CRISPR/Cas9 knockout (KO) of ß1 integrin (ΔITGB1) resulted in decreased cell area, which correlated with decreased YAP nuclear localization. ΔITGB1 did not significantly affect the slope of the correlation between YAP nuclear localization with area, but did decrease overall nuclear YAP independently of cell spreading. In contrast, ß4 integrin KO (ΔITGB4) cells showed no change in cell area and similarly decreased nuclear YAP. These results reveal proteins that differentially associate with YAP during activation, which may aid in regulating YAP nuclear translocation.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Matriz Extracelular/metabolismo , Integrina beta1/metabolismo , Integrina beta4/metabolismo , Mecanotransdução Celular , Fatores de Transcrição/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Células Cultivadas , Humanos , Integrina beta1/química , Integrina beta1/genética , Integrina beta4/química , Integrina beta4/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, ß4-integrin. Co-precipitation assays revealed that the central region (amino acids 330-1057) of Solo binds to the C-terminal region (1451-1752) of ß4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization.
Assuntos
Células Acinares/metabolismo , Células Epiteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hemidesmossomos/metabolismo , Animais , Linhagem Celular , Expressão Gênica , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Integrina beta4/química , Integrina beta4/metabolismo , Queratina-18/genética , Queratina-18/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de SinaisRESUMO
Integrin contributes to the maintenance of cell adhesion. In turn, cell adhesion triggers certain integrin signaling cascades, and influences cell biological behavior. In the present study, we explored the role and mechanism of integrin ß4 in the DNA damage response in colorectal cancer (CRC) using a threedimensional (3D) cell culture model. Under 3D culture condition, dispersed CRC cells automatically formed multicellular spheroids, which consisted of layers of cells with cell junctions commonly distributed. The expression level of integrin ß4 in HCT116 3D cultures was slightly higher compared with twodimensional (2D) cultures, while the expression level in LoVo 3D cultures was similar to or slightly lower than that in 2D cultures. Knockdown of integrin ß4 by lentiviral delivery of shRNA did not markedly change the architectural formation of 3D cultures under an inverted microscope or transmission electron microscope. Platinum increased p53 and pp53 (ser15) in a timedependent manner in 3D cultures. Knockdown of integrin ß4 increased sensitivity to cisplatin (CDDP) in 3D cultures. Under 3D culture condition, knockdown of integrin ß4 did not detectably change the basal p53 protein level but increased p53 and pp53 (ser15) protein accumulation induced by platinum. Integrin ß4 knockdown did not detectably change p53 protein level in HCT116 2D cultures with or without CDDP treatment. Knockdown of wildtype p53 decreased sensitivity to platinum in 3D cultures. Since it has been proven that platinum damages DNA to kill cells and p53 plays a key role in the DNA damage response, our results indicated that integrin ß4 reduced DNA damageinduced p53 activation to decrease chemosensitivity in CRC. This may be due to integrin ß4 activation in 3D cultures.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Integrina beta4/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Técnicas de Cultura de Células , Proliferação de Células , Cisplatino/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Humanos , Integrina beta4/química , Integrina beta4/genética , RNA Interferente Pequeno , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Although the regulation of hemidesmosome dynamics during processes such as epithelial migration, wound healing, and carcinoma invasion is important, the mechanisms involved are poorly understood. The integrin alpha 6 beta 4 is an essential component of the hemidesmosome and a target of such regulation. Epidermal growth factor (EGF) can induce hemidesmosome disassembly by a mechanism that involves serine phosphorylation of the beta 4 integrin subunit. Using a combination of biochemical and mutational analyses, we demonstrate that EGF induces the phosphorylation of three specific serine residues (S(1356), S(1360), and S(1364)) located within the connecting segment of the beta 4 subunit and that phosphorylation on these residues accounts for the bulk of beta 4 phosphorylation stimulated by EGF. Importantly, phosphorylation of these serines is critical for the ability of EGF to disrupt hemidesmosomes. Using COS-7 cells, which assemble hemidesmosomes type II upon exogenous expression of the alpha 6 beta 4 integrin, we observed that expression of a beta 4 construct containing Ser-->Ala mutations of S(1356), S(1360), and S(1364) reduced the ability of EGF to disrupt hemidesmosomes and that this effect appears to involve cooperation among these phosphorylation sites. Moreover, expression of Ser-->Asp mutants that mimic constitutive phosphorylation reduced hemidesmosome formation. Protein kinase C-alpha (PKC-alpha) is the kinase responsible for phosphorylating at least two of these serines, based on in vitro kinase assays, peptide mapping, and mutational analysis. Together, these results highlight the importance of serine phosphorylation in regulating type II hemidesmosome disassembly, implicate a cluster of serine residues within the connecting segment of beta 4, and argue for a key role for PKC-alpha in regulating these structures.
Assuntos
Hemidesmossomos/metabolismo , Integrina beta4/química , Integrina beta4/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Humanos , Integrina beta4/genética , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Fosforilação , Proteína Quinase C-alfa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Serina/química , TransfecçãoRESUMO
Integrin beta4 is a transmembrane protein expressed predominantly on epithelial cells. In human epidermis integrin beta4 associates with integrin beta6. Integrin alpha6beta4 is concentrated at the basement membrane zone, where it localizes to specialized adhesion structures called hemidesmosomes. In addition to its adhesive functions, keratinocyte integrin beta4 has been identified as an important regulator of epidermal homeostasis. This review summarizes the current knowledge regarding the role of integrin beta4 in keratinocyte adhesion, migration, as well as growth and differentiation. Changes in integrin beta4 expression in pathological conditions in skin and mucosa, especially those associated with human papillomavirus infection, are described.
Assuntos
Integrina beta4/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Papillomaviridae/fisiologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Animais , Desmossomos/metabolismo , Humanos , Integrina beta4/química , Integrina beta4/genética , Infecções por Papillomavirus/patologiaRESUMO
The mechanical stability of epithelial cells, which protect organisms from harmful external factors, is maintained by hemidesmosomes via the interaction between plectin 1a (P1a) and integrin α6ß4. Binding of calcium-calmodulin (Ca(2+)-CaM) to P1a together with phosphorylation of integrin ß4 disrupts this complex, resulting in disassembly of hemidesmosomes. We present structures of the P1a actin binding domain either in complex with the N-ter lobe of Ca(2+)-CaM or with the first pair of integrin ß4 fibronectin domains. Ca(2+)-CaM binds to the N-ter isoform-specific tail of P1a in a unique manner, via its N-ter lobe in an extended conformation. Structural, cell biology, and biochemical studies suggest the following model: binding of Ca(2+)-CaM to an intrinsically disordered N-ter segment of plectin converts it to an α helix, which repositions calmodulin to displace integrin ß4 by steric repulsion. This model could serve as a blueprint for studies aimed at understanding how Ca(2+)-CaM or EF-hand motifs regulate F-actin-based cytoskeleton.
Assuntos
Calmodulina/química , Hemidesmossomos/química , Integrina beta4/química , Plectina/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , RatosRESUMO
Simvastatin, an HMG-CoA reductase inhibitor, has lung vascular-protective effects that are associated with decreased agonist-induced integrin ß4 (ITGB4) tyrosine phosphorylation. Accordingly, we hypothesized that endothelial cell (EC) protection by simvastatin is dependent on these effects and sought to further characterize the functional role of ITGB4 as a mediator of EC protection in the setting of excessive mechanical stretch at levels relevant to ventilator-induced lung injury (VILI). Initially, early ITGB4 tyrosine phosphorylation was confirmed in human pulmonary artery EC subjected to excessive cyclic stretch (18% CS). EC overexpression of mutant ITGB4 with specific tyrosines mutated to phenylalanine (Y1440, Y1526 Y1640, or Y1422) resulted in significantly attenuated CS-induced cytokine expression (IL6, IL-8, MCP-1, and RANTES). In addition, EC overexpression of ITGB4 constructs with specific structural deletions also resulted in significantly attenuated CS-induced inflammatory cytokine expression compared to overexpression of wildtype ITGB4. Finally, mice expressing a mutant ITGB4 lacking a cytoplasmic signaling domain were found to have attenuated lung injury after VILI-challenge (VT = 40 ml/kg, 4 h). Our results provide mechanistic insights into the anti-inflammatory properties of statins and may ultimately lead to novel strategies targeted at ITGB4 signaling to treat VILI.
Assuntos
Integrina beta4/metabolismo , Estresse Mecânico , Animais , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Inflamação/prevenção & controle , Integrina beta4/química , Integrina beta4/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Permeabilidade/efeitos dos fármacos , Fosforilação , Estrutura Terciária de Proteína , Artéria Pulmonar/citologia , Sinvastatina/farmacologia , Sinvastatina/uso terapêutico , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controleRESUMO
Integrin alpha6beta4 is a hemidesmosomal transmembrane molecule involved in maintaining basal cell-matrix adhesion through interaction of the large intracytoplasmic tail of the beta4 subunit with the keratin intermediate filament network, at least in part through its binding with plectin and BP180/type XVII collagen. Here we report a patient with predominant features of epidermolysis bullosa simplex due to a mutation in the integrin beta4 gene. The patient, a 49-y-old female, had mild blistering of hands and feet from birth on, dystrophy of the nails with onychogryposis, and enamel hypoplasia. She had no alopecia and no history of pyloric atresia. Electron microscopy and antigen mapping of a skin blister revealed that the level of separation was intraepidermal, low in the basal keratinocytes through the attachment plaque of the hemidesmosome. Immuno-fluorescence microscopy revealed absent binding of monoclonal antibody 450-11 A against the third fibronectin III repeat on the intracellular domain of integrin beta4, whereas binding was reduced with monoclonal antibodies recognizing epitopes on amino-terminal and carboxy-terminal ends of the polypeptide. At the molecular level the phenotype was caused by a novel 2 bp deletion 4733delCT in ITGB4, resulting in in-frame skipping of exon 36 and a deduced 50 amino acid deletion (1450-1499) within the third fibronectin type III repeat in the cytoplasmic domain of the integrin beta4 polypeptide. Immunoblot analysis demonstrated a 5 kDa shorter beta4 polypeptide. The 4733delCT mutation was heterozygously present in the DNA. The patient is also expected to be heterozygous for a null allele, as no full-size protein was detected in vitro and the epitope 450-11 A was absent in vivo. These data show that deletion of the third fibronectin type III repeat in the cytoplasmic domain of integrin beta4, which is thought to interact with BP180/type XVII collagen, is clinically pathogenic and results in a mild phenotype with predominant features of epidermolysis bullosa simplex.