RESUMO
Interleukin-6 (IL-6) is a multifunctional cytokine involved in autoimmune thyroid diseases such as Hashimoto's thyroiditis and Graves' disease. IL-6 is produced by infiltrating immune cells and by thyrocytes. In the latter cell type, secretion of IL-6 is stimulated notably by interleukin-1 (IL-1), thyroid-stimulating hormone (TSH) or forskolin (Fk), a cAMP elevating agent. We report here that Fk and IL-1 synergistically enhance IL-6 mRNA expression in FRTL-5 thyroid cells by mechanisms involving the cAMP/PKA pathway, and both stabilization of the IL-6 mRNA and activation of the IL-6 promoter. Point mutations or deletions of the main transcription factor binding sites in the IL-6 promoter indicated that the synergistic effect was mainly mediated by the AP-1 site, and that the CRE site contributed to this effect. The DNA binding activity of AP-1 transcription factors and the expression of c-Fos and Fra-2 proteins, were all enhanced when the cAMP and IL-1 signalling pathways were both stimulated. These findings contribute to elucidating the synergistic mechanisms that regulate IL-6 secretion by thyroid cells, and suggest that such mechanisms may be involved in the development of thyroid autoimmune disorders.
Assuntos
AMP Cíclico/metabolismo , Antígeno 2 Relacionado a Fos/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais , Glândula Tireoide/metabolismo , Fator de Transcrição AP-1/metabolismo , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Linhagem Celular , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Genes Reporter , Interleucina-6/genética , Mutação , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/enzimologia , Fatores de Tempo , Transcrição Gênica , Transfecção , Regulação para CimaRESUMO
The expression pattern of CCAAT/enhancer binding protein-beta (C/EBP-beta) was investigated in thyroid cells and tissues. Translation of C/EBP-beta mRNA results in the production of two isoforms, liver-enriched transcriptional activating protein (LAP) and liver-enriched transcriptional inhibitory protein (LIP), the latter lacking the transactivation domain. We found that LAP and LIP are expressed in the rat thyroid gland and in the FRTL-5 and PCCL3 rat thyroid cell lines. Thyrotropin (TSH), insulin, and serum withdrawal from cultures of thyroid cells induced downregulation of LAP and LIP expression. Subsequent activation of the cyclic adenosine monophosphate (cAMP) and insulin signaling pathways reinduced both isoforms. Vectors expressing rat LAP and LIP were constructed to study the effect of C/EBP-beta isoforms on the activity of the sodium iodide symporter (NIS) promoter in PCCL3 cells. The cAMP-stimulated activity of the NIS promoter was decreased by overexpression of LAP, whereas LIP had no significant effect. Expression of C/EBP-beta was studied by immunohistochemistry in normal human thyroid and papillary cancer tissues. C/EBP-beta immunostaining was always restricted to the nuclei of the normal thyrocytes. In contrast, C/EBP-beta was expressed mainly in the cytoplasm of thyroid papillary carcinoma cells. These data suggest that this factor may play important roles in the regulation of thyroidspecific genes and processes, and that its functions are altered in human thyroid carcinoma.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Glândula Tireoide/metabolismo , Animais , Sequência de Bases , Colforsina/farmacologia , Primers do DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Insulina/farmacologia , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , RNA Mensageiro/genética , Ratos , Frações Subcelulares/metabolismo , Glândula Tireoide/citologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
The cAMP pathway activates p38-MAPKs in the FRTL-5 rat thyroid cell line, contributing to the increased expression of the Na+/I- symporter (NIS) mRNA. This study investigates the cAMP-dependent expression and transcriptional activity of the p38-MAPK substrate CCAAT/enhancer-binding protein-homologous protein (CHOP). CHOP is expressed in the rat thyroid gland and in confluent PCCL3 and FRTL-5 cells. In FRTL-5 cells, TSH withdrawal induced a rapid down-regulation of CHOP that could be prevented by forskolin (Fk). Moreover, TSH and Fk were able to reinduce CHOP expression. The use of pharmacological inhibitors indicated that cAMP-induced CHOP expression was dependent on protein kinase A (PKA), mammalian target of rapamycin pathway, and reactive oxygen species. Transfection of a CHOP trans- reporting system revealed strong stimulation of the transcriptional activity of CHOP by Fk, by chlorophenylthio-cAMP, and by the catalytic subunit of PKA. CHOP transcriptional activity was significantly reduced by the p38-MAPK inhibitor SB203580, by transfection of a dominant-negative variant of p38alpha-MAPK, or by mutation of two serine residues in CHOP targeted by p38-MAPKs. Finally, cAMP-induced NIS mRNA expression was higher in FRTL-5 cells stably transfected with CHOP cDNA than in control cells. Likewise, the activity of the NIS promoter was higher in cells overexpressing CHOP than in control cells. These findings suggest that the stimulation of CHOP expression and transcriptional activity by the cAMP pathway may contribute to the regulation of genes involved in thyroid cell differentiation.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Antioxidantes/metabolismo , Sequência de Bases , Proteínas de Transporte/farmacologia , Linhagem Celular , Colforsina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Simportadores/genética , Tireotropina/farmacologia , Fator de Transcrição CHOP , Transcrição Gênica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
AIMS: Multiple phosphodiesterases (PDEs) hydrolyze cAMP in cardiomyocytes, but the functional significance of this diversity is not well understood. Our goal here was to characterize the involvement of three different PDEs (PDE2-4) in cardiac excitation-contraction coupling (ECC). METHODS AND RESULTS: Sarcomere shortening and Ca(2+) transients were recorded simultaneously in adult rat ventricular myocytes and ECC protein phosphorylation by PKA was determined by western blot analysis. Under basal conditions, selective inhibition of PDE2 or PDE3 induced a small but significant increase in Ca(2+) transients, sarcomere shortening, and troponin I phosphorylation, whereas PDE4 inhibition had no effect. PDE3 inhibition, but not PDE2 or PDE4, increased phospholamban phosphorylation. Inhibition of either PDE2, 3, or 4 increased phosphorylation of the myosin-binding protein C, but neither had an effect on L-type Ca(2+) channel or ryanodine receptor phosphorylation. Dual inhibition of PDE2 and PDE3 or PDE2 and PDE4 further increased ECC compared with individual PDE inhibition, but the most potent combination was obtained when inhibiting simultaneously PDE3 and PDE4. This combination also induced a synergistic induction of ECC protein phosphorylation. Submaximal ß-adrenergic receptor stimulation increased ECC, and this effect was potentiated by individual PDE inhibition with the rank order of potency PDE4 = PDE3 > PDE2. Identical results were obtained on ECC protein phosphorylation. CONCLUSION: Our results demonstrate that PDE2, PDE3, and PDE4 differentially regulate ECC in adult cardiomyocytes. PDE2 and PDE3 play a more prominent role than PDE4 in regulating basal cardiac contraction and Ca(2+) transients. However, PDE4 becomes determinant when cAMP levels are elevated, for instance, upon ß-adrenergic stimulation or PDE3 inhibition.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Acoplamento Excitação-Contração/fisiologia , 3',5'-AMP Cíclico Fosfodiesterases/classificação , Animais , Cálcio/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/fisiologia , Masculino , Inibidores de Fosfodiesterase/farmacologia , Fosforilação , Ratos , Ratos WistarRESUMO
The sympathetic nervous system and pro-inflammatory cytokines play key roles in numerous cardiovascular disorders. Chronic beta-adrenergic receptor (beta-AR) stimulation in myocardium induces expression of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), which contribute to cardiac hypertrophy and failure. To evaluate the relationship between beta-AR stimulation and pro-inflammatory cytokines, we studied the effects of the beta-AR agonist isoprenaline (ISO) on IL-1-induced IL-6 production in adult rat ventricular myocytes (ARVMs). We report that ISO and IL-1 synergistically enhanced IL-6 gene expression and secretion. The synergistic effect of ISO was mimicked by cAMP elevating agents and involved the G(s) protein/cAMP/PKA signalling pathway, but not the exchange factor EPAC. To evaluate the contribution of IL-6 to cellular hypertrophy, we examined the signalling pathways stimulated by the membrane-bound IL-6 receptor (IL-6R), and the IL-6 soluble receptor (sIL-6R) involved in the mechanism named IL-6 trans-signalling. The IL-6/sIL-6R complex promoted a rapid and persistent phosphorylation of STAT3(Tyr705) in ARVMs. Moreover, IL-6 trans-signalling increased protein synthesis, c-fos gene expression and B-type natriuretic peptide secretion, three markers of cardiac hypertrophy. IL-6 trans-signalling also increased cell size. In contrast, IL-6 alone had no significant effect on either cell size or STAT3 phosphorylation although it induced phosphorylation of ERK1/2, AKT and S6K, demonstrating the presence of a functional IL-6R in ARVMs. Taken together, these results demonstrate that beta-AR stimulation synergises with IL-1 for IL-6 secretion in adult ventricular myocytes and indicate that IL-6 induces cardiac hypertrophy only via IL-6 trans-signalling. The IL-6 soluble receptor may thus serve as a switch for IL-6 to activate STAT3 phosphorylation and hypertrophy.