Cannabinoid 1 receptor and interleukin-6 receptor together induce integration of protein kinase and transcription factor signaling to trigger Neurite outgrowth

Activation of the G_o/i-coupled cannabinoid 1 receptor (CB1R) has been shown to induce neurite outgrowth in Neuro2A cells through activation of Src kinase and STAT3 transcription factor. Signaling by the interleukin 6 receptor (IL-6R) also activates STAT3 through Jak kinase. We studied if signals from the two pathways could be integrated in a synergistic manner to trigger neurite outgrowth in Neuro2A cells. At low concentrations, when agonist at either receptor by itself has no effect, we found that CB1R and IL-6R stimulation together induced synergistic neurite outgrowth. Signal integration requires activation of transcription factors by Src, Jak, and mitogen-activated protein kinases. Mitogen-activated protein kinase can be activated by both receptors and shows enhanced early activation in the presence of both ligands. CREB and STAT3 transcription factors are required for synergy and show enhanced DNA-binding activity when both receptors are activated. STAT3 plays a critical role in integration of the signals downstream of the two receptors. When both pathways are activated, STAT3 phosphorylation is sustained for 6 h. This prolonged activation of STAT3 requires deactivation of SHP2 phosphatase. Reduction of SHP2 levels by RNA interference results in greater synergy in neurite outgrowth. Simultaneous knockdown of both SHP2 and STAT3 blocks the synergistic triggering of neurite outgrowth, indicating that STAT3 is downstream of SHP2. CB1R and IL-6R costimulation enhanced the differentiation of rat cortical neuron primary cultures. These results provide a mechanism where multiple protein kinases and transcription factors interact to integrate signals from G protein-coupled and cytokine receptor to evoke neurite outgrowth in Neuro2A cells.