RESUMO
Talin (herein referring collectively to talin 1 and 2) couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix. Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades. Here, we demonstrate that the scaffold protein Caskin2 interacts directly with the R8 domain of talin through its C-terminal LD motif. Caskin2 also associates with the WAVE regulatory complex to promote cell migration in an Abi1-dependent manner. Furthermore, we demonstrate that the Caskin2-Abi1 interaction is regulated by growth factor-induced phosphorylation of Caskin2 on serine 878. In MCF7 and UACC893 cells, which contain an amplification of CASKIN2, Caskin2 localizes in plasma membrane-associated plaques and around focal adhesions in cortical microtubule stabilization complexes. Taken together, our results identify Caskin2 as a novel talin-binding protein that might not only connect integrin-mediated adhesion to actin polymerization but could also play a role in crosstalk between integrins and microtubules.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Movimento Celular , Proteínas do Citoesqueleto , Ligação Proteica , Talina , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Adesões Focais/metabolismo , Integrinas/metabolismo , Células MCF-7 , Microtúbulos/metabolismo , Fosforilação , Talina/metabolismoRESUMO
The vitronectin receptor integrin αVß5 can reside in two distinct adhesion structures - focal adhesions (FAs) and flat clathrin lattices (FCLs). Here, we investigate the mechanism that regulates the subcellular distribution of ß5 in keratinocytes and show that ß5 has approximately 7- and 5-fold higher affinity for the clathrin adaptors ARH (also known as LDLRAP1) and Numb, respectively, than for the talin 1 (TLN1); all proteins that bind to the membrane-proximal NPxY motif of the ß5 cytoplasmic domain. Using mass spectrometry, we identified ß5 interactors, including the Rho GEFs p115Rho-GEF and GEF-H1 (also known as ARHGEF1 and ARHGEF2, respectively), and the serine protein kinase MARK2, depletion of which diminishes the clustering of ß5 in FCLs. Replacement of two serine residues (S759 and S762) in the ß5 cytoplasmic domain with phospho-mimetic glutamate residues causes a shift in the localization of ß5 from FAs into FCLs without affecting the interactions with MARK2, p115Rho-GEF or GEF-H1. Instead, we demonstrate that changes in the actomyosin-based cellular contractility by ectopic expression of activated Rho or disruption of microtubules regulates ß5 localization. Finally, we present evidence that ß5 in either FAs or FCLs functions to promote adhesion to vitronectin, cell spreading, and proliferation.
Assuntos
Clatrina , Receptores de Vitronectina , Adesão Celular/fisiologia , Proliferação de Células , Clatrina/metabolismo , Adesões Focais/metabolismo , Receptores de Vitronectina/metabolismo , Serina/metabolismoRESUMO
Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.
Assuntos
Reparo do DNA , Neoplasias , Animais , Cisplatino/uso terapêutico , Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Medicina de Precisão , Antígeno Nuclear de Célula em Proliferação/metabolismo , UbiquitinaçãoRESUMO
Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype. We created a Fancg-KO (KO) mouse model using CRISPR/Cas9 to exclude these confounders. The entire Fancg locus was targeted and maintained on the immunological well-characterized C57BL/6J background. The intercrossing of heterozygous mice resulted in sub-Mendelian numbers of homozygous mice, suggesting the loss of FANCG can be embryonically lethal. KO mice displayed infertility and hypogonadism, but no other developmental problems. Bone marrow analysis revealed a defect in various hematopoietic stem and progenitor subsets with a bias towards myelopoiesis. Cell lines derived from Fancg-KO mice were hypersensitive to the crosslinking agents cisplatin and Mitomycin C, and Fancg-KO mouse embryonic fibroblasts (MEFs) displayed increased γ-H2AX upon cisplatin treatment. The reconstitution of these MEFs with Fancg cDNA corrected for the ICL hypersensitivity. This project provides a new, genetically, and immunologically well-defined Fancg-KO mouse model for further in vivo and in vitro studies on FANCG and ICL repair.
Assuntos
Cisplatino , Anemia de Fanconi , Humanos , Animais , Camundongos , Cisplatino/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Camundongos Endogâmicos C57BL , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Mitomicina , Fenótipo , Proteína do Grupo de Complementação G da Anemia de Fanconi/genéticaRESUMO
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.
Assuntos
Junções Célula-Matriz/metabolismo , Integrina alfa3beta1/metabolismo , Integrina alfa6beta4/metabolismo , Tetraspanina 24/metabolismo , Western Blotting , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Hemidesmossomos/metabolismo , Humanos , Imunoprecipitação , Integrina alfa3beta1/química , Integrina alfa6beta4/química , Queratinócitos/metabolismo , Plectina/metabolismo , Tetraspanina 24/químicaRESUMO
The family of integrin transmembrane receptors is essential for the normal function of multicellular organisms by facilitating cell-extracellular matrix adhesion. The vitronectin-binding integrin αVß5 localizes to focal adhesions (FAs) as well as poorly characterized flat clathrin lattices (FCLs). Here, we show that, in human keratinocytes, αVß5 is predominantly found in FCLs, and formation of the αVß5-containing FCLs requires the presence of vitronectin as ligand, Ca2+, and the clathrin adaptor proteins ARH (also known as LDLRAP1), Numb and EPS15/EPS15L1. Integrin chimeras, containing the extracellular and transmembrane domains of ß5 and the cytoplasmic domains of ß1 or ß3, almost exclusively localize in FAs. Interestingly, lowering actomyosin-mediated contractility promotes integrin redistribution to FLCs in an integrin tail-dependent manner, while increasing cellular tension favors αVß5 clustering in FAs. Our findings strongly indicate that clustering of integrin αVß5 in FCLs is dictated by the ß5 subunit cytoplasmic domain, cellular tension and recruitment of specific adaptor proteins to the ß5 subunit cytoplasmic domains.
Assuntos
Clatrina/metabolismo , Receptores de Vitronectina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cálcio/metabolismo , Células Cultivadas , Adesões Focais/metabolismo , Humanos , Queratinócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vitronectina/metabolismoRESUMO
BACKGROUND: HER2-driven breast cancer is correlated with poor prognosis, especially during its later stages. Numerous studies have shown the importance of the integrin α3ß1 during the initiation and progression of breast cancer; however, its role in this disease is complex and often opposite during different stages and in different types of tumors. In this study, we aim to elucidate the role of integrin α3ß1 in a genetically engineered mouse model of HER2-driven mammary tumorigenesis. METHODS: To investigate the role of α3ß1 in HER2-driven tumorigenesis in vivo, we generated a HER2-driven MMTV-cNeu mouse model of mammary tumorigenesis with targeted deletion of Itga3 (Itga3 KO mice). We have further used several established triple-negative and HER2-overexpressing human mammary carcinoma cell lines and generated ITGA3-knockout cells to investigate the role of α3ß1 in vitro. Invasion of cells was assessed using Matrigel- and Matrigel/collagen I-coated Transwell assays under static or interstitial fluid flow conditions. The role of α3ß1 in initial adhesion to laminin and collagen was assessed using adhesion assays and immunofluorescence. RESULTS: Tumor onset in mice was independent of the presence of α3ß1. In contrast, the depletion of α3ß1 reduced the survival of mice and increased tumor growth and vascularization. Furthermore, Itga3 KO mice were significantly more likely to develop lung metastases and had an increased metastatic burden compared to WT mice. In vitro, the deletion of ITGA3 caused a significant increase in the cellular invasion of HER2-overexpressing SKBR3, AU565, and BT474 cells, but not of triple-negative MDA-MB-231. This invasion suppressing function of α3ß1 in HER2-driven cells depended on the composition of the extracellular matrix and the interstitial fluid flow. CONCLUSION: Downregulation of α3ß1 in a HER2-driven mouse model and in HER2-overexpressing human mammary carcinoma cells promotes progression and invasiveness of tumors. The invasion-suppressive role of α3ß1 was not observed in triple-negative mammary carcinoma cells, illustrating the tumor type-specific and complex function of α3ß1 in breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Integrina alfa3beta1/deficiência , Receptor ErbB-2/genética , Animais , Biomarcadores Tumorais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunofenotipagem , Estimativa de Kaplan-Meier , Camundongos , Camundongos Knockout , Metástase Neoplásica , Receptor ErbB-2/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Hemidesmosomes have been extensively studied with immunofluorescence microscopy, but owing to its limited resolution, the precise organization of hemidesmosomes remains poorly understood. We studied hemidesmosome organization in cultured keratinocytes with two- and three-color super-resolution microscopy. We observed that, in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that ß4 integrin (also known as ITGB4) is distributed along, rather than under, keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with ß4 integrin and keratin. Furthermore, we show that BP180 (BPAG2, also known as collagen XVII) and BP230 (BPAG1e, an epithelial splice variant of dystonin) are characteristically arranged within hemidesmosomes with BP180 surrounding a central core of BP230 molecules. In skin cross-sections, hemidesmosomes of variable sizes could be distinguished with BP230 and plectin occupying a position in between ß4 integrin and BP180, and the intermediate filament system. In conclusion, our data provide a detailed view of the molecular architecture of hemidesmosomes in cultured keratinocytes and skin.
Assuntos
Autoantígenos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Hemidesmossomos/metabolismo , Integrina beta4/metabolismo , Queratinócitos/metabolismo , Queratinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Colágenos não Fibrilares/metabolismo , Pele/metabolismo , Autoantígenos/genética , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Distonina , Hemidesmossomos/genética , Hemidesmossomos/ultraestrutura , Humanos , Integrina beta4/genética , Queratinócitos/ultraestrutura , Queratinas/genética , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Colágenos não Fibrilares/genética , Pele/ultraestrutura , Colágeno Tipo XVIIRESUMO
In the functionally differentiated mammary gland, basal myoepithelial cells contract to eject the milk produced by luminal epithelial cells from the body. We report that conditional deletion of a laminin receptor, α3ß1 integrin, from myoepithelial cells leads to low rates of milk ejection due to a contractility defect but does not interfere with the integrity or functional differentiation of the mammary epithelium. In lactating mammary gland, in the absence of α3ß1, focal adhesion kinase phosphorylation is impaired, the Rho/Rac balance is altered and myosin light-chain (MLC) phosphorylation is sustained. Cultured mammary myoepithelial cells depleted of α3ß1 contract in response to oxytocin, but are unable to maintain the state of post-contractile relaxation. The expression of constitutively active Rac or its effector p21-activated kinase (PAK), or treatment with MLC kinase (MLCK) inhibitor, rescues the relaxation capacity of mutant cells, strongly suggesting that α3ß1-mediated stimulation of the Rac/PAK pathway is required for the inhibition of MLCK activity, permitting completion of the myoepithelial cell contraction/relaxation cycle and successful lactation. This is the first study highlighting the impact of α3ß1 integrin signalling on mammary gland function.
Assuntos
Epitélio/fisiologia , Integrina alfa3beta1/metabolismo , Glândulas Mamárias Animais/citologia , Células Musculares/fisiologia , Animais , Células Cultivadas , Deleção de Genes , Integrina alfa3beta1/genética , Camundongos , Contração Muscular , Relaxamento MuscularRESUMO
Progression through the various stages of skin tumorigenesis is correlated with an altered expression of the integrin α3ß1, suggesting that it plays an important role in the tumorigenic process. Using epidermis-specific Itga3 KO mice subjected to the 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis protocol, we demonstrate that efficient tumor development is critically dependent on the presence of α3ß1. In the absence of α3ß1, tumor initiation is dramatically decreased because of increased epidermal turnover, leading to a loss of DMBA-initiated label-retaining keratinocytes. Lineage tracing revealed emigration of α3-deficient keratinocytes residing in the bulge of the hair follicle toward the interfollicular epidermis. Furthermore, tumor growth and cell proliferation were strongly reduced in mice with an epidermis-specific deletion of Itga3. However, the rate of progression of α3ß1-null squamous cell carcinomas to undifferentiated, invasive carcinomas was increased. Therefore, α3ß1 critically affects skin carcinogenesis with opposing effects early and late in tumorigenesis.
Assuntos
Ciclo Celular , Transformação Celular Neoplásica/patologia , Epiderme/metabolismo , Epiderme/patologia , Integrina alfa3/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/prevenção & controle , 9,10-Dimetil-1,2-benzantraceno , Animais , Adesão Celular , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Progressão da Doença , Folículo Piloso/metabolismo , Folículo Piloso/patologia , Queratina-15/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Cutâneas/metabolismo , Coloração e RotulagemRESUMO
Mutations in SPINK5, encoding the serine protease inhibitor LEKTI, cause Netherton syndrome, a severe autosomal recessive genodermatosis. Spink5(-/-) mice faithfully replicate key features of Netherton syndrome, including altered desquamation, impaired keratinization, hair malformation and a skin barrier defect. LEKTI deficiency causes abnormal desmosome cleavage in the upper granular layer through degradation of desmoglein 1 due to stratum corneum tryptic enzyme and stratum corneum chymotryptic enzyme-like hyperactivity. This leads to defective stratum corneum adhesion and resultant loss of skin barrier function. Profilaggrin processing is increased and implicates LEKTI in the cornification process. This work identifies LEKTI as a key regulator of epidermal protease activity and degradation of desmoglein 1 as the primary pathogenic event in Netherton syndrome.
Assuntos
Caderinas/metabolismo , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/genética , Serpinas/genética , Dermatopatias Genéticas/metabolismo , Animais , Desmogleína 1 , Epiderme/patologia , Epiderme/ultraestrutura , Calicreínas/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Inibidor de Serinopeptidase do Tipo Kazal 5 , Inibidores de Serina Proteinase/metabolismo , Serpinas/metabolismo , Dermatopatias Genéticas/patologiaRESUMO
Fanconi anemia (FA) is a rare hereditary disease resulting from an inactivating mutation in the FA/BRCA pathway, critical for the effective repair of DNA interstrand crosslinks (ICLs). The disease is characterized by congenital abnormalities, progressing bone marrow failure, and an increased risk of developing malignancies early in life, in particular head and neck squamous cell carcinoma (HNSCC). While ICL-inducing cisplatin combined with radiotherapy is a mainstay of HNSCC treatment, cisplatin is contra-indicated for FA-HNSCC patients. This dilemma necessitates the identification of novel treatment modalities tolerated by FA-HNSCC patients. To identify druggable targets, an siRNA-based genetic screen was previously performed in HNSCC-derived cell lines from FA and non-FA tumor origin. Here, we report that the Ribonucleotide Reductase (RNR) complex, consisting of the RRM1 and RRM2 subunits, was identified as a therapeutic target for both, FA and non-FA HNSCC. While non-FA HNSCC cells responded differentially to RNR depletion, FA-HNSCC cells were consistently found hypersensitive. This insight was confirmed pharmacologically using 2', 2'-difluoro 2'deoxycytidine (dFdC), also known as gemcitabine, a clinically used nucleotide analog that is a potent inhibitor of the RNR complex. Importantly, while cisplatin exposure displayed severe, long-lasting toxicity on the hematopoietic stem and progenitor compartments in Fancg-/- mice, gemcitabine was well tolerated and had only a mild, transient impact. Taken together, our data implicate that gemcitabine-based chemoradiotherapy could serve as an alternative HNSCC treatment in Fanconi patients, and deserves clinical testing.
RESUMO
Integrins mediate cell adhesion by connecting the extracellular matrix to the intracellular cytoskeleton and orchestrate signal transduction in response to chemical and mechanical stimuli by interacting with many cytoplasmic proteins. We used BioID to interrogate the interactomes of ß1 and ß3 integrins in epithelial cells and identified PEAK1 as an interactor of the RGD-binding integrins α5ß1, αVß3, and αVß5 in focal adhesions. We demonstrate that the interaction between integrins and PEAK1 occurs indirectly through Tensin3, requiring both the membrane-proximal NPxY motif on the integrin ß tail and binding of the SH2 domain of Tensin3 to phosphorylated Tyr-635 on PEAK1. Phosphorylation of Tyr-635 is mediated by Src and regulates cell migration. Additionally, we found that Shc1 localizes in focal adhesions in a PEAK1 phosphorylated Tyr-1188-dependent fashion. Besides binding Shc1, PEAK1 also associates with a protein cluster that mediates late EGFR/Shc1 signaling. We propose a model in which PEAK1 binds Tensin3 and Shc1 to converge integrin and growth factor receptor signal transduction.
Assuntos
Adesão Celular , Integrinas , Proteínas Tirosina Quinases , Tensinas , Movimento Celular , Adesões Focais/metabolismo , Humanos , Integrina beta3/metabolismo , Integrinas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Tensinas/metabolismoRESUMO
The development and differentiation of B cells is intimately linked to cell proliferation and the generation of diverse immunoglobulin gene (Ig) repertoires. The ubiquitin E3 ligase HUWE1 controls proliferation, DNA damage responses, and DNA repair, including the base excision repair (BER) pathway. These processes are of crucial importance for B-cell development in the bone marrow, and the germinal center (GC) response, which results in the clonal expansion and differentiation of B cells expressing high affinity immunoglobulins. Here, we re-examined the role of HUWE1 in B-cell proliferation and Ig gene diversification, focusing on its involvement in somatic hypermutation (SHM) and class switch recombination (CSR). B-cell-specific deletion of Huwe1 resulted in impaired development, differentiation and maturation of B cells in the bone marrow and peripheral lymphoid organs. HUWE1 deficiency diminished SHM and CSR by impairing B-cell proliferation and AID expression upon activation in vitro and in vivo, and was unrelated to the HUWE1-dependent regulation of the BER pathway. Interestingly, we found that HUWE1-deficient B cells showed increased mRNA expression of Myc target genes upon in vitro activation despite diminished proliferation. Our results confirm that the E3 ligase HUWE1 is an important contributor in coordinating the rapid transition of antigen naïve, resting B cells into antigen-activated B cells and regulates mutagenic processes in B cells by controlling AID expression and the post-transcriptional output of Myc target genes.
Assuntos
Switching de Imunoglobulina , Hipermutação Somática de Imunoglobulina , Switching de Imunoglobulina/genética , Linfócitos B , Reparo do DNA , Diferenciação Celular/genéticaRESUMO
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
The tetraspanin CD151 is a cell-surface molecule known for its strong lateral interaction with the laminin-binding integrin alpha3beta1. Patients with a nonsense mutation in CD151 display end-stage kidney failure associated with regional skin blistering and sensorineural deafness, and mice lacking the integrin alpha3 subunit die neonatally because of severe abnormalities in the lung and kidney epithelia. We report the generation of Cd151-null mice that recapitulate the renal pathology of human patients, i.e., with age they develop massive proteinuria caused by focal glomerulosclerosis, disorganization of the glomerular basement membrane, and tubular cystic dilation. However, neither skin integrity nor hearing ability are impaired in the Cd151-null mice. Furthermore, we generated podocyte-specific conditional knockout mice for the integrin alpha3 subunit that show renal defects similar to those in the Cd151 knockout mice. Our results support the hypothesis that CD151 plays a key role in strengthening alpha3beta1-mediated adhesion in podocytes.
Assuntos
Antígenos CD/genética , Insuficiência Renal/genética , Animais , Adesão Celular , Genótipo , Membrana Basal Glomerular/patologia , Integrina alfa3/genética , Integrina alfa3beta1/fisiologia , Túbulos Renais/patologia , Camundongos , Mutação , Podócitos/citologia , Tetraspanina 24RESUMO
Integrin α3ß1 plays a crucial role in tumor formation in the two-stage chemical carcinogenesis model (DMBA and TPA treatment). However, the mechanisms whereby the expression of α3ß1 influences key oncogenic drivers of this established model are not known yet. Using an in vivo mouse model with epidermal deletion of α3ß1 and in vitro Matrigel cultures of transformed keratinocytes, we demonstrate the central role of α3ß1 in promoting the activation of several protumorigenic signaling pathways during the initiation of DMBA/TPAâdriven tumorigenesis. In transformed keratinocytes, α3ß1-mediated focal adhesion kinase/Src activation leads to in vitro growth of spheroids and to strong Akt and STAT 3 activation when the α3ß1-binding partner tetraspanin CD151 is present to stabilize cellâcell adhesion and promote Smad2 phosphorylation. Remarkably, α3ß1 and CD151 can support Akt and STAT 3 activity independently of α3ß1 ligation by laminin-332 and as such control the essential survival signals required for suprabasal keratin-10 expression during keratinocyte differentiation. These data demonstrate that α3ß1 together with CD151 regulate the signaling pathways that control the survival of differentiating keratinocytes and provide a mechanistic understanding of the essential role of α3ß1 in early stages of skin cancer development.
Assuntos
Transformação Celular Neoplásica/patologia , Integrina alfa3beta1/metabolismo , Queratinócitos/patologia , Neoplasias Experimentais/patologia , Neoplasias Cutâneas/patologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Carcinógenos/toxicidade , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Epiderme/efeitos dos fármacos , Epiderme/patologia , Humanos , Integrina alfa3beta1/genética , Queratinócitos/efeitos dos fármacos , Camundongos , Neoplasias Experimentais/induzido quimicamente , Transdução de Sinais , Neoplasias Cutâneas/induzido quimicamente , Esferoides Celulares , Acetato de Tetradecanoilforbol/toxicidade , Tetraspanina 24/metabolismo , CalininaRESUMO
In epithelial cancers, the epidermal growth factor receptor (EGFR) and integrin α6ß4 are frequently overexpressed and found to synergistically activate intracellular signaling pathways that promote cell proliferation and migration. In cancer cells, the ß4 subunit is phosphorylated at tyrosine residues not normally recognized as kinase substrates; however, the function of these phosphotyrosine residues in cancer cells is a subject of much debate. In EGFR-overexpressing carcinoma cells, we found that the Src family kinase (SFK) inhibitor PP2 reduces ß4 tyrosine phosphorylation following the activation of EGFR. However, siRNA mediated knockdown of the SFKs Src, Fyn, Yes and Lyn, individually or in combination, did not affect the EGF-induced phosphorylation of ß4. Using phospho-peptide affinity chromatography and mass spectrometry, we found that PLCγ1 binds ß4 at the phosphorylated residues Y1422/Y1440, but were unable to verify this interaction in A431 carcinoma cells that overexpress the EGFR. Furthermore, using A431 cells devoid of ß4 or reconstituted with phenylalanine specific mutants of ß4, the activation of several downstream signaling pathways, including PLCγ/PKC, MAPK and PI3K/Akt, were not substantially affected. We conclude that tyrosine-phosphorylated ß4 does not enhance EGFR-mediated signaling in EGFR-overexpressing cells, despite the fact that this integrin subunit is highly tyrosine phosphorylated in these cells.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Integrina beta4/metabolismo , Neoplasias Cutâneas/metabolismo , Tirosina/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Integrina beta4/fisiologia , Espectrometria de Massas , Fosforilação , Fosfotirosina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Neoplasias Cutâneas/genéticaRESUMO
An increased expression of the integrin alpha6beta4 is correlated with a poor prognosis in patients with squamous cell carcinomas. However, little is known about the role of alpha6beta4 in the early stages of tumor development. We have isolated cells from mouse skin (mouse tumor-initiating cells [mTICs]) that are deficient in both p53 and Smad4 and carry conditional alleles of the beta4 gene (Itgb4). The mTICs display many features of multipotent epidermal stem cells and produce well-differentiated tumors after subcutaneous injection into nude mice. Deletion of Itgb4 led to enhanced tumor growth, indicating that alpha6beta4 mediates a tumor-suppressive effect. Reconstitution experiments with beta4-chimeras showed that this effect is not dependent on ligation of alpha6beta4 to laminin-5, but on the recruitment by this integrin of the cytoskeletal linker protein plectin to the plasma membrane. Depletion of plectin, like that of beta4, led to increased tumor growth. In contrast, when mTICs had been further transformed with oncogenic Ras, alpha6beta4 stimulated tumor growth, as previously observed in human squamous neoplasms. Expression of different effector-loop mutants of Ras(V12) suggests that this effect depends on a strong activation of the Erk pathway. Together, these data show that depending on the mutations involved, alpha6beta4 can either mediate an adhesion-independent tumor-suppressive effect or act as a tumor promotor.
Assuntos
Epiderme/metabolismo , Epiderme/patologia , Integrina alfa6beta4/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Alelos , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Transformação Celular Neoplásica , Desmossomos/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Integrina alfa6beta4/deficiência , Integrina alfa6beta4/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Plectina/genética , Plectina/metabolismo , Ligação Proteica , Interferência de RNA , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas ras/genéticaRESUMO
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.