RESUMEN
Functional regeneration after nervous system injury requires transected axons to reconnect with their original target tissue. Axonal fusion, a spontaneous regenerative mechanism identified in several species, provides an efficient means of achieving target reconnection as a regrowing axon is able to contact and fuse with its own separated axon fragment, thereby re-establishing the original axonal tract. Here we report a molecular characterization of this process in Caenorhabditis elegans, revealing dynamic changes in the subcellular localization of the EFF-1 fusogen after axotomy, and establishing phosphatidylserine (PS) and the PS receptor (PSR-1) as critical components for axonal fusion. PSR-1 functions cell-autonomously in the regrowing neuron and, instead of acting in its canonical signalling pathway, acts in a parallel phagocytic pathway that includes the transthyretin protein TTR-52, as well as CED-7, NRF-5 and CED-6 (refs 9, 10, 11, 12). We show that TTR-52 binds to PS exposed on the injured axon, and can restore fusion several hours after injury. We propose that PS functions as a 'save-me' signal for the distal fragment, allowing conserved apoptotic cell clearance molecules to function in re-establishing axonal integrity during regeneration of the nervous system.
Asunto(s)
Apoptosis/fisiología , Axones/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Glicoproteínas de Membrana/metabolismo , Regeneración Nerviosa/fisiología , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Axones/patología , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/metabolismo , Conos de Crecimiento/metabolismo , Mutación , Fagocitos/metabolismo , Fagocitosis , Fosfatidilserinas/metabolismo , Fosfoproteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Espectrina/genética , Espectrina/metabolismoRESUMEN
Endonuclease G (EndoG) is a mitochondrial protein that traverses to the nucleus and participates in chromosomal DNA degradation during apoptosis in yeast, worms, flies, and mammals. However, it remains unclear how EndoG binds and digests DNA. Here we show that the Caenorhabditis elegans CPS-6, a homolog of EndoG, is a homodimeric Mg(2+)-dependent nuclease, binding preferentially to G-tract DNA in the optimum low salt buffer at pH 7. The crystal structure of CPS-6 was determined at 1.8 Å resolution, revealing a mixed αß topology with the two ßßα-metal finger nuclease motifs located distantly at the two sides of the dimeric enzyme. A structural model of the CPS-6-DNA complex suggested a positively charged DNA-binding groove near the Mg(2+)-bound active site. Mutations of four aromatic and basic residues: Phe(122), Arg(146), Arg(156), and Phe(166), in the protein-DNA interface significantly reduced the DNA binding and cleavage activity of CPS-6, confirming that these residues are critical for CPS-6-DNA interactions. In vivo transformation rescue experiments further showed that the reduced DNase activity of CPS-6 mutants was positively correlated with its diminished cell killing activity in C. elegans. Taken together, these biochemical, structural, mutagenesis, and in vivo data reveal a molecular basis of how CPS-6 binds and hydrolyzes DNA to promote cell death.
Asunto(s)
Apoptosis/fisiología , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/enzimología , ADN de Helmintos/química , Proteínas Mitocondriales/química , Modelos Moleculares , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografía por Rayos X , ADN de Helmintos/genética , ADN de Helmintos/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Hidrólisis , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación Missense , Relación Estructura-ActividadRESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world. While the role of NSAIDs as cyclooxygenase (COX) inhibitors is well established, other targets may contribute to anti-inflammation. Here we report caspases as a new pharmacological target for NSAID family drugs such as ibuprofen, naproxen, and ketorolac at physiologic concentrations both in vitro and in vivo. We characterize caspase activity in both in vitro and in cell culture, and combine computational modeling and biophysical analysis to determine the mechanism of action. We observe that inhibition of caspase catalysis reduces cell death and the generation of pro-inflammatory cytokines. Further, NSAID inhibition of caspases is COX independent, representing a new anti-inflammatory mechanism. This finding expands upon existing NSAID anti-inflammatory behaviors, with implications for patient safety and next-generation drug design.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Inhibidores de Caspasas/química , Caspasas/metabolismo , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Inhibidores de Caspasas/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas/química , Caspasas Iniciadoras/química , Caspasas Iniciadoras/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Ibuprofeno/química , Ibuprofeno/metabolismo , Ibuprofeno/farmacología , Concentración 50 Inhibidora , Ketorolaco/química , Ketorolaco/metabolismo , Ketorolaco/farmacología , Naproxeno/química , Naproxeno/metabolismo , Naproxeno/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Especificidad por SustratoRESUMEN
Endonuclease G (EndoG) is a mitochondrial protein that is released from mitochondria and relocated into the nucleus to promote chromosomal DNA fragmentation during apoptosis. Here, we show that oxidative stress causes cell-death defects in C. elegans through an EndoG-mediated cell-death pathway. In response to high reactive oxygen species (ROS) levels, homodimeric CPS-6-the C. elegans homolog of EndoG-is dissociated into monomers with diminished nuclease activity. Conversely, the nuclease activity of CPS-6 is enhanced, and its dimeric structure is stabilized by its interaction with the worm AIF homolog, WAH-1, which shifts to disulfide cross-linked dimers under high ROS levels. CPS-6 thus acts as a ROS sensor to regulate the life and death of cells. Modulation of the EndoG dimer conformation could present an avenue for prevention and treatment of diseases resulting from oxidative stress.
Asunto(s)
Apoptosis , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Endodesoxirribonucleasas/metabolismo , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/química , Cristalografía por Rayos X , Regulación hacia Abajo , Endodesoxirribonucleasas/química , Estabilidad de Enzimas , Proteínas Mitocondriales/química , Modelos Moleculares , Oxidación-Reducción , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
Caspases are cysteine proteases with critical roles in apoptosis. The Caenorhabditis elegans caspase CED-3 is activated by autocatalytic cleavage, a process enhanced by CED-4. Here we report that the CED-3 zymogen localizes to the perinuclear region in C. elegans germ cells and that CED-3 autocatalytic cleavage is held in check by C. elegans nuclei and activated by CED-4. The nuclear-pore protein NPP-14 interacts with the CED-3 zymogen prodomain, colocalizes with CED-3 in vivo and inhibits CED-3 autoactivation in vitro. Several missense mutations in the CED-3 prodomain result in stronger association with NPP-14 and decreased CED-3 activation by CED-4 in the presence of nuclei or NPP-14, thus leading to cell-death defects. Those same mutations enhance autocatalytic cleavage of CED-3 in vitro and increase apoptosis in vivo in the absence of npp-14. Our results reveal a critical role of nuclei and nuclear-membrane proteins in regulating the activation and localization of CED-3.
Asunto(s)
Apoptosis , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Caspasas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/genética , Caspasas/análisis , Caspasas/genética , Activación Enzimática , Células Germinativas/citología , Células Germinativas/metabolismo , Mutación Missense , Proteínas de Complejo Poro Nuclear/análisis , Proteínas de Complejo Poro Nuclear/genética , Dominios y Motivos de Interacción de ProteínasRESUMEN
Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , ADN Mitocondrial/metabolismo , Endodesoxirribonucleasas/metabolismo , Fertilización , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Animales , Autofagia , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/genética , Embrión no Mamífero/citología , Embrión no Mamífero/enzimología , Endodesoxirribonucleasas/genética , Masculino , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Espermatozoides/enzimología , Espermatozoides/ultraestructuraRESUMEN
Activation of caspases is an integral part of the apoptotic cell death program. Collectively, these proteases target hundreds of substrates, leading to the hypothesis that apoptosis is "death by a thousand cuts". Recent work, however, has demonstrated that caspase cleavage of only a subset of these substrates directs apoptosis in the cell. One such example is C. elegans CNT-1, which is cleaved by CED-3 to generate a truncated form, tCNT-1, that acquires a potent phosphoinositide-binding activity and translocates to the plasma membrane where it inactivates AKT survival signaling. We report here that ACAP2, a homolog of C. elegans CNT-1, has a pro-apoptotic function and an identical phosphoinositide-binding pattern to that of tCNT-1, despite not being an apparent target of caspase cleavage. We show that knockdown of ACAP2 blocks apoptosis in cancer cells in response to the chemotherapeutic antimetabolite 5-fluorouracil and that ACAP2 expression is down-regulated in some esophageal cancers, leukemias and lymphomas. These results suggest that ACAP2 is a functional homolog of C. elegans CNT-1 and its inactivation or downregulation in human cells may contribute to cancer development.
Asunto(s)
Apoptosis , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias/patología , Secuencia de Aminoácidos , Animales , Caspasas/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Activación Enzimática , Fluorouracilo/química , Proteínas Activadoras de GTPasa/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HCT116 , Humanos , Ligandos , Lípidos/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Neoplasias/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Inactivation of cell-survival factors is a crucial step in apoptosis. The phosphoinositide 3-kinase (PI3K)-AKT signaling pathway promotes cell growth, proliferation and survival, and its deregulation causes cancer. How this pathway is suppressed to promote apoptosis is poorly understood. Here we report the identification of a CED-3 caspase substrate in Caenorhabditis elegans, CNT-1, that is cleaved during apoptosis to generate an N-terminal phosphoinositide-binding fragment (tCNT-1). tCNT-1 translocates from the cytoplasm to the plasma membrane and blocks AKT binding to phosphatidylinositol (3,4,5)-trisphosphate, thereby disabling AKT activation and its prosurvival activity. Our findings reveal a new mechanism that negatively regulates AKT cell signaling to promote apoptosis and that may restrict cell growth and proliferation in normal cells.
Asunto(s)
Apoptosis , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Sitios de Unión , Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/química , Caspasas/metabolismo , Proteínas Activadoras de GTPasa/química , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Transporte de ProteínasRESUMEN
During C. elegans apoptosis, the dicer ribonuclease (DCR-1) is cleaved by the cell death protease CED-3 to generate a truncated DCR-1 (tDCR-1) with one and a half ribonuclease III (RNase III) domains, converting it into a deoxyribonuclease (DNase) that initiates apoptotic chromosome fragmentation. We performed biochemical and functional analyses to understand this unexpected RNase to DNase conversion. In full-length DCR-1, tDCR-1 DNase activity is suppressed by its N-terminal DCR-1 sequence. However, not all the sequence elements in the N-terminal DCR-1 are required for this suppression. Our deletion analysis reveals that a 20-residue α-helix sequence in DCR-1 appears to define a critical break point for the sequence required for suppressing tDCR-1 DNase activity through a structure-dependent mechanism. Removal of the N-terminal DCR-1 sequence from tDCR-1 activates a DNA-binding activity that also requires the one half RNase IIIa domain, and enables tDCR-1 to process DNA. Consistently, structural modeling of DCR-1 and tDCR-1 suggests that cleavage of DCR-1 by CED-3 may cause a conformational change that allows tDCR-1 to bind and process DNA, and may remove steric hindrance that blocks DNA access to tDCR-1. Moreover, a new DNase can be engineered using different RNase III domains, including the one from bacterial RNase III. Our results indicate that very distantly related RNase III enzymes have the potential to cleave DNA when processed proteolytically or paired with an appropriate partner that facilitates binding to DNA. We suggest the possibility that this phenomenon may be extrapolated to other ribonucleases.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caspasas/metabolismo , Desoxirribonucleasas/metabolismo , Ribonucleasa III/metabolismo , Animales , Apoptosis , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , ADN/metabolismo , Mutagénesis , Unión Proteica , Estructura Terciaria de Proteína , Ribonucleasa III/química , Ribonucleasa III/genéticaRESUMEN
Chromosome fragmentation is a hallmark of apoptosis, conserved in diverse organisms. In mammals, caspases activate apoptotic chromosome fragmentation by cleaving and inactivating an apoptotic nuclease inhibitor. We report that inactivation of the Caenorhabditis elegans dcr-1 gene, which encodes the Dicer ribonuclease important for processing of small RNAs, compromises apoptosis and blocks apoptotic chromosome fragmentation. DCR-1 was cleaved by the CED-3 caspase to generate a C-terminal fragment with deoxyribonuclease activity, which produced 3' hydroxyl DNA breaks on chromosomes and promoted apoptosis. Thus, caspase-mediated activation of apoptotic DNA degradation is conserved. DCR-1 functions in fragmenting chromosomal DNA during apoptosis, in addition to processing of small RNAs, and undergoes a protease-mediated conversion from a ribonuclease to a deoxyribonuclease.
Asunto(s)
Apoptosis , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Caspasas/metabolismo , Fragmentación del ADN , ADN de Helmintos/metabolismo , Desoxirribonucleasas/metabolismo , Ribonucleasa III/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Caspasas/genética , Dominio Catalítico , Etiquetado Corte-Fin in Situ , Interferencia de ARN , ARN Bicatenario/metabolismo , ARN de Helminto/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasa III/química , Ribonucleasa III/genéticaRESUMEN
Cell death related nuclease 4 (CRN-4) is one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. To understand how CRN-4 is involved in apoptotic DNA fragmentation, we analyzed CRN-4's biochemical properties, in vivo cell functions, and the crystal structures of CRN-4 in apo-form, Mn(2+)-bound active form, and Er(3+)-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn(2+)-bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4's function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn-domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis.
Asunto(s)
Apoptosis/fisiología , Proteínas de Caenorhabditis elegans/química , Fragmentación del ADN , Endodesoxirribonucleasas/química , Endonucleasas/química , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Calcio/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , ADN de Helmintos/metabolismo , Dimerización , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Concentración de Iones de Hidrógeno , Magnesio/metabolismo , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Zinc/metabolismoRESUMEN
Inhibitor of apoptosis (IAP) proteins have a crucial role in apoptosis, through negative regulation of caspases in species from fruitflies to mammals. In Caenorhabditis elegans, however, no IAP homolog or caspase inhibitor has been identified, calling into question how the cell-killing caspase CED-3 can be negatively regulated. Here we show that inactivation of the C. elegans csp-3 gene, which encodes a protein similar to the small subunit of the CED-3 caspase, causes cells that normally live to undergo apoptosis in a CED-3-dependent manner. Biochemical analysis reveals that CSP-3 associates with the large subunit of the CED-3 zymogen and inhibits zymogen autoactivation. However, CSP-3 does not block CED-3 activation induced by CED-4, nor does it inhibit the activity of the activated CED-3 protease. Therefore CSP-3 uses a previously unreported mechanism to protect cells from apoptosis.
Asunto(s)
Apoptosis , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/enzimología , Inhibidores de Caspasas , Caspasas/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Caspasas/química , Caspasas/genética , Secuencia Conservada , Activación Enzimática , Datos de Secuencia Molecular , Mutación/genética , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Alineación de SecuenciaRESUMEN
Natural and artificial substances present in the environment can affect our health. Testicular toxicants in particular are troublesome, because they disturb gonadal function of males. Translocation of substances into the seminiferous epithelium where sperm production proceeds is restricted due to the blood-testis barrier, but this permeability barrier temporarily disappears under physiological and sub-physiological conditions. This means that any substance could enter the seminiferous epithelium and disturb sperm production. To reduce the risk posed by such toxins, it is important to accurately determine which substances possess the toxicity. However, existing assay systems are not satisfactory in terms of both accuracy and sensitivity. Here, we report the establishment of such a system. We injected the androgen antagonists, flutamide and vinclozolin, directly into seminiferous tubules of live mice, which had been treated with busulfan for a temporal arrest of spermatogenesis, and the testes were histologically examined to see the effect of the injected materials on spermatogenesis that was in the process of recovery. The injection of either substance brought about a severe impairment of spermatogenesis at an amount over a million times smaller than that used in the previous assay systems where animals are administered with test substances outside of the testis. In contrast, these androgen antagonists at the same doses showed lesser effects when intratubularly or intraperitoneally administered into mice that had not been pretreated with busulfan. We propose that the method adopted in this study is a novel assay system to identify potential testicular toxicants.
Asunto(s)
Antagonistas de Andrógenos/toxicidad , Flutamida/toxicidad , Oxazoles/toxicidad , Túbulos Seminíferos/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Pruebas de Toxicidad , Animales , Antineoplásicos/farmacología , Barrera Hematotesticular , Busulfano/farmacología , Masculino , Ratones , MicroinyeccionesRESUMEN
Sertoli cells, a somatic cell type present within the seminiferous tubules of testes, are responsible for the phagocytic elimination of apoptotic spermatogenic cells. We here established an in vivo assay system that enables us to quantitatively analyze Sertoli cell phagocytosis of apoptotic cells in testes of live mice. Apoptotic cells were injected into the seminiferous tubules of spermatogenic cell-depleted mice, and the occurrence of phagocytosis by Sertoli cells was examined by histochemically analyzing testis sections or dispersed testicular cells. We reproducibly observed similar levels of phagocytosis in either examination, and the ratio of Sertoli cells that engulfed injected apoptotic cells was almost the same between the two examinations. These results indicated that a quantitative in vivo assay system was established using the seminiferous tubules of live mice as 'test tubes.' We then determined the requirements for Sertoli cell phagocytosis of apoptotic cells using this assay. For this purpose, apoptotic cells were injected together with various phagocytosis inhibitors, and the extent of phagocytosis by Sertoli cells was determined. The results revealed that Sertoli cells phagocytose apoptotic cells in a manner dependent on class B scavenger receptor type I (SR-BI) of Sertoli cells and phosphatidylserine exposed at the surface of target cells, as previously observed in vitro using primary cultures of dispersed rat testicular cells. Furthermore, the amount of SR-BI in Sertoli cells increased after injection of apoptotic cells into the seminiferous tubules, suggesting a positive feedback regulation of the expression of this phagocytosis receptor.
Asunto(s)
Apoptosis , Fagocitosis/fisiología , Células de Sertoli/fisiología , Espermatogénesis/fisiología , Animales , Humanos , Células Jurkat , Masculino , Ratones , Ratas , Células de Sertoli/citologíaRESUMEN
Class B scavenger receptor type I (SR-BI), a multiligand membrane protein, exists in various organs and cell types. In the testis, SR-BI is expressed in two somatic cell types: Leydig cells and Sertoli cells. Unlike interstitially localized Leydig cells, Sertoli cells present within the seminiferous tubules keep contact with spermatogenic cells and form the tight junction to divide the seminiferous epithelium into the basal and adluminal compartments. In this study, the expression and function of SR-BI in rat Sertoli cells were examined with respect to dependency on the spermatogenic cycle, the plasma membrane polarity, and the pituitary hormone follicle-stimulating hormone (FSH). When the expression of SR-BI was histochemically examined with testis sections, both protein and mRNA were already present in Sertoli cells during the first-round spermatogenesis and continued to be detectable thereafter. The level of SR-BI mRNA expression in Sertoli cells was lower at spermatogenic stages I-VI than at other stages. SR-BI was present and functional (in mediating cellular incorporation of lipids of high density lipoprotein) at both the apical and basolateral surfaces of polarized Sertoli cells. Finally, SR-BI expression at both the protein and mRNA levels was stimulated by FSH in cultured Sertoli cells. These results indicate that SR-BI functions on both the apical and basolateral plasma membranes of Sertoli cells, and that SR-BI expression in Sertoli cells changes during the spermatogenic cycle and is stimulated, at least in cultures, by FSH.
Asunto(s)
Membrana Celular/metabolismo , Receptores Inmunológicos/fisiología , Células de Sertoli/fisiología , Animales , Antígenos CD36 , Línea Celular , Polaridad Celular , Células Cultivadas , Hormona Folículo Estimulante/fisiología , Regulación de la Expresión Génica , Células Intersticiales del Testículo/fisiología , Masculino , Ratas , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Receptores Depuradores , Receptores Depuradores de Clase B , Espermatogénesis , Testículo/citología , Testículo/fisiología , Uniones Estrechas/metabolismoRESUMEN
Testicular Sertoli cells phagocytose apoptotic spermatogenic cells in a manner depending on the membrane phospholipid phosphatidylserine (PS) expressed at the surface of the latter cell type. Our previous studies have indicated that class B scavenger receptor type I (SR-BI) is responsible for the PS-mediated phagocytosis by Sertoli cells. We examined here whether SR-BI binds directly to PS. A cell line acquired the ability to bind to PS-exposing apoptotic cells and to incorporate PS-containing liposomes when it was forced to express SR-BI. Furthermore, the extracellular domain of rat SR-BI fused with human Fc (SRBIecd-Fc) bound to PS with a dissociation equilibrium constant of 2.4 x 10(-7) m in a cell-free solid-phase assay, whereas other phospholipids including phosphatidylethanolamine, phosphatidylinositol, and phosphatidylcholine were poor binding targets. The binding activity was enhanced when CaCl(2) was included in the assay or when SRBIecd-Fc was pre-treated with N-glycanase. A portion of the extracellular domain spanning amino acid positions 33 and 191 (numbered with respect to the amino terminus) fused with Fc (SRBI33-191-Fc) showed activity and phospholipid specificity equivalent to those of SRBIecd-Fc. Finally, SRBI33-191-Fc bound to the surface of apoptotic cells with externalized PS, and the injection of SRBI33-191-Fc into the seminiferous tubules of live mice increased the number of apoptotic spermatogenic cells. These results allowed us to conclude that SR-BI is a phagocytosis-inducing PS receptor of Sertoli cells.