Phosphorylation of signal transducer and activator of transcription-3 (Stat3) after focal cerebral ischemia in rats.
JAK-STAT is the major downstream signal pathway of interleukin-6 (IL-6) cytokine family and is regulated by Tyr705 phosphorylation of Stat3. The present study examined the extent and the localization of phosphorylated Stat3 protein in brain tissue after focal ischemia in rats. The localizations of unphosphorylated and phosphorylated Stat3 were immunohistochemically examined in rats after 0.5 to 168 h of reperfusion following 1.5 h of middle cerebral artery occlusion (MCAO), induced by the intraluminal suture method. Absolute phosphorylated Stat3 immunoreactive cell counts were made in the cerebral cortex (ischemic core, peri-ischemia region, and contralareral cortex) and lateral striatal regions on both the ischemic and the contralateral sides. Stat3 protein was localized diffusely in cortical and striatal neurons in the sham-operated animals. Although weak Stat3 staining was detected in damaged neurons in the ischemic region, activated microglia, astrocytes, and endothelial cells clearly expressed Stat3 in this region. On the other hand, the sham group showed no phosphorylated Stat3 immunoreactivity. Phosphorylated Stat3 immunoreactivity was first detected in neurons after 3.5 h of reperfusion in each cortical and striatal region. Thereafter, Stat3 phosphorylation was marked in neurons in the peri-infarct region, peaked at 24 h, and then gradually declined throughout the reperfusion period. Endothelial cells expressed phosphorylated Stat3 in the ischemic core at 48 h of reperfusion. To identify the cellular source of phosphorylated Stat3, lectin histochemical study and immunohistochemical study with anti-microtubule-associated proten-2 and anti-glial fibrillary acidic protein antibodies were carried out. Double-staining immunohistochemistry with these cellular makers revealed phosphorylated Stat3 to be present in neurons, but in neither astrocytes nor microglia/macrophages. These results were also confirmed be western blot analysis. The present results indicate that Stat3 activation occurs in neurons and endothelial cells only during post-ischemic reperfusion despite widespread expression of IL-6 cytokines.
Suzuki
S
S
Department of Neurology, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Tanaka
K
K
Nogawa
S
S
Dembo
T
T
Kosakai
A
A
Fukuuchi
Y
Y
D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western; D002545:Brain Ischemia / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D002452:Cell Count; D002540:Cerebral Cortex / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D003342:Corpus Striatum / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004195:Disease Models, Animal; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006131:Growth Inhibitors / Q000096:biosynthesis; D007150:Immunohistochemistry; D020244:Infarction, Middle Cerebral Artery / Q000150:complications / Q000378:metabolism; D015850:Interleukin-6 / Q000096:biosynthesis; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000096:biosynthesis; D008264:Macrophages / Q000378:metabolism / Q000473:pathology; D008297:Male; D017628:Microglia / Q000378:metabolism / Q000473:pathology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015424:Reperfusion; D050796:STAT3 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005904:Glial Fibrillary Acidic Protein; D006131:Growth Inhibitors; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines; D008869:Microtubule-Associated Proteins; D050796:STAT3 Transcription Factor; C494088:Stat3 protein, rat; D015534:Trans-Activators