RESUMO
Acyl-CoA synthetase 4 (Acsl4) is involved in several cellular functions including steroidogenesis, synaptic development and cancer metastasis. Although the expression of Acsl4 seems to be regulated by tissue- and cell-specific factors as well as pituitary hormones and growth factors, the transcriptional mechanisms involved remain unknown. We demonstrated hCG and cAMP regulation of Acsl4 mRNA in mouse steroidogenic MA-10 Leydig cells. We characterized the transcription initiation site and promoter of the Acsl4 mouse gene and identified three alternative splice variants present in MA-10 cells. Sequence analysis of a 1.5-kb fragment of the Acsl4 promoter revealed the absence of a TATA box and the presence of many putative binding sites for transcription factors including Sp1 and CREB. Functional characterization revealed that the specificity protein/Krüppel-like factor Sp1 binding site in the proximal promoter is involved in basal activity and that the cAMP response element-binding site is involved in cAMP stimulation of Acsl4 transcription.
Assuntos
Coenzima A Ligases/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fator de Transcrição Sp1/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Gonadotropina Coriônica/farmacologia , Clonagem Molecular , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , AMP Cíclico/farmacologia , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Transcrição Gênica/efeitos dos fármacosRESUMO
Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3'-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5' and 3' RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis.
Assuntos
Células Intersticiais do Testículo/metabolismo , Fosfoproteínas/genética , RNA Antissenso/genética , RNA Mensageiro/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Masculino , Camundongos , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
High doses of diazepam (10.0-20.0 mg/kg) were shown to reduce the volume of acute inflammatory paw edema in rats as a response to carrageenan administration. This effect was attributed to an action of diazepam on the peripheral-type benzodiazepine receptor (PBR) present in the adrenal and/or immune/inflammatory cells. The present study was undertaken to analyze the involvement of nitric oxide (NO) on the effects of diazepam on carrageenan-induced paw edema in rats (CIPE) and to look for the presence of PBR and inducible/constitutive NO synthases (NOS) on slices taken from the inflamed paws of diazepam-treated rats. For that, an acute inhibition of NO biosynthesis was achieved using 50.0 mg/kg No mega-nitro-L-arginine (L-NAME), L-arginine (300.0 mg/kg), the true precursor of NO, and D-arginine (300.0 mg/kg), its false substrate, were also used. The following results were obtained: (1) diazepam (10.0 and 20.0 mg/kg) decreased CIPE values in a dose- and time-dependent way; (2) diazepam effects on CIPE were increased by L-NAME pretreatment; (3) treatment with L-arginine but not with D-arginine reverted at least in part the decrements of CIPE values observed after diazepam administration; (4) PBR were found in endothelial and inflammatory cells that migrated to the inflammatory site at the rat paw; (5) confocal microscopy showed the presence of both PBR and NOS in endothelial and inflammatory cells taken from inflamed paw tissues of rats treated with diazepam a finding not observed in tissues provided from rats treated with diazepam's control solution. These results suggest an important role for NO on the effects of diazepam on CIPE. Most probably, these effects reflect a direct action of diazepam on PBR present in the endothelium of the microvascular ambient and/or on immune/inflammatory cells. An action like that would lead, among other factors, to a decrease in NO, generated by NO synthase, and thus in the mechanisms responsible for CIPE.