RESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Movimiento Celular , Proteínas del Citoesqueleto , Unión Proteica , Talina , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Adhesiones Focales/metabolismo , Integrinas/metabolismo , Células MCF-7 , Microtúbulos/metabolismo , Fosforilación , Talina/metabolismoRESUMEN
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.
Asunto(s)
Cisplatino , Anemia de Fanconi , Humanos , Animales , Ratones , Cisplatino/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Ratones Endogámicos C57BL , Sistemas CRISPR-Cas , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Mitomicina , Fenotipo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genéticaRESUMEN
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.
Asunto(s)
Reparación del ADN , Neoplasias , Animales , Cisplatino/uso terapéutico , Daño del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Medicina de Precisión , Antígeno Nuclear de Célula en Proliferación/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Adhesión Celular , Integrinas , Proteínas Tirosina Quinasas , Tensinas , Movimiento Celular , Adhesiones Focales/metabolismo , Humanos , Integrina beta3/metabolismo , Integrinas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Tensinas/metabolismoRESUMEN
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.
Asunto(s)
Clatrina , Receptores de Vitronectina , Adhesión Celular/fisiología , Proliferación Celular , Clatrina/metabolismo , Adhesiones Focales/metabolismo , Receptores de Vitronectina/metabolismo , Serina/metabolismoRESUMEN
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.
Asunto(s)
Cambio de Clase de Inmunoglobulina , Hipermutación Somática de Inmunoglobulina , Cambio de Clase de Inmunoglobulina/genética , Linfocitos B , Reparación del ADN , Diferenciación Celular/genéticaRESUMEN
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.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Integrina beta4/metabolismo , Neoplasias Cutáneas/metabolismo , Tirosina/metabolismo , Animales , Línea Celular Tumoral , Humanos , Integrina beta4/fisiología , Espectrometría de Masas , Fosforilación , Fosfotirosina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Neoplasias Cutáneas/genéticaRESUMEN
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.
Asunto(s)
Transformación Celular Neoplásica/patología , Integrina alfa3beta1/metabolismo , Queratinocitos/patología , Neoplasias Experimentales/patología , Neoplasias Cutáneas/patología , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Carcinógenos/toxicidad , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Epidermis/efectos de los fármacos , Epidermis/patología , Humanos , Integrina alfa3beta1/genética , Queratinocitos/efectos de los fármacos , Ratones , Neoplasias Experimentales/inducido químicamente , Transducción de Señal , Neoplasias Cutáneas/inducido químicamente , Esferoides Celulares , Acetato de Tetradecanoilforbol/toxicidad , Tetraspanina 24/metabolismo , KalininaRESUMEN
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.
Asunto(s)
Integrina alfa6beta4/metabolismo , Queratinocitos/metabolismo , Biotinilación , Línea Celular , Hemidesmosomas/metabolismo , Humanos , Microtúbulos/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas , Tetraspanina 24/metabolismoRESUMEN
Epidermal-specific deletion of integrin α3ß1 almost completely prevents the formation of papillomas during 7,12-Dimethylbenz[ a ]anthracene/12- O -tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage skin carcinogenesis. This dramatic decrease in tumorigenesis was thought to be due to an egress and premature differentiation of α3ß1-depleted hair bulge (HB) stem cells (SCs), previously considered to be the cancer cells-of-origin in the DMBA/TPA model. Using a reporter mouse line with inducible deletion of α3ß1 in HBs, we show that HB SCs remain confined to their niche regardless of the presence of α3ß1 and are largely absent from skin tumors. However, tumor formation was significantly decreased in mice deficient for α3ß1 in HB SCs. RNA sequencing of HB SCs isolated from short-term DMBA/TPA-treated skin showed α3ß1-dependent expression of the matricellular protein connective tissue growth factor (CCN2), which was confirmed in vitro, where CCN2 promoted colony formation and 3D growth of transformed keratinocytes. Together, these findings show that HBs contribute to skin tumorigenesis in an α3ß1-dependent manner and suggest a role of HB SCs in creating a permissive environment for tumor growth through the modulation of CCN2 secretion.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/genética , Regulación de la Expresión Génica , Folículo Piloso/citología , Integrina alfa3beta1/metabolismo , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/metabolismo , Células Madre/metabolismo , Animales , Biomarcadores , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epidermis/patología , Técnica del Anticuerpo Fluorescente , Expresión Génica , Inmunohistoquímica , Inmunofenotipificación , Integrina alfa3beta1/genética , Queratinocitos/metabolismo , Queratinocitos/patología , Ratones , Ratones Noqueados , Estadificación de Neoplasias , Neoplasias Cutáneas/patologíaRESUMEN
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.
Asunto(s)
Uniones Célula-Matriz/metabolismo , Integrina alfa3beta1/metabolismo , Integrina alfa6beta4/metabolismo , Tetraspanina 24/metabolismo , Western Blotting , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Hemidesmosomas/metabolismo , Humanos , Inmunoprecipitación , Integrina alfa3beta1/química , Integrina alfa6beta4/química , Queratinocitos/metabolismo , Plectina/metabolismo , Tetraspanina 24/químicaRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Integrina alfa3beta1/deficiencia , Receptor ErbB-2/genética , Animales , Biomarcadores de Tumor , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunofenotipificación , Estimación de Kaplan-Meier , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Receptor ErbB-2/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
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.
Asunto(s)
Distonina/química , Hemidesmosomas/metabolismo , Integrina alfa6beta4/química , Penfigoide Ampolloso/metabolismo , Dominios Proteicos , Secuencia de Aminoácidos , Membrana Basal/metabolismo , Sitios de Unión/genética , Cristalografía por Rayos X , Distonina/genética , Distonina/metabolismo , Hemidesmosomas/genética , Humanos , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Modelos Moleculares , Mutagénesis , Penfigoide Ampolloso/genética , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
Integrins, which bind laminin, a major component of the mammary basement membrane, are strongly expressed in basal stem cell-enriched populations, but their role in controlling mammary stem cell function remains unclear. We found that stem cell activity, as evaluated in transplantation and mammosphere assays, was reduced in mammary basal cells depleted of laminin receptors containing α3- and α6-integrin subunits. This was accompanied by low MDM2 levels, p53 stabilization, and diminished proliferative capacity. Importantly, disruption of p53 function restored the clonogenicity of α3/α6-integrin-depleted mammary basal stem cells, while inhibition of RHO or myosin II, leading to decreased p53 activity, rescued the mammosphere formation. These data suggest that α3/α6-integrin-mediated adhesion plays an essential role in controlling the proliferative potential of mammary basal stem/progenitor cells through myosin II-mediated regulation of p53 and indicate that laminins might be important components of the mammary stem cell niche.
RESUMEN
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.
Asunto(s)
Clatrina/metabolismo , Receptores de Vitronectina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Calcio/metabolismo , Células Cultivadas , Adhesiones Focales/metabolismo , Humanos , Queratinocitos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vitronectina/metabolismoRESUMEN
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.
Asunto(s)
Autoantígenos/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Hemidesmosomas/metabolismo , Integrina beta4/metabolismo , Queratinocitos/metabolismo , Queratinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Colágenos no Fibrilares/metabolismo , Piel/metabolismo , Autoantígenos/genética , Proteínas Portadoras/genética , Proteínas del Citoesqueleto/genética , Distonina , Hemidesmosomas/genética , Hemidesmosomas/ultraestructura , Humanos , Integrina beta4/genética , Queratinocitos/ultraestructura , Queratinas/genética , Microscopía Fluorescente , Proteínas del Tejido Nervioso/genética , Colágenos no Fibrilares/genética , Piel/ultraestructura , Colágeno Tipo XVIIRESUMEN
Epidermolysis bullosa simplex associated with late-onset muscular dystrophy (EBS-MD) is an autosomal recessive disorder resulting from mutations in the plectin gene. The majority of these mutations occur within the large exon 31 encoding the central rod domain and leave the production of a low-level rodless plectin splice variant unaffected. To investigate the function of the rod domain, we generated rodless plectin mice through conditional deletion of exon 31. Rodless plectin mice develop normally without signs of skin blistering or muscular dystrophy. Plectin localization and hemidesmosome organization are unaffected in rodless plectin mice. However, superresolution microscopy revealed a closer juxtaposition of the C-terminus of plectin to the integrin ß4 subunit in rodless plectin keratinocytes. Wound healing occurred slightly faster in rodless plectin mice than in wild-type mice, and keratinocytes migration was increased in the absence of the rod domain. The faster migration of rodless plectin keratinocytes is not due to altered biochemical properties because, like full-length plectin, rodless plectin is a dimeric protein. Our data demonstrate that rodless plectin can functionally compensate for the loss of full-length plectin in mice. Thus the low expression level of plectin rather than the absence of the rod domain dictates the development of EBS-MD.
Asunto(s)
Epidermólisis Ampollosa Simple/metabolismo , Distrofia Muscular de Cinturas/metabolismo , Plectina/metabolismo , Animales , Citoesqueleto/metabolismo , Citoesqueleto/patología , Epidermólisis Ampollosa Simple/genética , Epidermólisis Ampollosa Simple/patología , Femenino , Hemidesmosomas/metabolismo , Integrina beta4/metabolismo , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/patología , Plectina/genética , Isoformas de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Nesprin-3 is a nuclear envelope protein that connects the nucleus to intermediate filaments by interacting with plectin. To investigate the role of nesprin-3 in the perinuclear localization of plectin, we generated nesprin-3-knockout mice and examined the effects of nesprin-3 deficiency in different cell types and tissues. Nesprin-3 and plectin are coexpressed in a variety of tissues, including peripheral nerve and muscle. The expression level of nesprin-3 in skeletal muscle is very low and decreases during myoblast differentiation in vitro. Of interest, plectin was concentrated at the nuclear envelope in only a few cell types. This was most prominent in Sertoli cells of the testis, in which nesprin-3 is required for the localization of both plectin and vimentin at the nuclear perimeter. Testicular morphology and the position of the nucleus in Sertoli cells were normal, however, in the nesprin-3-knockout mice and the mice were fertile. Furthermore, nesprin-3 was not required for the polarization and migration of mouse embryonic fibroblasts. Thus, although nesprin-3 is critical for the localization of plectin to the nuclear perimeter of Sertoli cells, the resulting link between the nuclear envelope and the intermediate filament system seems to be dispensable for normal testicular morphology and spermatogenesis.