RESUMO
Epididymal Cysteine Rich Secretory Proteins 1 and 4 (CRISP1 and CRISP4) associate with sperm during maturation and play different roles in fertilization. However, males lacking each of these molecules individually are fertile, suggesting compensatory mechanisms between these homologous proteins. Based on this, in the present work, we generated double CRISP1/CRISP4 knockout (DKO) mice and examined their reproductive phenotype. Our data showed that the simultaneous lack of the two epididymal proteins results in clear fertility defects. Interestingly, whereas most of the animals exhibited specific sperm fertilizing ability defects supportive of the role of CRISP proteins in fertilization, one third of the males showed an unexpected epididymo-orchitis phenotype with altered levels of inflammatory molecules and non-viable sperm in the epididymis. Further analysis showed that DKO mice exhibited an immature epididymal epithelium and abnormal luminal pH, supporting these defects as likely responsible for the different phenotypes observed. These observations reveal that CRISP proteins are relevant for epididymal epithelium differentiation and male fertility, contributing to a better understanding of the fine-tuning mechanisms underlying sperm maturation and immunotolerance in the epididymis with clear implications for human epididymal physiology and pathology.
Assuntos
Diferenciação Celular , Epididimo/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Glicoproteínas de Membrana/deficiência , Proteínas de Plasma Seminal/genética , Animais , Epididimo/patologia , Epitélio/metabolismo , Epitélio/patologia , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
Even though many extracellular factors have been identified as promoters of general dendritic growth and branching, little is known about the cell-intrinsic modulators that allow neurons to sculpt distinctive patterns of dendrite arborization. Here, we identify Lrig1, a nervous system-enriched LRR protein, as a key physiological regulator of dendrite complexity of hippocampal pyramidal neurons. Lrig1-deficient mice display morphological changes in proximal dendrite arborization and defects in social interaction. Specifically, knockdown of Lrig1 enhances both primary dendrite formation and proximal dendritic branching of hippocampal neurons, two phenotypes that resemble the effect of BDNF on these neurons. In addition, we show that Lrig1 physically interacts with TrkB and attenuates BDNF signaling. Gain and loss of function assays indicate that Lrig1 restricts BDNF-induced dendrite morphology. Together, our findings reveal a novel and essential role of Lrig1 in regulating morphogenic events that shape the hippocampal circuits and establish that the assembly of TrkB with Lrig1 represents a key mechanism for understanding how specific neuronal populations expand the repertoire of responses to BDNF during brain development.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dendritos/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hipocampo/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Técnicas de Inativação de Genes , Células HEK293 , Hipocampo/citologia , Humanos , Glicoproteínas de Membrana/deficiência , Camundongos , Morfogênese , Proteínas do Tecido Nervoso/deficiência , Neurônios/metabolismo , Polissacarídeos , Transdução de SinaisRESUMO
Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major components, collagen IV and laminin. We used this model to demonstrate that antibody targeted blockade of CTLD2, the second of eight C-type lectin-like domains in Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) that can recognize glycosylated collagens, reversed actinomyosin-based contractility [myosin-light chain-2 (MLC2) phosphorylation], loss of cell polarity, loss of cell-cell junctions, luminal infiltration and basal invasion induced by AGE-modified basal lamina matrix in PEC acini. Our in vitro results were concordant with luminal occlusion of acini in the prostate glands of adult Endo180(Δ) (Ex2-6/) (Δ) (Ex2-6) mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE-dependent modification of the basal lamina induces invasive behaviour in non-transformed PECs via a molecular mechanism linked to cancer progression. This study provides a rationale for targeting CTLD2 in Endo180 in prostate cancer and other pathologies in which increased basal lamina thickness and tissue stiffness are driving factors. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Membrana Basal/metabolismo , Movimento Celular , Células Epiteliais/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Mitogênicos/metabolismo , Animais , Membrana Basal/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Elasticidade , Humanos , Estimativa de Kaplan-Meier , Lectinas Tipo C/metabolismo , Masculino , Mecanotransdução Celular , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos Knockout , Invasividade Neoplásica , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Estrutura Terciária de Proteína , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Fatores de TempoRESUMO
Cell death implies morphological changes that may contribute to the progression of this process. In astrocytes, the mechanisms involving the cytoskeletal changes during cell death are not well explored. Although NADPH oxidase (NOX) has been described as being a critical factor in the production of ROS, not much information is available about the participation of NOX-derived ROS in the cell death of astrocytes and their role in the alterations of the cytoskeleton during the death of astrocytes. In this study, we have evaluated the participation of ROS in the death of cultured cerebellar astrocytes using staurosporine (St) as death inductor. We found that astrocytes express NOX1, NOX2, and NOX4. Also, St induced an early ROS production and NOX activation that participate in the death of astrocytes. These findings suggest that ROS produced by St is generated through NOX1 and NOX4. Finally, we showed that the reorganization of tubulin and actin induced by St is ROS independent and that St did not change the level of expression of these cytoskeletal proteins. We conclude that ROS produced by a NOX is required for cell death in astrocytes, but not for the morphological alterations induced by St.
Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estaurosporina/farmacologia , Actinas/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Células Cultivadas , Cerebelo/citologia , Citoesqueleto/efeitos dos fármacos , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Tubulina (Proteína)/metabolismoRESUMO
(â¢)NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91(phox) (-/-) or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with (â¢)NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.
Assuntos
Doença de Chagas/imunologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/imunologia , NADPH Oxidases/deficiência , NADPH Oxidases/imunologia , Fagócitos/enzimologia , Fagócitos/imunologia , Choque , Trypanosoma cruzi/imunologia , Animais , Células Cultivadas , Doença de Chagas/mortalidade , Modelos Animais de Doenças , Feminino , Interferon gama/sangue , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , Parasitemia/imunologia , Baço/imunologia , Análise de Sobrevida , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismoRESUMO
To define the role of CD38 in the migration of neutrophils to the liver and consequently in the induction of an innate immune response during murine hepatic amoebiasis by Entamoeba histolytica, we examined amoebic liver abscess development (ALA), presence of amoebae and neutrophils, and expression levels of cytokines and other inflammation mediators mRNA, in infected wild-type and CD38 Knockout (CD38KO) C57BL/6J mice. Results showed that CD38KO mice undergo a delay in ALA development in comparison with the wild-type strain. The presence of amoebae lasted longer in CD38(-/-), and although neutrophils arrived to the liver in both strains, there was a clear difference in the time between the two strains; whereas in the wild-type strain, neutrophils arrived at early times (6-12 h), in the CD38KO strain, neutrophils arrived later (48-72 h). Cytokines profile during the innate immune response development (TNF-α, IL-1ß, IL-6) was, for WT mice concomitant with, and preceded, for CD38KO mice, the time in which neutrophils were present in the liver lesion. In conclusion, CD38 is important for neutrophils migration during hepatic amoebiasis, and in turn, these cells play an important role in the innate immune response.
Assuntos
ADP-Ribosil Ciclase 1/deficiência , Entamoeba histolytica/imunologia , Imunidade Inata , Abscesso Hepático Amebiano/imunologia , Fígado/imunologia , Glicoproteínas de Membrana/deficiência , Neutrófilos/imunologia , ADP-Ribosil Ciclase 1/imunologia , Animais , Citocinas/biossíntese , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Mediadores da Inflamação/imunologia , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de TempoRESUMO
Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. Epididymal protein CRISP1, a member of the Cysteine-RIch Secretory Protein (CRISP) family, was identified by our laboratory and postulated to participate in both sperm-zona pellucida (ZP) interaction and gamete fusion by binding to egg-complementary sites. To elucidate the functional role of CRISP1 in vivo, we disrupted the Crisp1 gene and evaluated the effect on animal fertility and several sperm parameters. Male and female Crisp1(-/-) animals exhibited no differences in fertility compared to controls. Sperm motility and the ability to undergo a spontaneous or progesterone-induced acrosome reaction were neither affected in Crisp1(-/-) mice. However, the level of protein tyrosine phosphorylation during capacitation was clearly lower in mutant sperm than in controls. In vitro fertilization assays showed that Crisp1(-/-) sperm also exhibited a significantly reduced ability to penetrate both ZP-intact and ZP-free eggs. Moreover, when ZP-free eggs were simultaneously inseminated with Crisp1(+/+) and Crisp1(-/-) sperm in a competition assay, the mutant sperm exhibited a greater disadvantage in their fusion ability. Finally, the finding that the fusion ability of Crisp1(-/-) sperm was further inhibited by the presence of CRISP1 or CRISP2 during gamete co-incubation, supports that another CRISP cooperates with CRISP1 during fertilization and might compensate for its lack in the mutant mice. Together, these results indicate that CRISP proteins are players in the mammalian fertilization process. To our knowledge this is the first knockout mice generated for a CRISP protein. The information obtained might have important functional implications for other members of the widely distributed and evolutionarily conserved CRISP family.
Assuntos
Fertilização/fisiologia , Glicoproteínas de Membrana/deficiência , Espermatozoides/fisiologia , Reação Acrossômica , Animais , Fertilidade , Marcação de Genes , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Capacitação EspermáticaRESUMO
TLRs function as pattern recognition receptors in mammals and play an essential role in the recognition of microbial components. We found that the injection of glycoinositolphospholipids (GIPLs) from Trypanosoma cruzi into the peritoneal cavity of mice induced neutrophil recruitment in a TLR4-dependent manner: the injection of GIPL in the TLR4-deficient strain of mice (C57BL/10ScCr) caused no inflammatory response. In contrast, in TLR2 knockout mice, neutrophil chemoattraction did not differ significantly from that seen in wild-type controls. GIPL-induced neutrophil attraction and MIP-2 production were also severely affected in TLR4-mutant C3H/HeJ mice. The role of TLR4 was confirmed in vitro by testing genetically engineered mutants derived from TLR2-deficient Chinese hamster ovary (CHO)-K1 fibroblasts that were transfected with CD14 (CHO/CD14). Wild-type CHO/CD14 cells express the hamster TLR4 molecule and the mutant line, in addition, expresses a nonfunctional form of MD-2. In comparison to wild-type cells, mutant CHO/CD14 cells failed to respond to GIPLs, indicating a necessity for a functional TLR4/MD-2 complex in GIPL-induced NF-kappaB activation. Finally, we found that TLR4-mutant mice were hypersusceptible to T. cruzi infection, as evidenced by a higher parasitemia and earlier mortality. These results demonstrate that natural resistance to T. cruzi is TLR4 dependent, most likely due to TLR4 recognition of their GIPLs.
Assuntos
Doença de Chagas/imunologia , Doença de Chagas/patologia , Citocinas/biossíntese , Glicolipídeos/fisiologia , Mediadores da Inflamação/fisiologia , Glicoproteínas de Membrana/fisiologia , Fosfolipídeos/fisiologia , Receptores de Superfície Celular/fisiologia , Trypanosoma cruzi/imunologia , Animais , Células CHO , Doença de Chagas/genética , Quimiocina CXCL2 , Quimiocinas/biossíntese , Cricetinae , Citocinas/fisiologia , Glicolipídeos/administração & dosagem , Glicolipídeos/farmacologia , Imunidade Inata/genética , Mediadores da Inflamação/metabolismo , Injeções Intraperitoneais , Interleucina-10/biossíntese , Cinética , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Fosfolipídeos/administração & dosagem , Fosfolipídeos/farmacologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/biossínteseRESUMO
We demonstrate here that neutrophils from chronic granulomatous disease (CGD) patients release larger amounts of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) than neutrophils from control subjects. Incremental cytokine production was observed under both basal and stimulated conditions in neutrophils from two CGD (gp 91phox) patients. The basal production of IL-8 was over seven-fold greater in CGD patients. The two samples assayed showed 3- and 10-fold increases in TNF-alpha. Basically, the same magnitude of increment was observed in lypopolysaccharide (LPS) and serum amyloid A protein (SAA)-stimulated cells. We also found that the levels of SAA and IL-8 were higher in the serum of CGD patients than the levels found in the serum of healthy donors. The increased responsiveness of neutrophils from CGD patients may be closely related with a deficiency in the assembly of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme system, or it may be due to a frequent inflammatory condition in these patients. In the latter case, the increased serum levels of systemic inflammatory factors, among them SAA, would contribute to the sustained accumulation and activation of phagocytes. Whatever the origin, the excessive production of cytokines may lead to inappropriate activation and tissue injury and even to increased susceptibility to invasive microorganisms, impairing the quality life of CGD patients.
Assuntos
Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/imunologia , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/deficiência , NADPH Oxidases/deficiência , Neutrófilos/imunologia , Proteína Amiloide A Sérica/farmacologia , Adulto , Criança , Feminino , Doença Granulomatosa Crônica/genética , Humanos , Interleucina-8/análise , Interleucina-8/sangue , Masculino , Glicoproteínas de Membrana/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , Neutrófilos/química , Neutrófilos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/análiseRESUMO
Studies performed in vitro suggest that activation of Toll-like receptors (TLRs) by parasite-derived molecules may initiate inflammatory responses and host innate defense mechanisms against Trypanosoma cruzi. Here, we evaluated the impact of TLR2 and myeloid differentiation factor 88 (MyD88) deficiencies in host resistance to infection with T. cruzi. Our results show that macrophages derived from TLR2 (-/-) and MyD88(-/-) mice are less responsive to GPI-mucin derived from T. cruzi trypomastigotes and parasites. In contrast, the same cells from TLR2(-/-) still produce TNF-alpha, IL-12, and reactive nitrogen intermediates (RNI) upon exposure to live T. cruzi trypomastigotes. Consistently, we show that TLR2(-/-) mice mount a robust proinflammatory cytokine response as well as RNI production during the acute phase of infection with T. cruzi parasites. Further, deletion of the functional TLR2 gene had no major impact on parasitemia nor on mortality. In contrast, the MyD88(-/-) mice had a diminished cytokine response and RNI production upon acute infection with T. cruzi. More importantly, we show that MyD88(-/-) mice are more susceptible to infection with T. cruzi as indicated by the higher parasitemia and accelerated mortality, as compared with the wild-type mice. Together, our results indicate that T. cruzi parasites elicit an alternative inflammatory pathway independent of TLR2. This pathway is partially dependent on MyD88 and necessary for mounting optimal inflammatory and RNI responses that control T. cruzi replication during the early stages of infection.