RESUMEN
Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9-EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9-EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9-EWI-F with potential CD9 homo-dimerization at either end provides a "concatenation model" for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.
Asunto(s)
Tetraspanina 29/metabolismo , Tetraspanina 29/ultraestructura , Antígenos CD/química , Antígenos CD/metabolismo , Antígenos CD/ultraestructura , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Glicoproteínas de Membrana/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Tetraspanina 28/metabolismo , Tetraspanina 28/ultraestructura , Tetraspanina 29/fisiología , Tetraspaninas/metabolismo , Tetraspaninas/fisiología , Tetraspaninas/ultraestructura , Factores de Transcripción/metabolismoAsunto(s)
Plaquetas/química , Receptores Fc/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Tetraspanina 29/deficiencia , Trombocitopenia/tratamiento farmacológico , Trombopoyetina/uso terapéutico , Benzoatos/uso terapéutico , Médula Ósea/patología , Terapia Combinada , Consanguinidad , Sustitución de Medicamentos , Equimosis/etiología , Femenino , Humanos , Hidrazinas/uso terapéutico , Inmunofenotipificación , Lactante , Transfusión de Plaquetas , Pirazoles/uso terapéutico , Tetraspanina 29/fisiología , Trombocitopenia/sangre , Trombocitopenia/patología , Trombocitopenia/terapia , Secuenciación del ExomaRESUMEN
Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short and large extracellular loops, and associate with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2. The reversed cone-like molecular shape of CD9 generates membrane curvature in the crystalline lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The molecular interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the critical involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins.
Asunto(s)
Tetraspanina 29/química , Tetraspanina 29/fisiología , Animales , Antígenos CD/química , Adhesión Celular/fisiología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Femenino , Fertilización/fisiología , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Modelos Moleculares , Conformación Proteica , Tetraspanina 29/genéticaRESUMEN
Purpose: Cd9 is a tetraspanin membrane protein that plays various roles in tissue development and disease pathogenesis, especially in cancer, but the expression patterns and function of Cd9 in retinal development and disease are not well understood. We asked its roles during retinal photoreceptor degeneration by using CD9-knockout mice. Methods: Cd9 knockout mice and rd1 mice were used to examine roles of Cd9 for progression of photoreceptor degeneration. Reverse transcription-polymerase chain reaction and immunohistochemistry were mainly used as analytical methods. Results: Cd9 transcripts were only weakly expressed in retina at embryonic day 14, but its expression level subsequently increased and peaked at around postnatal day 12. In 6-week-old female mice derived retina, mRNA expression decreased slightly but was maintained at a significant level. Published RNA-sequencing data and immunohistochemistry indicated that Cd9 was expressed abundantly in Müller glia and weakly in other retinal neurons. Notably, when photoreceptors were damaged, Cd9 expression was increased in rod photoreceptors and decreased in Müller glia. Cd9 knockout mice retinas developed normally; however, once the retina suffered damage, degeneration of photoreceptors was more severe in Cd9 knockout retinas than control retinas. Induction of Edn2, which is known to protect against photoreceptor damage, was severely hampered. In addition, induction of Socs3, which is downstream of gp130 (Il6st), was weaker in Cd9 knockout retinas. Conclusions: Taken together, these findings indicate that, although Cd9 was dispensable for normal gross morphological development, it protected rod photoreceptors and enhanced Edn2 expression, possibly through modulation of gp130 signaling.
Asunto(s)
Endotelina-2/metabolismo , Degeneración Retiniana/prevención & control , Células Fotorreceptoras Retinianas Bastones/metabolismo , Tetraspanina 29/fisiología , Animales , Receptor gp130 de Citocinas/fisiología , Células Ependimogliales/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Ratones Endogámicos ICR , Ratones Noqueados , ARN Mensajero/genética , Retina/crecimiento & desarrollo , Retina/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/patología , Transducción de Señal/fisiología , Tetraspanina 29/deficiencia , Tetraspanina 29/genéticaRESUMEN
The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and ß1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.
Asunto(s)
Enfermedades Renales/patología , Tetraspanina 29/fisiología , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Femenino , Glomerulonefritis/genética , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Enfermedades Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Tetraspanina 29/genética , Tetraspanina 29/metabolismoRESUMEN
Integrin α6ß4 is an essential, dynamic adhesion receptor for laminin 332 found on epithelial cells, required for formation of strong cell-extracellular matrix (ECM) adhesion and induced migration, and coordinated by regions of the ß4C cytoplasmic domain. ß4E, a unique splice variant of ß4 expressed in normal tissue, contains a cytoplasmic domain of 231 amino acids with a unique sequence of 114 amino acids instead of ß4C's canonical 1089 amino acids. We determined the distribution of α6ß4E within normal human glandular epithelium and its regulation and effect on cellular biophysical properties. Canonical α6ß4C expressed in all basal cells, as expected, while α6ß4E expressed within a subset of luminal cells. α6ß4E expression was induced by three-dimensional culture conditions, activated Src, was reversible, and was stabilized by bortezomib, a proteasome inhibitor. α6ß4C expressed in all cells during induced migration, whereas α6ß4E was restricted to a subset of cells with increased kinetics of cell-cell and cell-ECM resistance properties. Interestingly, α6ß4E presented in "ringlike" patterns measuring â¼1.75 × 0.72 microns and containing actin and CD9 at cell-ECM locations. In contrast, α6ß4C expressed only within hemidesmosome-like structures containing BP180. Integrin α6ß4E is an inducible adhesion isoform in normal epithelial cells that can alter biophysical properties of cell-cell and cell-ECM interactions.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Integrina alfa6beta4/fisiología , Actinas/metabolismo , Actinas/fisiología , Línea Celular Tumoral , Desmosomas/metabolismo , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Hemidesmosomas/metabolismo , Humanos , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Integrinas/metabolismo , Laminina/metabolismo , Isoformas de Proteínas , Tetraspanina 29/metabolismo , Tetraspanina 29/fisiologíaRESUMEN
Tetraspanin CD9 is essential for sperm-egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility.
Asunto(s)
Tetraspanina 29 , Útero , Animales , Secreciones Corporales/química , Secreciones Corporales/citología , Línea Celular , Ciclo Estral , Exosomas/química , Exosomas/metabolismo , Femenino , Humanos , Infertilidad Femenina , Ratones , Ratones Endogámicos C57BL , Mitocondrias/química , Mitocondrias/metabolismo , Tetraspanina 29/química , Tetraspanina 29/metabolismo , Tetraspanina 29/fisiología , Útero/química , Útero/citología , Útero/metabolismo , Útero/fisiologíaRESUMEN
Chronic obstructive pulmonary disease (COPD) is frequently associated with extrapulmonary complications, including cardiovascular disease, diabetes, and osteoporosis. Persistent, low-grade, systemic inflammation underlies these comorbid disorders. Tetraspanins, which have a characteristic structure spanning the membrane four times, facilitate lateral organization of molecular complexes and thereby form tetraspanin-enriched microdomains that are distinct from lipid rafts. Recent basic research has suggested a preventive role of tetraspanin CD9 in COPD. CD9-enriched microdomains negatively regulate LPS-induced receptor formation by preventing CD14 from accumulating into the rafts, and decreased CD9 in macrophages enhances inflammation in mice. Mice doubly deficient in CD9 and a related tetraspanin, CD81, show pulmonary emphysema, weight loss, and osteopenia, a phenotype akin to human COPD. A therapeutic approach to up-regulating CD9 in macrophages might improve the clinical course of patients with COPD with comorbidities.
Asunto(s)
Enfermedad Pulmonar Obstructiva Crónica/inmunología , Tetraspanina 29/fisiología , Animales , Humanos , Activación de Macrófagos , Microdominios de Membrana/metabolismo , Regulación hacia ArribaRESUMEN
Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the "bad seed in bad soil" hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis.
Asunto(s)
Megacariocitos/metabolismo , Mielofibrosis Primaria/metabolismo , Células del Estroma/metabolismo , Tetraspanina 29/fisiología , Trombopoyesis/fisiología , Técnicas de Cocultivo , Humanos , Megacariocitos/patología , Mielofibrosis Primaria/patología , Células del Estroma/patologíaRESUMEN
Given its tumor-specific expression, including liver cancer, OY-TES-1 is a potential molecular marker for the diagnosis and immunotherapy of liver cancers. However, investigations of the mechanisms and the role of OY-TES-1 in liver cancer are rare. In the present study, based on a comprehensive bioinformatic analysis combined with RNA interference (RNAi) and oligonucleotide microarray, we report for the first time that downregulation of OY-TES-1 resulted in significant changes in expression of NANOG, CD9, CCND2 and CDCA3 in the liver cancer cell line BEL-7404. NANOG, CD9, CCND2 and CDCA3 may be involved in cell proliferation, migration, invasion and apoptosis, yet also may be functionally related to each other and OY-TES-1. Among these molecules, we identified that NANOG, containing a Kazal-2 binding motif and homeobox, may be the most likely candidate protein interacting with OY-TES-1 in liver cancer. Thus, the present study may provide important information for further investigation of the roles of OY-TES-1 in liver cancer.
Asunto(s)
Carcinoma Hepatocelular/genética , Proteínas Portadoras/fisiología , Ciclina D2/fisiología , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/fisiología , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/fisiología , Tetraspanina 29/fisiología , Secuencias de Aminoácidos , Apoptosis , Carcinoma Hepatocelular/patología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Adhesión Celular , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , División Celular , Movimiento Celular , Biología Computacional , Ciclina D2/biosíntesis , Ciclina D2/genética , Bases de Datos Genéticas , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Humanos , Neoplasias Hepáticas/patología , Proteína Homeótica Nanog , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , ARN Bacteriano , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Tetraspanina 29/biosíntesis , Tetraspanina 29/genética , TranscriptomaRESUMEN
Understanding how the immune response is activated and amplified requires detailed knowledge of the stages in the formation of the immunological synapse (IS) between T lymphocytes and antigen-presenting cells (APCs). We show that tetraspanins CD9 and CD151 congregate at the T-cell side of the IS. Silencing of CD9 or CD151 blunts the IL-2 secretion and expression of the activation marker CD69 by APC-conjugated T lymphocytes, but does not affect the accumulation of CD3 or actin to the IS, or the translocation of the microtubule-organizing center toward the T-B contact area. CD9 or CD151 silencing diminishes the relocalization of α4ß1 integrin to the IS and reduces the accumulation of high-affinity ß1 integrins at the cell-cell contact. These changes are accompanied by diminished phosphorylation of the integrin downstream targets FAK and ERK1/2. Our results suggest that CD9 and CD151 support integrin-mediated signaling at the IS.
Asunto(s)
Sinapsis Inmunológicas/inmunología , Integrinas/fisiología , Transducción de Señal/fisiología , Linfocitos T/inmunología , Tetraspanina 24/fisiología , Tetraspanina 29/fisiología , Humanos , Células Jurkat , Activación de LinfocitosRESUMEN
Multinucleated giant cells, formed by the fusion of monocytes/macrophages, are features of chronic granulomatous inflammation associated with infections or the persistent presence of foreign material. The tetraspanins CD9 and CD81 regulate multinucleated giant cell formation: soluble recombinant proteins corresponding to the large extracellular domain (EC2) of human but not mouse CD9 can inhibit multinucleated giant cell formation, whereas human CD81 EC2 can antagonise this effect. Tetraspanin EC2 are all likely to have a conserved three helix sub-domain and a much less well-conserved or hypervariable sub-domain formed by short helices and interconnecting loops stabilised by two or more disulfide bridges. Using CD9/CD81 EC2 chimeras and point mutants we have mapped the specific regions of the CD9 EC2 involved in multinucleated giant cell formation. These were primarily located in two helices, one in each sub-domain. The cysteine residues involved in the formation of the disulfide bridges in CD9 EC2 were all essential for inhibitory activity but a conserved glycine residue in the tetraspanin-defining 'CCG' motif was not. A tyrosine residue in one of the active regions that is not conserved between human and mouse CD9 EC2, predicted to be solvent-exposed, was found to be only peripherally involved in this activity. We have defined two spatially-distinct sites on the CD9 EC2 that are required for inhibitory activity. Agents that target these sites could have therapeutic applications in diseases in which multinucleated giant cells play a pathogenic role.
Asunto(s)
Células Gigantes/metabolismo , Tetraspanina 29/fisiología , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Secuencia Conservada , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Puntual , Estructura Terciaria de Proteína , Alineación de Secuencia , Tetraspanina 29/química , Tetraspanina 29/genéticaRESUMEN
Although the membrane fusion of spermatozoon and egg cells is the central event of fertilization, the underlying molecular mechanism remains virtually unknown. Gene disruption studies have showed that IZUMO1 on spermatozoon and CD9 on oocyte are essential transmembrane proteins in sperm-egg fusion. In this study, we dissected IZUMO1 protein to determine the domains that were required for the function of sperm-egg fusion. We found that a fragment of the N terminus (Asp5 to Leu113) interacts with fertilization inhibitory antibodies. It also binds to the egg surface and effectively inhibits fusion in vitro. We named this fragment 'IZUMO1 putative functional fragment (IZUMO1PFF)'. Surprisingly, IZUMO1PPF still maintains binding ability on the egg surface of Cd9(-/-) eggs. A series of biophysical measurements using circular dichroism, sedimentation equilibrium and small angle X-ray scattering revealed that IZUMO1PFF is composed of an N-terminal unfolded structure and a C-terminal ellipsoidal helix dimer. Egg binding and fusion inhibition were not observed in the IZUMO1PFF derivative, which was incapable of helix formation. These findings suggest that the formation of a helical dimer at the N-terminal region of IZUMO1 is required for its function. Cos-7 cells expressing the whole IZUMO1 molecule bound to eggs, and IZUMO1 accumulated at the interface between the two cells, but fusion was not observed. These observations suggest that IZUMO1 alone cannot promote sperm-egg membrane fusion, but it works as a factor that is related to the cellular surface interaction, such as the tethering of the membranes by a helical region corresponding to IZUMO1PFF-core.
Asunto(s)
Inmunoglobulinas/fisiología , Proteínas de la Membrana/fisiología , Interacciones Espermatozoide-Óvulo/fisiología , Animales , Anticuerpos Monoclonales , Sitios de Unión , Fenómenos Biofísicos , Femenino , Inmunoglobulinas/química , Inmunoglobulinas/genética , Masculino , Fusión de Membrana/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Estructura Cuaternaria de Proteína , Tetraspanina 29/deficiencia , Tetraspanina 29/genética , Tetraspanina 29/fisiologíaRESUMEN
Skeletal muscle regeneration after injury follows a remarkable sequence of synchronized events. However, the mechanisms regulating the typical organization of the regenerating muscle at different stages remain largely unknown. Here we show that muscle regeneration in mice lacking either CD9 or CD81 is abnormal and characterized by the formation of discrete giant dystrophic myofibres, which form more quickly in the absence of both tetraspanins. We also show that, in myoblasts, these two tetraspanins associate with the immunoglobulin domain molecule CD9P-1 (EWI-F/FPRP), and that grafting of CD9P-1-depleted myoblasts in regenerating muscles also leads to abnormal regeneration. In vitro myotubes lacking CD9P-1 or both CD9 and CD81 fuse with a higher frequency than normal myotubes. Our study unveils a mechanism preventing inappropriate fusion of myotubes that has an important role in the restitution of normal muscle architecture during muscle regeneration.
Asunto(s)
Fusión Celular , Músculo Esquelético/fisiología , Regeneración , Tetraspanina 28/fisiología , Tetraspanina 29/fisiología , Animales , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/citología , Regulación hacia ArribaRESUMEN
Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca(2+) response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)(2) or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcεRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)(2) fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcεRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/metabolismo , Mastocitos/citología , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores de IgE/metabolismo , Tetraspanina 29/fisiología , Animales , Antígenos/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Quimiotaxis , Citoesqueleto/metabolismo , Glucuronidasa/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Fosforilación , Unión Proteica , Ratas , Ratas Wistar , Tirosina/químicaRESUMEN
Tetraspanins have emerged as key players in malignancy and inflammatory diseases, yet little is known about their roles in angiogenesis, and nothing is known about their involvement in lymphangiogenesis. We found here that tetraspanins are abundantly expressed in human lymphatic endothelial cells (LEC). After intrathoracic tumor implantation, metastasis to lymph nodes was diminished and accompanied by decreased angiogenesis and lymphangiogenesis in tetraspanin CD9-KO mice. Moreover, lymphangiomas induced in CD9-KO mice were less pronounced with decreased lymphangiogenesis compared with those in wild-type mice. Although mouse LEC isolated from CD9-KO mice showed normal adhesion, lymphangiogenesis was markedly impaired in several assays (migration, proliferation, and cable formation) in vitro and in the lymphatic ring assay ex vivo. Consistent with these findings in mouse LEC, knocking down CD9 in human LEC also produced decreased migration, proliferation, and cable formation. Immunoprecipitation analysis demonstrated that deletion of CD9 in LEC diminished formation of functional complexes between VEGF receptor-3 and integrins (α5 and α9). Therefore, knocking down CD9 in LEC attenuated VEGF receptor-3 signaling, as well as downstream signaling such as Erk and p38 upon VEGF-C stimulation. Finally, double deletion of CD9/CD81 in mice caused abnormal development of lymphatic vasculature in the trachea and diaphragm, suggesting that CD9 and a closely related tetraspanin CD81 coordinately play an essential role in physiological lymphangiogenesis. In conclusion, tetraspanin CD9 modulates molecular organization of integrins in LEC, thereby supporting several functions required for lymphangiogenesis.
Asunto(s)
Linfangiogénesis/genética , Tetraspanina 29/genética , Tetraspaninas/química , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Progresión de la Enfermedad , Células Endoteliales/citología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunoprecipitación , Técnicas In Vitro , Inflamación , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neovascularización Patológica , Tetraspanina 28/genética , Tetraspanina 28/metabolismo , Tetraspanina 29/fisiologíaRESUMEN
UNLABELLED: Recognition of hepatitis C virus (HCV)-infected hepatocyes and interferon (IFN) induction are critical in antiviral immune response. We hypothesized that cell-cell contact between plasmacytoid dendritic cells (pDCs) and HCV-infected cells was required for IFN-α induction through the involvement of cell-surface molecules. Coculture of human peripheral blood mononuclear cells (PBMCs) with genotype 1a full-length (FL) HCV genomic replicon cells or genotype 2a Japanese fulminant hepatitis type 1 (JFH-1) virus-infected hepatoma cells (JFH-1), and not with uninfected hepatoma cells (Huh7.5), induced IFN-α production. Depletion of pDCs from PBMCs attenuated IFN-α release, and purified pDCs produced high levels of IFN-α after coculture with FL replicons or JFH-1-infected cells. IFN-α induction by HCV-containing hepatoma cells required viral replication, direct cell-cell contact with pDCs, and receptor-mediated endocytosis. We determined that the tetraspanin proteins, CD81 and CD9, and not other HCV entry receptors, were required for IFN-α induction in pDCs by HCV-infected hepatoma cells. Disruption of cholesterol-rich membrane microdomains, the localization site of CD81, or inhibition of the CD81 downstream molecule, Rac GTPase, inhibited IFN-α production. IFN-α induction involved HCV RNA and Toll-like receptor (TLR) 7. IFN-α production by HCV-infected hepatoma cells was decreased in pDCs from HCV-infected patients, compared to healthy controls. We found that preexposure of healthy PBMCs to HCV viral particles attenuated IFN-α induction by HCV-infected hepatoma cells or TLR ligands, and this inhibitory effect could be prevented by an anti-HCV envelope glycoprotein 2-blocking antibody. CONCLUSION: Our novel data show that recognition of HCV-infected hepatoma cells by pDCs involves CD81- and CD9-associated membrane microdomains and induces potent IFN-α production.
Asunto(s)
Células Dendríticas/patología , Hepacivirus/aislamiento & purificación , Hepatocitos/metabolismo , Hepatocitos/patología , Interferón-alfa/metabolismo , Tetraspanina 28/fisiología , Tetraspanina 29/fisiología , Tetraspaninas/fisiología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Estudios de Casos y Controles , Comunicación Celular/fisiología , Línea Celular Tumoral , Técnicas de Cocultivo , Células Dendríticas/fisiología , Endocitosis/fisiología , Hepatitis C/metabolismo , Hepatitis C/patología , Hepatitis C/virología , Hepatocitos/virología , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/virología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Microdominios de Membrana/fisiología , Replicación Viral/fisiologíaRESUMEN
Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis of in vitro exchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists in Cd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.
Asunto(s)
Membrana Celular/fisiología , Fertilización/fisiología , Oocitos/fisiología , Oocitos/ultraestructura , Espermatozoides/ultraestructura , Tetraspanina 29/fisiología , Reacción Acrosómica , Animales , Membrana Celular/química , Femenino , Masculino , Ratones , Microscopía Electrónica , Capacitación Espermática , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/fisiología , Tetraspanina 29/deficiencia , Tetraspaninas/análisisRESUMEN
OBJECTIVE: To determine the physiological role of IGSF8 for fertility. DESIGN: Experimental prospective study. SETTING: Academic basic research laboratory. ANIMAL(S): C57BL/6J and hybrid B6D2F1 mice, as well as Cd9 and Igsf8 knockout mice (C57BL/6J and 129/SvJ mix background), were used for this study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): In vitro and in vivo fertility tests of Igsf8 knockout mice. RESULT(S): Tetraspanin family member CD9 plays an important role in sperm-egg fusion. Recently, some researchers have reported that CD9 tightly associates with the immunoglobulin superfamily member IGSF8 on the egg surface and that IGSF8 is undetectable on the surface of Cd9-deficient eggs. This led us to hypothesize that IGSF8 participates in sperm-egg fusion together with CD9. To examine the physiological role of IGSF8 in vivo, we generated Igsf8-deficient mice by homologous recombination and examined the fertility of the females. CONCLUSION(S): The Igsf8-deficient female mice showed no fertilization defect in vitro or in vivo. We observed that Igsf8-deficient eggs retained the normal level and localization of CD9, resulting in normal microvilli formation, which indicates that IGSF8 is dispensable in fertility.
Asunto(s)
Proteínas Portadoras/metabolismo , Fertilidad/fisiología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Tetraspaninas/metabolismo , Animales , Proteínas Portadoras/fisiología , Femenino , Humanos , Masculino , Proteínas de la Membrana/fisiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Estudios Prospectivos , Tetraspanina 29/deficiencia , Tetraspanina 29/metabolismo , Tetraspanina 29/fisiología , Tetraspaninas/fisiologíaRESUMEN
Cutaneous injury triggers a cascade of signaling events essential for wound re-epithelialization. CD9, a cell-surface protein, has been implicated in a number of cellular processes by coupling to intracellular signaling; however, its exact role in wound healing remains unidentified. We reported that CD9 was downregulated in migrating epidermis, and reelevated to basal level when re-epithelialization was completed. Although low level of CD9 appears to be required for normal wound healing, a significant healing delay was found in CD9-null mice, with wounds gaping wider on day 5 and day 7 post wounding. Further analysis showed that re-epithelialization was adversely affected in CD9-null mice, due to impaired migration of epidermis. Notably, CD9 deficiency caused a persistent enhancement of C-JUN NH2 terminal kinase (JNK) signaling primarily in migrating epidermis with abnormal elevation of matrix metalloproteinase (MMP)-9 detected in CD9-null wounds, leading to excessive degradation of type IV collagen, and thus a defective basement membrane at the wound site. JNK suppression reduced MMP-9 production and therefore ameliorated the healing delay with the appearance of significantly elongated migrating epidermis in CD9-null mice. Our study demonstrated the importance of CD9 in wound re-epithelialization, linking this molecule directly to basement membrane formation and epidermal migration through participating in the regulation of the JNK/MMP-9 pathway.