Abstract
The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway.
Highlights
Astrocytes are the predominant supportive glial cells in the brain, where they participate in the formation of the blood-brain barrier and contribute to the maintenance of homeostasis in the central nervous system (CNS)
We examined whether LPS activated the JNK and nuclear factor-kB (NF-kB) signaling pathways and as shown in Fig. 1A, only low levels of NF-kB were found in control astrocytes, but a significant increase in NF-kB levels was detected at 6–18 h of LPS treatment (Fig. 1A and B)
A previous study demonstrated that activation of the NFkB and PI3 kinase pathways, but not JNK pathway, by TLR3 leads to a reduction in Cx43 expression in astrocytes [12]
Summary
Astrocytes are the predominant supportive glial cells in the brain, where they participate in the formation of the blood-brain barrier and contribute to the maintenance of homeostasis in the central nervous system (CNS). Astroglial networks are interconnected through clusters of intercellular channels named gap junctions, which allow diffusion of second messengers, ions, and small metabolites between adjacent astrocytes [1]. Each gap junction channel is generated by the docking of two end-to-end hemichannels, termed connexons, in the opposing plasma membranes [2]. Connexon is composed of six membranespanning proteins named connexins (Cxs). Despite several Cx members that have been detected in astrocytes in many brain regions and in the hippocampus such as Cx43, Cx30, and Cx26, the major gap junction protein present in astrocytes in vitro is Cx43 [3,4]. Evidence indicates that pathological and inflammatory stimuli affect Cx43 mRNA and protein levels and its phosphorylation state in astrocytes and thereby regulate gap junction intercellular communication (GJIC) [5]. Deletion of astrocyte Cx43 and Cx30 in double knock-out mice leads to myelin pathology, hippocampal vacuolation, and functional impairments in sensorimotor and spatial memory [6,7]
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