mTOR- and SGK-Mediated Connexin 43 Expression Participates in Lipopolysaccharide-Stimulated Macrophage Migration through the iNOS/Src/FAK Axis

Abstract

Connexin 43 (Cx43) deficiency was found to increase mortality in a mouse model of bacterial peritonitis, and Cx43 is upregulated in macrophages by LPS treatment. In this study, we characterized a novel signaling pathway for LPS-induced Cx43 expression in RAW264.7 cells and thioglycolate-elicited peritoneal macrophages (TGEMs). LPS alone or LPS-containing conditioned medium (CM) upregulated Cx43. Overexpression or silencing of Cx43 led to the enhancement or inhibition, respectively, of CM-induced TGEM migration. This response involved the inducible NO synthase (iNOS)/focal adhesion kinase (FAK)/Src pathways. Moreover, CM-induced migration was compromised in TGEMs from Cx43 +/2 mice compared with TGEMs from Cx43 +/+ littermates. Cx43 was upregulated by a serum/glucocorticoid-regulated kinase 1 (SGK) activator and downregulated, along with inhibition of CMinduced TGEM migration, by knockdown of the SGK gene or blockade of the SGK pathway. LPS-induced SGK activation was abrogated by Torin2, whereas LPS-induced Cx43 was downregulated by both Torin2 and rapamycin. Analysis of the effects of FK506 and methylprednisolone, common immunosuppressive agents following organ transplantation, suggested a link between these immunosuppressive drugs and impaired macrophage migration via the Cx43/iNOS/Src/FAK pathway. In a model of Escherichia coli infectious peritonitis, GSK650349-, an SGK inhibitor, or Torin2-treated mice showed less accumulation of F4/80 + CD11b + macrophages in the peritoneal cavity, with a delay in the elimination of bacteria. Furthermore, following pretreatment with Gap19, a selective Cx43 hemichannel blocker, the survival of model mice was significantly reduced. Taken together, our study suggested that Cx43 in macrophages was associated with macrophage migration, an important immune process in host defense to infection.

Angiotensin II induces RAW264.7 macrophage polarization to the M1‑type through the connexin 43/NF‑κB pathway

“…Immunofluorescence staining of Cx43, CD86 and iNOS was used to investigate the effect of AngII on macrophage polarization and it was discovered that Cx43 was expressed in RAW264.7 macrophages. Moreover, some of these results are consistent with the Shen et al (36) study. In addition, AngII treatment increased the expression levels of CD86, iNOS and Cx43, which indicated that Cx43 may be involved in the M1-type macrophage polarization process.…”

“…Cx43 deficiency has been reported to increase mortality in a mouse model of bacterial peritonitis, and Cx43 is reportedly upregulated in macrophages following LPS treatment (36). Shen et al (36) suggested that Cx43 expression in macrophages was associated with macrophage migration, an important immune process in the host defence against infection. In addition, nie et al (37) demonstrated that the expression levels of Cx43 in mouse dendritic cells were significantly increased following AngII treatment.…”

Serine Supports IL-1β Production in Macrophages Through mTOR Signaling

“…Isolation of the peritoneal macrophages from mice was conducted as described in previous studies with some minor modifications (28). Briefly, 3 days after the injection of 4% thioglycolate, macrophages were flushed from peritoneal cavity with cold Dulbecco's modified Eagle's medium (DMEM) (Gibco), and cultured in complete DMEM medium (10% FBS, 1% penicillin/streptomycin).…”

Function of Connexin-43 in Macrophages

“…Lastly, it is important to note, that the investigations into Cx43 in relation to Mϕs are limited, since the homozygous Cx43 knockout is lethal in mice due to heart failure [16,23]. These circumstances have led to studies making use of different circumventions, such as heterozygous mice [24,25], Mϕ-Cx43 specific knockout mice [9,18] and fetal liver cell transplantation from homozygous Cx43 knockout mice into irradiated recipient-mice [26], as discussed below.…”