科研之友
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摘要
Previous studies have implicated a role of G alpha(i) proteins as co-regulators of Toll-like receptor (TLR) activation. These studies largely derived from examining the effect of G alpha(i) protein inhibitors or genetic deletion of G alpha(i) proteins. However, the effect of increased G alpha(i) protein function or G alpha(i) protein expression on TLR activation has not been investigated. We hypothesized that gain of function or increased expression of G alpha(i) proteins suppresses TLR2- and TLR4-induced inflammatory cytokines. Novel transgenic mice with genomic "knock-in" of a regulator of G protein signaling (RGS)-insensitive Gnai2 allele (G alpha (i2) (G184S/G184S) ; GS/GS) were employed. These mice express essentially normal levels of G alpha(i2) protein; however, the G alpha(i2) is insensitive to its negative regulator RGS thus rendering more sustained G alpha(i2) protein activation following ligand/receptor binding. In subsequent studies, we generated Raw 264.7 cells that stably overexpress G alpha(i2) protein (Raw G alpha(i2)). Peritoneal macrophages, splenocytes, and mouse embryonic fibroblasts (MEF) were isolated from WT and GS/GS mice and were stimulated with LPS, Pam3CSK4, or Poly (I:C). We also subjected WT and GS/GS mice to endotoxic shock (LPS, 25 mg/kg i.p.) and plasma tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-6 production were determined. We found that in vitro LPS and Pam3CSK4-induced TNF-alpha, and IL-6 production are decreased in macrophages from GS/GS mice compared with WT mice (p < 0.05). In vitro, LPS and Pam3CSK4 induced IL-6 production in splenocytes, and in vivo, LPS-induced IL-6 were suppressed in GS/GS mice. Poly (I:C)-induced TNF-alpha, and IL-6 in vitro demonstrated no difference between GS/GS mice and WT mice. LPS-induced IL-6 production was inhibited in MEFs from GS/GS mice similarly to macrophage and splenocytes. In parallel studies, Raw G alpha(i2) cells also exhibit decreased TNF-alpha and IL-6 production in response to LPS and Pam3CSK4. These studies support our hypothesis that G alpha(i2) proteins are novel negative regulators of TLR activation.
