Lipopolysaccharide-induced inhibition of connexin43 gap junction communication in astrocytes is mediated by downregulation of caveolin-3.
Astrocytes play a crucial role in maintaining the homeostasis of the brain. Changes to gap junctional intercellular communication (GJIC) in astrocytes and excessive inflammation may trigger brain damage and neurodegenerative diseases. In this study, we investigated the effect of lipopolysaccharide (LPS) on connexin43 (Cx43) gap junctions in rat primary astrocytes. Following LPS treatment, dose- and time-dependent inhibition of Cx43 expression was seen. Moreover, LPS induced a reduction in Cx43 immunoreactivity at cell-cell contacts and significantly inhibited GJIC, as revealed by the fluorescent dye scrape loading assay. Toll-like receptor 4 (TLR4) protein expression was increased 2-3-fold following LPS treatment. To study the pathways underlying these LPS-induced effects, we examined downstream effectors of TLR4 signaling and found that LPS induced a significant increase in phosphorylated extracellular signal-regulated kinase (pERK) levels up to 6 h, followed by signal attenuation and downregulation of caveolin-3 expression. Interestingly, LPS treatment also induced a dramatic increase in inducible nitric oxide synthase (iNOS) levels at 6 h, which were sustained up to 18-24 h. The LPS-induced downregulation of Cx43 and caveolin-3 was prevented by co-treatment of astrocytes with the iNOS cofactor inhibitor 1400W, but not the ERK inhibitor PD98059. Specific knockdown of caveolin-3 using siRNA had a significant inhibitory effect on GJIC and resulted in a downregulation of Cx43. Our results suggest that long-term LPS treatment of astrocytes leads to inhibition of Cx43 gap junction communication by the activation of iNOS and downregulation of caveolin-3 via a TLR4-mediated signaling pathway.
Liao
Chih-Kai
CK
Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan.
Wang
Seu-Mei
SM
Chen
Yuh-Lien
YL
Wang
Hwai-Shi
HS
Wu
Jiahn-Chun
JC
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000473:pathology / Q000502:physiology*; D051244:Caveolin 3 / Q000235:genetics / Q000378:metabolism*; D002450:Cell Communication; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D017629:Gap Junctions / Q000473:pathology / Q000502:physiology*; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D015854:Up-Regulation
C495549:Cav3 protein, rat; D051244:Caveolin 3; D018031:Connexin 43; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat