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Mtor- and SGK-Mediated Connexin 43 Expression Participates in Lipopolysaccharide-Stimulated Macrophage Migration Through the Inos/Src/FAK Axis

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Mtor- and SGK-Mediated Connexin 43 Expression Participates in Lipopolysaccharide-Stimulated Macrophage Migration Through the Inos/Src/FAK Axis The Journal of Immunology mTOR- and SGK-Mediated Connexin 43 Expression Participates in Lipopolysaccharide-Stimulated Macrophage Migration through the iNOS/Src/FAK Axis Chen Shen,* Jin Hong Chen,* Youngyi Lee,† Md. Mehedi Hassan,‡ Su Jin Kim,* Eun Young Choi,x Seong-Tshool Hong,‡ Byung-Hyun Park,† and Ji Hyun Park* 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 CM- induced 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 pre- treatment 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. The Journal of Immunology, 2018, 201: 2986–2997. acrophages, which are present in almost all tissues, for TLR4 (2). Upon binding TLR4, LPS triggers common constitute an essential component of the innate im- downstream signaling pathways, most notably PI3K/protein M mune system and form the first line of defense against kinase B (Akt) and NF-kB, leading to upregulation of proin- pathogens. Under physiological conditions, macrophage recruit- flammatory mediators, such as cytokines and NO (3). ment to sites of infection is an important immune process in host Cellular Src is the prototype of Src family kinases (SFKs) of defenses (1). highly conserved proteins, including Blk, Fgr, Fyn, Hck, Lck, Lyn, LPS, a major constituent of the Gram-negative bacterial endo- and Yes (4). In resting macrophages, Src is barely detectable; toxin, is also an important pathogen-associated molecular pattern however, LPS treatment leads to upregulation of Src, resulting in activation of focal adhesion kinase (FAK) and cell motility (5). *Division of Endocrinology and Metabolism, Department of Internal Medicine, FAK, a Src substrate, plays a critical role in macrophage adhesion Chonbuk National University Medical School, Research Institute of Clinical Medi- and motility (6). Accumulating evidence has indicated that LPS- cine of Chonbuk National University–Biomedical Research Institute of Chonbuk elicited macrophage motility requires the participation of Src and National University Hospital, Jeonju 54907, Republic of Korea; †Department of Biochemistry, Chonbuk National University Medical School, Jeonju 54907, Republic FAK and is dependent on inducible NO synthase (iNOS) (7). of Korea; ‡Department of Biomedical Sciences and Institute for Medical Science, Hence, targeting the iNOS/Src/FAK axis may directly reflect Chonbuk National University Medical School, Jeonju 54907, Republic of Korea; and xDepartment of Biomedical Sciences, University of Ulsan College of Medicine, Seoul LPS-triggered macrophage motility. 05505, Republic of Korea Gap junctions (GJs) are intercellular channels that allow ORCIDs: 0000-0001-7026-3461 (C.S.); 0000-0002-0417-6236 (J.H.C.); 0000-0003- communication between contacting cells by mediating recipro- 3768-4285 (B.-H.P.). cal exchange of ions and small molecules (8). Connexin 43 Received for publication July 3, 2017. Accepted for publication September 12, 2018. (Cx43), the most ubiquitous GJ protein subunit, interacts with This work was supported by research funds from the Research Institute of Clinical many proteins and has channel-independent functions (9). Medicine of Chonbuk National University–Biomedical Research Institute of Chonbuk Moreover, Cx43 is expressed by almost all immune cells and can National University Hospital. be upregulated when the immune cells become exposed to in- Address correspondence and reprint requests to Dr. Ji Hyun Park, Division of Endo- crinology and Metabolism, Department of Internal Medicine, Chonbuk National flammatory factors (10). However, the role of Cx43 in signaling- University Medical School, 20 Geonji-Ro, Deokjin-Gu, Jeonju 54907, Republic of mediated immune regulation is poorly defined. Importantly, Korea. E-mail address: [email protected] several recent studies have reported the correlation between Abbreviations used in this article: bFGF, basic fibroblast growth factor; CM, con- Cx43 and invasion or migration in cancer and embryonic stem ditioned medium; Cx43, connexin 43; FAK, focal adhesion kinase; GJ, gap junc- tion; iNOS, inducible NO synthase; MCP-1, monocyte chemotactic protein-1; cells (11, 12). mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; SGK, Therefore, in this study, we investigated the impact of Cx43 serum/glucocorticoid-regulated kinase 1; si, small interfering; si-Scr, scrambled on LPS-induced macrophage migration to provide a theoreti- siRNA; TGEM, thioglycolate-elicited peritoneal macrophage. cal basis for Cx43 gene therapy in the treatment of infection or Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 immunosuppression. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700954 The Journal of Immunology 2987 FIGURE 1. Effect of CM on Cx43 expression in RAW264.7 cells and TGEMs. RAW264.7 cells (A and B)andTGEMs(C and D)weretreatedwith CM for 6 and 24 h. Protein levels of Cx43 from treated cell lysates were analyzed by Western blotting and normalized against GAPDH (n =4).Cx43 mRNA expression was measured by real-time PCR and normalized to 36B4 expression (n =5).(E) The concentration of TNF-a,IL-1b, IL-6, and MCP-1 from CM was analyzed by ELISA kit. (F) TGEMs were treated with 10 ng/ml TNF-a,IL-1b, IL-6, and MCP-1 for 24 h. Protein levels of Cx43 and p-serum/ glucocorticoid-regulated kinase 1 (SGK) from treated cell lysates were analyzed by Western blotting and normalized against GAPDH and SGK, respectively. Values are presented as means 6 SEMs of three or four (indicated with n =4)orfive(indicatedwithn = 5) independent experiments. *p , 0.05, **p , 0.01 versus vehicle by one-way ANOVA (A–D) or Student t test. Materials and Methods methylprednisolone, and GSK650394 were obtained from Cayman a Animals Chemical (Ann Arbor, MI). Recombinant mouse TNF- monocyte che- motactic protein-1 (MCP-1), IL-1b, and IL-6 were purchased from Seven-week-old male C57BL/6 mice were purchased from Samtako ProSpec (Ness-Ziona, Israel). Gap19 was obtained from Apexbio Tech- Bio (Osan, Korea). Wild-type mice and Cx43-knockout mice were nology (Houston, TX). obtained from mating of mice heterozygous for the mutant allele (Gja1+/Gja1–), which were purchased from The Jackson Laboratory Cell culture (Bar Harbor, ME). Because homozygous Cx43-knockout mice Murine RAW 264.7 macrophages were purchased from American Type (Cx432/2) are embryonic, lethal, heterozygous mice (Cx43+/2)and +/+ Culture Collection (Manassas, VA). Cells were thawed and grown in age-matched wild-type littermates (Cx43 ) older than 8 wk were phenol-red DMEM (Lonza, Walkersville, MD). All cell cultures were used for experiments. The animals were housed under pathogen-free supplemented with 10% FBS (GenDEPOT, Barker, TX) and kept at 37˚C conditions and fed a standard laboratory normal chow diet ad libi- in a humidified 5% CO incubator. For treatment with FBS (Fig. 6A), tum. There were no significant differences in average body weight 2 +/+ +/2 cells were starved for 2 h and treated with indicated concentration of between Cx43 littermates and Cx43 mice (26.2 versus 25.8 g, the serum. respectively; p = 0.36). All experimental procedures were approved by the Institutional Animal Care and Use Committee (approval no. Isolation of primary macrophages CBNU-2016-0080). Thioglycolate-elicited peritoneal macrophages (TGEMs) were obtained Materials from 8-wk-old C57BL/6 mice 3 d after i.p. injection of Brewer thio- glycollate (3 ml of 3% p/v; BD Life Sciences, Franklin Lakes, NJ). Routine chemicals, laboratory ware, and reagents were obtained from Peritoneal exudates were washed with PBS and plated in 10% FBS- Invitrogen Life Technologies (Carlsbad, CA) or Sigma-Aldrich (St. Louis, containing RPMI 1640 medium (Life Technologies, Grand Island, NY) MO). Anti–p-serum/glucocorticoid-regulated kinase 1 (SGK), anti-SGK, for 2 h. Exudates were then washed with medium to remove nonadherent and anti-iNOS Abs were purchased from Santa Cruz Biotechnology cells. The resulting macrophage
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