Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
1.
Development ; 143(6): 1005-15, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26893349

RESUMO

Insulin inhibits transcription factor Forkhead box O (FoxO) activity, and the steroid hormone 20-hydroxyecdysone (20E) activates FoxO; however, the mechanism is unclear. We hypothesized that 20E upregulates phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN) expression to activate FoxO, thereby promoting proteolysis during molting in the lepidopteran insect Helicoverpa armigera. FoxO expression is increased during molting and metamorphosis. The knockdown of FoxO in fifth instar larvae results in larval molting failure. 20E inhibits FoxO phosphorylation, resulting in FoxO nuclear translocation. Insulin, via Akt, induces FoxO phosphorylation and cytoplasmic localization. 20E represses insulin-induced Akt phosphorylation and FoxO phosphorylation. 20E, via ecdysone receptor B1 (EcRB1) and the ultraspiracle protein (USP1), upregulates PTEN expression, which represses Akt phosphorylation, thereby repressing FoxO phosphorylation. The non-phosphorylated FoxO enters the nucleus and attaches to a FoxO-binding element in the upstream region of the Broad isoform 7 (BrZ7) gene to regulate BrZ7 transcription under 20E induction. 20E upregulates FoxO expression via EcRB1 and USP1. FoxO regulation of BrZ7 expression regulates Carboxypeptidase A expression for final proteolysis during insect molting. Hence, 20E activates FoxO via upregulating PTEN expression to counteract insulin activity and promote proteolysis.


Assuntos
Ecdisterona/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Muda/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Proteólise/efeitos dos fármacos , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sequência Conservada , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas de Insetos/metabolismo , Insulina/farmacologia , Larva/fisiologia , Modelos Biológicos , Mariposas/efeitos dos fármacos , Mariposas/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos
2.
Lipids Health Dis ; 18(1): 138, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186008

RESUMO

BACKGROUND: Hepatitis B virus (HBV) infection remains a global health issue associated with substantial morbidity and mortality. Serum apolipoprotein C3 (ApoC3) and apolipoprotein A5 (ApoA5) levels were decreased in chronic hepatitis B (CHB) patients, however the relationship between ApoC3 or ApoA5 and HBV DNA load remains elusive. METHODS: A total of 384 CHB patients including 194 HBsAg(+) HBeAg(-) and 190 HBsAg(+) HBeAg(+) and 154 healthy individuals were recruited in our study. Serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST), total cholesterol (Chol), triglycerides (TG), apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB), high-density lipoproteins cholesterol (HDL-C), low-density lipoproteins cholesterol (LDL-C) and lipoprotein a (Lpa) were examined in an automatic biochemical analyzer. Apolipoprotein A5 (ApoA5) and apolipoprotein C3 (ApoC3) were detected via ELISA. RESULTS: Serum ApoA1, ApoB, ApoC3 and ApoA5 levels were reduced in CHB patients. In HBeAg(-) CHB patients, plasma ApoC3 levels were negatively associated with HBV DNA load (r = 0.219, P < 0.001). But no correlation between ApoA5 and HBV DNA load was observed in CHB patients. CONCLUSIONS: These data showed that HBV infection inhibits lipid metabolism and ApoC3 is negatively associated with HBV DNA load in HBeAg (-) CHB patients. These findings provided new evidence about the link between ApoC3-related lipid metabolism and immune response.


Assuntos
HDL-Colesterol/sangue , LDL-Colesterol/sangue , DNA Viral/genética , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/sangue , Hepatite B Crônica/virologia , Triglicerídeos/sangue , Adulto , Apolipoproteína A-V/sangue , Apolipoproteína C-III , Apolipoproteínas B/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
3.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 39(4): 499-505, 2017 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-28877827

RESUMO

Objective To investigate the effects of gender and age on the prevalence and complications of nonalcoholic fatty liver disease(NAFLD). Methods A total of 8429 NAFLD patients were selected from the Health Check-up Center and Outpatient Departments of Qilu Hospital of Shandong University(Qingdao).The questionnaire-based survey,physical examinations,biochemical tests,and liver ultrasonography were performed for all cases.Patients were divided into young group(<45 years),middle aged group(45 years≤age<60 years),and old group(≥60 years)according to age,and the clinical features and laboratory findings were analyzed. Results The proportion of male patients gradually decreased with age,while the proportion of female patients increased(P<0.01);The incidences of metabolic diseases showed significant difference among young group,middle aged group,and old group(P<0.01).Except for hyperlipidemia,the proportion of male patients with NAFLD-accompanied metabolic symdrome was significantly higher than that of female patients in all three age groups(all P<0.01). Conclusions The prevalence of NAFLD-accompanied metabolic syndrome disease is associated with age and gender.This finding is useful for the prevention and treatment of NAFLD.


Assuntos
Fatores Etários , Síndrome Metabólica/epidemiologia , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Fatores Sexuais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de Risco , Ultrassonografia
4.
J Biol Chem ; 289(19): 13026-41, 2014 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-24692553

RESUMO

In addition to the classical nuclear receptor pathway, there is a nongenomic pathway in the cell membrane that regulates gene expression in animal steroid hormone signaling; however, this mechanism is unclear. Here, we report that the insect steroid hormone 20-hydroxyecdysone (20E) regulates calcium influx via phospholipase Cγ1 (PLCG1) to modulate the protein kinase C phosphorylation of the transcription factor ultraspiracle (USP1) in the lepidopteran insect Helicoverpa armigera. The PLCG1 mRNA levels are increased during the molting and metamorphic stages. The depletion of PLCG1 by RNA interference can block 20E-enhanced pupation, cause larvae death and pupation defects, and repress 20E-induced gene expression. 20E may induce the tyrosine phosphorylation of PLCG1 at the cytosolic tyrosine kinase (Src) homology 2 domains and then determine the migration of PLCG1 toward the plasma membrane. The G-protein-coupled receptor (GPCR) inhibitor suramin, Src family kinase inhibitor PP2, and the depletions of ecdysone-responsible GPCR (ErGPCR) and Gαq restrain the 20E-induced tyrosine phosphorylation of PLCG1. PLCG1 participates in the 20E-induced Ca(2+) influx. The inhibition of GPCR, PLC, inositol 1,4,5-trisphosphate receptor, and calcium channels represses the 20E-induced Ca(2+) influx. Through calcium signaling, PLCG1 mediates the transcriptional activation driven by the ecdysone-response element. Through PLCG1 and calcium signaling, 20E regulates PKC phosphorylation of USP1 at Ser-21 to determine its ecdysone-response element binding activity. These results suggest that 20E activates PLCG1 via the ErGPCR and Src family kinases to regulate Ca(2+) influx and PKC phosphorylation of USP1 to subsequently modulate gene transcription for metamorphosis.


Assuntos
Sinalização do Cálcio/fisiologia , Ecdisterona/metabolismo , Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Fosfolipase C gama/metabolismo , Receptores de Esteroides/metabolismo , Animais , Antinematódeos/farmacologia , Sequência de Bases , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Ecdisterona/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Insetos/genética , Dados de Sequência Molecular , Mariposas/genética , Fosfolipase C gama/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Esteroides/genética , Suramina/farmacologia
5.
J Biol Chem ; 289(38): 26630-26641, 2014 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-25096576

RESUMO

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7.


Assuntos
Ecdisterona/farmacologia , Proteínas de Insetos/metabolismo , Hormônios Juvenis/fisiologia , Mariposas/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Células Cultivadas , Ecdisterona/fisiologia , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Hormônios Juvenis/farmacologia , Larva/crescimento & desenvolvimento , Metamorfose Biológica/efeitos dos fármacos , Dados de Sequência Molecular , Controle de Pragas , Fosforilação , Filogenia , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Transcrição Gênica
6.
Biochim Biophys Acta ; 1830(11): 5184-92, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23850472

RESUMO

BACKGROUND: Heat shock protein 90 (Hsp90) interacts with steroid hormone receptors, signaling kinases, and various transcription factors. However, the mechanism by which Hsp90 interacts with different proteins in various pathways remains unclear. METHODS: Western blot was used to study Hsp90 expression profile in Helicoverpa armigera (Lepidoptera). RNA interference was performed to investigate the function of Hsp90 in 20-hydroxyecdysone (20E) and juvenile hormone (JH) signal pathways. The binding of Hsp90 to the transcription factor ultraspiracle protein (USP1) and JH candidate receptor methoprene-tolerant (Met1) was analyzed by co-immunoprecipitation. Phospho-(Ser) PKC substrate antibody was used to detect Hsp90 phosphorylation. RESULTS: Hsp90 participated in 20E- or JH-induced gene expression. 20E induced the interaction between Hsp90 and USP1, whereas JH III and methoprene induced the interaction between Hsp90 and Met1, respectively. 20E and JH counteracted each other for these protein interactions. Both JH III and methoprene induced protein kinase C (PKC) phosphorylation of Hsp90. This process could be inhibited by phospholipase C (PLC) and PKC inhibitors. 20E suppressed JH III- or methoprene-induced PKC phosphorylation of Hsp90. CONCLUSION: 20E maintained the non-PKC-phosphorylation status of Hsp90. Hsp90 interacted with USP1 to induce gene expression in the 20E pathway. JH regulated the PKC-phosphorylation status of Hsp90. Hsp90 also interacted with Met1 to induce gene expression in the JH pathway. GENERAL SIGNIFICANCE: Our study describes a novel mechanism of Hsp90 action by altering phosphorylation and protein interaction in various hormonal signaling pathways.


Assuntos
Ecdisterona/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Insetos/metabolismo , Hormônios Juvenis/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Animais , Ecdisterona/genética , Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos/genética , Hormônios Juvenis/genética , Lepidópteros/genética , Lepidópteros/metabolismo , Metoprene/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
7.
Cell Commun Signal ; 12: 9, 2014 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-24507557

RESUMO

BACKGROUND: Animal steroid hormones are conventionally known to initiate signaling via a genomic pathway by binding to the nuclear receptors. The mechanism by which 20E initiates signaling via a nongenomic pathway is unclear. RESULTS: We illustrate that 20E triggered the nongenomic pathway through a plasma membrane G-protein-coupled receptor (named ErGPCR) in the lepidopteran insect Helicoverpa armigera. The transcript of ErGPCR was increased at the larval molting stage and metamorphic molting stage by 20E regulation. Knockdown of ErGPCR via RNA interference in vivo blocked larval-pupal transition and suppressed 20E-induced gene expression. ErGPCR overexpression in the H. armigera epidermal cell line increased the 20E-induced gene expression. Through ErGPCR, 20E modulated Calponin nuclear translocation and phosphorylation, and induced a rapid increase in cytosolic Ca2+ levels. The inhibitors of T-type voltage-gated calcium channels and canonical transient receptor potential calcium channels repressed the 20E-induced Ca2+ increase. Truncation of the N-terminal extracellular region of ErGPCR inhibited its localization on the plasma membrane and 20E-induced gene expression. ErGPCR was not detected to bind with the steroid hormone analog [3H]Pon A. CONCLUSION: These results suggest that ErGPCR participates in 20E signaling on the plasma membrane.


Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Ecdisterona/metabolismo , Proteínas de Insetos/metabolismo , Lepidópteros/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Proteínas de Insetos/genética , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Receptores Acoplados a Proteínas G/genética , Calponinas
8.
Dev Biol ; 371(1): 13-22, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22824427

RESUMO

The insulin and 20-hydroxyecdysone (20E) pathways coordinately regulate insect growth and metamorphosis. However, the molecular mechanism of the interaction of these two pathways in regulating insect development is not well understood. In the present study, we found that a small GTPase Rab4b from a lepidopteran insect Helicoverpa armigera participates in gene transcription in the two pathways. The results show that RNA interference of Rab4b in larvae results in a decrease in glycogen levels, small pupae, abnormal metamorphic transition, or larval death. The molecular mechanisms are demonstrated that knockdown of Rab4b in the larvae suppresses the transcription of glycogen synthase (GS), as well as the metamorphic-initiating factor (Br) and hormone receptor 3 (HR3), but increases the transcription of Forkhead box class O (FOXO). Further studies in the cell line confirm that Rab4b is necessary for gene transcription in the insulin and 20E pathways. Rab4b locates in the cytoplasm and takes part in regulation on FOXO cytoplasmic location by insulin induction, but travels toward the cell membrane upon 20E induction without affecting the FOXO location. The transcription of Rab4b could be upregulated by insulin injection or glucose feeding to the larvae, but not by 20E or juvenile hormone analogy methoprene. Our data suggest that Rab4b takes part in metamorphosis by regulating gene transcription and glycogen level in the insulin and 20E pathways.


Assuntos
Ecdisterona/metabolismo , Glicogênio/metabolismo , Insulina/metabolismo , Metamorfose Biológica/fisiologia , Mariposas/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Transcrição Gênica/fisiologia , Proteínas rab4 de Ligação ao GTP/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Primers do DNA/genética , Fatores de Transcrição Forkhead/metabolismo , Glicogênio Sintase/metabolismo , Imuno-Histoquímica , Larva/crescimento & desenvolvimento , Larva/metabolismo , Dados de Sequência Molecular , Mariposas/metabolismo , Filogenia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Proteínas rab4 de Ligação ao GTP/genética
9.
Apoptosis ; 17(12): 1327-39, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23011181

RESUMO

The insect midgut undergoes programmed cell death (PCD) during metamorphosis, but the molecular basis for this phenomenon has not been demonstrated. We report a mod(mdg4) protein [designated as mod(mdg4)1A] that is involved in hormonally regulated insect midgut PCD, from the lepidopteran Helicoverpa armigera. Mod(mdg4)1A is localized in the larval midgut and is highly expressed during metamorphosis. Knockdown of mod(mdg4)1a by feeding dsRNA to the larvae suppressed midgut PCD and delayed metamorphosis. The mechanism is that mod(mdg4)1a knockdown decreased the transcript levels of genes involved in PCD and metamorphosis, but increased the transcript level of inhibitor of apoptosis survivin. The transcript level of mod(mdg4)1a is independently upregulated by 20-hydroxyecdysone (20E) or juvenile hormone (JH) analog methoprene. Overlapped 20E and methoprene counteractively regulate the transcript level of mod(mdg4)1a. 20E upregulates the mod(mdg4)1a transcript level not through its nuclear receptor EcRB1. Methoprene upregulates the mod(mdg4)1a transcript level through the juvenile hormone candidate receptor Met. These findings indicate that mod(mdg4)1a participates in midgut PCD and metamorphosis by regulating the transcript levels of a network of genes via different pathways under 20E and JH regulation.


Assuntos
Apoptose , Hormônios de Inseto/metabolismo , Metamorfose Biológica , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Animais , Sistema Digestório/citologia , Sistema Digestório/crescimento & desenvolvimento , Sistema Digestório/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mariposas/citologia , Mariposas/genética
10.
Gene ; 702: 66-74, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-30930224

RESUMO

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver with high mortality and frequent recurrence. Although various therapies provide potential cure for HCC patients, unfortunately the five-year survival rate of advanced HCC remains dismal. It is critical to explore the pathogenesis of HCC and identify novel biomarkers for early HCC diagnosis. PSMD4 is a major receptor of the 26S proteasome involved in ubiquitindependent and proteasome-mediated protein degradation. In our study, PSMD4 was overexpressed in HCC tissues and cell lines determined by Northern blot, western blot and immunohistochemistry. The silencing of PSMD4 blocked cell proliferation and tumor growth, induced cell apoptosis and inhibited the proteasome activity. Western blot results showed that the knockdown of PSMD4 blocked the expression of cyclooxygenase 2 (COX2), phosphorylated Sarcoma tyrosine kinase (P-SRC) and Bcl-2, but improved the levels of p53 and Bax in HCC, lung cancer, colorectal cancer, breast cancer and endometrial cancer cell lines. Taken together, these findings indicated that the subunit of 26S proteasome PSMD4 exerts as an oncogene in HCC and other cancers via regulating the expression p53, Bcl-2 and Bax. These findings enriched the pathogenesis of HCC, and provided a new biomarker for cancers diagnosis and a new target for cancers therapy.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Animais , Apoptose , Carcinogênese , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos Nus , Complexo de Endopeptidases do Proteassoma/biossíntese , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA
11.
Gene ; 677: 17-23, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30036656

RESUMO

Endometrial cancer (EC) is the fourth most common cancer in women and exhibits increasing incidence and mortality. Some reports showed that the 5-year survival rate of EC was closely associated with the diagnosed stage. It is urgent to screen for sensitive and specific targets to improve early detection and EC therapy. In our study, we found that zinc finger protein like 1 (ZFPL1) was highly expressed in EC tissues and the EC cell line RL95-2, as detected via RT-qPCR and western blot analysis. Immunocytochemistry results showed that ZFPL1 was localized in the Golgi complex dependent on the C-terminal transmembrane domain. The MTT and EdU stains were employed to examine the effect of ZFPL1 on cell proliferation. We found that the silencing of ZFPL1 blocked cell proliferation and the expression of p-Akt308 and p-Akt473 but improved the protein level of PTEN. The overexpression of ZFPL1 and ZFPL1ΔTMD (deletion of the transmembrane domain) promoted cell proliferation and induced the expression of p-Akt308 and p-Akt473. However, the overexpression of ZFPL1ΔRING (deletion of the RING domain) caused loss of the function of ZFPL1 in cell proliferation and the PI3K/Akt pathway. In summary, ZFPL1 induced RL95-2 cell proliferation and was involved in PI3K/Akt pathway, suggesting the oncogenic role of ZFPL1 during EC development. Additionally, the RING domain was essential for the function of ZFPL1. These findings provided a new biomarker for EC diagnosis and therapy.


Assuntos
Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Domínios Proteicos/fisiologia , Linhagem Celular Tumoral , Endométrio/metabolismo , Endométrio/patologia , Feminino , Complexo de Golgi , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia
12.
Cell Calcium ; 68: 24-33, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29129205

RESUMO

Animal steroid hormones stimulate extracellular Ca2+ influx into cells; however, the mechanism remains unclear. In this study, we determined that the Ca2+ influx induced by steroid hormone 20-hydroxyecdysone (20E) is mediated by the calcium release-activated calcium channel modulator 1 (CRACM1/Orai1). The Orai1 mRNA is highly expressed during midgut programmed cell death in the lepidopteran insect Helicoverpa armigera. 20E upregulated the expression of Orai1 in H. armigera larvae and in an epidermal cell line (HaEpi). Knockdown of Orai1 in HaEpi cells blocked 20E-induced Ca2+ influx, and the inhibitor of inositol 1, 4, 5-trisphosphate receptor (IP3R) Xestospongin (XeC) blocked 20E-induced Ca2+ influx, suggesting that 20E, via Orai1, induces stored-operated Ca2+ influx. Orai1 interacts with stromal interaction molecule 1(Stim1) to exert its function in 20E-induced Ca2+ influx. 20E promotes Orai1 aggregation through G-protein-coupled receptors, phospholipase C gamma 1, and Stim1. Knockdown of Orai1 in the HaEpi cell line repressed apoptosis and maintained autophagy under 20E regulation. Knockdown of Orai1 in larvae delayed pupation, repressed midgut apoptosis, maintained the midgut in an autophagic state, and repressed 20E-pathway gene expression. These results revealed that steroid hormone 20E, via Orai1, induces Ca2+ influx to promote the transition of midgut from autophagy to apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Sistema Digestório/citologia , Ecdisterona/farmacologia , Mariposas/citologia , Proteína ORAI1/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Modelos Biológicos , Muda/efeitos dos fármacos , Proteína ORAI1/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
13.
Sci Rep ; 5: 8675, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25728569

RESUMO

G-protein-coupled receptors (GPCRs) are involved in animal steroid hormone signaling, but their mechanism is unclear. In this research, we report that a GPCR called ErGPCR-2 controls steroid hormone 20-hydroxyecdysone (20E) signaling in the cell membrane of the lepidopteran insect Helicoverpa armigera. ErGPCR-2 was highly expressed during molting and metamorphosis. 20E, via ErGPCR-2, regulated rapid intracellular calcium increase, protein phosphorylation, gene transcription, and insect metamorphosis. ErGPCR-2 was located in the cell surface and was internalized by 20E induction. GPCR kinase 2 participated in 20E-induced ErGPCR-2 phosphorylation and internalization. The internalized ErGPCR-2 was degraded by proteases to desensitize 20E signaling. ErGPCR-2 knockdown suppressed the entrance of 20E analog [(3)H] ponasterone A ([(3)H]Pon A) into the cells. ErGPCR-2 overexpression or blocking of ErGPCR-2 internalization increased the entrance of [(3)H]Pon A into the cells. However, ErGPCR-2 did not bind to [(3)H]Pon A. Results suggest that ErGPCR-2 transmits steroid hormone 20E signaling and controls 20E entrance into cells in the cell membrane.


Assuntos
Membrana Celular/metabolismo , Hormônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esteroides/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Ecdisona/farmacologia , Endocitose/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lepidópteros/efeitos dos fármacos , Lepidópteros/genética , Lepidópteros/crescimento & desenvolvimento , Lepidópteros/metabolismo , Modelos Biológicos , Muda/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Trítio/metabolismo , Regulação para Cima/efeitos dos fármacos
14.
Cell Signal ; 27(4): 878-86, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25660147

RESUMO

The steroid hormone 20-hydroxyecdysone (20E) plays a critical role in insect development, particularly in larval molting and larval-pupal transition. Studies have indicated that 20E transmits its signal via a G protein-coupled receptor (GPCR)-mediated non-genomic pathway before a genomic pathway is initiated. However, the mechanism by which a 20E signal is desensitized remains unclear. We proposed that ß-arrestin1 interacts with ecdysone-responsible GPCR (ErGPCR1) to desensitize a 20E signal in the lepidopteran insect Helicoverpa armigera. Results showed that ß-arrestin1 was highly expressed in various tissues during metamorphosis. ß-Arrestin1 knockdown by RNA interference in larvae caused advanced pupation and a larval-pupal chimera. The mRNA levels of 20E-response genes were increased after ß-arrestin1 was knocked down but were decreased after ß-arrestin1 was overexpressed. 20E induced the migration of ß-arrestin1 from the cytosol to the cytoplasmic membrane to interact with ErGPCR1. The inhibitors suramin and chelerythrine chloride repressed 20E-induced ß-arrestin1 phosphorylation and membrane migration. With ErGPCR1, 20E regulated ß-arrestin1 phosphorylation on serines at positions 170 and 234. The double mutation of the amino acids Ser170 and Ser234 to asparagine inhibited phosphorylation and membrane migration of ß-arrestin1 in 20E induction. Therefore, 20E via ErGPCR1 and PKC signaling induces ß-arrestin1 phosphorylation; phosphorylated ß-arrestin1 migrates to the cytoplasmic membrane to interact with ErGPCR1 to block 20E signaling via a feedback mechanism.


Assuntos
Arrestinas/metabolismo , Ecdisterona/metabolismo , Proteínas de Insetos/metabolismo , Lepidópteros/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Arrestinas/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Proteínas de Insetos/genética , Lepidópteros/genética , Lepidópteros/crescimento & desenvolvimento , Metamorfose Biológica , Dados de Sequência Molecular , Fosforilação , Proteína Quinase C/metabolismo , Transdução de Sinais , beta-Arrestinas
15.
Endocrinology ; 155(5): 1738-50, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24517229

RESUMO

The insect steroid hormone 20-hydroxyecdysone (20E) regulates gene transcription via a genomic pathway by forming a transcription complex that binds to DNA with the help of the chaperone proteins, heat shock proteins (Hsps) Hsc70 and Hsp90. However, the nongenomic mechanisms by which 20E regulates gene expression remain unclear. In this study, we found that 20E regulated the phosphorylation of serine/threonine protein kinase cyclin-dependent kinase 10 (CDK10) through a nongenomic pathway to mediate gene transcription in the lepidopteran Helicoverpa armigera. The down-regulation of CDK10 by RNA interference in larvae and the epidermal cell line delayed development and suppressed 20E-induced gene transcription. CDK10 was localized to the nucleus via its KKRR motif, and this nuclear localization and the ATPase motif were necessary for the efficient expression of the 20E-inducible gene. The rapid phosphorylation of CDK10 was induced by 20E, whereas it was repressed by the inhibitors of G-protein-coupled receptors, phospholipase C, and Ca²âº channels. Phosphorylated CDK10 exhibited increased interactions with Hsps Hsc70 and Hsp90 and then promoted the interactions between Hsps and ecdysone receptor EcRB1 and the binding of the Hsps-EcRB1 complex to the 20E response element for the regulation of gene transcription. CDK10 depletion suppressed the formation of the Hsps-EcRB1 complex at the hormone receptor 3 promoter. These results suggest that 20E induces CDK10 phosphorylation via a nongenomic pathway to regulate gene transcription in the nucleus.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ecdisterona/farmacologia , Epigênese Genética , Proteínas de Insetos/metabolismo , Lepidópteros/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/metabolismo , Lepidópteros/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Metamorfose Biológica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Receptores de Esteroides/metabolismo , Elementos de Resposta/efeitos dos fármacos
16.
J Steroid Biochem Mol Biol ; 144 Pt B: 313-23, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25125388

RESUMO

The nuclear receptor-mediated genomic pathways of the animal steroid hormones are well known. However, the cell membrane receptor-mediated nongenomic pathways of the animal steroid hormones are little understood. In this study, we report the participation of a G-protein alpha q (Gαq)(1) subunit in the 20E nongenomic pathway in the cell membrane and regulating gene expression during molting and metamorphosis in a lepidopteran insect, Helicoverpa armigera. 20E-induced phosphorylation of Gαq was detected using two-dimensional electrophoresis techniques. Knockdown of Gαq by injecting double-stranded RNA suppressed the development of larvae, delayed metamorphosis, and inhibited 20E-induced gene expression. Gαq was distributed throughout the cell, and migrated toward the plasma membrane upon 20E induction. Gαq was necessary in the 20E-induced intracellular Ca(2+) release and extracellular Ca(2+) influx. The protein kinase C (PKC) inhibitor could repress 20E-induced phosphorylation of cyclin-dependent kinase 10 (CDK10) and transcription factor ultraspiracle (USP1). PKC inhibitor could repress the Gαq phosphorylation and membrane trafficking. These results suggest that Gαq participates in 20E signaling in the cell membrane at the pre-genomic stage by modulating the increase of the intracellular Ca(2+) and phosphorylation of CDK10 and USP1 in 20E transcription complex to regulate gene transcription.


Assuntos
Ecdisterona/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Insetos/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Proteínas de Insetos/genética , Lepidópteros , Metamorfose Biológica/fisiologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Proteases Específicas de Ubiquitina/metabolismo
17.
Insect Biochem Mol Biol ; 43(4): 328-35, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23416133

RESUMO

During the metamorphic stage of holometabolous insects, the biosynthetic precursors needed for the synthesis of a large number of adult proteins are acquired from the selective absorption of storage proteins. The very-high-density lipoprotein (VHDL), a non-hexameric storage protein, is consumed by the fat body from the hemolymph through VHDL receptor (VHDL-R)-mediated endocytosis. However, the mechanism of the uptake of VHDL by a VHDL-R remains unclear. In this study, a VHDL-R from Helicoverpa armigera was found to be involved in 20E-regulated VHDL uptake through the regulation of steroid hormone 20-hydroxyecdysone (20E). The transcripts of VHDL-R were detected mainly in the fat body and integument during the wandering stage. The transcription of VHDL-R was upregulated by 20E through the ecdysteroid receptor (EcRB1) and Ultraspiracle (USP1). In addition, 20E stimulates the phosphorylation of VHDL-R through protein kinase C for ligand binding. VHDL-R knockdown in larvae results the inhibition of development to adulthood. These data imply that 20E regulates VHDL-R on both transcriptional and posttranslational levels for VHDL absorption.


Assuntos
Ecdisterona/metabolismo , Hormônios de Inseto/metabolismo , Proteínas de Insetos/metabolismo , Lipoproteínas HDL/metabolismo , Mariposas/metabolismo , Animais , Transporte Biológico , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lipoproteínas HDL/genética , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Fosforilação , Regulação para Cima
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA