Ischemia induces regulator of G protein signaling 2 (RGS2) protein upregulation and enhances apoptosis in astrocytes.
Regulator of G protein signaling (RGS) family members, such as RGS2, interact with Galpha subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and attenuating the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. They are also reported to regulate G protein-effector interactions and form multiprotein signaling complexes. Ischemic stress-induced changes in RGS2 expression have been described in astrocytes, and these changes are associated with intracellular signaling cascades, suggesting that RGS2 upregulation may be an important mechanism by which astrocytes may regulate RGS2 function in response to physiological stress. However, information on the functional roles of stress-induced modulation of RGS2 protein expression in astrocyte function is limited. We report the role of ischemic stress in RGS2 protein expression in rat C6 astrocytoma cells and primary mouse astrocytes. A marked increase in RGS2 occurred after ischemic stress induced by chemicals (sodium azide and 2-deoxyglucose) or oxygen-glucose deprivation (OGD, real ischemia). RGS2 mRNA expression was markedly enhanced by 1 h of exposure to chemical ischemia or 6 h of OGD followed by 2 or 6 h of recovery, respectively. This enhanced expression in primary astrocytes and C6 cells was restored to baseline levels after 12 h of recovery from chemically induced ischemic stress or 4-6 h of recovery from OGD. RGS2 protein was also significantly expressed at 12-24 h of recovery from ischemic insult. Ischemia-induced RGS2 upregulation was associated with enhanced apoptosis. It significantly increased annexin V-positive cells, cleaved caspase-3, and enhanced DNA ladder formation and cell cycle arrest. However, a small interfering RNA (siRNA)-mediated RGS2 knockdown reversed the apoptotic cell death associated with ischemia-induced RGS2 upregulation. Upregulated RGS2 was significantly inhibited by SB-203580, a p38 MAPK inhibitor. Rottlerin, a potent inhibitor of PKCdelta, completely abrogated the increased RGS2 expression. We also examine whether ischemia-induced RGS2-mediated apoptosis is affected by siRNA-targeted endogenous PKCdelta downregulation or its phosphorylation. Although RGS2 upregulation was not affected, siRNA transfection significantly suppressed endogenous PKCdelta mRNA and protein expressions. Ischemia-induced PKCdelta phosphorylation and caspase-3 cleavage were dose dependently inhibited by PKCdelta knockdown, and this endogenous PKCdelta suppression reversed ischemia-induced annexin V-positive cells. This study suggests that ischemic stress increases RGS2 expression and that this condition contributes to enhanced apoptosis in C6 cells and primary astrocytes. The signaling it follows may involve PKCdelta and p38 MAPK pathways.
Endale
Mehari
M
Laboratory of Physiology and Signaling, College of Veterinary Medicine, Kyungpook National Univ., Daegu 702-701, Republic of Korea.
Kim
Sung Dae
SD
Lee
Whi Min
WM
Kim
Sangseop
S
Suk
Kyoungho
K
Cho
Jae Youl
JY
Park
Hwa Jin
HJ
Wagley
Yadav
Y
Kim
Suk
S
Oh
Jae-Wook
JW
Rhee
Man Hee
MH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D005947:Glucose / Q000172:deficiency; D006801:Humans; D051379:Mice; D018384:Oxidative Stress; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D047428:Protein Kinase Inhibitors; D020710:RGS Proteins; C087182:RGS2 protein, human; C514563:RGS2 protein, rat; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C105345:Rgs2 protein, mouse; D051745:Protein Kinase C-delta; D048051:p38 Mitogen-Activated Protein Kinases; D053148:Caspase 3; D005947:Glucose