RESUMEN
BACKGROUND: Tetraspanin CD151 is highly expressed in endothelia and reinforces cell adhesion, but its role in vascular inflammation remains largely unknown. METHODS: In vitro molecular and cellular biological analyses on genetically modified endothelial cells, in vivo vascular biological analyses on genetically engineered mouse models, and in silico systems biology and bioinformatics analyses on CD151-related events. RESULTS: Endothelial ablation of Cd151 leads to pulmonary and cardiac inflammation, severe sepsis, and perilous COVID-19, and endothelial CD151 becomes downregulated in inflammation. Mechanistically, CD151 restrains endothelial release of proinflammatory molecules for less leukocyte infiltration. At the subcellular level, CD151 determines the integrity of multivesicular bodies/lysosomes and confines the production of exosomes that carry cytokines such as ANGPT2 (angiopoietin-2) and proteases such as cathepsin-D. At the molecular level, CD151 docks VCP (valosin-containing protein)/p97, which controls protein quality via mediating deubiquitination for proteolytic degradation, onto endolysosomes to facilitate VCP/p97 function. At the endolysosome membrane, CD151 links VCP/p97 to (1) IFITM3 (interferon-induced transmembrane protein 3), which regulates multivesicular body functions, to restrain IFITM3-mediated exosomal sorting, and (2) V-ATPase, which dictates endolysosome pH, to support functional assembly of V-ATPase. CONCLUSIONS: Distinct from its canonical function in strengthening cell adhesion at cell surface, CD151 maintains endolysosome function by sustaining VCP/p97-mediated protein unfolding and turnover. By supporting protein quality control and protein degradation, CD151 prevents proteins from (1) buildup in endolysosomes and (2) discharge through exosomes, to limit vascular inflammation. Also, our study conceptualizes that balance between degradation and discharge of proteins in endothelial cells determines vascular information. Thus, the IFITM3/V-ATPase-tetraspanin-VCP/p97 complexes on endolysosome, as a protein quality control and inflammation-inhibitory machinery, could be beneficial for therapeutic intervention against vascular inflammation.
Asunto(s)
COVID-19 , Endosomas , Lisosomas , Tetraspanina 24 , Animales , Lisosomas/metabolismo , Tetraspanina 24/metabolismo , Tetraspanina 24/genética , Humanos , Ratones , COVID-19/metabolismo , COVID-19/inmunología , COVID-19/patología , Endosomas/metabolismo , Ratones Noqueados , Vasculitis/metabolismo , Ratones Endogámicos C57BL , SARS-CoV-2 , Inflamación/metabolismo , Inflamación/patología , Sepsis/metabolismoRESUMEN
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.
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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
Integrin-dependent cell-extracellular matrix adhesion is essential for wound healing, embryonic development, immunity, and tissue organization. Here, we present a protocol for the imaging and quantitative analysis of integrin-dependent cell-matrix adhesions. We describe steps for cell culture; virus preparation; lentiviral transduction; imaging with widefield, confocal, and total internal reflection fluorescence microscopy; and using a script for their quantitative analysis. We then detail procedures for analyzing adhesion dynamics by live-cell imaging and fluorescence recovery after photobleaching (FRAP). For complete details on the use and execution of this protocol, please refer to Margadant et al. (2012),1 van der Bijl et al. (2020),2 Amado-Azevedo et al. (2021).3.
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Técnicas de Cultivo de Célula , Microscopía , Femenino , Embarazo , Humanos , Uniones Célula-Matriz , Desarrollo Embrionario , IntegrinasRESUMEN
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.
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Clatrina , Receptores de Vitronectina , Adhesión Celular/fisiología , Proliferación Celular , Clatrina/metabolismo , Adhesiones Focales/metabolismo , Receptores de Vitronectina/metabolismo , Serina/metabolismoRESUMEN
The main laminin-binding integrins α3ß1, α6ß1 and α6ß4 are co-expressed in the developing kidney collecting duct system. We previously showed that deleting the integrin α3 or α6 subunit in the ureteric bud, which gives rise to the kidney collecting system, caused either a mild or no branching morphogenesis phenotype, respectively. To determine whether these two integrin subunits cooperate in kidney collecting duct development, we deleted α3 and α6 in the developing ureteric bud. The collecting system of the double knockout phenocopied the α3 integrin conditional knockout. However, with age, the mice developed severe inflammation and fibrosis around the collecting ducts, resulting in kidney failure. Integrin α3α6-null collecting duct epithelial cells showed increased secretion of pro-inflammatory cytokines and displayed mesenchymal characteristics, causing loss of barrier function. These features resulted from increased nuclear factor kappa-B (NF-κB) activity, which regulated the Snail and Slug (also known as Snai1 and Snai2, respectively) transcription factors and their downstream targets. These data suggest that laminin-binding integrins play a key role in the maintenance of kidney tubule epithelial cell polarity and decrease pro-inflammatory cytokine secretion by regulating NF-κB-dependent signaling.
Asunto(s)
Integrinas , Túbulos Renales Colectores , Animales , Células Epiteliales , Inflamación/genética , Integrina alfa3beta1 , Integrinas/genética , Laminina/genética , Ratones , FN-kappa B/genéticaRESUMEN
Hemidesmosomes (HDs) are specialized multiprotein complexes that connect the keratin cytoskeleton of epithelial cells to the extracellular matrix (ECM). In the skin, these complexes provide stable adhesion of basal keratinocytes to the underlying basement membrane. Integrin α6ß4 is a receptor for laminins and plays a vital role in mediating cell adhesion by initiating the assembly of HDs. In addition, α6ß4 has been implicated in signal transduction events that regulate diverse cellular processes, including proliferation and survival. In this Review, we detail the role of α6ß4 in HD assembly and beyond, and we discuss the molecular mechanisms that regulate its function.
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Hemidesmosomas , Integrina alfa6beta4 , Adhesión Celular , Integrina alfa6beta4/genética , Queratinocitos , Transducción de SeñalRESUMEN
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.
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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.
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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
Physical forces regulate numerous biological processes during development, physiology, and pathology. Forces between the external environment and intracellular actin cytoskeleton are primarily transmitted through integrin-containing focal adhesions and cadherin-containing adherens junctions. Crosstalk between these complexes is well established and modulates the mechanical landscape of the cell. However, integrins and cadherins constitute large families of adhesion receptors and form multiple complexes by interacting with different ligands, adaptor proteins, and cytoskeletal filaments. Recent findings indicate that integrin-containing hemidesmosomes oppose force transduction and traction force generation by focal adhesions. The cytolinker plectin mediates this crosstalk by coupling intermediate filaments to the actin cytoskeleton. Similarly, cadherins in desmosomes might modulate force generation by adherens junctions. Moreover, mechanotransduction can be influenced by podosomes, clathrin lattices, and tetraspanin-enriched microdomains. This review discusses mechanotransduction by multiple integrin- and cadherin-based cell adhesion complexes, which together with the associated cytoskeleton form an integrated network that allows cells to sense, process, and respond to their physical environment.
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Uniones Adherentes , Mecanotransducción Celular , Cadherinas , Adhesión Celular , Citoesqueleto , Humanos , IntegrinasRESUMEN
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
Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding ß1 and ß3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal ß1/ß3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal ß1/ß3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis.
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Integrina beta1/metabolismo , Integrina beta3/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células Neuroepiteliales/citología , Proteína de Unión al GTP rhoA/metabolismo , Movimiento Celular , Plasticidad de la Célula , Citoplasma/metabolismo , Desarrollo Embrionario , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Neuroepiteliales/metabolismo , FenotipoRESUMEN
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.
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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
Hemidesmosomes are specialized cell-matrix adhesion structures that are associated with the keratin cytoskeleton. Although the adhesion function of hemidesmosomes has been extensively studied, their role in mechanosignaling and transduction remains largely unexplored. Here, we show that keratinocytes lacking hemidesmosomal integrin α6ß4 exhibit increased focal adhesion formation, cell spreading, and traction-force generation. Moreover, disruption of the interaction between α6ß4 and intermediate filaments or laminin-332 results in similar phenotypical changes. We further demonstrate that integrin α6ß4 regulates the activity of the mechanosensitive transcriptional regulator YAP through inhibition of Rho-ROCK-MLC- and FAK-PI3K-dependent signaling pathways. Additionally, increased tension caused by impaired hemidesmosome assembly leads to a redistribution of integrin αVß5 from clathrin lattices to focal adhesions. Our results reveal a novel role for hemidesmosomes as regulators of cellular mechanical forces and establish the existence of a mechanical coupling between adhesion complexes.
Asunto(s)
Hemidesmosomas/genética , Integrina alfa6beta4/genética , Queratinas/genética , Mecanotransducción Celular/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Moléculas de Adhesión Celular/genética , Movimiento Celular/genética , Uniones Célula-Matriz/genética , Uniones Célula-Matriz/metabolismo , Células Cultivadas , Citoesqueleto/genética , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Humanos , Filamentos Intermedios/genética , Filamentos Intermedios/metabolismo , Queratinocitos/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Proteínas Señalizadoras YAP , Quinasas Asociadas a rho/genética , KalininaRESUMEN
Integrin dimers α3/ß1, α6/ß1 and α6/ß4 are the mammary epithelial cell receptors for laminins, which are major components of the mammary basement membrane. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated using the Cre-Lox approach. During pregnancy, mutant mice displayed decreased luminal progenitor activity and retarded lobulo-alveolar development. Mammary glands appeared functional at the onset of lactation in mutant mice; however, myoepithelial cell morphology was markedly altered, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. Notably, lactation was not sustained in mutant females, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. These results suggest that the p53 pathway is involved in the control of mammary cell proliferation and survival downstream of laminin-binding integrins, and underline an essential role of cell interactions with laminin for lactogenic differentiation.
Asunto(s)
Integrinas/fisiología , Lactancia , Glándulas Mamarias Animales/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Citoesqueleto/fisiología , Progresión de la Enfermedad , Femenino , Eliminación de Gen , Hormonas/fisiología , Integrina alfa3/fisiología , Integrina alfa6/fisiología , Integrina beta1/fisiología , Integrina beta4/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones Mutantes , Mutación , Células Madre Neoplásicas/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ovario/fisiología , Fenotipo , Embarazo , Preñez , Pronóstico , Unión Proteica , Multimerización de ProteínaRESUMEN
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.
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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
E-cadherin (CDH1) is a master regulator of epithelial cell adherence junctions and a well-established tumor suppressor in Invasive Lobular Carcinoma (ILC). Intriguingly, somatic inactivation of E-cadherin alone in mouse mammary epithelial cells (MMECs) is insufficient to induce tumor formation. Here we show that E-cadherin loss induces extrusion of luminal MMECs to the basal lamina. Remarkably, E-cadherin-deficient MMECs can breach the basal lamina but do not disseminate into the surrounding fat pad. Basal lamina components laminin and collagen IV supported adhesion and survival of E-cadherin-deficient MMECs while collagen I, the principle component of the mammary stromal micro-environment did not. We uncovered that relaxation of actomyosin contractility mediates adhesion and survival of E-cadherin-deficient MMECs on collagen I, thereby allowing ILC development. Together, these findings unmask the direct consequences of E-cadherin inactivation in the mammary gland and identify aberrant actomyosin contractility as a critical barrier to ILC formation.
Asunto(s)
Actomiosina/metabolismo , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Carcinoma Lobular/patología , Neoplasias Mamarias Experimentales/patología , Animales , Neoplasias de la Mama/genética , Cadherinas/genética , Carcinoma Lobular/genética , Adhesión Celular/genética , Supervivencia Celular/genética , Células Cultivadas , Células Epiteliales , Femenino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Transgénicos , Cultivo Primario de CélulasRESUMEN
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.