Louis Stokes Cleveland VA Medical Center, Research Sec, Rm: K217, 10701 East BLVD, Cleveland, Ohio 44106, USA.
Exp Cell Res. 2012 Oct 1;318(16):2004-13. doi: 10.1016/j.yexcr.2012.04.015. Epub 2012 May 29.
Deficiencies in brain orexins and components of mitogen activated protein kinase (MAPK) signaling pathway have been reported in either human depression or animal model of depression. Brain administration of orexins affects behaviors toward improvement of depressive symptoms. However, the documentation of endogenous linkage between orexin receptor activation and MAPK signaling pathway remains to be insufficient. In this study, we report the effects of orexin 2 receptor (OX2R) activation on cell signaling in CHO cells over-expressing OX2R and in mouse hypothalamus cell line CLU172. Short-term extracellular signal-regulated kinase (ERK) phosphorylation and long-term cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) phosphorylation were subsequently observed in CHO cells that over-express OX2R while 20 min of ERK phosphorylation was significantly detected in mouse adult hypothalamus neuron cell line CLU172. Orexin A, which can also activate OX2R, mediated ERK phosphorylation was as the same as orexin B in CHO cells. A MAPK inhibitor eliminated ERK phosphorylation but not CREB phosphorylation in CHO cells. Also, ERK and CREB phosphorylation was not mediated by protein kinase A (PKA) or calmodulin kinase (CaMK). However, inhibition of protein kinase C (PKC) by GF 109203X eliminated the phosphorylation of ERK and CREB in CHO cells. A significant decrease in ERK and CREB phosphorylation was observed with 1 μM GF 109203X pre-treatment indicating that the conventional and novel isoforms of PKC are responsible for CREB phosphorylation after OX2R activation. In contrast, ERK phosphorylation induced by orexin B in CLU172 cells cannot be inhibited by 1 μM of protein kinase C inhibitor. From above observation we conclude that OX2R activation by orexin B induces ERK and CREB phosphorylation and orexin A played the same role as orexin B. Several isoforms of PKC may be involved in prolonged CREB phosphorylation. Orexin B induced ERK phosphorylation in mouse hypothalamus neuron cells differs from CHO cell line and cannot be inhibited by PKC inhibitor GF 109203X. And hypothalamus neuron cells may use different downsteam pathway for orexin B induced ERK phosphorylation. This result supports findings that orexins might have anti-depressive roles.
在人类抑郁症或动物抑郁症模型中，已经报道了大脑食欲素和丝裂原活化蛋白激酶（MAPK）信号通路的组成部分的缺乏。脑内食欲素的给药会影响改善抑郁症状的行为。然而，食欲素受体激活与 MAPK 信号通路之间的内源性联系的记录仍然不足。在这项研究中，我们报告了食欲素 2 受体（OX2R）激活对过表达 OX2R 的 CHO 细胞和小鼠下丘脑细胞系 CLU172 中的细胞信号的影响。在过表达 OX2R 的 CHO 细胞中，随后观察到短期细胞外信号调节激酶（ERK）磷酸化和长期环腺苷酸反应元件结合蛋白（CREB）磷酸化，而在小鼠成年下丘脑神经元细胞系 CLU172 中，20 分钟的 ERK 磷酸化明显检测到。同样，也可以激活 OX2R 的食欲素 A 介导了 CHO 细胞中的 ERK 磷酸化，与食欲素 B 相同。MAPK 抑制剂消除了 CHO 细胞中的 ERK 磷酸化，但不消除 CREB 磷酸化。此外，ERK 和 CREB 磷酸化不是由蛋白激酶 A（PKA）或钙调蛋白激酶（CaMK）介导的。然而，GF 109203X 抑制蛋白激酶 C（PKC）消除了 CHO 细胞中 ERK 和 CREB 的磷酸化。用 1 μM GF 109203X 预处理显著减少了 ERK 和 CREB 的磷酸化，表明在 OX2R 激活后，传统和新型 PKC 同工型负责 CREB 的磷酸化。相反，在 CLU172 细胞中，由食欲素 B 诱导的 ERK 磷酸化不能被 1 μM 蛋白激酶 C 抑制剂抑制。从以上观察结果，我们得出结论，食欲素 B 通过 OX2R 激活诱导 ERK 和 CREB 磷酸化，并且食欲素 A 发挥与食欲素 B 相同的作用。几种 PKC 同工型可能参与延长 CREB 磷酸化。在小鼠下丘脑神经元细胞中，食欲素 B 诱导的 ERK 磷酸化与 CHO 细胞系不同，不能被 PKC 抑制剂 GF 109203X 抑制。并且下丘脑神经元细胞可能用于食欲素 B 诱导的 ERK 磷酸化的不同下游途径。该结果支持食欲素可能具有抗抑郁作用的发现。
