RESUMO
The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Receptor Smoothened/fisiologia , Animais , Animais Geneticamente Modificados , Domínio Catalítico/genética , Células Cultivadas , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Embrião não Mamífero , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Camundongos , Domínios e Motivos de Interação entre Proteínas/genética , Transdução de Sinais/genética , Receptor Smoothened/metabolismo , Peixe-ZebraRESUMO
Recently, a mutation was discovered in the gene PRKACB encoding the catalytic subunit ß of PKA (PKAcß) from a patient with severe Cushing's syndrome. This mutation, S54L, leads to a structural change in the glycine-rich loop of the protein. In the present study, an inhibitor with six-fold selectivity toward S54L-PKAcß mutant over the wild-type enzyme was constructed. Moreover, we developed a fluorescent assay allowing to determine side by side the affinity of commercially available PKA inhibitors, newly synthesized compounds, and fluorescent probes toward PKAcß and S54L-PKAcß.
Assuntos
Adenoma/genética , Adenoma/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Hidrocortisona/biossíntese , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Humanos , MutaçãoRESUMO
The chimeric DnaJ-PKAc enzymeresulting from an approximately 400-kb deletion of chromosome 19 is a primary contributor to the oncogenic transformation that occurs in fibrolamellar hepatocellular carcinoma, also called fibrolamellar carcinoma (FLC). This oncogenic deletion juxtaposes exon 1 of the DNAJB1 heat shock protein gene with exon 2 of the PRKACA gene encoding the protein kinase A catalytic subunit, resulting in DnaJ-PKAc fusion under the transcriptional control of the DNAJB1 promoter. The expression of DnaJ-PKAc is approximately 10 times that of wild-type (wt) PKAc catalytic subunits, causing elevated and dysregulated kinase activity that contributes to oncogenic transformation. In normal cells, PKAc activity is regulated by a group of endogenous proteins, termed protein kinase inhibitors (PKI) that competitively inhibit PKAc and assist with the nuclear export of the enzyme. Currently, it is scarcely known whether interactions with PKI are perturbed in DnaJ-PKAc. In this report, we survey existing data sets to assess the expression levels of the various PKI isoforms that exist in humans to identify those that are candidates to encounter DnaJ-PKAc in both normal liver and FLC tumors. We then compare inhibition profiles of wtPKAc and DnaJ-PKAc against PKI and demonstrate that extensive structural homology in the active site clefts of the two enzymes confers similar kinase activities and inhibition by full-length PKI and PKI-derived peptides.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico , Proteínas de Choque Térmico HSP40 , Proteínas de Fusão Oncogênica , Peptídeos/química , Inibidores de Proteínas Quinases/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas de Choque Térmico HSP40/antagonistas & inibidores , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/genética , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genéticaRESUMO
Fibrolamellar hepatocellular carcinoma (FLC) is a rare form of primary liver cancer that affects adolescents and young adults without underlying liver disease. Surgery remains the mainstay of therapy; however, most patients are either not surgical candidates or suffer from recurrence. There is no approved systemic therapy and the overall survival remains poor. Historically classified as a subtype of hepatocellular carcinoma (HCC), FLC has a unique clinical, histological, and molecular presentation. At the genomic level, FLC contains a single 400kB deletion in chromosome 19, leading to a functional DNAJB1-PRKACA fusion protein. In this review, we detail the recent advances in our understanding of the molecular underpinnings of FLC and outline the current knowledge gaps.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Transformação Celular Neoplásica/genética , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cromossomos Humanos Par 19 , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Fusão Gênica , Predisposição Genética para Doença , Proteínas de Choque Térmico HSP40/genética , Humanos , Terapia de Alvo Molecular , Recidiva Local de Neoplasia , Fenótipo , Inibidores de Proteínas Quinases/uso terapêutico , Resultado do TratamentoRESUMO
The use of synovial fluid-derived mesenchymal stem cells (SFMSCs) obtained from patients with degenerative arthropathy may serve as an alternative therapeutic strategy in osteoarthritis (OA) and rheumatoid arthritis (RA). For treatment of OA and RA patients, autologous transplantation of differentiated MSCs has several beneficial effects for cartilage regeneration including immunomodulatory activity. In this study, we induced chondrogenic differentiation of SFMSCs by inhibiting protein kinase A (PKA) with a small molecule and microRNA (miRNA). Chondrogenic differentiation was confirmed by PCR and immunocytochemistry using probes specific for aggrecan, the major cartilaginous proteoglycan gene. Absorbance of alcian blue stain to detect chondrogenic differentiation was increased in H-89 and/or miRNA-23btransfected cells. Furthermore, expression of matrix metalloproteinase (MMP)-9 and MMP-2 was decreased in treated cells. Therefore, differentiation of SFMSCs into chondrocytes through inhibition of PKA signaling may be a therapeutic option for OA or RA patients.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Isoquinolinas/farmacologia , Transplante de Células-Tronco Mesenquimais/métodos , MicroRNAs/genética , Osteoartrite/terapia , Sulfonamidas/farmacologia , Líquido Sinovial/citologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/metabolismo , Colagenases/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Osteoartrite/genéticaRESUMO
BACKGROUND AND PURPOSE: We previously reported that adenosine, acting at adenosine A(2A) receptors (A(2A)R), inhibits osteoclast (OC) differentiation in vitro (A(2A)R activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A(2A)R stimulation regulates OC differentiation, we dissected the signalling pathways involved in A(2A)R signalling. EXPERIMENTAL APPROACH: OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A(2A)R agonist CGS21680, the A(2A)R antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR. KEY RESULTS: A(2A)R stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A(2A)R stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A(2A)R stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A(2A)R stimulation inhibited OC expression of differentiation markers by a PKA-mechanism. CONCLUSIONS AND IMPLICATIONS: A(2A)R activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like rheumatoid arthritis.
Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Núcleo Celular/efeitos dos fármacos , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Subunidade p50 de NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Receptor A2A de Adenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Células Cultivadas , AMP Cíclico/agonistas , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Camundongos , Camundongos Endogâmicos C57BL , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Osteoclastos/citologia , Osteoclastos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Receptor A2A de Adenosina/química , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismoRESUMO
Lung cancer is the leading cause of cancer-related death worldwide. Hypoxia is known to increase cancer cell migration and invasion. We have previously reported that hypoxia induces epithelial-mesenchymal transition (EMT) in lung cancer cells. However, it is unknown whether hypoxia promotes lung cancer cell migration and invasion via EMT and whether cyclic AMP (cAMP) dependent protein kinase (PKA) plays a role in this process. We found that hypoxia increased PKA activity and induced mRNA and protein expression of PKA catalytic subunit α (PKACA), and regulatory subunits R1A and R1B. Knockdown of HIF-1/2α prevented hypoxia-mediated induction of PKACA mRNA expression and PKA activity. Inhibition of PKA activity with chemical inhibitors prevented EMT induced by hypoxia and tumor growth factor ß1. However, activation of PKA by forskolin and 8-Br-cAMP did not induce EMT. Furthermore, treatment with H89 and knockdown of PKACA prevented hypoxia-mediated, EMT, cell migration, and invasion, whereas overexpression of mouse PKACA rescued hypoxia-mediated migration and invasion in PKACA deficient cancer cells. Our results suggest that hypoxia enhances PKA activity by upregulating PKA gene expression in a HIF dependent mechanism and that PKA plays a key role in hypoxia-mediated EMT, migration, and invasion in lung cancer cells.
Assuntos
Hipóxia Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colforsina/farmacologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoquinolinas/farmacologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sulfonamidas/farmacologia , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Using a newly developed competitive binding assay dependent upon the reassembly of a split reporter protein, we have tested the promiscuity of a panel of reported kinase inhibitors against the AGC group. Many non-AGC targeted kinase inhibitors target multiple members of the AGC group. In general, structurally similar inhibitors consistently exhibited activity toward the same target as well as toward closely related kinases. The inhibition data was analyzed to test the predictive value of either using identity scores derived from residues within 6 Å of the active site or identity scores derived from the entire kinase domain. The results suggest that the active site identity in certain cases may be a stronger predictor of inhibitor promiscuity. The overall results provide general guidelines for establishing inhibitor selectivity as well as for the future design of inhibitors that either target or avoid AGC kinases.
Assuntos
Luciferases/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Ligação Competitiva , Domínio Catalítico , Sistema Livre de Células , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Bases de Dados Factuais , Genes Reporter , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/química , Coelhos , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-AtividadeRESUMO
Inflammation is increasingly recognized as a critical mediator of angiogenesis, and unregulated angiogenic response is involved in human diseases, including cancer. Proinflammatory prostaglandin E2 (PGE2) is secreted by many cell types and plays important roles in the process of angiogenesis via activation of cognate EP1-4 receptors. Here, we provide evidence that PGE2 promotes the in vitro tube formation of human microvascular endothelial cells, ex vivo vessel outgrowth of aortic rings, and actual in vivo angiogenesis. Use of EP subtype-selective agonists and antagonists suggested EP4 mediates the prostaglandin-induced tube formation, and this conclusion was substantiated with small interfering RNA to specifically knockdown the EP4 expression. EP4 couples to Gαs, leading to activation of protein kinase A (PKA). Inhibition of PKA activity or knockdown of PKA catalytic subunit γ with RNAi attenuates the PGE2-induced tube formation. Further, knocking down the expression of Rap1A, HSPB6, or endothelial NO synthase, which serve as PKA-activatable substrates, inhibits the tube formation, whereas knockdown of RhoA or glycogen synthase kinase 3ß that are inactivated after phosphorylation by PKA increases the tube formation. These results support the existence of EP4-to-PKA angiogenic signal and provide rationale for use of selective EP4 signal inhibitors as a probable strategy to control pathologic angiogenesis.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/fisiologia , Dinoprostona/fisiologia , Neovascularização Fisiológica/fisiologia , Receptores de Prostaglandina E Subtipo EP4/fisiologia , Animais , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Sequência de Bases , Embrião de Galinha , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Dinoprostona/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , RNA Interferente Pequeno/genética , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/genéticaRESUMO
Capacitation encompasses the molecular changes sperm undergo to fertilize an oocyte, some of which are postulated to occur via a cAMP-PRKACA (protein kinase A)-mediated pathway. Due to the recent discovery of cAMP-activated guanine nucleotide exchange factors RAPGEF3 and RAPGEF4, we sought to investigate the separate roles of PRKACA and RAPGEF3/RAPGEF4 in modulating capacitation and acrosomal exocytosis. Indirect immunofluorescence localized RAPGEF3 to the acrosome and subacrosomal ring and RAPGEF4 to the midpiece in equine sperm. Addition of the RAPGEF3/RAPGEF4-specific cAMP analogue 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8pCPT) to sperm incubated under both noncapacitating and capacitating conditions had no effect on protein tyrosine phosphorylation, thus supporting a PRKACA-mediated event. Conversely, activation of RAPGEF3/RAPGEF4 with 8pCPT induced acrosomal exocytosis in capacitated equine sperm at rates (34%) similar (P > 0.05) to those obtained in progesterone- and calcium ionophore-treated sperm. In the mouse, capacitation-dependent hyperpolarization of the sperm plasma membrane has been shown to recruit low voltage-activated T-type Ca(2+) channels, which later open in response to zona pellucida-induced membrane depolarization. We hypothesized that RAPGEF3 may be inducing acrosomal exocytosis via depolarization-dependent Ca(2+) influx, as RAPGEF3/RAPGEF4 have been demonstrated to play a role in the regulation of ion channels in somatic cells. We first compared the membrane potential (E(m)) of noncapacitated (-37.11 mV) and capacitated (-53.74 mV; P = 0.002) equine sperm. Interestingly, when sperm were incubated (6 h) under capacitating conditions in the presence of 8pCPT, E(m) remained depolarized (-32.06 mV). Altogether, these experiments support the hypothesis that RAPGEF3/RAPGEF4 activation regulates acrosomal exocytosis via its modulation of E(m), a novel role for RAPGEF3/RAPGEF4 in the series of events required to achieve fertilization.
Assuntos
Acrossomo/fisiologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Exocitose , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Cavalos/metabolismo , Capacitação Espermática , Animais , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Diclororribofuranosilbenzimidazol/análogos & derivados , Masculino , Potenciais da Membrana , Camundongos , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , TionucleotídeosRESUMO
The retinoic acid related orphan receptor RORα positively regulates the transcription of genes important for cerebellar development, immune function, lipid metabolism, and circadian rhythm. In the present study, we identified protein kinase A (PKA) as RORα4 phosphorylating kinase in vitro. The primary sequence of RORα4 contains a PKA recognition motif (R-D-S99) within the c-terminal extension of the DNA-binding domain, and mutation of Ser-99 to Ala prevents RORα4 phosphorylation by PKA. Activation of PKA by dBcAMP results in a marked induction of RORα4 activity. Inhibition of PKA with the selective kinase inhibitor H89 inhibits dBcAMP mediated as well as CaMK-IV triggered increase in RORα4 transcriptional activity. The regulation of RORα activity by PKA as well as CaMK-IV provides a new link in the signalling network that regulates metabolic processes such as glycogen and lipid metabolism.
Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Glicogênio/metabolismo , Células HeLa , Humanos , Metabolismo dos Lipídeos , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Serina/genética , Serina/metabolismo , Especificidade por SubstratoRESUMO
Intracellular signaling mechanisms within the pedunculopontine tegmental (PPT) nucleus for the regulation of recovery rapid eye movement (REM) sleep following REM sleep deprivation remain unknown. This study was designed to determine the role of PPT intracellular cAMP-dependent protein kinase A (cAMP-PKA) in the regulation of recovery REM sleep in freely moving rats. The results show that a brief period (3 h) of selective REM sleep deprivation caused REM sleep rebound associated with increased PKA activity and expression of the PKA catalytic subunit protein (PKA-CU) in the PPT. Local application of a cAMP-PKA-activation-selective inhibitor, RpCAMPS (0.55, 1.1, and 2.2 nmol/100 nl; n = 8 rats/group), bilaterally into the PPT, reduced PKA activity and PKA-CU expression in the PPT, and suppressed the recovery REM sleep, in a dose-dependent manner. Regression analyses revealed significant positive relationships between: PPT levels of PKA activity and the total percentages of REM sleep recovery (Rsqr = 0.944; n = 40 rats); PPT levels of PKA-CU expression and the total percentages of REM sleep recovery (Rsqr = 0.937; n = 40 rats); PPT levels of PKA-CU expression and PKA activity (Rsqr = 0.945; n = 40 rats). Collectively, these results provide evidence that activation of intracellular PKA in the PPT contributes to REM sleep recovery following REM sleep deprivation.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/fisiologia , Núcleo Tegmental Pedunculopontino/enzimologia , Subunidades Proteicas/fisiologia , Privação do Sono/enzimologia , Sono REM/fisiologia , Animais , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática/fisiologia , Líquido Intracelular/enzimologia , Masculino , Neurônios/enzimologia , Núcleo Tegmental Pedunculopontino/citologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/fisiologia , Privação do Sono/fisiopatologiaRESUMO
The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-kappaB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-kappaB activity. In this study, we have demonstrated the effect of PKA on NF-kappaB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-kappaB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-kappaB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-kappaB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-kappaB activation; PKA-activating agents enhanced NF-kappaB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-kappaB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-kappaB-induced breast cancer cell proliferation and multiple NF-kappaB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-kappaB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Apoptose/genética , Neoplasias da Mama/patologia , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Serina/metabolismo , Fator de Transcrição RelA/química , Transcrição Gênica/efeitos dos fármacosRESUMO
OBJECTIVE: In this study we investigated the activity of the nitric oxide synthase (NOS) in parotid glands from rats with experimental periodontitis and controls. METHODS: Periodontitis was produced by a ligature placed around the cervix of the two lower first molar. Experiments were carried out 22 days after the ligature. RESULTS: Ligation caused an increase in parotid NOS activity. The selective blocker of the inducible isoform of the enzyme partially inhibited its activity in parotid glands from rat with ligature. In controls, the activity was partially inhibited by the antagonists of the selective neural and endothelial isoforms. NOS activity in rats with ligature was cyclic adenosine monophosphate (cAMP)-dependent while in controls it was calcium-dependent. Prostaglandin E2 concentration was increased in parotid gland from rats with ligature. The inhibitor of prostaglandin production, FR 122047, diminished both, prostaglandin production and NOS activity. In rats with ligature unstimulated amylase released is increased. Both, prostaglandin and NOS were involved in the increment of amylase release. CONCLUSION: It can be concluded that in parotid glands from ligated rats, prostaglandin E2 production is increased and, through cAMP accumulation, activates the inducible NOS isoform. The increment of nitric oxide production participates in the increase in basal amylase release.
Assuntos
Óxido Nítrico Sintase/metabolismo , Glândula Parótida/enzimologia , Periodontite/enzimologia , Proteínas e Peptídeos Salivares/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Amilases/metabolismo , Animais , Cálcio/farmacologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/antagonistas & inibidores , Dinoprostona/metabolismo , Modelos Animais de Doenças , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Indazóis/farmacologia , Indometacina/farmacologia , Masculino , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Tamanho do Órgão , Ornitina/análogos & derivados , Ornitina/farmacologia , Glândula Parótida/efeitos dos fármacos , Piperazinas/farmacologia , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Ratos , Ratos Wistar , Proteínas e Peptídeos Salivares/efeitos dos fármacos , Tiazóis/farmacologia , ômega-N-Metilarginina/farmacologiaRESUMO
Activation of the protease-activated receptor 2 (PAR2) or the transient receptor potential vanilloid type 1 (TRPV1) channels expressed in cardiac sensory afferents containing calcitonin gene-related peptide (CGRP) and/or substance P (SP) has been proposed to play a protective role in myocardial ischemia-reperfusion (I/R) injury. However, the interaction between PAR2 and TRPV1 is largely unknown. Using gene-targeted TRPV1-null mutant (TRPV1(-/-)) or wild-type (WT) mice, we test the hypothesis that TRPV1 contributes to PAR2-mediated cardiac protection via increasing the release of CGRP and SP. Immunofluorescence labeling showed that TRPV1 coexpressed with PAR2, PKC-epsilon, or PKAc in cardiomyocytes, cardiac blood vessels, and perivascular nerves in WT but not TRPV1(-/-) hearts. WT or TRPV1(-/-) hearts were Langendorff perfused with the selective PAR2 agonist, SLIGRL, in the presence or absence of various antagonists, followed by 35 min of global ischemia and 40 min of reperfusion (I/R). The recovery rate of coronary flow, the maximum rate of left ventricular pressure development, left ventricular end-diastolic pressure, and left ventricular developed pressure were evaluated after I/R. SLIGRL improved the recovery of hemodynamic parameters, decreased lactate dehydrogenase release, and reduced the infarct size in both WT and TRPV1(-/-) hearts (P < 0.05). The protection of SLIGRL was significantly surpassed for WT compared with TRPV1(-/-) hearts (P < 0.05). CGRP(8-37), a selective CGRP receptor antagonist, RP67580, a selective neurokinin-1 receptor antagonist, PKC-epsilon V1-2, a selective PKC-epsilon inhibitor, or H-89, a selective PKA inhibitor, abolished SLIGRL protection by inhibiting the recovery of the rate of coronary flow, maximum rate of left ventricular pressure development, and left ventricular developed pressure, and increasing left ventricular end-diastolic pressure in WT but not TRPV1(-/-) hearts. Radioimmunoassay showed that SLIGRL increased the release of CGRP and SP in WT but not TRPV1(-/-) hearts (P < 0.05), which were prevented by PKC-epsilon V1-2 and H-89. Thus our data show that PAR2 activation improves cardiac recovery after I/R injury in WT and TRPV1(-/-) hearts, with a greater effect in the former, suggesting that PAR2-mediated protection is TRPV1 dependent and independent, and that dysfunctional TRPV1 impairs PAR2 action. PAR2 activation of the PKC-epsilon or PKA pathway stimulates or sensitizes TRPV1 in WT hearts, leading to the release of CGRP and SP that contribute, at least in part, to PAR2-induced cardiac protection against I/R injury.
Assuntos
Cardiotônicos/farmacologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Oligopeptídeos/farmacologia , Receptor PAR-2/agonistas , Canais de Cátion TRPV/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Circulação Coronária/efeitos dos fármacos , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Imunofluorescência , Isoindóis/farmacologia , Isoquinolinas/farmacologia , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Antagonistas dos Receptores de Neurocinina-1 , Fragmentos de Peptídeos/farmacologia , Proteína Quinase C-épsilon/antagonistas & inibidores , Proteína Quinase C-épsilon/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Radioimunoensaio , Receptor PAR-2/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores da Neurocinina-1/metabolismo , Recuperação de Função Fisiológica , Substância P/metabolismo , Sulfonamidas/farmacologia , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacosRESUMO
We report herein the design and synthesis of 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-amine derivatives as inhibitors of p70S6 kinase. Screening hits containing the 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-ylamine scaffold were optimized for p70S6K potency and selectivity against related kinases. Structure-based design employing an active site homology model derived from PKA led to the preparation of benzimidazole 5-substituted compounds 26 and 27 as highly potent inhibitors (K(i) <1nM) of p70S6K, with >100-fold selectivity against PKA, ROCK and GSK3.
Assuntos
Benzimidazóis/síntese química , Oxidiazóis/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Benzimidazóis/química , Benzimidazóis/farmacologia , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Desenho de Fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Oxidiazóis/síntese química , Oxidiazóis/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Relação Estrutura-Atividade , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
Studies on cAMP signaling and protein kinase A (PKA) function in vivo are limited by the lack of highly specific inhibitors that can be used in primary cell culture and whole animals. Previously we reported that a mutation in the ATP binding pocket of a catalytic subunit (Calpha) of PKA confers sensitivity to the pyrazolo[3,4-d]pyrimidine inhibitor, 1NM-PP1. We have now engineered the mouse Pkraca gene such that after Cre-mediated recombination in vivo, the CalphaM120A mutant protein is expressed and the wild-type Calpha is turned off. We demonstrate the utility of this approach by examining the requirement for PKA activity during capacitation of sperm from mice that express CalphaM120A mutant protein. For CalphaM120A sperm, 10 microM of 1NM-PP1 prevented PKA-dependent phosphorylation and the activation of motility that are both rapidly (<90 s) evoked by the HCO(3)(-) anion. A continuous (90 min) inhibition with 10 microM of 1NM-PP1 prevented the protein tyrosine phosphorylation of late-stage capacitation. Delayed application of 1NM-PP1 demonstrated that PKA activity was required for at least the initial 30 min of capacitation to produce subsequent protein tyrosine phosphorylation. Acute application of 1NM-PP1 rapidly slowed the accelerated beat of activated motility but did not affect the established waveform asymmetry of hyperactivated sperm. Our results demonstrate that PKA in CalphaM120A mutant sperm is rapidly and reversibly inhibited by 1NM-PP1 and that this blockade has selective and time-dependent effects on multiple aspects of capacitation. The conditional CalphaM120A-expressing mouse lines will be valuable tools for studying PKA function in vivo.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/enzimologia , Animais , Sítios de Ligação/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Capacitação Espermática/genética , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/genéticaRESUMO
This study examined the effects of three different cellular stresses on oocyte maturation in meiotically arrested mouse oocytes. Cumulus-cell enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured for 17-18 h in dbcAMP-containing medium plus increasing concentrations of the metabolic poison, sodium arsenite, or the free radical-generating agent, menadione. Alternatively, oocytes were exposed to osmotic stress by pulsing with sorbitol and returned to control inhibitory conditions for the duration of culture. Arsenite and menadione each dose-dependently induced germinal vesicle breakdown (GVB) in both DO and CEO. DO, but not CEO, pulsed for 60 min with 500 mM sorbitol were stimulated to resume maturation. The lack of effect in CEO suggests that the cumulus cells may be playing a protective role in osmotic stress-induced GVB. The AMP-activated protein kinase (PRKA; formerly known as AMPK) inhibitors, compound C and araA, completely blocked the meiosis-stimulating effects of all the tested stresses. Western blots showed that acetyl-CoA carboxylase, an important substrate of PRKA, was phosphorylated before GVB, supporting a role for PRKA in stress-induced maturation. Together, these data show that a variety of stresses stimulate GVB in meiotically arrested mouse oocytes in vitro and suggest that this effect is mediated through activation of PRKA.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Meiose/fisiologia , Oócitos/citologia , Oócitos/enzimologia , Estresse Fisiológico/fisiologia , Acetil-CoA Carboxilase/metabolismo , Animais , Antimetabólitos/farmacologia , Arsenitos/farmacologia , Western Blotting , Bucladesina/farmacologia , Catárticos/farmacologia , Células Cultivadas , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Sorbitol/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Teratogênicos/farmacologia , Vidarabina/farmacologia , Vitamina K 3/farmacologia , Vitaminas/farmacologiaRESUMO
Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.