P2 purinergic receptors signal to STAT3 in astrocytes: Difference in STAT3 responses to P2Y and P2X receptor activation

Extracellular ATP, released upon tissue damage to the CNS, can evoke reactive astrogliosis. The released ATP activates P2 purinergic receptors associated with the proliferation of normally quiescent astrocytes, although the underlying mechanisms remain to be fully elucidated. Signal transducer and activator of transcription 3 (STAT3) has been implicated in reactive astrogliosis and plays an important role in cell cycle regulation. Therefore, we investigated whether extracellular ATP and purinergic receptors regulate STAT3 signaling. Using immunoblot analysis, we found that addition of ATP to primary cultures of rat cortical astrocytes increased Ser-727 phosphorylation of STAT3 in a time-sensitive and concentration-dependent manner. ATP-stimulated Ser-727 STAT3 phosphorylation was mediated through P2 receptor activation since suramin, an antagonist of P2 receptors, diminished this response, whereas 8-(para-sulfo-phenyl)-theophylline (8PSTP), an antagonist of P1 receptors, did not. We found that UTP, an agonist of P2Y2/4/6 receptors, stimulated rapid and robust phosphorylation of Ser727-STAT3, whereas BzATP, an agonist for P2X receptors, exhibited a delayed and weaker response. In contrast, both P2Y and P2X agonists stimulated phosphorylation of Tyr705-STAT3 to a similar extent. P2 receptors can couple to extracellular signal-regulated protein kinases (ERK) and we found that inhibition of ERK signaling blocked phosphorylation of Ser727-STAT3. Further characterization of the Ser727-STAT3 phosphorylation response to P2Y receptor activation supported a role for P2Y2 and P2Y4, but not P2Y6, receptors as well as a partial role for P2Y1 receptors. Because phosphorylation of Ser727-STAT3 can promote DNA transcriptional activity of cell cycle regulatory genes, the differences in phosphorylation of Ser727-STAT3 may contribute to the mechanism by which P2Y receptors promote, whereas P2X receptors inhibit, astrocyte proliferation. In support of this hypothesis, inhibition of STAT3 activation by cucurbitacin I prevented ATP-stimulated mitogenesis. We conclude that P2 receptors stimulate STAT3 activation and suggest that P2 receptor/STAT3 signaling may play an important role in astrocyte proliferation and reactive astrogliosis.