Molecular basis of Period 1 regulation by adrenergic signaling in the heart.

The cardiac circadian clock is responsible for the modulation of different myocardial processes, and its dysregulation has been linked to disease development. How this clock machinery is regulated in the heart remains an open question. Because noradrenaline (NE) can act as a zeitgeber in cardiomyocytes, we tested the hypothesis that adrenergic signaling resets cardiac clock gene expression in vivo. In its anti-phase with Clock and Bmal1, cardiac Per1 abundance increased during the dark phase, concurrent with the rise in heart rate and preceded by an increase in NE levels. Sympathetic denervation altered Bmal1 and Clock amplitude, while Per1 was affected in both amplitude and oscillatory pattern. We next treated mice with a ß-adrenergic receptor (ß-AR) blocker. Strikingly, the ß-AR blockade during the day suppressed the nocturnal increase in Per1 mRNA, without altering Clock or Bmal1. In contrast, activating ß-AR with isoproterenol (ISO) promoted an increase in Per1 expression, demonstrating its responsiveness to adrenergic input. Inhibitors of ERK1/2 and CREB attenuated ISO-induced Per1 expression. Upstream of ERK1/2, PI3Kγ mediated ISO induction of Per1 transcription, while activation of ß2-AR, but not ß1-AR induced increases in ERK1/2 phosphorylation and Per1 expression. Consistent with the ß2-induction of Per1 mRNA, ISO failed to activate ERK1/2 and elevate Per1 in the heart of ß2-AR-/- mice, whereas a ß2-AR antagonist attenuated the nocturnal rise in Per1 expression. Our study established a link between NE/ß2-AR signaling and Per1 oscillation via the PI3Ky-ERK1/2-CREB pathway, providing a new framework for understanding the physiological mechanism involved in resetting cardiac clock genes.

Migração e invasão do câncer de boca via ativação de receptor beta 2 adrenérgico por mediador do estresse / Cell migration and invasion of oral cancer via activation of beta 2 adrenergic receptor by stress mediator

A ativação do receptor beta 2 adrenérgico (β2-AR), pelos mediadores químicos do estresse, pode induzir efeitos estimuladores ou inibidores na migração e invasão celular, dependendo do tipo de tumor maligno. A importância deste receptor na evolução do câncer de boca não está totalmente esclarecida. O objetivo deste estudo foi verificar a expressão do β2-AR em linhagens de carcinomas espinocelular de boca (SCC-9 e SCC-25), e investigar o papel da ativação deste receptor pela norepinefrina e de seu bloqueio por um antagonista na migração e invasão destas células neoplásicas. As células SCC-9 e SCC-25 foram investigadas quanto à expressão gênica e proteica do β2-AR, respectivamente, pelo RT-qPCR e pelo Western blot. A migração e a invasão celular foram analisadas pelo ensaio de cicatrização de feridas e pelo sistema de câmeras de invasão Transwell, respectivamente. Diferentes concentrações (0,1; 1 e 10μM) de norepinefrina foram utilizadas para estimular e 1μM de propranolol foi empregado para bloquear os receptores beta adrenérgicos nas células neoplásicas. As diferenças das médias obtidas nos experimentos de invasão e migração de SCC-9 e SCC-25 e da expressão proteica do β2-AR, foram comparadas pelo teste t de Student com nível de significância de 5%. Os resultados mostraram que a expressão gênica e proteica do β2-AR foi verificada em ambas as linhagens de câncer de boca. A concentração de 10μM de norepinefrina inibiu, significativamente (p≤0,05), a migração e invasão celular de SCC-9 e SCC-25, sendo este efeito mais acentuado nas células SCC-25. Além disso, houve uma redução significativa (p≤0,05) do efeito da norepinefrina na migração celular quando os β2-AR foram inibidos pelo propranolol. Adicionalmente, o bloqueio dos β-ARs pelo propranolol reverteu parcialmente o efeito da norepinefrina na capacidade invasiva de SCC-9 e SCC-25. Estes resultados comprovam que a norepinefrina, via ativação do β2-AR, reduziu a migração e a invasão das...

The activation of beta 2 adrenergic receptor (β2-AR), by chemical mediators of stress, can induce stimulatory or inhibitory effects on cell migration and invasion, depending on the type of malignancy. The importance of this receptor in the oral cancer outcome is not fully understood. The aim of this study was to verify β2- AR expression in oral squamous cell carcinoma cell lines (SCC-9 and SCC-25), and to investigate the role of activation of this receptor by norepinephrine and its blockade by an antagonist in migration and invasion of these neoplastic cells. SCC-9 and SCC-25 cells were investigated for gene and protein expression of β2-AR, respectively, by RT-qPCR and Western blot. The cell migration and invasion were analyzed by wound healing assay and Transwell invasion camera system, respectively. Different concentrations (0.1, 1 and 10μM) of norepinephrine were used to stimulate and 1μM propranolol was used to block the beta adrenergic receptors on cancer cells. Differences in mean values of the invasion and migration assays of SCC-9 and SCC-25 and β2-AR protein expression were compared by the Student t test with 5% significance level. The results showed that β2-AR gene and protein expression was verified in both oral cancer cell lines. The concentration of 10μM of norepinephrine inhibited significantly (p≤0.05), cell migration and invasion of SCC-9 and SCC-25, being the most pronounced effect in SCC-25 cells. Furthermore, there was a significant reduction (p≤0.05) of norepinephrine effect on cell migration when the β2-AR was inhibited by propranolol. In addition, blockade of β-ARs by propranolol partially reversed the effect of norepinephrine on the invasiveness of SCC-9 and SCC-25. These results show that norepinephrine via β2-AR activation, reduced the migration and invasion of oral squamous cell carcinoma cells and, therefore, the use of beta-adrenergic receptors agonists could become an adjuvant therapeutic target in the treatment of this malignancy...

Migração e invasão do câncer de boca via ativação de receptor beta 2 adrenérgico por mediador do estresse / Cell migration and invasion of oral cancer via activation of beta 2 adrenergic receptor by stress mediator

A ativação do receptor beta 2 adrenérgico (β2-AR), pelos mediadores químicos do estresse, pode induzir efeitos estimuladores ou inibidores na migração e invasão celular, dependendo do tipo de tumor maligno. A importância deste receptor na evolução do câncer de boca não está totalmente esclarecida. O objetivo deste estudo foi verificar a expressão do β2-AR em linhagens de carcinomas espinocelular de boca (SCC-9 e SCC-25), e investigar o papel da ativação deste receptor pela norepinefrina e de seu bloqueio por um antagonista na migração e invasão destas células neoplásicas. As células SCC-9 e SCC-25 foram investigadas quanto à expressão gênica e proteica do β2-AR, respectivamente, pelo RT-qPCR e pelo Western blot. A migração e a invasão celular foram analisadas pelo ensaio de cicatrização de feridas e pelo sistema de câmeras de invasão Transwell, respectivamente. Diferentes concentrações (0,1; 1 e 10μM) de norepinefrina foram utilizadas para estimular e 1μM de propranolol foi empregado para bloquear os receptores beta adrenérgicos nas células neoplásicas. As diferenças das médias obtidas nos experimentos de invasão e migração de SCC-9 e SCC-25 e da expressão proteica do β2-AR, foram comparadas pelo teste t de Student com nível de significância de 5%. Os resultados mostraram que a expressão gênica e proteica do β2-AR foi verificada em ambas as linhagens de câncer de boca. A concentração de 10μM de norepinefrina inibiu, significativamente (p≤0,05), a migração e invasão celular de SCC-9 e SCC-25, sendo este efeito mais acentuado nas células SCC-25. Além disso, houve uma redução significativa (p≤0,05) do efeito da norepinefrina na migração celular quando os β2-AR foram inibidos pelo propranolol. Adicionalmente, o bloqueio dos β-ARs pelo propranolol reverteu parcialmente o efeito da norepinefrina na capacidade invasiva de SCC-9 e SCC-25. Estes resultados comprovam que a norepinefrina, via ativação do β2-AR, reduziu a migração e a invasão das...

The activation of beta 2 adrenergic receptor (β2-AR), by chemical mediators of stress, can induce stimulatory or inhibitory effects on cell migration and invasion, depending on the type of malignancy. The importance of this receptor in the oral cancer outcome is not fully understood. The aim of this study was to verify β2- AR expression in oral squamous cell carcinoma cell lines (SCC-9 and SCC-25), and to investigate the role of activation of this receptor by norepinephrine and its blockade by an antagonist in migration and invasion of these neoplastic cells. SCC-9 and SCC-25 cells were investigated for gene and protein expression of β2-AR, respectively, by RT-qPCR and Western blot. The cell migration and invasion were analyzed by wound healing assay and Transwell invasion camera system, respectively. Different concentrations (0.1, 1 and 10μM) of norepinephrine were used to stimulate and 1μM propranolol was used to block the beta adrenergic receptors on cancer cells. Differences in mean values of the invasion and migration assays of SCC-9 and SCC-25 and β2-AR protein expression were compared by the Student t test with 5% significance level. The results showed that β2-AR gene and protein expression was verified in both oral cancer cell lines. The concentration of 10μM of norepinephrine inhibited significantly (p≤0.05), cell migration and invasion of SCC-9 and SCC-25, being the most pronounced effect in SCC-25 cells. Furthermore, there was a significant reduction (p≤0.05) of norepinephrine effect on cell migration when the β2-AR was inhibited by propranolol. In addition, blockade of β-ARs by propranolol partially reversed the effect of norepinephrine on the invasiveness of SCC-9 and SCC-25. These results show that norepinephrine via β2-AR activation, reduced the migration and invasion of oral squamous cell carcinoma cells and, therefore, the use of beta-adrenergic receptors agonists could become an adjuvant therapeutic target in the treatment of this malignancy.