OX2R activation induces PKC-mediated ERK and CREB phosphorylation

Louis Stokes Cleveland VA Medical Center, Research Sec, Rm: K217, 10701 East BLVD, Cleveland, Ohio 44106, USA.
Experimental Cell Research (Impact Factor: 3.37). 05/2012; 318(16):2004-13. DOI: 10.1016/j.yexcr.2012.04.015
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Early onset of invasive and metastatic progression of pancreatic ductal adenocarcinoma (PDAC) makes it one of the most lethal human cancers. In this study, we investigated the role of the pro-metastatic actin bundling protein fascin in PDAC progression. Expression levels of fascin were higher in cancer tissues than in normal tissues and fascin overexpression correlated with PDAC differentiation and prognosis. Fascin overexpression promoted PDAC cells migration and invasion by elevating MMP-2 expression via PKC and ERK. Importantly, our results showed that hypoxia-induced fascin overexpression in PDAC cells by promoting HIF-1 binding to the fascin promoter and transactivating fascin mRNA transcription. Intriguingly, HIF-1alpha expression levels in PDAC patient specimens correlated with fascin expression. Moreover, immunohistochemical staining of consecutive sections demonstrated HIF-1alpha and fascin colocalization in PDAC specimens, suggesting that hypoxia and HIF-1alpha were responsible for fascin overexpression in PDAC. When ectopically expressed, fascin rescued PDAC cell invasion after HIF-1alpha silencing. Our results demonstrated that fascin is a direct target gene of HIF-1. Further, they suggested that the hypoxic tumor microenvironment might promote invasion and metastasis of PDAC via fascin overexpression, highlighting fascin as a target to block PDAC progression.
    Cancer Research 03/2014; 74(9). DOI:10.1158/0008-5472.CAN-13-3009 · 9.28 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Orexin-A and Orexin-B play important roles in many physiological processes in which Orexins orchestrate diverse downstream effects via two G-protein coupled receptors: Orexin1R and Orexin2R. Two alternative C-terminus splice variants of the mouse Orexin receptors mOX2αR and mOX2βR have recently been identified. This study explored the possibility of heterodimerization between mOX2αR and mOX2βR, and investigated novel signal transduction characteristics after stimulation. The dimerization of mOX2αR and mOX2βR was confirmed by BRET and co-immunoprecipitation assays. Meanwhile, in HEK293 cells, co-expression of mOX2αR and mOX2βR resulted in a strengthened increase in activation of ERK1/2, with maximal activation at 5 mins and 100 nM. Furthermore, heterodimerization also elicits stronger intracellular Ca(2+) elevation after orexin(s) stimulation, followed by a slower decline in intracellular Ca(2+) to a steady endpoint. Protein Kinase C inhibitor significantly inhibited these downstream effects. In addition, the cAMP response element reporter activities were significantly reduced, whereas the serum response element luciferase and the T-lymphocyte activation of nuclear factor-responsive element reporter activity were significantly up-regulated after Orexin(s) stimulation. Besides, Orexin-A/-B induced a significantly higher rate of HEK293 cell proliferation in cells co-expressing mOX2αR/mOX2βR compared to the control group. Taken together, we provide conclusive evidence that mOX2αR can form a functional heterodimer with mOX2βR and this leads to increased PKC and decreased protein kinase A activity by ERK signal pathway leading to a significant increase in cell proliferation. The nature of this signaling pathway has significant implications for the role of Orexin in the regulation of physiological processes including the homeostasis of feeding.
    Biochimica et Biophysica Acta 12/2013; 1843(3). DOI:10.1016/j.bbamcr.2013.12.010 · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Diurnal release of the orexin neuropeptides, orexin-A (ox-A, hypocretin-1) and orexin-B (ox-B, hypocretin-2), stabilises arousal, regulates energy homeostasis and contributes to cognition and learning. However, whether cellular correlates of brain plasticity are regulated through orexins, and whether they do so in a time-of-day-dependent manner, has never been assessed. Immunohistochemically, we found sparse but widespread innervation of hippocampal subfields through ox-A- and ox-B-containing fibres in young adult rats. The actions of ox-A were studied on NMDA receptor (NMDAR)-mediated excitatory synaptic transmission in acute hippocampal slices prepared around the trough (Zeitgeber Time (ZT) 4–8, corresponding to 4–8 h into the resting phase) and peak (ZT 23) of intracerebroventricular orexin levels. At ZT 4–8, exogenous ox-A (100 nm in bath) inhibited NMDA-EPSCs at mossy fibre (MF)-CA3 (to 55.6 ± 6.8% of control, P = 0.0003) and at Schaffer collateral-CA1 synapses (70.8 ± 6.3%, P = 0.013), whereas it remained ineffective at non-MF excitatory synapses in CA3. Ox-A actions were mediated postsynaptically and blocked by the orexin-2 receptor (OX2R) antagonist JNJ10397049 (1 μm), but not by OX1R inhibition (SB334867, 1 μm) or by adrenergic and cholinergic antagonists. At ZT 23, inhibitory effects of exogenous ox-A were absent (97.6 ± 2.9%, P = 0.42), but reinstated (87.2 ± 3.3%, P = 0.002) when endogenous orexin signalling was attenuated for 5 hours through I.P. injections of almorexant (100 mg kg−1), a dual orexin receptor antagonist. In conclusion, endogenous orexins modulate hippocampal NMDAR function in a time-of-day-dependent manner, suggesting that they may influence cellular plasticity and consequent variations in memory performance across the sleep-wake cycle.This article is protected by copyright. All rights reserved
    The Journal of Physiology 07/2014; 592(19). DOI:10.1113/jphysiol.2014.272757 · 4.54 Impact Factor