RESUMEN
Human herpes virus 8 (HHV-8) or Kaposi sarcoma-associated herpes virus is the etiologic agent of Kaposi sarcoma, primary effusion lymphoma, and plasma cell-type multicentric Castleman disease (MCD). HHV-8 encodes a viral homolog of human IL-6, called viral IL-6 (vIL-6), which does not require the cellular IL-6 receptor for binding to the ubiquitously expressed gp130 receptor subunit and subsequent JAK-STAT signaling. Thus, in contrast to IL-6, vIL-6 can stimulate virtually all cells in the body. To elucidate the mechanism by which vIL-6 drives human diseases, we generated transgenic mice that constitutively express vIL-6 under control of the MHC class I promoter. The mice were found to exhibit vIL-6 serum levels comparable with those observed in HHV-8-infected patients, to contain elevated amounts of phosphorylated STAT3 in spleen and lymph nodes, where vIL-6 was produced, and to spontaneously develop key features of human plasma cell-type MCD, including splenomegaly, multifocal lymphadenopathy, hypergammaglobulinemia, and plasmacytosis. Transfer of the vIL-6 transgene onto an IL-6-deficient genetic background abrogated MCD-like phenotypes, indicating that endogenous mouse IL-6 is a crucial cofactor in the natural history of the disease. Our results in mice suggest that human IL-6 plays an important role in the pathogenesis of HHV-8-associated MCD.
Asunto(s)
Enfermedad de Castleman/inmunología , Enfermedad de Castleman/patología , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 8/inmunología , Interleucina-6/inmunología , Proteínas Virales/inmunología , Animales , Enfermedad de Castleman/genética , Enfermedad de Castleman/virología , Receptor gp130 de Citocinas/genética , Receptor gp130 de Citocinas/inmunología , Infecciones por Herpesviridae/genética , Herpesvirus Humano 8/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Interleucina-6/genética , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/inmunología , Proteínas Virales/genéticaRESUMEN
Ligand-independent constitutively active gp130 mutants were described to be responsible for the development of inflammatory hepatocellular adenomas (IHCAs). These variants had gain-of-function somatic mutations within the extracellular domain 2 (D2) of the gp130 receptor chain. Cytokine-dependent Ba/F3 cells were transduced with the constitutively active variant of gp130 featuring a deletion in the domain 2 from Tyr-186 to Tyr-190 (gp130ΔYY). These cells showed constitutive phosphorylation of signal transducer and activator of transcription-3 (STAT3) and cytokine-independent proliferation. Deletion of the Ig-like domain 1 (D1) of gp130, but not anti-gp130 mAbs directed against D1, abolished constitutive activation of gp130ΔYY, highlighting that this domain is involved in ligand-independent activation of gp130ΔYY. Moreover, soluble variants of gp130 were not able to inhibit the constitutive activation of gp130ΔYY. However, the inhibition of constitutive activation of gp130ΔYY was achieved by the anti-gp130 mAb B-P4, which specifically inhibits gp130 signaling by IL-11 but not by other IL-6 type cytokines. IL-11 but not IL-6 levels were found previously to be up-regulated in IHCAs, suggesting that mutations in gp130 are leading to IL-11-like signaling. The mAb B-P4 might be a valuable tool to inhibit the constitutive activation of naturally occurring gp130 mutants in IHCAs and rare cases of gp130-associated hepatocellular carcinoma.
Asunto(s)
Adenoma de Células Hepáticas/metabolismo , Receptor gp130 de Citocinas/metabolismo , Interleucina-11/metabolismo , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/farmacología , Células COS , Proliferación Celular , Chlorocebus aethiops , Receptor gp130 de Citocinas/inmunología , Citocinas/metabolismo , Citometría de Flujo/métodos , Eliminación de Gen , Humanos , Interleucina-6/metabolismo , Ligandos , Ratones , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
N-Linked glycosylation is an important determinant of protein structure and function. The interleukin-6 signal transducer glycoprotein 130 (gp130) is a common co-receptor for cytokines of the interleukin (IL)-6 family and is N-glycosylated at 9 of 11 potential sites. Whereas N-glycosylation of the extracellular domains D1-D3 of gp130 has been shown to be dispensable for binding of the gp130 ligand IL-6 and its cognate receptor in vitro, the role of the N-linked glycans on domains D4 and D6 is still unclear. We have mutated the asparagines of all nine functional N-glycosylation sites of gp130 to glutamine and systematically analyzed the consequences of deleted N-glycosylation (dNG) in both cellular gp130 and in a soluble gp130-IgG1-Fc fusion protein (sgp130Fc). Our results show that sgp130Fc-dNG is inherently unstable and degrades rapidly under conditions that do not harm wild-type sgp130Fc. Consistently, the bulk of cellular gp130-dNG is not transported to the plasma membrane but is degraded in the proteasome. However, the small quantities of gp130-dNG, which do reach the cell surface, are still able to activate the key gp130 signaling target signal transducer and activator of transcription-3 (STAT3) upon binding of the agonistic complex of IL-6 and soluble IL-6 receptor. In conclusion, N-linked glycosylation is required for the stability but not the signal-transducing function of gp130.
Asunto(s)
Receptor gp130 de Citocinas/química , Receptor gp130 de Citocinas/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Interleucina-6/metabolismo , Nitrógeno/metabolismo , Transducción de Señal , Animales , Línea Celular , Cricetinae , Cricetulus , Receptor gp130 de Citocinas/genética , Glicoproteínas/genética , Glicosilación , Humanos , Inmunoglobulina G/metabolismo , Ligandos , Ratones , Oxígeno/metabolismo , Estabilidad Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción STAT3/metabolismo , Eliminación de SecuenciaRESUMEN
Human herpesvirus 8 encodes a viral version of interleukin-6 (vIL-6) which shows 25% sequence homology with human IL-6. In contrast to human IL-6, which first binds to the IL-6 receptor (IL-6R) and only subsequently associates with the signal transducing receptor subunit gp130, vIL-6 has been shown to directly bind to gp130 without the need of IL-6R. As a functional consequence, vIL-6 can activate far more target cells in the body since all cells express gp130, but only cells such as hepatocytes and some leukocytes express IL-6R. We sought to understand which amino acid sequences within the vIL-6 protein were responsible for its ability to bind and activate gp130 independent of IL-6R. As a first approach, we constructed chimeric IL-6 proteins in which all known gp130 interacting sites (sites II and III) were sequentially transferred from vIL-6 into the human IL-6 protein. To our surprise, human IL-6 carrying all gp130 interacting sites from vIL-6 did not show IL-6R-independent gp130 activation. Even more surprisingly, the loop between helix B and C of vIL-6, clearly shown in the crystal structure not to be in contact with gp130, is indispensable for direct binding to and activation of gp130. This points to an IL-6R induced change of site III conformation in human IL-6, which is already preformed in vIL-6. These data indicate a novel activation mechanism of human IL-6 by the IL-6R that will be important for the construction of novel hyperactive cytokine variants.
Asunto(s)
Receptor gp130 de Citocinas/metabolismo , Herpesvirus Humano 8/metabolismo , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Herpesvirus Humano 8/genética , Humanos , Ratones , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/genéticaRESUMEN
Shrew-1 was previously isolated from an endometriotic cell line in our search for invasion-associated genes. It proved to be a membrane protein that targets to the basolateral membrane of polarized epithelial cells, interacting with E-cadherin-catenin complexes of adherens junctions. Paradoxically, the existence of adherens junctions is incompatible with invasion. To investigate whether shrew-1 can indeed influence cellular invasion, we overexpressed it in HT1080 fibrosarcoma cells. This resulted in enhanced invasiveness, accompanied by an increased matrix metalloprotease (MMP)-9 level in the supernatant, raising the question about the role of shrew-1 in this process. Logic suggested we looked for an interaction with CD147, a known promoter of invasiveness and MMP activity. Indeed, genetics-based, biochemical, and microscopy experiments revealed shrew-1- and CD147-containing complexes in invasive endometriotic cells and an interaction in epithelial cells, which was stronger in MCF7 tumor cells, but weaker in Madin-Darby canine kidney cells. In contrast to the effect mediated by overexpression, small interfering RNA-mediated down-regulation of either shrew-1 or CD147 in HeLa cells decreased invasiveness without affecting the proliferation behavior of HeLa cells, but the knockdown cells displayed decreased motility. Altogether, our results imply that shrew-1 has a function in the regulation of cellular invasion, which may involve its interaction with CD147.
Asunto(s)
Basigina/metabolismo , Movimiento Celular , Proteínas de la Membrana/metabolismo , Animales , Secuencia de Bases , Basigina/genética , Moléculas de Adhesión Celular , Células Cultivadas , Perros , Endometriosis/patología , Femenino , Humanos , Inmunoprecipitación , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de la Membrana/genética , Microscopía Fluorescente/métodos , Datos de Secuencia Molecular , Invasividad Neoplásica , Fragmentos de Péptidos/metabolismo , ARN Interferente Pequeño , Ubiquitina/metabolismo , Levaduras/genética , Levaduras/metabolismoRESUMEN
Viral Interleukin-6 (vIL-6) is encoded by Human herpes virus 8 (HHV8), also known as Kaposi's sarcoma (KS)-associated herpes virus (KSHV). HHV8 infection is found in patients with KS, primary effusion lymphoma (PEL) and plasma cell-type of multicentric Castleman's disease (MCD), with a high incidence observed in HIV infected individuals. vIL-6 shares about 25% identity with its human counterpart. Human IL-6 (hIL-6) binds to the human IL-6 receptor (hIL-6R) and the hIL-6/hIL-6R complex associates with the signaling receptor subunit gp130. Upon dimerization of gp130 intracellular signaling is initiated. All cells in the body express gp130 but only some cell types express the hIL-6R. Human IL-6 does not stimulate cells, which do not express hIL-6R. However, a naturally occurring soluble form of the hIL-6R (shIL-6R) can bind hIL-6 and the complex of hIL-6/shIL-6R can stimulate cells, which only express gp130 but no hIL-6R. This process, which has been named trans-signaling, leads to a dramatic increase in the spectrum of hIL-6 target cells during inflammation and cancer. vIL-6, in contrast to hIL-6, can directly bind to and activate gp130 without the need of the hIL-6R. Therefore, at least in theory, vIL-6 can stimulate every cell in the human body. This review highlights the properties of vIL-6 regarding structural features, implications for pathophysiology, and strategies of neutralization. Furthermore, mechanisms of activation of gp130 by hIL-6, vIL-6, and by forced dimerization will be discussed.
Asunto(s)
Herpesvirus Humano 8/metabolismo , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Animales , Receptor gp130 de Citocinas/inmunología , Receptor gp130 de Citocinas/metabolismo , Herpesvirus Humano 8/química , Herpesvirus Humano 8/inmunología , Humanos , Interleucina-6/química , Interleucina-6/inmunología , Ratones , Receptores de Interleucina-6/química , Receptores de Interleucina-6/inmunología , Transducción de SeñalRESUMEN
Naturally ligand independent constitutively active gp130 variants were described to be responsible for inflammatory hepatocellular adenomas. Recently, we genetically engineered a ligand-independent constitutively active gp130 variant based on homodimerization of Jun leucine zippers. Because also heterodimeric complexes within the gp130 family may have tumorigenic potential, we seek to generate ligand-independent constitutively active heterodimers for all known gp130-receptor complexes based on IL-15/IL-15R alpha-sushi fusion proteins. Ligand-independent heterodimerization of gp130 with WSX-1, LIFR, and OSMR and of OSMR with GPL led to constitutive, ligand-independent STAT1 and/or STAT3 and ERK1/2 phosphorylation. Moreover, these receptor combinations induced transcription of the STAT3 target genes c-myc and Pim-1 and factor-independent growth of stably transduced Ba/F3-gp130 cells. Here, we establish the IL-15/IL-15R alpha-sushi system as a new system to mimic constitutive and ligand-independent activation of homo- and heterodimeric receptor complexes, which might be applicable to other heterodimeric receptor families. A mutated IL-15 protein, which was still able to bind the IL-15R alpha-sushi domain, but not to beta- and gamma-receptor chains, in combination with the 2A peptide technology may be used to translate our in vitro data into the in vivo situation to assess the tumorigenic potential of gp130-heterodimeric receptor complexes.