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

Human prostacyclin receptor (hIP) stimulates STAT3 via pertussis toxin-insensitive G proteins in human erythroleukemia (HEL) cells. Since hIP can utilize Gs and Gq 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 Tyr705 and Ser727 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 Gα16-mediated regulation of STAT3 in the same cells, cicaprost-induced STAT3 Tyr705 phosphorylation was resistant to inhibition of Src and MEK while STAT3 Ser727 phosphorylation distinctly required phosphatidylinositol-3 kinase. This unique inhibitor-sensitivity pattern of STAT3 phosphorylation was reproduced in HEL cells by stimulating the G16-coupled C5a receptor in the presence of dibutyryl-cAMP, suggesting that the change in inhibitor-sensitivity was due to activation of the Gs pathway. This postulation was confirmed by expressing constitutively active Gα16QL and GαsQL in human embryonic kidney 293 cells and the inhibitor-sensitivity of Gα16QL-induced STAT3 phosphorylations could be converted by the mere presence of GαsQL to resemble that obtained with cicaprost in HEL cells. In addition, the restoration of the Gα16-mediated inhibitor-sensitivity upon cicaprost induction in Gαs-knocked down HEL cells again verified the pivotal role of Gs signal. Taken together, our observations illustrate that co-stimulation of Gs and Gq 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.