RESUMO
Histamine H1 receptor (H1R) is one of the targets of histamine in the nervous system and the peripheral tissues. Protein kinase Cδ (PKCδ) signaling is involved in histamine-induced upregulation of H1R gene expression in HeLa cells. Histamine also upregulates H1R gene expression in U-373 MG cells. However, the molecular signaling of this upregulation is still unclear. Here, we investigated the molecular mechanism of histamine-induced H1R gene upregulation in U-373 MG cells. Histamine-induced H1R gene upregulation was inhibited by H1R antagonist d-chlorpheniramine, but not by ranitidine, ciproxifan, or JNJ77777120, and H2R, H3R, or H4R antagonists, respectively. Ro-31-8220 and Go6976 also suppressed this upregulation, however, the PKCδ selective inhibitor rottlerin and the PKCß selective inhibitor Ly333531 did not. Time-course studies showed distinct kinetics of H1R gene upregulation in U-373 MG cells from that in HeLa cells. A promoter assay revealed that the promoter region responsible for H1R gene upregulation in U-373 MG cells was different from that of HeLa cells. These data suggest that the H1R-activated H1R gene expression signaling pathway in U-373 MG cells is different from that in HeLa cells, possibly by using different promoters. The involvement of PKCα also suggests that compounds that target PKCδ could work as peripheral type H1R-selective inhibitors without a sedative effect.
Assuntos
Regulação da Expressão Gênica , Histamina/metabolismo , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Histamina/farmacologia , Humanos , Proteína Quinase C-alfa/metabolismo , Splicing de RNA , Ratos , Transdução de Sinais/efeitos dos fármacos , Transcrição GênicaRESUMO
Histamine H(1) receptor (H1R) gene is up-regulated in patients with allergic rhinitis, and its expression level strongly correlates with the severity of symptoms. However, the mechanism underlying this remains unknown. Here we report the mechanism of H1R gene up-regulation. The luciferase assay revealed the existence of two promoter regions, A and B1. Two AP-1 and one Ets-1 bound to region A, while Ku86, Ku70, and PARP-1 bound to region B1. Ku86 was responsible for DNA binding and poly(ADP-ribosyl)ated in response to phorbol-12-myristate-13-acetate stimulation, inducing its dissociation from region B1 that is crucial for promoter activity. Knockdown of Ku86 gene enhanced up-regulation of H1R gene expression. Experiments using inhibitors for MEK and PARP-1 indicate that regions A and B1 are downstream regulatory elements of the PKCδ/ERK/PARP-1 signaling pathway. Data suggest a novel mechanism for the up-regulation of H1R gene expression.
Assuntos
Antígenos Nucleares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Receptores Histamínicos H1/genética , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Humanos , Autoantígeno Ku , Masculino , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C-delta/metabolismo , Ratos , Receptores Histamínicos H1/metabolismo , Elementos de Resposta , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
The histamine H(1) receptor (H1R) gene is up-regulated in patients with allergic rhinitis. However, the mechanism and reason underlying this up-regulation are still unknown. Recently, we reported that the H1R expression level is strongly correlated with the severity of allergic symptoms. Therefore, understanding the mechanism of this up-regulation will help to develop new anti-allergic drugs targeted for H1R gene expression. Here we studied the molecular mechanism of H1R up-regulation in HeLa cells that express H1R endogenously in response to histamine and phorbol 12-myristate 13-acetate (PMA). In HeLa cells, histamine stimulation caused up-regulation of H1R gene expression. Rottlerin, a PKCδ-selective inhibitor, inhibited up-regulation of H1R gene expression, but Go6976, an inhibitor of Ca(2+)-dependent PKCs, did not. Histamine or PMA stimulation resulted in PKCδ phosphorylation at Tyr(311) and Thr(505). Activation of PKCδ by H(2)O(2) resulted in H1R mRNA up-regulation. Overexpression of PKCδ enhanced up-regulation of H1R gene expression, and knockdown of the PKCδ gene suppressed this up-regulation. Histamine or PMA caused translocation PKCδ from the cytosol to the Golgi. U0126, an MEK inhibitor, and DPQ, a poly(ADP-ribose) polymerase-1 inhibitor, suppressed PMA-induced up-regulation of H1R gene expression. These results were confirmed by a luciferase assay using the H1R promoter. Phosphorylation of ERK and Raf-1 in response to PMA was also observed. However, real-time PCR analysis showed no inhibition of H1R mRNA up-regulation by a Raf-1 inhibitor. These results suggest the involvement of the PKCδ/ERK/poly(ADP-ribose) polymerase-1 signaling pathway in histamine- or PMA-induced up-regulation of H1R gene expression in HeLa cells.
