Effects of P2Y1 receptor on glial fibrillary acidic protein and glial cell line-derived neurotrophic factor production of astrocytes under ischemic condition and the related signaling pathways.
OBJECTIVE
The present study aimed to explore the role of P2Y(1) receptor in glial fibrillary acidic protein (GFAP) production and glial cell line-derived neurotrophic factor (GDNF) secretion of astrocytes under ischemic insult and the related signaling pathways.


METHODS
Using transient right middle cerebral artery occlusion (tMCAO) and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, enzyme linked immunosorbent assay (ELISA) were used to investigate location of P2Y(1) receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules.


RESULTS
Blockage of P2Y(1) receptor with the selective antagonist N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate diammonium (MRS2179) reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y(1) receptor caused elevation of phosphorylated Akt and cAMP response element binding protein (CREB), and reduction of phosphorylated Janus kinase2 (JAK2) and signal transducer and activator of transcription3 (STAT3, Ser727). After blockage of P2Y(1) receptor and deprivation of oxygen-glucose-serum, AG490 (inhibitor of JAK2) reduced phosphorylation of STAT3 (Ser727) as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase1/2 (MEK1/2) U0126, an important molecule of Ras/extracellular signal-regulated kinase (ERK) signaling pathway, decreased the phosphorylation of JAK2, STAT3 (Ser727), Akt and CREB.


CONCLUSIONS
These results suggest that P2Y(1) receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.
Sun
Jing-Jun
JJ
Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, China.
Liu
Ying
Y
Ye
Zhu-Rong
ZR
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000096:biosynthesis*; D005904:Glial Fibrillary Acidic Protein / Q000096:biosynthesis*; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*
D051100:Glial Cell Line-Derived Neurotrophic Factor; D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1