RESUMEN
BACKGROUND: The suppressor of cytokine signaling 1 (SOCS1) gene is repressed in prostate cancer (PCa) by epigenetic silencing and microRNA miR30d. Increased expression of the SOCS1-targeting miR30d correlates with higher biochemical recurrence, suggesting a tumor suppressor role of SOCS1 in PCa, but the underlying mechanisms are unclear. We have shown that SOCS1 inhibits MET receptor kinase signaling, a key oncogenic pathway in cancer progression. Here we evaluated the role of SOCS1 in attenuating MET signaling in PCa cells and tumor growth in vivo. METHODS: MET-overexpressing human DU145 and PC3 PCa cell lines were stably transduced with SOCS1, and their growth, migration and invasion of collagen matrix were evaluated in vitro. Cells expressing SOCS1 or the control vector were evaluated for tumor growth in NOD.scid.gamma mice as xenograft or orthotopic tumors. RESULTS: HGF-induced MET signaling was attenuated in SOCS1-expressing DU145 and PC3 cells. Compared with vector control cells, SOCS1-expressing cells showed reduced proliferation and impaired migration following HGF stimulation. DU145 and PC3 cells showed marked ability to invade the collagen matrix following HGF stimulation and this was attenuated by SOCS1. As xenografts, SOCS1-expressing PCa cells showed significantly reduced tumor growth compared with vector control cells. In the orthotopic tumor model, SOCS1 reduced the growth of primary tumors and metastatic spread. Intriguingly, the SOCS1-expressing DU145 and PC3 tumors showed increased collagen deposition, associated with increased frequency of myofibroblasts. CONCLUSIONS: Our findings support the tumor suppressor role of SOCS1 in PCa and suggest that attenuation of MET signaling is one of the underlying mechanisms. SOCS1 in PCa cells also appears to prevent the tumor-promoting functions of cancer-associated fibroblasts.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Células del Estroma/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Colágeno/metabolismo , Metilación de ADN , Modelos Animales de Enfermedad , Epigénesis Genética , Expresión Génica , Factor de Crecimiento de Hepatocito/metabolismo , Xenoinjertos , Humanos , Masculino , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Células del Estroma/patología , Proteína 1 Supresora de la Señalización de Citocinas/genética , Carga Tumoral , Microambiente TumoralRESUMEN
The SOCS1 gene coding for suppressor of cytokine signaling 1 is frequently repressed in hepatocellular carcinoma (HCC), and hence SOCS1 is considered a tumor suppressor in the liver. However, the tumor-suppressor mechanisms of SOCS1 are not yet well understood. SOCS1 is known to inhibit pro-inflammatory cytokine production and signaling and to promote activation of the p53 tumor suppressor. However, we observed that SOCS1-deficient mice developed numerous and large liver tumor nodules following treatment with the hepatocarcinogen diethylnitrosamine (DEN) without showing increased interleukin-6 production or activation of p53. On the other hand, the livers of DEN-treated Socs1-null mice showed elevated levels of p21(CIP1/WAF1) protein (p21). Even though p21 generally functions as a tumor suppressor, paradoxically many cancers, including HCC, are known to express elevated levels of p21 that correlate with poor prognosis. We observed elevated p21 expression also in the regenerating livers of SOCS1-deficient mice and in cisplatin-treated Socs1-null hepatocytes, wherein the p21 protein showed increased stability. We show that SOCS1 interacts with p21 and promotes its ubiquitination and proteasomal degradation. Besides, the DEN-treated livers of Socs1-null mice showed increased nuclear and cytosolic p21 staining, and the latter was associated with growth factor-induced, phosphatidylinositol 3-kinase-dependent phosphorylation of p21 in SOCS1-deficient hepatocytes. Cytosolic p21 is often associated with malignancy and chemo-resistance in many cancers. Accordingly, SOCS1-deficient hepatocytes showed increased resistance to apoptosis that was reversed by shRNA-mediated p21 knockdown. In the regenerating livers of Socs1-null mice, increased p21 expression coincided with elevated cyclinD levels. Correspondingly, SOCS1-deficient hepatocytes showed increased proliferation to growth factor stimulation that was reversed by p21 knockdown. Overall, our findings indicate that the tumor-suppressor functions of SOCS1 in the liver could be mediated, at least partly, via regulation of the expression, stability and subcellular distribution of p21 and its paradoxical oncogenic functions, namely, resistance to apoptosis and increased proliferation.
Asunto(s)
Carcinoma Hepatocelular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Oncogenes , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , ADN/biosíntesis , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Estabilidad Proteica , Transporte de Proteínas/efectos de los fármacos , Proteína 1 Supresora de la Señalización de Citocinas/deficiencia , Proteína 1 Supresora de la Señalización de Citocinas/genética , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
Suppressor of cytokine signaling 1 (SOCS1) is considered as a tumor suppressor protein in hepatocellular carcinoma (HCC), but the underlying mechanisms remain unclear. Previously, we have shown that SOCS1-deficient hepatocytes displayed increased responsiveness to hepatocyte growth factor (HGF) due to enhanced signaling via the MET receptor tyrosine kinase. As aberrant MET activation occurs in many tumors including HCC, here we elucidated the mechanisms of SOCS1-mediated regulation. SOCS1 attenuated HGF-induced proliferation of human and mouse HCC cell lines and their growth as tumors in NOD.scid.gamma mice. Tumors formed by SOCS1 expressing HCC cells showed significantly reduced MET expression, indicating that SOCS1 not only attenuates MET signaling but also regulates MET expression. Mechanistically, SOCS1 interacted with MET via the Src homology 2 domain and this interaction was promoted by MET tyrosine kinase activity. The SOCS1-mediated reduction in MET expression does not require the juxtamembrane Y1003 residue implicated in Cbl-mediated downmodulation. Moreover, the proteasome inhibitor MG-132, but not the inhibitors of lysosomal degradation bafilomycin and chloroquine, reversed the SOCS1-mediated reduction in MET expression, indicating that this process is distinct from Cbl-mediated downmodulation. Accordingly, SOCS1 promoted polyubiquitination of MET via K48-dependent but not K63-mediated ubiquitin chain elongation. Furthermore, siRNA-mediated downmodulation of Cbl did not abolish SOCS1-mediated reduction in MET expression in HCC cells. SOCS1-dependent ubiquitination of endogenous MET receptor occurred rapidly following HGF stimulation in HCC cells, leading to proteasomal degradation of phosphorylated MET receptor. These findings indicate that SOCS1 mediates its tumor suppressor functions, at least partly, by binding to MET and interfering with downstream signaling pathways as well as by promoting the turnover of the activated MET receptor. We propose that loss of this control mechanism due to epigenetic repression of SOCS1 could contribute to oncogenic MET signaling in HCC and other cancers, and that MET inhibitors might be useful in treating these patients.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Células COS , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular , Chlorocebus aethiops , Cloroquina/farmacología , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células Hep G2 , Humanos , Leupeptinas/farmacología , Neoplasias Hepáticas/patología , Ratones , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 1 Supresora de la Señalización de CitocinasRESUMEN
Non-obese diabetic (NOD) mice lacking interleukin (IL)-21 or IL-21 receptor do not develop autoimmune type 1 diabetes (T1D). We have shown recently that IL-21 may promote activation of autoreactive CD8(+) T cells by increasing their antigen responsiveness. To investigate the role of IL-21 in activating diabetogenic CD8(+) T cells in the NOD mouse, we generated IL-21-deficient NOD mice expressing the highly pathogenic major histocompatibility complex (MHC) class-I-restricted 8.3 transgenic T cell receptor (TCR). IL-21 deficiency protected 8.3-NOD mice completely from T1D. CD8(+) T cells from the 8.3-NOD.Il21(-/-) mice showed decreased antigen-induced proliferation but displayed robust antigen-specific cytolytic activity and production of effector cytokines. IL-21-deficient 8.3 T cells underwent efficient homeostatic proliferation, and previous antigen stimulation enabled these cells to cause diabetes in NOD.Scid recipients. The 8.3 T cells that developed in an IL-21-deficient environment showed impaired antigen-specific proliferation in vivo even in IL-21-sufficient mice. These cells also showed impaired IL-2 production and Il2 gene transcription following antigen stimulation. However, IL-2 addition failed to reverse their impaired proliferation completely. These findings indicate that IL-21 is required for efficient initial activation of autoreactive CD8(+) T cells but is dispensable for the activated cells to develop effector functions and cause disease. Hence, therapeutic targeting of IL-21 in T1D may inhibit activation of naive autoreactive CD8(+) T cells, but may have to be combined with other strategies to inhibit already activated cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Interleucinas/inmunología , Animales , Autoantígenos/inmunología , Proliferación Celular , Células Cultivadas , Citotoxicidad Inmunológica/genética , Interleucinas/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Transgénicos , Terapia Molecular Dirigida , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
AIMS/HYPOTHESIS: IL-15, induced by innate immune stimuli, promotes rheumatoid arthritis and inflammatory bowel disease. However, its role in autoimmune type 1 diabetes is unclear. Our aim is to define the role of IL-15 in the pathogenesis of diabetes in the NOD mouse model. METHODS: We generated NOD.Il15(-/-) mice expressing a polyclonal repertoire of T cell antigen receptor (TCR) or a transgenic TCR and monitored diabetes onset and insulitis. NOD Scid.Il15(-/-) (full name NOD.CB17-Prkdc (scid)/NCrCrl) and NOD Scid.gamma (full name NOD.Cg-Prkdc(scid) Il2rg ( tm1Wjl )/SzJ) mice were used to distinguish the requirement for IL-15 signalling in CD8(+) T cells and antigen-presenting cells (APCs) to induce disease. We examined the effect of blocking IL-15 signalling on diabetes onset in NOD mice. RESULTS: At 7 months of age, more than 75% of the NOD Il15(-/-) female mice remained diabetes free compared with only 30% in the control group. Diabetes incidence was also decreased in 8.3-NOD (full name NOD Cg-Tg[TcraTcrbNY8.3]-1Pesa/DvsJ).Il15(-/-) mice expressing a highly pathogenic transgenic TCR on CD8(+) T cells. Adoptive transfer of splenocytes from diabetic NOD and 8.3-NOD donors induced disease in NOD Scid recipients but not in NOD Scid.Il15(-/-) or NOD Scid.gamma mice. Transient blockade of IL-15 signalling at the onset of insulitis prevented diabetes in NOD mice. CONCLUSIONS/INTERPRETATION: Our results show that IL-15 is needed for the initial activation of diabetogenic CD8(+) T cells as well as for sustaining the diabetogenic potential of antigen-stimulated cells, acting on both CD8(+) T cells and on APCs. Our findings demonstrate a critical role for IL-15 in the pathogenesis of autoimmune diabetes and suggest that IL-15 is a promising therapeutic target.
Asunto(s)
Autoinmunidad/inmunología , Diabetes Mellitus Tipo 1/inmunología , Interleucina-15/genética , Interleucina-15/inmunología , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Comunicación Celular/inmunología , Supervivencia Celular/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animales de Enfermedad , Femenino , Interleucina-15/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Estado Prediabético/inmunología , Estado Prediabético/metabolismo , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/citologíaRESUMEN
Lipoarabinomannans (LAMs) are major lipoglycans of the mycobacterial envelope and constitute immunodominant epitopes of mycobacteria. In this paper, we show that mannose-capped (ManLAM) and non-mannose-capped (PILAM) mycobacterial lipoglycans insert into T helper cell rafts without apparent binding to known receptors. T helper cells modified by the insertion of PILAM responded to CD3 cross-linking by decreasing type 1 (IL-2 and IFN-gamma) and increasing type 2 (IL-4 and IL-5) cytokine production. Modification by the mannose-capped ManLAMs had similar, but more limited effects on T helper cell cytokine production. When incorporated into isolated rafts, PILAMs modulated membrane-associated kinases in a dose-dependent manner, inducing increased phosphorylation of Src kinases and Cbp/PAG in Th1 rafts, while decreasing phosphorylation of the same proteins in Th2 rafts. Mycobacterial lipoglycans thus modify the signalling machineries of rafts/microdomains in T helper cells, a modification of the membrane organization that eventually leads to an overall enhancement of type 2 and inhibition of type 1 cytokine production.
Asunto(s)
Citocinas/biosíntesis , Lipopolisacáridos/farmacología , Microdominios de Membrana/metabolismo , Mycobacterium/inmunología , Células TH1/inmunología , Células Th2/inmunología , Secuencia de Bases , Humanos , Interleucinas/metabolismo , Manosa/química , Manosa/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Microdominios de Membrana/química , Datos de Secuencia Molecular , Mycobacterium/química , Transducción de Señal , Células TH1/química , Células Th2/químicaRESUMEN
On the basis of clinical features and bacteriological status, macular skin lesions of nine cases of leprosy were classified as falling within a spectrum between the tuberculoid at one end and the lepromatous at the other. While histologic correlation was seen in 60% of cases, humoral and cellular systemic immunologic features were found to be uncharacteristic. It is suggested that macular lesions form an early stage in the development of leprosy where the systemic immunological response is yet to set in fully.
Asunto(s)
Antígenos Bacterianos , Lepra/inmunología , Lepra/patología , Adulto , Femenino , Glucolípidos/inmunología , Humanos , Hipopigmentación/patología , Lepra Tuberculoide/inmunología , Lepra Tuberculoide/patología , Masculino , Persona de Mediana Edad , Piel/patología , Nervio Cubital/patologíaRESUMEN
Suppressor of cytokine signaling-1 (SOCS1) is an inducible Src homology 2 (SH2)-containing protein that negatively regulates cytokine and growth factor signaling required during thymic development. Recent evidence indicates that SOCS1 interacts with elongins B and C, which are components of a ubiquitin ligase complex, VCB (VHL/elonginC/B), based on the VHL (von Hippel Lindau) tumor suppressor protein. SOCS1 has previously been shown to operate as an inhibitor of Janus kinases. Here we show that SOCS1 has the distinct function of targeting the hematopoietic specific guanine nucleotide exchange factor, VAV, for ubiquitin-mediated protein degradation. VAV and SOCS1 form a protein complex through interactions between the VAV NH(2)-terminal regulatory region and the SH2 domain of SOCS1 in a phosphotyrosine-independent manner. SOCS1 decreases the steady state levels of cotransfected VAV and onco-VAV and reduces the focus forming activity of onco-VAV. SOCS1 stimulates the polyubiquitination of VAV proteins in vivo, which was stabilized by proteasomal inhibitors. These results suggest that SOCS1 programs VAV degradation by acting as a substrate-specific recognition component of a VCB-like ubiquitin ligase complex.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Represoras , Transducción de Señal , Animales , Línea Celular , Humanos , Hidrólisis , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-vav , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Ubiquitinas/metabolismoRESUMEN
Transmembrane signaling requires modular interactions between signaling proteins, phosphorylation or dephosphorylation of the interacting protein partners and temporary elaboration of supramolecular structures, to convey the molecular information from the cell surface to the nucleus. Such signaling complexes at the plasma membrane are instrumental in translating the extracellular cues into intracellular signals for gene activation. In the most straightforward case, ligand binding promotes homodimerization of the transmembrane receptor which facilitates modular interactions between the receptor's cytoplasmic domains and intracellular signaling and adaptor proteins. For example, most growth factor receptors contain a cytoplasmic protein tyrosine kinase (PTK) domain and ligand-mediated receptor dimerization leads to cross phosphorylation of tyrosines in the receptor's cytoplasmic domains, an event that initiates the signaling cascade. In other signaling pathways where the receptors have no intrinsic kinase activity, intracellular nonreceptor PTKs (i.e. Src family PTKs, JAKs) are recruited to the cytoplasmic domain of the engaged receptor. Execution of these initial phosphorylations and their translation into efficient cellular stimulation requires concomitant activation of diverse signaling pathways. Availability of stable, preassembled matrices at the plasma membrane would facilitate scaffolding of a large array of receptors, coreceptors, tyrosine kinases and other signaling and adapter proteins, as it is the case in signaling via the T cell antigen receptor. The concept of the signaling platform has gained usage to characterize the membrane structure where many different membrane-bound components need to be assembled in a coordinated manner to carry out signaling. The structural basis of the signaling platform lies in preferential assembly of certain classes of lipids into distinct physical and functional compartments within the plasma membrane. These membrane microdomains or rafts (Figure 1) serve as privileged sites where receptors and proximal signaling molecules optimally interact. In this review, we shall discuss first how signaling platforms are assembled and how receptors and their signaling machinery could be functionally linked in such structures. The second part of our review will deal with selected examples of raft-based signaling pathways in T lymphocytes and NK cells to illustrate the ways in which rafts may facilitate signaling.
Asunto(s)
Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Transducción de Señal , Esfingolípidos/metabolismo , Animales , Humanos , Células Asesinas Naturales/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
CD44 is the principal cell surface receptor for extracellular matrix glycosaminoglycan hyaluronan. CD44-hyaluronan mediated cell adhesion is important in several pathophysiological processes such as inflammation and metastatic spread of cancer cells. It has been recently recognized that CD44 also functions as a signaling receptor in a variety of cell types. Cell stimulation by monoclonal anti-CD44 antibody or natural CD44 ligands activate several signaling pathways that culminate in cell proliferation, cytokine secretion, chemokine gene expression and cytolytic effector functions. One of the earliest signaling events following stimulation via CD44 is tyrosine phosphorylation of intracellular proteins substrates, and CD44 mediated cellular activation could be abolished by protein tyrosine kinase (PTK) inhibitors. The Src-family non-receptor PTKs such as Lck, Fyn, Lyn and Hck were shown to be coupled to CD44 via sphingolipid-rich microdomains (lipid rafts) of the plasma membrane. Studies on T cell receptor and IgE receptor mediated signaling in lymphocytes and mast cells have consolidated the notion that microdomains consist of signaling platforms where components of multiple signaling pathways are assembled. Co-isolation of CD44 with microdomains strongly suggests that CD44 generates cellular activation signals utilizing the signaling machinery of the plasma membrane microdomains.
Asunto(s)
Receptores de Hialuranos/fisiología , Proteínas de la Membrana/fisiología , Transducción de Señal/fisiología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Adhesión Celular/efectos de los fármacos , Citocinas/fisiología , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Predicción , Humanos , Receptores de Hialuranos/inmunología , Ácido Hialurónico/farmacología , Ácido Hialurónico/fisiología , Ligandos , Proteínas de la Membrana/química , Ratones , Proteínas de Neoplasias/fisiología , Neoplasias/inmunología , Neoplasias/fisiopatología , Fosforilación , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/fisiología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Superficie Celular/fisiología , Receptores de IgE/inmunología , Solubilidad , Linfocitos T Citotóxicos/metabolismoRESUMEN
Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/fisiología , Animales , Anticuerpos Monoclonales , Membrana Celular/fisiología , Dipeptidil Peptidasa 4/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Cinética , Antígenos Comunes de Leucocito/metabolismo , Linfoma de Células T , Lípidos de la Membrana/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-fyn , Ratas , Transducción de Señal , Esfingolípidos/metabolismo , Células Tumorales CultivadasRESUMEN
Sphingolipid microdomains are thought to result from the organization of plasma membrane sphingolipids and cholesterol into a liquid ordered phase, wherein the glycosylphosphatidylinositol (GPI)-anchored proteins are enriched. These domains, resistant to extraction by cold Triton X-100, can be isolated as buoyant membrane complexes (detergent-resistant membranes) in isopycnic density gradients. Here the effects of methyl-beta-cyclodextrin (MBCD), a specific cholesterol-binding agent that neither binds nor inserts into the plasma membrane, were investigated on the sphingolipid microdomains of lymphocytes. MBCD released substantial quantities of GPI-anchored Thy-1 and glycosphingolipid GM1, and also other surface proteins including CD45, and intracellular Lck and Fyn kinases. From endothelial cells, MBCD released GPI-anchored CD59, and CD44, but only a negligible amount of caveolin. Most MBCD-released Thy-1 and CD59 were not sedimentable and thus differed from Thy-1 released by membrane-active cholesterol-binding agents such as saponin and streptolysin O, or Triton X-100. Unlike that released by Triton X-100, only part of the Thy-1 molecules released by MBCD was buoyant in density gradients and co-isolated with GM1. Finally, treatment of Triton X-100-isolated detergent-resistant membranes with MBCD extracted most of the cholesterol without affecting the buoyant properties of Thy-1 or GM1. We suggest that (1) MBCD preferentially extracts cholesterol from outside, rather than within the sphingolipid microdomains and (2) this partly solubilizes GPI-anchored and transmembrane proteins from the glycerophospholipid-rich membrane and releases sphingolipid microdomains in both vesicular and non-vesicular form.
Asunto(s)
Caveolinas , Membrana Celular/metabolismo , Colesterol/metabolismo , Ciclodextrinas/farmacología , Esfingolípidos/metabolismo , beta-Ciclodextrinas , Animales , Antígenos CD59/metabolismo , Caveolina 1 , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Ciclodextrinas/metabolismo , Detergentes/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Gangliósido G(M1)/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Linfocitos/química , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Esfingolípidos/química , Antígenos Thy-1/metabolismoRESUMEN
CD44 is the major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is implicated in a variety of biological events that include embryonic morphogenesis, lymphocyte recirculation, inflammation, and tumor metastasis. CD44 delivers activation signals to T lymphocytes, B lymphocytes, natural killer cells, polymorphonuclear leukocytes, and macrophages by stimulating protein tyrosine phosphorylation and calcium influx. The mechanism of signal transduction via CD44 remains undefined, although CD44 was shown to physically associate with intracellular protein tyrosine kinase Lck in T lymphocytes. In the present report, we show that a significant proportion of CD44 in human peripheral blood T lymphocytes and endothelial cells is associated with low-density plasma membrane fractions that represent specialized plasma membrane domains enriched in glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins. CD44 and the GPI-anchored CD59 do not appear to directly interact in the low-density membrane fractions. In human peripheral blood T lymphocytes, 20% to 30% of the Src family protein tyrosine kinases, Lck and Fyn, are recovered from these fractions. CD44-associated protein kinase activity was selectively recovered from the low-density membrane fractions, corresponding to glycosphingolipid-rich plasma membrane microdomains. Reprecipitation of the in vitro phosphorylated proteins showed that CD44 associates not only with Lck but also with Fyn kinase in these membrane domains. Our results suggest that cellular stimulation via CD44 may proceed through the signaling machinery of glycosphingolipid-enriched plasma membrane microdomains and, hence, depend on the functional integrity of such domains.
Asunto(s)
Membrana Celular/metabolismo , Glicoesfingolípidos/metabolismo , Receptores de Hialuranos/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Linfocitos T/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Gangliósido G(M1)/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Humanos , Activación de Linfocitos/efectos de los fármacos , Sustancias Macromoleculares , Fosforilación , Fitohemaglutininas/farmacología , Proteínas Proto-Oncogénicas c-fyn , Linfocitos T/efectos de los fármacosRESUMEN
Glycosylphosphatidylinositol (GPI)-anchored glycoproteins are enriched in sphingolipid-rich plasma membrane domains, which are often isolated as low-density membrane complexes. This association is believed to arise from the interactions between the GPI-acyl chains and sphingolipids, but is not fully understood. In this study, we compared the physical properties of GPI-anchored glycoproteins from a non-polarized (murine T-lymphocyte) and a polarized (human endothelial) cell by equilibrium density gradient centrifugation after extraction by detergents under identical conditions. Unlike those on epithelial cells, the GPI-anchored proteins of lymphocytes (Thy-1 and the heat stable antigen CD24) were enriched in the floating fractions after extraction over a wide range of octylglucoside concentrations. In contrast, the floatability of endothelial GPI-anchored CD59 was markedly diminished, not only by octylglucoside, but also by increasing concentrations of Triton X-100. Distribution of cholera toxin binding ganglioside GM1 in the sucrose gradient fractions closely followed that of the GPI-anchored proteins in both lymphocytes and endothelial cells under most extraction conditions. Analysis of the intracellular acylated molecules revealed that a significant amount of p56(lck) was always associated with the floating GPI-rich fractions of lymphocytes when extracted by Triton X-100 or octylglucoside at 4 degrees C, while the behaviour of endothelial cell caveolin was comparable to that of CD59. The transmembrane glycoproteins CD45 in lymphocytes and MHC class I antigen in endothelial cells interacted weakly with GPI domains, whereas endothelial CD44 and lymphocyte CD26 displayed a strong association. These results show that: (1) the physical properties of different GPI-anchored proteins may vary significantly; and (2) transmembrane and acylated intracellular proteins could be associated with GPI domains to a variable extent. These differences probably reflect cell type-specific interactions of GPI anchors with the sphingolipid framework of plasma membranes, as well as extracellular interactions of GPI-anchored glycoproteins with neighbouring cell surface molecules.
Asunto(s)
Membrana Celular/química , Endotelio Vascular/química , Glicosilfosfatidilinositoles/análisis , Linfocitos/química , Proteínas de la Membrana/análisis , Esfingolípidos/análisis , Animales , Polaridad Celular , Centrifugación por Gradiente de Densidad , Endotelio Vascular/citología , Gangliósido G(M1)/análisis , Humanos , Linfocitos/citología , Ratas , Fracciones Subcelulares/químicaRESUMEN
The association of glycosylphosphatidylinositol (GPI)-anchored cell surface glycoproteins with Src-family protein tyrosine kinases was analysed in intact T lymphocyte plasma membranes. Following subcellular fractionation without detergent, 25% of the recovered plasma membranes were light density vesicles enriched in GPI-anchored glycoproteins and sphingolipids (GPI domains), while the remainder behaved as heavier density vesicles containing equal amounts of lipids and proteins. Qualitatively similar lipids were found in both vesicle types, but only light density vesicles made of 65-75% lipids yielded a Triton X-100 resistant, sedimentable fraction containing GPI-linked glycoproteins and sphingolipids. The GPI-rich vesicles phosphotyrosylated an exogenous substrate as efficiently as the denser vesicles, despite a low Lck and Fyn kinase content. Likewise, these kinases were more efficiently phosphorylated in GPI domains than in denser vesicles. GPI domains thus could constitute plasma membrane "hot spots" where associated Src kinases assume an optimally active conformation that contributes to signaling via GPI-anchored cell surface glycoproteins.
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Glicosilfosfatidilinositoles/metabolismo , Linfocitos T/metabolismo , Familia-src Quinasas/metabolismo , Animales , Fraccionamiento Celular , Membrana Celular/química , Membrana Celular/metabolismo , Glicosilfosfatidilinositoles/análisis , Lípidos de la Membrana/análisis , Lípidos de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Transducción de SeñalRESUMEN
Purified GPI-linked molecules incorporate spontaneously in vitro into mammalian cell plasma membranes. Recent evidence suggests that the transferred molecules insert stably into the external leaflet of the acceptor cell plasma membrane through their acyl chains and behave subsequently in a way similar to endogenous GPI-linked molecules. Transfer of GPI-linked proteins between cells has also been documented in vivo and may explain the uptake by host cells o f pathogen-derived virulence factors carrying a GPI anchor. In this comment article, Subburaj Ilangumaran, Peter Robinson and Daniel Hoessli review what is known about GPI transfer and discuss the use of GPI transfer for transient cell-surface expression of foreign proteins.
RESUMEN
In this study, we measured simultaneously the in vitro and in vivo T lymphocyte reactivities and the antibody responses of leprosy patients and healthy family contacts (HFC) toward Mycobacterium leprae antigens. The in vitro lymphoproliferative response of the HFC to leprosin A was comparable to that of tuberculoid leprosy patients. However, their skin-test reactivity to Dharmendra lepromin was considerably higher compared to the in vitro response to leprosin A. A significant number of HFC failed to respond to M. leprae antigens, both in vitro and in vivo, and the unresponsiveness to either test was not related to the type of leprosy patients in the household. A marginal correlation was observed between the skin-test reactivity of HFC and the age of the individuals. Even though a significant proportion of HFC showed positive anti-PGL-I IgM levels, none showed a positive titer in the serum antibody competition test toward the M. leprae-specific epitope My2. A positive anti-PGL-I IgM response together with a negative lepromin skin-test reactivity showed a clear downward trend from the lepromatous pole toward the tuberculoid pole. A small number of HFC, all contacts of lepromatous patients, were lepromin skin-test negative with positive anti-PGL-I IgM levels, but the majority among them showed T-cell reactivity to mycobacterial antigens in vitro. These results are discussed in relation to immunological correlates of the susceptibility to M. leprae infection.
Asunto(s)
Antígenos Bacterianos/inmunología , Lepra/inmunología , Mycobacterium leprae/inmunología , Anticuerpos Antibacterianos/sangre , Glucolípidos/inmunología , Humanos , Inmunoglobulina M/sangre , Lepromina/inmunología , Lepra/transmisión , Linfocitos T/inmunologíaRESUMEN
The dot-immunoassay has been adapted for rapid detection and partial characterization of glycosylphosphatidylinositol (GPI)-linked, transmembrane, and intracellular proteins in Triton X-100 (TX-100) extracts of lymphoma cells and intestinal tissue. The GPI-anchored proteins tend to concentrate into specialized plasma membrane domains enriched in glycosphingolipids. The dot-immunoassay has been successfully used to demonstrate the differential distribution of GPI-linked and transmembrane surface glycoproteins of T lymphocytes in sucrose density gradient fractions of TX-100 lysate. The type II transmembrane protein CD26 and the intracellular tyrosine kinase p56lck partially cofractionated with GPI-linked glycoproteins, and the extent to which they partition into GPI-rich plasma membrane domains could be evaluated. Preferential association of the acidic glycosphingolipid GM1 with these domains could be demonstrated by cholera toxin binding directly to the dot-blotted sucrose density gradient fractions. Treatment of whole cell TX-100 lysates or sucrose gradient fractions dotted onto nitrocellulose filter strips with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) proved to be an efficient method to assay for the presence of a GPI-anchor in Thy-1 and Ly6 surface glycoproteins. We have used three criteria, namely flotation to light density fractions in sucrose gradients, colocalization with GM1, and sensitivity to PI-PLC cleavage, to assess the presence of a GPI modification in a putative GPI-linked protein in intestinal tissue extract. It is envisaged that the techniques described in this report would find a wider application to rapidly assess the contents of GPI-rich plasma membrane domains in different cells and tissues.