PI3Kγ as a nodal point in μ-opioid receptor signaling
Phosphoinositide-3-kinases (PI3Ks) are enzymes that catalyze the phosphorylation of 3 position of the inositol ring of the phosphoinositol lipids. By this way they are implicated in many cellular processes like cell cycle progression, cell growth, survival and migration. Whereas PI3Kα, β and PI3Kδ isoforms were involved in receptor tyrosine kinase signaling, PI3Kβ and PI3Kγ are the PI3K isoforms involved in GPCR mediated signaling. μOR is a Gi coupled GPCR. Morphine, an opioid analgesic drug, acts as an agonist at μOR to exert pain relief in moderate to severe pain conditions. Loss of analgesic effectiveness of morphine following repeated use is mainly attributed to the desensitization of the μOR and cellular adaptations that takes place following stimulation of μOR. The molecular mechanisms associated with desensitization of μOR need further understanding. In order to investigate the role of PI3Kγ in μOR mediated signaling we generated suitable model cell systems. At first, SK-N-LO cells endogenously expressing PI3Kγ were transfected with a plasmid encoding μOR to generate a cell line stably expressing μOR (m-μOR SK-N-LO cells). Then, PI3Kγ gene was stably down regulated in m-μOR SK-N-LO cells using shRNA approach to generate γ5-m-μOR SK-N-LO cells with down-regulated PI3Kγ (~ 70 % suppression was observed) and Ctrl-m-μOR SK-N-LO cells expressing normal levels of PI3Kγ (shRNA control). Stimulation of m-μOR SK-N-LO cells with morphine resulted in activation of AKT and ERK1/2. Maximum activation was observed at 2.5 min post stimulation. Activation of AKT was evidenced by phosphorylation of AKT at serine 473 and threonine 308. Pertussis toxin (Gi inactivator), naloxone (specific μOR antagonist) and wortmannin (pan PI3Ks inhibitor) pretreatment inhibited the morphine induced activation of AKT and ERK1/2 in m-μOR SK-N-LO cells, suggesting the involvement of Gi-coupled GPCRs, in particular μOR and a pathway dependent on activation of PI3Ks. Pharmacological inhibition of PI3Kβ or PI3Kγ isoform impaired the morphine induced activation of AKT in m-μOR SK-N-LO cells. However, pharmacological inhibition of PI3Kα or PI3Kδ isoform did not interfere with morphine induced activation of AKT in m-μOR SK-N-LO cells. Moreover, in response to morphine, γ5-m-μOR SK-N-LO 82 PI3Kγ down-regulated cells showed significantly reduced phosphorylation of AKT at serine 473 and threonine 308 sites indicating the requirement of PI3Kγ for the morphine induced activation of AKT. Preincubation of m-μOR SK-N-LO cells with intracellular cAMP elevating agents, forskolin or IBMX, inhibited the activation of AKT in response to morphine. In addition, inhibition of PKA by H-89 enhanced the morphine induced phosphorylation of AKT at threonine 308 site. Together, these data indicated a crosstalk between cAMP-AKT pathways in m-μOR SK-N-LO cells. Further, H-89 pretreatment resulted in significantly reduced enhancement of morphine induced AKT activation in γ5-m-μOR SK-N-LO down-regulated cells compared to Ctrl-m-μOR SK-N-LO cells indicating the requirement of PI3Kγ for the crosstalk between cAMP-AKT pathways. Here we showed for the first time, morphine induced activation of AKT as measured by the phosphorylation of AKT at threonine 308 site. Taking this as a marker, the crosstalk between cAMP-AKT pathways was explored. The novel observations included the enhancement of morphine induced phosphorylation of AKT at threonine 308 site following inhibition of PKA by H-89 and its dependence on PI3Kγ. Because the activation of AKT might be considered as a marker for the lipid kinase activity of PI3Kγ, we could propose that the inhibition of PKA might have enhanced the lipid kinase activity of PI3Kγ, as measured by the phosphorylation of AKT at threonine 308 site. Finally, the crosstalk between cAMP-AKT pathways has been explored in primary neuronal cells from wild type and PI3Kγ knockout mice. Morphine induced activation of AKT was observed in cultured DRG neurons of wild type but not PI3Kγ knockout mice as measured by the phosphorylation of AKT at threonine 308 site. Moreover, forskolin pretreatment of DRG neurons of wild type mice resulted in inhibition of morphine induced AKT activation which was not observed in DRG neurons from PI3Kγ knockout mice. Inhibition of PKA by H-89 resulted in enhanced morphine induced activation of AKT in wild type DRG neurons but not in PI3Kγ knockout DRG neurons.