RESUMEN
Neuromedin U (NmU) is a neuropeptide belonging to the neuromedin family. Recently, we reported a significant association between NmU and breast cancer, particularly correlating with increased aggressiveness, resistance to HER2-targeted therapies and overall significantly poorer outcome for patients, although the mechanism through which it exerts this effect remained unexplained. Investigating this, here we found that ectopic over-expression of NmU in HER2-positive breast cancer cells induced aberrant metabolism, with increased glycolysis, likely due to enhanced pyruvate dehydrogenase kinase activity. Similar results were observed in HER2-targeted drug-resistant cell variants, which we had previously shown to display increased levels of NmU. Overexpression of NmU also resulted in upregulation of epithelial-mesenchymal transition markers and increased IL-6 secretion which, together with aberrant metabolism, have all been associated with the cancer stem cell (CSC) phenotype. Flow cytometry experiments confirmed that NmU-overexpressing and HER2-targeted drug-resistant cells showed an increased proportion of cells with CSC phenotype (CD44+ /CD24- ). Taken together, our results report a new mechanism of action for NmU in HER2-overexpressing breast cancer that enhances resistance to HER2-targeted drugs through conferring CSC characteristics and expansion of the CSC phenotype.
Asunto(s)
Metabolismo Energético , Células Madre Neoplásicas/metabolismo , Neuropéptidos/metabolismo , Receptor ErbB-2/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Neuropéptidos/genética , FenotipoRESUMEN
BACKGROUND: Neuromedin U (NmU) belongs to the neuromedin family, comprising a series of neuropeptides involved in the gut-brain axis and including neuromedins B and C (bombesin-like), K (neurokinin B), L (neurokinin A or neurotensin), N, S, and U. CONTENT: Although initially isolated from porcine spinal cord on the basis of their ability to induce uterine smooth muscle contraction, these peptides have now been found to be expressed in several different tissues and have been ascribed numerous functions, from appetite regulation and energy balance control to muscle contraction and tumor progression. NmU has been detected in several species to date, particularly in mammals (pig, rat, rabbit, dog, guinea pig, human), but also in amphibian, avian, and fish species. The NmU sequence is highly conserved across different species, indicating that this peptide is ancient and plays an important biological role. Here, we summarize the main structural and functional characteristics of NmU and describe its many roles, highlighting the jack-of-all-trades nature of this neuropeptide. SUMMARY: NmU involvement in key processes has outlined the possibility that this neuropeptide could be a novel target for the treatment of obesity and cancer, among other disorders. Although the potential for NmU as a therapeutic target is obvious, the multiple functions of this molecule should be taken into account when designing an approach to targeting NmU and/or its receptors.
Asunto(s)
Neuropéptidos/fisiología , Secuencia de Aminoácidos , Animales , Circulación Sanguínea/fisiología , Presión Sanguínea/fisiología , Conducta Alimentaria/fisiología , Homeostasis/fisiología , Humanos , Datos de Secuencia Molecular , Contracción Muscular/fisiología , Músculo Liso/fisiología , Neuropéptidos/química , Neuropéptidos/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Distribución TisularRESUMEN
Gram-positive bacteria cause a broad spectrum of infection-related diseases in both immunocompetent and immunocompromised hosts, ranging from localized infections to severe systemic conditions such as septic and toxic shock syndromes. This situation has been aggravated by the recent emergence of multidrug-resistant strains, thus stressing the need for alternative therapeutic approaches. One such possibility would be modulating the host's immune response. Herein, the potential use of a soluble form of the scavenger-like human lymphocyte receptor CD6 (shCD6) belonging to an ancient family of innate immune receptors has been evaluated. shCD6 can bind to a broad spectrum of gram-positive bacteria thanks to the recognition of highly conserved cell wall components (lipoteichoic acid [LTA] and peptidoglycan [PGN]), which are essential for their viability and pathogenicity and are not amenable to antibiotic resistance. shCD6 has in vitro inhibitory effects on both bacterial growth and Toll-like receptor-mediated inflammatory response induced by LTA plus PGN. In vivo infusion of shCD6 improves survival on mouse models of septic shock by Staphylococcus aureus (either multidrug-resistant or -sensitive) or their endotoxins (LTA + PGN) or exotoxins (TSST-1). These results support the use of shCD6 and/or other scavenger-like immune receptors in the treatment of severe gram-positive-induced infectious conditions.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Productos Biológicos/inmunología , Peptidoglicano/inmunología , Staphylococcus aureus/inmunología , Ácidos Teicoicos/inmunología , Factores de Virulencia/inmunología , Animales , Antígenos CD/uso terapéutico , Antígenos de Diferenciación de Linfocitos T/uso terapéutico , Productos Biológicos/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Peptidoglicano/metabolismo , Unión Proteica , Choque Séptico/tratamiento farmacológico , Infecciones Estafilocócicas/tratamiento farmacológico , Ácidos Teicoicos/metabolismo , Factores de Virulencia/metabolismoRESUMEN
The scavenger receptor cysteine-rich superfamily (SRCR-SF) members are transmembrane and/or secreted receptors exhibiting one or several repeats of a cysteine-rich protein module of â¼100 aa, named scavenger receptor cysteine-rich (SRCR). Two types of SRCR domains (A or B) have been reported, which differ in the number of coding exons and intradomain cysteines. Although no unifying function has been reported for SRCR-SF members, recognition of pathogen-associated molecular patterns (PAMPs) was recently shown for some of them. In this article, we report the structural and functional characterization of mouse S5D-SRCRB, a new group B member of the SRCR-SF. The s5d-srcrb gene maps at mouse chromosome 7 and encompasses 14 exons extending over 15 kb. The longest cDNA sequence found is 4286 bp in length and encodes a mature protein of 1371 aa, with a predicted M(r) of 144.6 kDa. Using an episomal mammalian-expression system, a glycosylated soluble recombinant form >200 kDa was obtained and used as immunogen for the generation of specific rat mAbs. Subsequent immunohistochemical and real-time PCR analysis showed significant S5D-SRCRB expression in murine genitourinary and digestive tracts. S5D-SRCRB was shown to bind endogenous extracellular matrix proteins (laminin and galectin-1), as well as PAMPs present on Gram-positive and Gram-negative bacteria and fungi. PAMP binding by S5D-SRCRB induced microbial aggregation and subsequent inhibition of PAMP-induced cytokine release. These abilities suggest that S5D-SRCRB might play a role in the innate defense and homeostasis of certain specialized epithelial surfaces.
Asunto(s)
Cisteína/metabolismo , Regulación de la Expresión Génica/inmunología , Familia de Multigenes/inmunología , Receptores Depuradores de Clase B/química , Receptores Depuradores de Clase B/fisiología , Secuencia de Aminoácidos , Animales , Células Epiteliales/química , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Células HEK293 , Homeostasis/genética , Homeostasis/inmunología , Humanos , Inmunidad Innata/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Unión Proteica/inmunología , Estructura Terciaria de Proteína/genética , Ratas , Ratas Sprague-Dawley , Receptores de Reconocimiento de Patrones/química , Receptores de Reconocimiento de Patrones/metabolismo , Receptores de Reconocimiento de Patrones/fisiología , Receptores Depuradores de Clase B/biosíntesis , Receptores Depuradores de Clase B/genéticaRESUMEN
BACKGROUND: Radical prostatectomy cures the majority of men with clinically localized disease, but up to 30% of men relapse with rising serum PSA levels. Stage, Gleason grade, and pre-operative PSA levels are associated with outcome but do not accurately predict which individuals will relapse. MicroRNA (miRNA) levels are altered in cancer and are associated with progression of disease. The miR-200 family has roles in prostate cancer. METHODS: miR-200a levels were measured in 18 radical prostatectomy samples from men who did not relapse and from 18 who did relapse, matched for stage (all T3), grade, and PSA levels. A pair of cancer and normal prostate cell lines derived from the same radical prostatectomy specimen were transfected with miR-200a to determine the effects on growth, wound healing, and invasion. RESULTS: Comparing the matched samples, 11 of the relapsers contained lower, 2 higher and 5 similar levels to the non-relapsers. Transient transfection of miR-200a significantly reduced cell proliferation in prostate cancer cell lines but did not affect invasiveness. CONCLUSION: miR-200a overexpression reduced prostate cancer cell growth and may have potential, in combination with other markers, in stratifying prostate cancer patients for more intensive monitoring and therapy.
Asunto(s)
MicroARNs/genética , Recurrencia Local de Neoplasia/genética , Antígeno Prostático Específico/sangre , Próstata/metabolismo , Neoplasias de la Próstata/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Humanos , Masculino , MicroARNs/biosíntesis , Recurrencia Local de Neoplasia/sangre , Prostatectomía , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/cirugía , TransfecciónRESUMEN
Due to limited availability of pharmacological therapies, triple-negative breast cancer (TNBC) is the subtype with worst outcome. We hypothesised that 2-Deoxy-D-Glucose (2-DG), a glucose analogue, may hold potential as a therapy for particularly aggressive TNBC. We investigated 2-DG's effects on TNBC cell line variants, Hs578T parental cells and their isogenic more aggressive Hs578Ts(i)8 variant, using migration, invasion and anoikis assays. We assessed their bioenergetics by Seahorse. We evaluated metabolic alterations using a Seahorse XF Analyzer, citrate synthase assay, immunoblotting and flow cytometry. We assessed the cancer stem cell (CSC) phenotype of the variants and 2-DG's effects on CSCs. 2-DG significantly inhibited migration and invasion of Hs578Ts(i)8 versus Hs578T and significantly decreased their ability to resist anoikis. Investigating 2-DG's preferential inhibitory effect on the more aggressive cells, we found Hs578Ts(i)8 also had significantly decreased oxidative phosphorylation and increased glycolysis compared to Hs578T. This is likely due to mitochondrial dysfunction in Hs578Ts(i)8, shown by their significantly decreased mitochondrial membrane potential. Furthermore, Hs578Ts(i)8 had a significantly increased proportion of cells with CSC phenotype, which was significantly decreased by 2-DG. 2-DG may have benefit as a therapy for TNBC with a particularly aggressive phenotype, by targeting increased glycolysis. Studies of more cell lines and patients' specimens are warranted.
Asunto(s)
Desoxiglucosa/farmacología , Glucólisis/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Anoicis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Citrato (si)-Sintasa/metabolismo , Femenino , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/metabolismoRESUMEN
Activity of cytochromes P450 is highly dependent on cytochrome P450 NADPH reductase (P450R), but this enzyme can also metabolise drugs on its own. MDA 231 breast adenocarcinoma cells transfected with human P450R (MDA R4) or an empty vector (MDA EV) were exposed to a series of commonly used chemotherapeutic drugs. Overexpression of P450R did not affect cell sensitivity to cisplatin, mitoxantrone, paclitaxel, docetaxel, vincristine or etoposide. However, MDA R4 cells showed increased sensitivity to mitomycin C (6.6-fold) and also to 5-fluorouracil (2.8-fold). In vitro toxicity assays where mitomycin C, 5-fluorouracil and vincristine were preincubated with microsomes expressing recombinant P450R showed that this effect was not a result of direct metabolism by P450R. Levels of NADPH were considerably decreased in MDA R4 as compared to MDA EV cells, while reactive oxygen species (ROS) production was increased in MDA R4 cells in basal conditions, showing no significant further increase after treatment with mitomycin C or 5-fluorouracil. P450R overexpression appears therefore to be detrimental to MDA 231 cells, depleting NADPH and increasing ROS levels; the increased oxidative stress observed in MDA R4 cells might explain the enhanced sensitivity to 5-fluorouracil. Expression of this enzyme in tumour cells might therefore modulate response to 5-fluorouracil.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Fluorouracilo/farmacología , NADPH-Ferrihemoproteína Reductasa/biosíntesis , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacología , Antimetabolitos Antineoplásicos/metabolismo , Antineoplásicos Fitogénicos/metabolismo , Antineoplásicos Fitogénicos/farmacología , Western Blotting , Neoplasias de la Mama/genética , Línea Celular Tumoral , Supervivencia Celular , Femenino , Fluorouracilo/metabolismo , Glutatión/metabolismo , Humanos , Microsomas/metabolismo , Mitomicina/metabolismo , Mitomicina/farmacología , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transfección , Vincristina/metabolismo , Vincristina/farmacologíaRESUMEN
Neuromedin U (NmU) -a neuropeptide belonging to the neuromedin family- plays a substantial role in HER2-positive breast cancer, correlating with increased aggressiveness, resistance to HER2-targeted therapies and overall significantly poorer outcome for patients. However, the mechanism through which it exerts these effects remains unclear. To elucidate this, initially we used HER2-positive breast cancer cells stably over-expressing NmU. These cells and their released extracellular vesicles (EVs) had increased amounts of the immunosuppressive cytokine TGFß1 and the lymphocyte activation inhibitor PD-L1. Furthermore, these cells also showed enhanced resistance to antibody-dependent cell cytotoxicity (ADCC) mediated by trastuzumab, indicating a role of NmU in enhancing immune evasion. All these features were also found in HER2-targeted drug-resistant cells which we previously found to express higher levels of NmU than their drug-sensitive counterparts. Interestingly, EVs from drug-resistant cells were able to increase levels of TGFß1 in drug-sensitive cells. In our neo-adjuvant clinical trial, TGFß1 levels were significantly higher in EVs isolated from the serum of patients with HER2-overexpressing breast cancers who went on to not respond to HER2-targeted drug treatment, compared with those who experienced complete or partial response. Taken together, our results report a new mechanism-of-action for NmU in HER2-overexpressing breast cancer that enhances resistance to the anti-tumor immune response. Furthermore, EV levels of TGFß1 correlating with patients' response versus resistance to HER2-targeted drugs suggests a potential use of EV-TGFß1 as a minimally-invasive companion diagnostic for such treatment in breast cancer.
RESUMEN
Modulation of antitumor immune responses by targeting immune checkpoint regulators has been proven successful in the treatment of many different tumors. Recent evidence shows that the lymphocyte receptor CD5 -a negative regulator of TCR-mediated signaling- may play a role in the anti-tumor immune response. To explore such an issue, we developed transgenic C57BL/6 mice expressing a soluble form of human CD5 (shCD5EµTg), putatively blocking CD5-mediated interactions ("decoy receptor" effect). Homozygous shCD5EµTg mice showed reduced growth rates of tumor cells of melanoma (B16-F0) and thymoma (EG7-OVA) origin. Concomitantly, increased CD4+ and CD8+ T cell numbers, as well as reduced proportion of CD4+CD25+FoxP3+ (Treg) cells were observed in tumor draining lymph nodes (TdLN). TdLN cell suspensions from tumor-bearing shCD5EµTg mice showed increased both tumor specific and non-specific cytolitic activity. Moreover, subcutaneous peritumoral (p.t.) injection of recombinant shCD5 to wild-type (WT) mice slowed B16-F0 tumor growth, and reproduced the above mentioned TdLN cellular changes. Interestingly, lower intratumoral IL-6 levels -an inhibitor of Natural Killer (NK) cell cytotoxity- were observed in both transgenic and rshCD5-treated WT mice and the anti-tumor effect was abrogated by mAb-induced NK cell depletion. Taken together, the results further illustrate the putative regulatory role of CD5-mediated interactions in anti-tumor immune responses, which would be at least in part fostered by NK cells.
RESUMEN
Exosomes (EVs) have relevance in cell-to-cell communication carrying pro-tumorigenic factors that participate in oncogenesis and drug resistance and are proposed to have potential as self-delivery systems. Advancing on our studies of EVs in triple-negative breast cancer, here we more comprehensively analysed isogenic cell line variants and their EV populations, tissues cell line variants and their EV populations, as well as breast tumour and normal tissues. Profiling 384 miRNAs showed EV miRNA content to be highly representative of their cells of origin. miRNAs most substantially down-regulated in aggressive cells and their EVs originated from 14q32. Analysis of miR-134, the most substantially down-regulated miRNA, supported its clinical relevance in breast tumours compared to matched normal breast tissue. Functional studies indicated that miR-134 controls STAT5B which, in turn, controls Hsp90. miR-134 delivered by direct transfection into Hs578Ts(i)8 cells (in which it was greatly down-regulated) reduced STAT5B, Hsp90, and Bcl-2 levels, reduced cellular proliferation, and enhanced cisplatin-induced apoptosis. Delivery via miR-134-enriched EVs also reduced STAT5B and Hsp90, reduced cellular migration and invasion, and enhanced sensitivity to anti-Hsp90 drugs. While the differing effects achieved by transfection or EV delivery are likely to be, at least partly, due to specific amounts of miR-134 delivered by these routes, these EV-based studies identified miRNA-134 as a potential biomarker and therapeutic for breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Exosomas/metabolismo , MicroARNs/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Apoptosis/efectos de los fármacos , Estudios de Casos y Controles , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Biología Computacional , Relación Dosis-Respuesta a Droga , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Apoptosis inhibitor of macrophages (AIMs), a homologue of human Spα, is a mouse soluble member of the scavenger receptor cysteine-rich superfamily (SRCR-SF). This family integrates a group of proteins expressed by innate and adaptive immune cells for which no unifying function has yet been described. Pleiotropic functions have been ascribed to AIM, from viability support in lymphocytes during thymic selection to lipid metabolism and anti-inflammatory effects in autoimmune pathologies. In the present report, the pathogen binding properties of AIM have been explored. By using a recombinant form of AIM (rAIM) expressed in mammalian cells, it is shown that this protein is able to bind and aggregate Gram-positive and Gram-negative bacteria, as well as pathogenic and saprophytic fungal species. Importantly, endogenous AIM from mouse serum also binds to microorganisms and secretion of AIM was rapidly induced in mouse spleen macrophages following exposure to conserved microbial cell wall components. Cytokine release induced by well-known bacterial and fungal Toll-like receptor (TLR) ligands on mouse splenocytes was also inhibited in the presence of rAIM. Furthermore, mouse models of pathogen-associated molecular patterns (PAMPs)-induced septic shock of bacterial and fungal origin showed that serum AIM levels changed in a time-dependent manner. Altogether, these data suggest that AIM plays a general homeostatic role by supporting innate humoral defense during pathogen aggression.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Hongos/fisiología , Bacterias Grampositivas/fisiología , Macrófagos/inmunología , Receptores Inmunológicos/metabolismo , Receptores Depuradores/metabolismo , Choque Séptico/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Adhesión Bacteriana , Pared Celular/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Hongos/patogenicidad , Regulación de la Expresión Génica , Bacterias Grampositivas/patogenicidad , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Receptores Inmunológicos/genética , Receptores Depuradores/genética , Receptores Depuradores de Clase B/genética , Homología de Secuencia de Aminoácido , Choque Séptico/inducido químicamente , Choque Séptico/microbiologíaRESUMEN
CD6 is a lymphocyte glycoprotein receptor that physically associates with the antigen-specific receptor complex at the center of the immunological synapse, where it interacts with its ligand CD166/ALCAM. The present work reports the carbohydrate-dependent interaction of CD6 and CD166/ALCAM with Galectin-1 and -3, two well-known soluble mammalian lectins. Both galectins interfered with superantigen-induced T cell proliferation and cell adhesion phenomena mediated by the CD6-CD166/ALCAM pair, while CD6 expression protected cells from galectin-induced apoptosis. The results suggest that interaction of Galectin-1 and -3 with CD6 and CD166/ALCAM might modulate some relevant aspects of T cell physiology.
Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Galectina 1/metabolismo , Galectina 3/metabolismo , Apoptosis , Linfocitos B/citología , Metabolismo de los Hidratos de Carbono , Adhesión Celular , Membrana Celular/metabolismo , Células Dendríticas/inmunología , Humanos , Unión Proteica , Superantígenos/inmunología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
CD5 is a lymphoid-specific transmembrane glycoprotein constitutively expressed on thymocytes and mature T and B1a lymphocytes. Current data support the view that CD5 is a negative regulator of antigen-specific receptor-mediated signaling in these cells, and that this would likely be achieved through interaction with CD5 ligand/s (CD5L) of still undefined nature expressed on immune or accessory cells. To determine the functional consequence of loss of CD5/CD5L interaction in vivo, a new transgenic mouse line was generated (shCD5EµTg), expressing a circulating soluble form of human CD5 (shCD5) as a decoy to impair membrane-bound CD5 function. These shCD5EµTg mice showed an enhanced response to autologous antigens, as deduced from the presentation of more severe forms of experimentally inducible autoimmune disease (collagen-induced arthritis, CIA; and experimental autoimmune encephalitis, EAE), as well as an increased anti-tumoral response in non-orthotopic cancer models (B16 melanoma). This enhancement of the immune response was in agreement with the finding of significantly reduced proportions of spleen and lymph node Treg cells (CD4+CD25+FoxP3+), and of peritoneal IL-10-producing and CD5+ B cells, as well as an increased proportion of spleen NKT cells in shCD5EµTg mice. Similar changes in lymphocyte subpopulations were observed in wild-type mice following repeated administration of exogenous recombinant shCD5 protein. These data reveal the relevant role played by CD5/CD5L interactions on the homeostasis of some functionally relevant lymphocyte subpopulations and the modulation of immune responses to autologous antigens.
Asunto(s)
Artritis Experimental/inmunología , Antígenos CD5/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Neoplasias Experimentales/inmunología , Animales , Secuencia de Bases , Antígenos CD5/genética , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Reacción en Cadena de la PolimerasaRESUMEN
CD6 is a transmembrane receptor expressed by all T and a subset of B lymphocytes, where it physically associates with the antigen-specific receptor to modulate activation and differentiation processes through still poorly understood mechanisms. Its cytoplasmic tail lacks intrinsic catalytic activity but presents several consensus motifs for phosphorylation. The present work reports on the identification of two constitutively phosphorylated serine clusters (S480/482/484 and S560/562/565/567/568), which are embedded into Casein Kinase 2 consensus motifs, and are indispensable for proper mitogen-activated protein kinase activation following CD6 ligation. The data point to a novel level of regulation of CD6 function by intracytoplasmic serine phosphorylation.
Asunto(s)
Antígenos CD/química , Antígenos de Diferenciación de Linfocitos T/química , Células Jurkat/metabolismo , Procesamiento Proteico-Postraduccional , Serina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Citoplasma/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Células Jurkat/inmunología , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Fosforilación , Transducción de SeñalRESUMEN
Galectin-1 (Gal-1), a beta galactoside-binding lectin, is involved in multiple biological functions, such as cell adhesion, apoptosis, and metastasis. On the basis of its ability to interact with extracellular matrix (ECM) glycoproteins, we investigated the Gal-1 effect on Leydig cells, which express and are influenced by ECM proteins. In this study, Gal-1 was identified in Leydig cell cultures by immunofluorescence. To gain insight into its biological role, Gal-1 was added to purified rat Leydig cells, under both basal and human chorionic gonadotrophin-stimulated conditions. Substantial morphological changes were observed, and cell viability showed an 80% decrease after 24 h culture. As a functional consequence of Gal-1 addition, testosterone production was reduced in a dose-dependent fashion, reaching a minimum of 26% after 24 h compared with basal values. cAMP showed a similar variation after 3 h. Assessment of DNA hypodiploidy and caspase activity determinations indicated that the reduction in viability and in steroidogenesis was caused by apoptosis induced by Gal-1. Besides, addition of Gal-1 caused Leydig cell detachment. Presence of laminin-1 or lactose prevented the effect of Gal-1, suggesting that the carbohydrate recognition domain is involved in inducing apoptosis. These findings demonstrate a novel mechanism, based on Gal-1 and laminin-1 interaction, which could help us better understand the molecular basis of Leydig cell function and survival control.