RESUMO
Lysophosphatidic acid (LPA) induces a wide range of cellular processes and its signaling is increased in several cancers including glioblastoma (GBM), a high-grade astrocytoma, which is the most common malignant brain tumor. LPA1 receptor is expressed in GBM cells and its signaling pathways activate protein kinases C (PKCs). A downstream target of PKC, involved in GBM progression, is the intracellular progesterone receptor (PR), which can be phosphorylated by this enzyme, increasing its transcriptional activity. Interestingly, in GBM cells, PKCα isotype translocates to the nucleus after LPA stimulation, resulting in an increase in PR phosphorylation. In this study, we determined that LPA1 receptor activation induces protein-protein interaction between PKCα and PR in human GBM cells; this interaction increased PR phosphorylation in serine400. Moreover, LPA treatment augmented VEGF transcription, a known PR target. This effect was blocked by the PR selective modulator RU486; also, the activation of LPA1/PR signaling promoted migration of GBM cells. Interestingly, using TCGA data base, we found that mRNA expression of LPAR1 increases according to tumor malignancy and correlates with a lower survival in grade III astrocytomas. These results suggest that LPA1/PR pathway regulates GBM progression.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína Quinase C-alfa/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Lisofosfolipídeos/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The central autonomic network, which is connected to the limbic system structures including the amygdala (AMY) and anterior hippocampus (aHIP), regulates the sympathetic and parasympathetic modulation of visceromotor, neuroendocrine, pain, and behavior manifestations during stress responses. Heart rate variability (HRV) is useful to estimate the cardiac autonomic tone. The levels of phosphorylation on the Ser831 and Ser845 sites of the GluA1 subunit of the AMPAr (P-GluA1-Ser845 and P-GluA1-Ser831) are useful markers of synaptic plasticity. The relation between synaptic plasticity in the human limbic system structures and autonomic regulation in humans is unknown. This study investigated the association between HRV and neurochemistry biomarkers of synaptic plasticity in AMY and aHIP. HRV indices were obtained from the resting state electrocardiogram of patients with drug-resistant mesial temporal lobe epilepsy (MTLE, n = 18) and the levels of P-GluA1-Ser845 and P-GluA1-Ser831 in the AMY and aHIP resected during the epilepsy surgery. A backward stepwise multiple linear regression models were used to analyze the association between HRV and synaptic plasticity biomarkers controlling for imbalances in the distribution of sociodemographic, clinical, neuroimaging, and neurosurgical variables. P-GluA1-Ser845 levels in AMY show a negative association (p < 0.05) with the 3 investigated parasympathetic autonomic HRV indices (SDNN, rMSSD, and HF) predicting 24 to 40% of their variation. The final multiple linear regression models include disease duration and levels of P-GluA1-Ser845 and predict 24 to 56% of cardiac autonomic tone variation (p < 0.01). P-GluA1-Ser845 levels in AMY and aHIP are negatively associated with the resting HRV in MTLE-HS indicating that increased synaptic efficiency in amygdala is associated with a parasympathetic cardiac autonomic tone impairment. The results suggest that specific changes in synaptic plasticity may be involved in the brain-heart axis regulation by the limbic system.
Assuntos
Sistema Nervoso Autônomo/metabolismo , Coração/inervação , Sistema Límbico/metabolismo , Fosfosserina/metabolismo , Receptores de AMPA/metabolismo , Tonsila do Cerebelo/metabolismo , Biomarcadores/metabolismo , Feminino , Frequência Cardíaca , Hipocampo/metabolismo , Humanos , Masculino , Plasticidade Neuronal , FosforilaçãoRESUMO
To relieve endoplasmic reticulum (ER) stress, IRE1 splices XBP1 messenger RNA (mRNA) or engages regulated IRE1-dependent decay (RIDD) of other mRNAs. Upon XBP1 deficiency, IRE1 switches to perform RIDD. We examined IRE1 in XBP1-deficient B cells and discovered that IRE1 undergoes phosphorylation at S729. We generated an anti-phospho-S729 antibody to investigate such phosphorylation. Compared with pharmacological ER stress inducers or Toll-like receptor ligands, the bacterial subtilase cytotoxin has an unusual capability in causing rapid and strong phosphorylation at S729 and triggering B cells to express spliced XBP1. To assess the function of S729 in IRE1, we generated S729A knock-in mice and found S729 is critically important for lipopolysaccharide-stimulated plasmablasts to respond to additional ER stress and for antibody production in response to immunization. We further crossed mice carrying an S729A mutation or ΔIRE1 (missing the kinase domain) with B cell-specific XBP1-deficient mice to trigger RIDD and discovered a critical role for S729 in regulating RIDD in B cells.
Assuntos
Formação de Anticorpos , Linfócitos B/metabolismo , Imunização , Proteínas de Membrana/metabolismo , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade de RNA , Sequência de Aminoácidos , Animais , Ditiotreitol/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lipopolissacarídeos , Proteínas de Membrana/química , Camundongos Endogâmicos C57BL , Modelos Animais , Modelos Biológicos , Fosforilação , Proteínas Serina-Treonina Quinases/química , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Tamoxifen is a standard endocrine therapy for estrogen receptor positive breast cancer patients. Despite its success, development of resistance mechanisms is still a serious clinical problem. Deregulation of survival signaling pathways play a key role in tamoxifen resistance, being upregulation of Rac1-PAK1 signaling pathway one of the most important. Here, we report the development of the breast cancer cell model MCF7::C1199 having Rac1 enhanced activity with the aim of evaluating the role of Rac1 in acquired endocrine resistance. These cells not only showed distinctive features of Rac1-regulated process as increased migration and proliferation rates, but also showed that upregulation of Rac1 activity triggered a hormonal-independent and tamoxifen resistant phenotype. We also demonstrated that PAK1 activity increases in response to Tamoxifen, increasing phosphorylation levels of estrogen receptor at Ser305, a key phosphorylation site involved in tamoxifen resistance. Finally, we evaluated the effect of 1A-116, a specific Rac1 inhibitor developed by our group, in tamoxifen-resistant cells. 1A-116 effectively restored tamoxifen anti-proliferative effects, switched off PAK1 activity and decreased estrogen receptor phospho-Ser305 levels. Since combination schemes of novel targeted agents with endocrine therapy could be potential new strategies to restore tamoxifen sensibility, these results show that inhibition of Rac1-PAK1 signaling pathway may provides benefits to revert resistance mechanisms in endocrine therapies.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estrogênios/farmacologia , Feminino , Humanos , Células MCF-7 , Modelos Biológicos , Fenótipo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Regulação para Cima/efeitos dos fármacos , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Vasodilator-stimulated phosphoprotein (VASP) and Zyxin are interacting proteins involved in cellular adhesion and motility. PKA phosphorylates VASP at serine 157, regulating VASP cellular functions. VASP interacts with ABL and is a substrate of the BCR-ABL oncoprotein. The presence of BCR-ABL protein drives oncogenesis in patients with chronic myeloid leukemia (CML) due to a constitutive activation of tyrosine kinase activity. However, the function of VASP and Zyxin in BCR-ABL pathway and the role of VASP in CML cells remain unknown. In vitro experiments using K562 cells showed the involvement of VASP in BCR-ABL signaling. VASP and Zyxin inhibition decreased the expression of anti-apoptotic proteins, BCL2 and BCL-XL. Imatinib induced an increase in phosphorylation at Ser157 of VASP and decreased VASP and BCR-ABL interaction. VASP did not interact with Zyxin in K562 cells; however, after Imatinib treatment, this interaction was restored. Corroborating our data, we demonstrated the absence of phosphorylation at Ser157 in VASP in the bone marrow of CML patients, in contrast to healthy donors. Phosphorylation of VASP on Ser157 was restored in Imatinib responsive patients though not in the resistant patients. Therefore, we herein identified a possible role of VASP in CML pathogenesis, through the regulation of BCR-ABL effector proteins or the absence of phosphorylation at Ser157 in VASP.
Assuntos
Benzamidas/farmacologia , Moléculas de Adesão Celular/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Zixina/metabolismo , Apoptose/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proliferação de Células/efeitos dos fármacos , Células Clonais , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Células K562 , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína bcl-X/metabolismoRESUMO
The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Fosfoproteínas/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , Mitocôndrias/enzimologia , Dinâmica Mitocondrial/genética , Modelos Biológicos , Fosfoproteínas/genética , Fosforilação , Fosfosserina/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esteroides/biossíntese , Transcrição GênicaRESUMO
Leptin, a peripheral signal synthetized by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work, we aimed to study the molecular mechanisms that mediate the survival effect of leptin in placenta. We used the human placenta choriocarcinoma BeWo and first trimester Swan-71 cell lines, as well as human placental explants. We tested the late phase of apoptosis, triggered by serum deprivation, by studying the activation of Caspase-3 and DNA fragmentation. Recombinant human leptin added to BeWo cell line and human placental explants, showed a decrease on Caspase-3 activation. These effects were dose dependent. Maximal effect was achieved at 250 ng leptin/ml. Moreover, inhibition of endogenous leptin expression with 2 µM of an antisense oligonucleotide, reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. We analyzed the presence of low DNA fragments, products from apoptotic DNA cleavage. Placental explants cultivated in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. Taken together these results reinforce the survival effect exerted by leptin on placental cells. To improve the understanding of leptin mechanism in regulating the process of apoptosis we determined the expression of different intermediaries in the apoptosis cascade. We found that under serum deprivation conditions, leptin increased the anti-apoptotic BCL-2 protein expression, while downregulated the pro-apoptotic BAX and BID proteins expression in Swan-71 cells and placental explants. In both models leptin augmented BCL-2/BAX ratio. Moreover we have demonstrated that p53, one of the key cell cycle-signaling proteins, is downregulated in the presence of leptin under serum deprivation. On the other hand, we determined that leptin reduced the phosphorylation of Ser-46 p53 that plays a pivotal role for apoptotic signaling by p53. Our data suggest that the observed anti-apoptotic effect of leptin in placenta is in part mediated by the p53 pathway. In conclusion, we provide evidence that demonstrates that leptin is a trophic factor for trophoblastic cells.
Assuntos
Apoptose , Regulação para Baixo , Leptina/metabolismo , Placenta/citologia , Placenta/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Fosforilação , Fosfosserina/metabolismo , Gravidez , Trofoblastos/citologia , Trofoblastos/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
This study sought to evaluate the effects of a single session of exercise on the expression of Hsp70, of c-jun N-terminal kinase (JNK), and insulin receptor substrate 1 serine 612 (IRS(ser612)) phosphorylation in the skeletal muscle of obese and obese insulin-resistant patients. Twenty-seven volunteers were divided into three experimental groups (eutrophic insulin-sensitive, obese insulin-sensitive, and obese insulin-resistant) according to their body mass index and the presence of insulin resistance. The volunteers performed 60 min of aerobic exercise on a cycle ergometer at 60 % of peak oxygen consumption. M. vastus lateralis samples were obtained before and after exercise. The protein expressions were evaluated by Western blot. Our findings show that compared with paired eutrophic controls, obese subjects have higher basal levels of p-JNK (100 ± 23 % vs. 227 ± 67 %, p = 0.03) and p-IRS-1(ser612) (100 ± 23 % vs. 340 ± 67 %, p < 0.001) and reduced HSP70 (100 ± 16 % vs. 63 ± 12 %, p < 0.001). The presence of insulin resistance results in a further increase in p-JNK (460 ± 107 %, p < 0.001) and a decrease in Hsp70 (46 ± 5 %, p = 0.006), but p-IRS-1(ser612) levels did not differ from obese subjects (312 ± 73 %, p > 0.05). Exercise reduced p-JNK in obese insulin-resistant subjects (328 ± 33 %, p = 0.001), but not in controls or obese subjects. Furthermore, exercise reduced p-IRS-1(ser612) for both obese (122 ± 44 %) and obese insulin-resistant (185 ± 36 %) subjects. A main effect of exercise was observed in HSP70 (p = 0.007). We demonstrated that a single session of exercise promotes changes that characterize a reduction in cellular stress that may contribute to exercise-induced increase in insulin sensitivity.
Assuntos
Exercício Físico , Resistência à Insulina , Músculo Esquelético/patologia , Estresse Fisiológico , Adulto , Jejum/sangue , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Insulina/sangue , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/enzimologia , Fosforilação , Fosfosserina/metabolismoRESUMO
PURPOSE: Retinal degeneration caused by a defect in the phototransduction cascade leads to the apoptosis of photoreceptor cells, although the precise molecular mechanism is still unknown. In addition, constant low light exposure produces photoreceptor cell death through the activation of downstream phototransduction. The authors investigated the time course and molecular mechanisms of death and the rhodopsin phosphorylation occurring during retinal degeneration after exposure to continuous low-intensity light. METHODS: Wistar rats were exposed to constant cool white 200 lx intensity LED light (LL) for one to ten days and compared with animals kept in the dark (DD) or controls exposed to a regular 12:12 h (LD) cycle. One eye from each rat was used for histological and quantitative outer nuclear layer (ONL) analysis and the other for biochemical assays. RESULTS: The histological analysis showed a significant reduction in the ONL of LL-exposed rats after seven days compared with LD- or DD-exposed rats. Retinal analysis by flow cytometer and the TUNEL assay revealed an increase in cell death in the ONL, the in vitro enzymatic activity assay and western blot analysis showing no caspase-3 activation. The rhodopsin analysis demonstrated more phosphorylation in serine 334 residues (Ser(334)) in LL-exposed than in LD- or DD-exposed rats. However, for all times studied, rhodopsin was completely dephosphorylated after four days of DD treatment. CONCLUSIONS: Constant light exposure for seven days produces ONL reduction by photoreceptor cell death through a capase-3-independent mechanism. Increases in rhodopsin-phospho-Ser(334) levels were observed, supporting the notion that changes in the regulation of the phototransduction cascade occur during retinal degeneration.
Assuntos
Luz , Mamíferos/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Degeneração Retiniana/patologia , Animais , Anexina A5/metabolismo , Caspase 3/metabolismo , Morte Celular/efeitos da radiação , Modelos Animais de Doenças , Marcação In Situ das Extremidades Cortadas , Fosforilação/efeitos da radiação , Fosfosserina/metabolismo , Células Fotorreceptoras de Vertebrados/enzimologia , Propídio/metabolismo , Ratos , Ratos Wistar , Degeneração Retiniana/enzimologia , Rodopsina/metabolismoRESUMO
Polycystic ovary syndrome (PCOS) is a common hyperandrogenic disorder associated with insulin resistance. Insulin exerts its metabolic function by activating the PI3K/Akt pathway and favoring glucose uptake. Caveolin-1 is a scaffolding protein which increases insulin receptor (IR) stability. Alternatively, activation of IR increases caveolin-1 phosphorylation on tyrosine-14. Furthermore, endometrial tissue from PCOS patients is proposed to be insulin resistant; however, the particular role of testosterone in modulating the metabolic effects of insulin remains unexplored in endometrial stromal cells. To evaluate whether androgens modulate the response to insulin, T-HESCs cells were stimulated with 100 nM testosterone for 24 h and changes in the protein levels of caveolin-1, IR, and Akt were determined by Western blotting (WB). After testosterone treatment, the consequences of acute insulin stimulation were evaluated by WB analysis of phospho-S473Akt and phospho-Y14Caveolin-1, as well as by measuring glucose incorporation analyzing 2-deoxyglucose (2-DOG) uptake. For cells pretreated with testosterone, higher IR, IRS-1, and caveolin-1 protein levels compared with control conditions were detected. However, in testosterone treated cells acute insulin stimulation did not increase phospho-S473Akt and phospho-Y14caveolin-1 levels and reduced 2-DOG uptake was observed compared to control cells. Our results suggest that testosterone may have a detrimental role on the metabolic effects of insulin in endometrial stromal cell cultures. Thus, the high androgen levels in patients with PCOS may favor insulin resistance observed in endometria from these women.