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: 3.25). 05/2012; 318(16):2004-13. DOI: 10.1016/j.yexcr.2012.04.015
Source: PubMed


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.

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    • "Immortalized murine microglial cells (BV2) and adult murine hypothalamic cells (mHypoA-1/2, cited elsewhere as CLU172; CEL- Lutions Biosystems) [16] [17] [18] were grown in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum and 1% penicillin , streptomycin, and neomycin (Invitrogen) and maintained at 37 • C with 5% CO 2 . Orexin A peptide (American Peptides) was suspended in phosphate buffered saline (PBS, Invitrogen) and diluted to 300 nM in DMEM. "
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