Prostacyclin receptor-induced STAT3 phosphorylation in human erythroleukemia cells is mediated via Gαs and Gα16 hybrid signaling

Abstract Human prostacyclin receptor (hIP) stimulates STAT3 via pertussis toxin-insensitive G proteins in human erythroleukemia (HEL) cells. Since hIP can utilize G s and G q proteins for signal transduction and that both G proteins can induce STAT3 phosphorylation and activation via complex signaling networks, we sought to determine if one of them is predominant in mediating the hIP signal. Stimulation of STAT3 Tyr 705 and Ser 727 phosphorylations by the IP-specific agonist, cicaprost, was sensitive to inhibition of protein kinase A, phospholipase Cβ, protein kinase C, calmodulin-dependent protein kinase II and Janus kinase 2/3. Unlike Gα 16 -mediated regulation of STAT3 in the same cells, cicaprost-induced STAT3 Tyr 705 phosphorylation was resistant to inhibition of Src and MEK while STAT3 Ser 727 phosphorylation distinctly required phosphatidylinositol-3 kinase. This unique inhibitor-sensitivity pattern of STAT3 phosphorylation was reproduced in HEL cells by stimulating the G 16 -coupled C5a receptor in the presence of dibutyryl-cAMP, suggesting that the change in inhibitor-sensitivity was due to activation of the G s pathway. This postulation was confirmed by expressing constitutively active Gα 16 QL and Gα s QL in human embryonic kidney 293 cells and the inhibitor-sensitivity of Gα 16 QL-induced STAT3 phosphorylations could be converted by the mere presence of Gα s QL to resemble that obtained with cicaprost in HEL cells. In addition, the restoration of the Gα 16 -mediated inhibitor-sensitivity upon cicaprost induction in Gα s -knocked down HEL cells again verified the pivotal role of G s signal. Taken together, our observations illustrate that co-stimulation of G s and G q can result in the fine-tuning of STAT3 activation status, and this may provide the basis for cell type-specific responses following activation of hIP.