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• W4280608766 abstract "Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis–induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1β, and IL-8 in P. gingivalis–stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre–mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis–induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection–induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis–induced inflammatory immune responses and mapped out a novel SGK1–TRAF2/3–c-Jun–NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity. Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis–induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1β, and IL-8 in P. gingivalis–stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre–mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis–induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection–induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis–induced inflammatory immune responses and mapped out a novel SGK1–TRAF2/3–c-Jun–NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity. Homeostasis between proinflammatory and anti-inflammatory response is critical to the outcome of immune responses and progression of many inflammatory diseases. Concomitant to initiation of proinflammatory responses, anti-inflammatory mechanisms are also ignited to restrain the overwhelming inflammation (1Murray P.J. Smale S.T. Restraint of inflammatory signaling by interdependent strata of negative regulatory pathways.Nat. Immunol. 2012; 13: 916-924Crossref PubMed Scopus (123) Google Scholar, 2Fullerton J.N. Gilroy D.W. Resolution of inflammation: a new therapeutic frontier.Nat. Rev. Drug Discov. 2016; 15: 551-567Crossref PubMed Scopus (443) Google Scholar). In this regard, our and other studies have demonstrated that activation of a variety of signaling molecules, such as PI3K, PKB/Akt, mammalian target of rapamycin complex (mTORC) 1, tumor necrosis factor α–induced protein 3 (TNFAIP3/A20), glycogen synthase kinase 3 beta (GSK3β), Janus kinase 3, and wingless-related integration site 3a, restrains the magnitude and intensity of inflammation (3Adamowicz K. Wang H. Jotwani R. Zeller I. Potempa J. Scott D.A. Inhibition of GSK3 abolishes bacterial-induced periodontal bone loss in mice.Mol. Med. 2012; 18: 1190-1196Crossref PubMed Scopus (26) Google Scholar, 4Li Y. Mooney E.C. Holden S.E. Xia X.J. Cohen D.J. Walsh S.W. et al.A20 orchestrates inflammatory response in the oral mucosa through restraining NF-kappaB activity.J. Immunol. 2019; 202: 2044-2056Crossref PubMed Scopus (14) Google Scholar, 5Lu L. Yakoumatos L. Ren J. Duan X. Zhou H. Gu Z. et al.JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling.FASEB J. 2020; 34: 9120-9140Crossref PubMed Scopus (7) Google Scholar, 6Yang D. Li S. Duan X. Ren J. Liang S. Yakoumatos L. et al.TLR4 induced Wnt3a-Dvl3 restrains the intensity of inflammation and protects against endotoxin-driven organ failure through GSK3beta/beta-catenin signaling.Mol. Immunol. 2020; 118: 153-164Crossref PubMed Scopus (10) Google Scholar, 7Nakayama M. Inoue T. Naito M. Nakayama K. Ohara N. Attenuation of the phosphatidylinositol 3-kinase/Akt signaling pathway by Porphyromonas gingivalis gingipains RgpA, RgpB, and Kgp.J. Biol. Chem. 2015; 290: 5190-5202Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). With advances in understanding the mechanism of anti-inflammatory machinery, it is becoming evident that anti-inflammatory response is almost always activated concurrently with proinflammatory response, lasts from several seconds to several years, and acts as a positive process rather than a negative one (1Murray P.J. Smale S.T. Restraint of inflammatory signaling by interdependent strata of negative regulatory pathways.Nat. Immunol. 2012; 13: 916-924Crossref PubMed Scopus (123) Google Scholar, 2Fullerton J.N. Gilroy D.W. Resolution of inflammation: a new therapeutic frontier.Nat. Rev. Drug Discov. 2016; 15: 551-567Crossref PubMed Scopus (443) Google Scholar). Periodontitis is a chronic inflammatory disease characterized by severe gingival inflammation and destruction of alveolar bone and connective tissues surrounding the teeth. It is estimated that over half of the adult population in the United States has periodontitis (8Eke P.I. Dye B.A. Wei L. Thornton-Evans G.O. Genco R.J. CDC Periodontal Disease Surveillance Workgroup: James Beck, G.D.R.P.Prevalence of periodontitis in adults in the United States: 2009 and 2010.J. Dent Res. 2012; 91: 914-920Crossref PubMed Scopus (1093) Google Scholar). In oral cavity, the balance of proinflammatory and anti-inflammatory responses ensures a state of immune homeostasis, which is critical for the maintenance of gingival health. Tissue destruction is primarily a consequence of excessive intensity and/or prolonged duration of host immune inflammatory responses to dysbiotic microbiota. It was reported that the increased proinflammatory cytokines, including TNFα, IL-12, and IL-1β (9Bostanci N. Belibasakis G.N. Porphyromonas gingivalis: an invasive and evasive opportunistic oral pathogen.FEMS Microbiol. Lett. 2012; 333: 1-9Crossref PubMed Scopus (322) Google Scholar, 10Gibson 3rd, F.C. Ukai T. Genco C.A. Engagement of specific innate immune signaling pathways during Porphyromonas gingivalis induced chronic inflammation and atherosclerosis.Front. Biosci. 2008; 13: 2041-2059Crossref PubMed Scopus (82) Google Scholar, 11Hajishengallis G. Krauss J.L. Liang S. McIntosh M.L. Lambris J.D. Pathogenic microbes and community service through manipulation of innate immunity.Adv. Exp. Med. Biol. 2012; 946: 69-85Crossref PubMed Scopus (38) Google Scholar), and decreased anti-inflammatory cytokines, including IL-10, IL-4, and IL-1Ra, are relevant to the periodontal health (12Berker E. Kantarci A. Hasturk H. Van Dyke T.E. Blocking proinflammatory cytokine release modulates peripheral blood mononuclear cell response to Porphyromonas gingivalis.J. Periodontol. 2013; 84: 1337-1345Crossref PubMed Scopus (10) Google Scholar). Porphyromonas gingivalis is considered as a typical pathogenic bacterium, and its colonization in the oral community induces dysregulation of the inflammatory immune response and, ultimately causing irreversible damage to the periodontal tissues (13Hajishengallis G. Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response.Trends Immunol. 2014; 35: 3-11Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 14Hajishengallis G. Periodontitis: from microbial immune subversion to systemic inflammation.Nat. Rev. Immunol. 2015; 15: 30-44Crossref PubMed Scopus (1184) Google Scholar, 15Hajishengallis G. Lamont R.J. Breaking bad: manipulation of the host response by Porphyromonas gingivalis.Eur. J. Immunol. 2014; 44: 328-338Crossref PubMed Scopus (207) Google Scholar). Apart from periodontal inflammation, P. gingivalis infection was also reported to increase the risk of several serious systemic diseases, such as atherosclerosis, diabetes mellitus, rheumatoid arthritis, and oral cancer (16Cullinan M.P. Ford P.J. Seymour G.J. Periodontal disease and systemic health: current status.Aust. Dent. J. 2009; 54 Suppl 1: S62-S69Crossref PubMed Scopus (137) Google Scholar, 17Meyer M.S. Joshipura K. Giovannucci E. Michaud D.S. A review of the relationship between tooth loss, periodontal disease, and cancer.Cancer Causes Control. 2008; 19: 895-907Crossref PubMed Scopus (216) Google Scholar), highlighting the importance of elucidating regulatory mechanisms of P. gingivalis–mediated inflammation. While P. gingivalis was found to induce proinflammatory responses through binding to pattern recognition receptors on host immune cells, the P. gingivalis–mediated anti-inflammatory immune mechanisms and their functional role in the overall periodontal disease process remain less explored. P. gingivalis frequently exploits host machinery, especially the amount and/or activity of protein kinases, such as β-catenin, mTORC1, or Akt, and by which it can modulate the course and magnitude of inflammatory responses (7Nakayama M. Inoue T. Naito M. Nakayama K. Ohara N. Attenuation of the phosphatidylinositol 3-kinase/Akt signaling pathway by Porphyromonas gingivalis gingipains RgpA, RgpB, and Kgp.J. Biol. Chem. 2015; 290: 5190-5202Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 18Stafford P. Higham J. Pinnock A. Murdoch C. Douglas C.W. Stafford G.P. et al.Gingipain-dependent degradation of mammalian target of rapamycin pathway proteins by the periodontal pathogen Porphyromonas gingivalis during invasion.Mol. Oral Microbiol. 2013; 28: 366-378Crossref PubMed Scopus (23) Google Scholar, 19Zhou Y. Sztukowska M. Wang Q. Inaba H. Potempa J. Scott D.A. et al.Noncanonical activation of beta-catenin by Porphyromonas gingivalis.Infect. Immun. 2015; 83: 3195-3203Crossref PubMed Scopus (31) Google Scholar). Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a ubiquitous serine/threonine kinase that was originally considered to function in regulation of sodium homeostasis. Recent studies found that SGK1 was associated to many other diseases, such as hypertension, diabetes, recurrent pregnancy loss, multiple sclerosis, and periodontitis (20Lu X. Crowley S.D. Inflammation in salt-sensitive hypertension and renal damage.Curr. Hypertens. Rep. 2018; 20: 103Crossref PubMed Scopus (28) Google Scholar, 21Lang F. Gorlach A. Vallon V. Targeting SGK1 in diabetes.Expert Opin. Ther. Targets. 2009; 13: 1303-1311Crossref PubMed Scopus (81) Google Scholar, 22Ren J. Han X. Lohner H. Liang R. Liang S. Wang H. Serum- and glucocorticoid-inducible kinase 1 promotes alternative macrophage polarization and restrains inflammation through FoxO1 and STAT3 signaling.J. Immunol. 2021; 207: 268-280Crossref PubMed Scopus (3) Google Scholar, 23Wu C. Yosef N. Thalhamer T. Zhu C. Xiao S. Kishi Y. et al.Induction of pathogenic TH17 cells by inducible salt-sensing kinase SGK1.Nature. 2013; 496: 513-517Crossref PubMed Scopus (674) Google Scholar, 24Matthias J. Maul J. Noster R. Meinl H. Chao Y.Y. Gerstenberg H. et al.Sodium chloride is an ionic checkpoint for human TH2 cells and shapes the atopic skin microenvironment.Sci. Transl. Med. 2019; 11eaau0683Crossref PubMed Scopus (37) Google Scholar). Activation of SGK1 is through PI3K pathway with being phosphorylated by phosphoinositide-dependent kinase 1 at threonine 256 within the kinase domain and mTORC2 at serine 422 within the C-terminal hydrophobic motif and thereby engages in regulation of innate and adaptive immune responses (23Wu C. Yosef N. Thalhamer T. Zhu C. Xiao S. Kishi Y. et al.Induction of pathogenic TH17 cells by inducible salt-sensing kinase SGK1.Nature. 2013; 496: 513-517Crossref PubMed Scopus (674) Google Scholar, 25Garcia-Martinez J.M. Alessi D.R. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1).Biochem. J. 2008; 416: 375-385Crossref PubMed Scopus (684) Google Scholar, 26Di Cristofano A. SGK1: the dark side of PI3K signaling.Curr. Top. Dev. Biol. 2017; 123: 49-71Crossref PubMed Scopus (59) Google Scholar, 27Zhou H. Gao S. Duan X. Liang S. Scott D.A. Lamont R.J. et al.Inhibition of serum- and glucocorticoid-inducible kinase 1 enhances TLR-mediated inflammation and promotes endotoxin-driven organ failure.FASEB J. 2015; 29: 3737-3749Crossref PubMed Scopus (21) Google Scholar, 28Heikamp E.B. Patel C.H. Collins S. Waickman A. Oh M.H. Sun I.H. et al.The AGC kinase SGK1 regulates TH1 and TH2 differentiation downstream of the mTORC2 complex.Nat. Immunol. 2014; 15: 457-464Crossref PubMed Scopus (130) Google Scholar). Our and other recent studies have demonstrated that SGK1 negatively regulates Toll-like receptor (TLR)–mediated inflammatory responses and acts as a key regulator in macrophage polarization (22Ren J. Han X. Lohner H. Liang R. Liang S. Wang H. Serum- and glucocorticoid-inducible kinase 1 promotes alternative macrophage polarization and restrains inflammation through FoxO1 and STAT3 signaling.J. Immunol. 2021; 207: 268-280Crossref PubMed Scopus (3) Google Scholar, 27Zhou H. Gao S. Duan X. Liang S. Scott D.A. Lamont R.J. et al.Inhibition of serum- and glucocorticoid-inducible kinase 1 enhances TLR-mediated inflammation and promotes endotoxin-driven organ failure.FASEB J. 2015; 29: 3737-3749Crossref PubMed Scopus (21) Google Scholar, 29Yang M. Zheng J. Miao Y. Wang Y. Cui W. Guo J. et al.Serum-glucocorticoid regulated kinase 1 regulates alternatively activated macrophage polarization contributing to angiotensin II-induced inflammation and cardiac fibrosis.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1675-1686Crossref PubMed Scopus (97) Google Scholar). However, the role of SGK1 in P. gingivalis–induced inflammation and the underlying molecular mechanisms remain largely unknown. Tumor necrosis factor receptor–associated factors (TRAFs) are a large family of intracellular (IC) signaling molecules that include six typical members (TRAF1–6) and one atypical member (TRAF7) in mammalian cells (30Dhillon B. Aleithan F. Abdul-Sater Z. Abdul-Sater A.A. The evolving role of TRAFs in mediating inflammatory responses.Front. Immunol. 2019; 10: 104Crossref PubMed Scopus (29) Google Scholar, 31Xie P. TRAF molecules in cell signaling and in human diseases.J. Mol. Signal. 2013; 8: 7Crossref PubMed Scopus (285) Google Scholar). Originally identified as signaling adaptors, the TRAF molecules are now known to act as ubiquitin E3 ligases (E3s) and mediate a large variety of signaling transduction upon activation of immune receptors, such as pattern-recognition receptors, antigen receptors, cytokine receptors and, importantly, TNF receptor superfamily members (32Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78: 399-434Crossref PubMed Scopus (1809) Google Scholar, 33Etemadi N. Chopin M. Anderton H. Tanzer M.C. Rickard J.A. Abeysekera W. et al.TRAF2 regulates TNF and NF-kappaB signalling to suppress apoptosis and skin inflammation independently of Sphingosine kinase 1.Elife. 2015; 4e10592Crossref PubMed Scopus (63) Google Scholar). All TRAF members but TRAF1 have been revealed to contain a RING domain located in the N-terminal region. This is key to mediate protein ubiquitination as well as oligomerization and association of TRAF members with their upstream receptors, adaptors, and downstream effector proteins. Through the lysine (K)-48 and K63-linked polyubiquitin chain, TRAF members control multiple signaling pathways that in turn are important for the induction of genes associated with innate immunity, inflammation, and cell survival (34Skaug B. Jiang X. Chen Z.J. The role of ubiquitin in NF-kappaB regulatory pathways.Annu. Rev. Biochem. 2009; 78: 769-796Crossref PubMed Scopus (402) Google Scholar, 35Yang X.D. Sun S.C. Targeting signaling factors for degradation, an emerging mechanism for TRAF functions.Immunol. Rev. 2015; 266: 56-71Crossref PubMed Scopus (75) Google Scholar). Recent studies demonstrated that some TRAF proteins, especially TRAF2 and TRAF3, could function as negative regulators in NF-κB signaling pathways, in which their E3 activity is key for their regulatory roles (35Yang X.D. Sun S.C. Targeting signaling factors for degradation, an emerging mechanism for TRAF functions.Immunol. Rev. 2015; 266: 56-71Crossref PubMed Scopus (75) Google Scholar, 36Jin J. Xiao Y. Hu H. Zou Q. Li Y. Gao Y. et al.Proinflammatory TLR signalling is regulated by a TRAF2-dependent proteolysis mechanism in macrophages.Nat. Commun. 2015; 6: 5930Crossref PubMed Scopus (56) Google Scholar, 37Zarnegar B. Yamazaki S. He J.Q. Cheng G. Control of canonical NF-kappaB activation through the NIK-IKK complex pathway.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 3503-3508Crossref PubMed Scopus (148) Google Scholar). On the other hand, our and other studies found that some protein serine and threonine kinases such as SGK1 and Janus kinase 3 regulate the activity of an ubiquitin E3 ligase, Nedd4 (neural precursor cell–expressed developmentally downregulated 4)-2, and thereby control the activity of downstream inflammatory signaling pathways (5Lu L. Yakoumatos L. Ren J. Duan X. Zhou H. Gu Z. et al.JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling.FASEB J. 2020; 34: 9120-9140Crossref PubMed Scopus (7) Google Scholar, 38Grimsey N.J. Narala R. Rada C.C. Mehta S. Stephens B.S. Kufareva I. et al.A tyrosine switch on NEDD4-2 E3 ligase transmits GPCR inflammatory signaling.Cell Rep. 2018; 24: 3312-3323.e5Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 39Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. et al.Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression.EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (565) Google Scholar). Moreover, P. gingivalis infection was closely associated with activation of SGK1 and multiple E3 ligases such as Nedd4-2 and Smurf1 (Smad ubiquitin regulatory factor 1) (5Lu L. Yakoumatos L. Ren J. Duan X. Zhou H. Gu Z. et al.JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling.FASEB J. 2020; 34: 9120-9140Crossref PubMed Scopus (7) Google Scholar, 40Maekawa T. Krauss J.L. Abe T. Jotwani R. Triantafilou M. Triantafilou K. et al.Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis.Cell Host Microbe. 2014; 15: 768-778Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Surprisingly, there are no studies about the possible activation of TRAF2/3 in P. gingivalis–induced inflammatory responses nor about whether SGK1 affects E3 activity of TRAF members, and thereby functions in the regulation of inflammation. In this study, we demonstrated for the first time that SGK1 acts as a gatekeeper to restrain the inflammatory immune response in cultured cells and a P. gingivalis–induced periodontal bone loss model. Moreover, our mechanistic data identified that SGK1 inhibition enhances expression of c-Jun and phosphorylation of TRAF2, which in turn elevates the degradation of TRAF3, diminishes its suppressive effect on NF-κB, and ultimately aggravates P. gingivalis–mediated inflammatory immune responses. In addition, SGK1 inhibition aggravates P. gingivalis–induced periodontal inflammation in mice gingiva and exacerbates subsequent alveolar bone loss, suggesting that the anti-inflammatory signaling axis, SGK1–TRAF2/3–c-Jun/NF-κB, could be targeted for the development of novel interventional therapeutics to control periodontitis and other inflammatory diseases beyond the oral cavity. Our previous studies have demonstrated that SGK1 promotes macrophage polarization to M2 and restrains secretion of TLR-mediated inflammatory cytokines (22Ren J. Han X. Lohner H. Liang R. Liang S. Wang H. Serum- and glucocorticoid-inducible kinase 1 promotes alternative macrophage polarization and restrains inflammation through FoxO1 and STAT3 signaling.J. Immunol. 2021; 207: 268-280Crossref PubMed Scopus (3) Google Scholar, 27Zhou H. Gao S. Duan X. Liang S. Scott D.A. Lamont R.J. et al.Inhibition of serum- and glucocorticoid-inducible kinase 1 enhances TLR-mediated inflammation and promotes endotoxin-driven organ failure.FASEB J. 2015; 29: 3737-3749Crossref PubMed Scopus (21) Google Scholar, 29Yang M. Zheng J. Miao Y. Wang Y. Cui W. Guo J. et al.Serum-glucocorticoid regulated kinase 1 regulates alternatively activated macrophage polarization contributing to angiotensin II-induced inflammation and cardiac fibrosis.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1675-1686Crossref PubMed Scopus (97) Google Scholar). P. gingivalis is a key pathogenic bacterium that triggers periodontitis and was associated with multiple inflammatory diseases, such as arthritis, atherosclerosis, and Alzheimer’s disease (41Hajishengallis G. Chavakis T. Local and systemic mechanisms linking periodontal disease and inflammatory comorbidities.Nat. Rev. Immunol. 2021; 21: 426-440Crossref PubMed Scopus (131) Google Scholar, 42Olsen I. Taubman M.A. Singhrao S.K. Porphyromonas gingivalis suppresses adaptive immunity in periodontitis, atherosclerosis, and Alzheimer's disease.J. Oral Microbiol. 2016; 8: 33029Crossref PubMed Scopus (78) Google Scholar, 43Olsen I. Yilmaz O. Modulation of inflammasome activity by Porphyromonas gingivalis in periodontitis and associated systemic diseases.J. Oral Microbiol. 2016; 8: 30385Crossref PubMed Scopus (65) Google Scholar). To examine if SGK1 also functions in P. gingivalis–induced inflammation and elucidate the molecular mechanisms involved, we first examined activation of SGK1 in response to the challenge of P. gingivalis in different innate cells. We found that P. gingivalis infection enhanced phosphorylation of SGK1 at serine 422 in human monocytes (Fig. 1, A and D), bone marrow–derived macrophages (BMDMs) (Fig. 1, B and E), and primary oral epithelial cells (Fig. 1, C and F) throughout all the time points measured (up to 24 h). In contrast, other oral bacteria including Streptococcus gordonii and Streptococcus sanguinis failed to do so (Fig. 1, A and D). To further confirm if P. gingivalis activated SGK1, we next examined phosphorylation of N-myc downstream regulated gene 1 (NDRG1), a bona fide substrate of SGK1 (44Murray J.T. Campbell D.G. Morrice N. Auld G.C. Shpiro N. Marquez R. et al.Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3.Biochem. J. 2004; 384: 477-488Crossref PubMed Scopus (260) Google Scholar, 45Schmid E. Xuan N.T. Zahir N. Russo A. Yang W. Kuhl D. et al.Serum- and glucocorticoid-inducible kinase 1 sensitive NF-kappaB signaling in dendritic cells.Cell. Physiol. Biochem. 2014; 34: 943-954Crossref PubMed Scopus (28) Google Scholar). Like the phosphorylation of SGK1, infection of P. gingivalis indeed elevated phosphorylation of NDRG1 at threonine 346/356/366 in all cells we investigated (Fig. 1). Moreover, the expression of total SGK1 and NDRG1 was not substantially changed in human monocytes or BMDM cells (Fig. S1) after culture for 24 h, which substantiated the effect of SGK1 inhibition on the phosphorylation of SGK1 and NDRG1. Taken together, these results demonstrated that infection with P. gingivalis activates SGK1 in innate cells. Since P. gingivalis infection activates SGK1 in innate immune cells, we next examined if SGK1 affects production of inflammatory cytokines in response to P. gingivalis challenge. Using EMD638683, a specific SGK1 inhibitor, we found that pretreatment with SGK1 inhibitor drastically decreased phosphorylation of NDRG1 in response to the challenge of P. gingivalis, indicating the inhibitory efficacy of EMD638683 on SGK1 (Fig. 2A). Moreover, inhibition of SGK1 with EMD638683 significantly increased the secretion of TNFα, IL-6, IL-1β, and IL-8 in human monocytes upon the challenge with P. gingivalis (Fig. 2B). We also examined the possible effect of SGK1 on the secretion of IL-10, a prototypical anti-inflammatory cytokine, in P. gingivalis–stimulated monocytes. By using IC IL-10 staining and analysis with flow cytometry, we found that SGK1 inhibition significantly decreased IL-10 in P. gingivalis–stimulated human monocytes, represented by a decrease in positive cells and mean fluorescence intensity (Fig. 2, C–E), suggesting SGK1 may differentially regulate proinflammatory and anti-inflammatory cytokines. To exclude the possible unspecific effects of chemical inhibitor, we next utilized specific sgk1 siRNA to verify the effects of SGK1 on the secretion of inflammatory cytokines. We found that silencing of SGK1 (Fig. 2F) significantly elevated the levels of TNFα, IL-6, IL-1β, and IL-8 production in P. gingivalis–stimulated human monocytes (Fig. 2G). Given the possible off-target effects of siRNA, we next utilized BMDMs from LysM-Cre+sgk1fl/fl mice, in which the expression of SGK1 was efficiently expunged (Fig. 2H), represented by the disappearance of p-NDRG1, to confirm the influences of SGK1 on P. gingivalis–induced inflammatory cytokines. We found that SGK1 deficiency significantly elevated the secretion of proinflammatory cytokines, including TNFα, IL-1β, IL-6 (Fig. 2I), and macrophage inflammatory protein 2 (MIP-2) (Fig. 2J) in P. gingivalis–stimulated cells. Taken together, these results demonstrated that inhibition of SGK1 promotes production of P. gingivalis–induced proinflammatory cytokines while concurrently decreasing IL-10 secretion in innate immune cells, suggesting SGK1 may act as a rheostat to restrain P. gingivalis–mediated inflammatory immune responses. Since we have demonstrated that SGK1 restrains P. gingivalis–mediated inflammatory responses, we next sought to delineate how SGK1 affects inflammatory responses in innate immune cells upon the challenge of P. gingivalis. As shown in Figure 3A, we found that SGK1 inhibition robustly promotes the activity of NF-κB, represented by the phosphorylation of NF-κB P65 at serine 536, in P. gingivalis–stimulated monocytes. Notably, SGK1 inhibition–mediated increases of p-NF-κB were more evident for the samples with prolonged treatment (Fig. 3A, right panel). The incapability of the SGK1 inhibitor at early stage of treatments like 30 min might result from the proteolytic effect of gingipain, which could directly cleave NF-κB P65 and thus decrease its phosphorylation level at the early treated period. The results were consolidated by SGK1-deficient BMDMs (Fig. 3B). However, we did not observe the substantial changes of other prototypical inflammatory signaling pathways including phosphorylation of P38 and extracellular signal–regulated kinases in monocytes pretreated with EMD638683 (Fig. 3C) or SGK1-deficient BMDMs (Fig. 3B). In addition, SGK1 inhibition or sgk1 gene deficiency drastically increased expression of c-Jun in P. gingivalis–stimulated human monocytes and BMDMs (Fig. 3, D and E). Given both NF-κB and c-Jun are key transcriptional factors that bind to the promoters of inflammatory cytokines, these results suggest that SGK1 inhibition–enhanced proinflammatory cytokine production in P. gingivalis–stimulated cells could arise from the increased NF-κB activity and c-Jun expression. Next we tested whether and how P. gingivalis–activated SGK1 affects TRAF2/3 in immune cells. As shown in Figure 4, A and B, SGK1 deficiency led to a drastic reduction of TRAF2 and TRAF3 in P. gingivalis–stimulated BMDMs, especially at 2 h postinfection. Since phosphorylation of TRAF2 is essential for degradation of TRAF3 and subsequently increased activity of NF-κB signaling (46Workman L.M. Zhang L. Fan Y. Zhang W. Habelhah H. TRAF2 ser-11 phosphorylation promotes cytosolic translocation of the CD40 complex to regulate downstream signaling pathways.Mol. Cell. Biol. 2020; 40e00429-19Crossref PubMed Scopus (3) Google Scholar), we next examined the effect of SGK1 activation on phosphorylation of TRAF2. As expected, SGK1 deficiency robustly increased pho" @default.
• W4280608766 created "2022-05-22" @default.
• W4280608766 creator A5006251408 @default.
• W4280608766 creator A5018188465 @default.
• W4280608766 creator A5022173802 @default.
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• W4280608766 date "2022-06-01" @default.
• W4280608766 modified "2023-09-30" @default.
• W4280608766 title "SGK1 negatively regulates inflammatory immune responses and protects against alveolar bone loss through modulation of TRAF3 activity" @default.
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