RESUMO
Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression.
Assuntos
Ceramidase Ácida/metabolismo , Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/enzimologia , Ceramidase Ácida/antagonistas & inibidores , Ceramidase Ácida/genética , Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Endopeptidases/química , Endopeptidases/genética , Indução Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ubiquitina TiolesteraseRESUMO
Although coordinated molecular signaling through excitatory and modulatory neurotransmissions is critical for the induction of immediate early genes (IEGs), which lead to effective changes in synaptic plasticity, the intracellular mechanisms responsible remain obscure. Here we measured the expression of IEGs and used bioluminescence imaging to visualize the expression of Bdnf when GPCRs, major neuromodulator receptors, were stimulated. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor (PAC1), a Gαs/q-protein-coupled GPCR, with PACAP selectively activated the calcineurin (CN) pathway that is controlled by calcium signals evoked via NMDAR. This signaling pathway then induced the expression of Bdnf and CN-dependent IEGs through the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Intracerebroventricular injection of PACAP and intraperitoneal administration of MK801 in mice demonstrated that functional interactions between PAC1 and NMDAR induced the expression of Bdnf in the brain. Coactivation of NMDAR and PAC1 synergistically induced the expression of Bdnf attributable to selective activation of the CN pathway. This CN pathway-controlled expression of Bdnf was also induced by stimulating other Gαs- or Gαq-coupled GPCRs, such as dopamine D1, adrenaline ß, CRF, and neurotensin receptors, either with their cognate agonists or by direct stimulation of the protein kinase A (PKA)/PKC pathway with chemical activators. Thus, the GPCR-induced expression of IEGs in coordination with NMDAR might occur via the selective activation of the CN/CRTC1/CREB pathway under simultaneous excitatory and modulatory synaptic transmissions in neurons if either the Gαs/adenylate cyclase/PKA or Gαq/PLC/PKC-mediated pathway is activated.
Assuntos
Calcineurina/metabolismo , Cálcio/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Calcineurina/genética , Inibidores de Calcineurina/farmacologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between -49bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.
Assuntos
DNA Ligases/genética , Proteína Quinase Ativada por DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Imunoglobulinas/metabolismo , Leucemia/tratamento farmacológico , Proteínas Nucleares/genética , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Ligase Dependente de ATP , Reparo do DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Daunorrubicina/administração & dosagem , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Imunoglobulinas/genética , Células K562 , Leucemia/genética , Leucemia/patologia , Proteínas Nucleares/metabolismo , Fosforilação , RNA Mensageiro/biossínteseRESUMO
Although dynorphins are widely involved in the control of not only nociceptive neurotransmission but also a variety of brain functions such as memory and emotion, no natural regulator for inducing the mRNA expression of prodynorphin (Pdyn), a precursor protein of dynorphins, is known. Using primary cultures of rat cortical neurons, we found that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon neuropeptide family, markedly induces Pdyn mRNA expression. PACAP was much more effective than VIP, indicating a major role for PAC1 in the PACAP-induced Pdyn mRNA expression. The increase in Pdyn mRNA expression was independent of de novo protein synthesis. Administration of forskolin, an activator for adenylate cyclase/protein kinase A (PKA), but not TPA, an activator for protein kinase C (PKC), induced Pdyn mRNA expression, suggesting a major role for PKA. The involvement of PKA was supported by the inhibition of PACAP-induced Pdyn mRNA expression upon addition of H89, an inhibitor for PKA. The PACAP-induced potentiation of NMDA-R was involved in the mRNA expression of Bdnf or c-fos but not Pdyn. These results suggest PACAP to be an upstream regulator for inducing Pdyn mRNA expression through PKA.