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ER Stress Stimulates Production of the Key Antimicrobial Peptide, Cathelicidin, by Forming a Previously Unidentified Intracellular S1P Signaling Complex

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ER Stress Stimulates Production of the Key Antimicrobial Peptide, Cathelicidin, by Forming a Previously Unidentified Intracellular S1P Signaling Complex ER stress stimulates production of the key antimicrobial peptide, cathelicidin, by forming a previously unidentified intracellular S1P signaling complex Kyungho Parka,b, Hiroko Ikushiroc,1, Ho Seong Seod,1, Kyong-Oh Shine, Young il Kima,b, Jong Youl Kimb,f, Yong-Moon Leee, Takato Yanoc, Walter M. Hollerana,b, Peter Eliasa,b, and Yoshikazu Uchidaa,b,2 aDepartment of Dermatology, School of Medicine, University of California, San Francisco, CA 94158; bNorthern California Institute for Research and Education, Veterans Affairs Medical Center, San Francisco, CA 94121; cDepartment of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan; dRadiation Research Division, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Korea; eCollege of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea; and fDepartment of Endocrinology, School of Medicine, University of California, San Francisco, CA 94121 Edited by Sen-itiroh Hakomori, Pacific Northwest Research Institute, Seattle, WA, and approved January 19, 2016 (received for review March 5, 2015) We recently identified a previously unidentified sphingosine-1- synthesis under basal conditions (3, 7). However, CAMP produc- phosphate (S1P) signaling mechanism that stimulates production tion is not only always beneficial: Although transient increases of a key innate immune element, cathelicidin antimicrobial pep- defend against microbial pathogens, sustained production of this tide (CAMP), in mammalian cells exposed to external perturbations, AMP can stimulate downstream inflammatory responses, and such as UVB irradiation and other oxidative stressors that provoke even tumorigenesis (1). subapoptotic levels of endoplasmic reticulum (ER) stress, independent S1P modulates a variety of cellular functions (e.g., cell pro- of the well-known vitamin D receptor-dependent mechanism. ER liferation, differentiation and motility) through well-known G pro- stress increases cellular ceramide and one of its distal metabolites, tein coupled, S1P receptor-dependent mechanisms (8). However, S1P, which activates NF-κBfollowedbyC/EBPα activation, leading to we showed recently that ER stress-stimulated CAMP production CAMP production, but in a S1P receptor-independent fashion. We is likely receptor-independent (7). Although prior studies showed now show that S1P activates NF-κB through formation of a previ- that ER stress activates NF-κB via plasma membrane-localized ously unidentified signaling complex, consisting of S1P, TRAF2, and S1P1, S1P2, and S1P3 receptor activation (9, 10), using specific RIP1 that further associates with three stress-responsive proteins; i.e., activators/agonists and inhibitors/antagonists of all five identified heat shock proteins (GRP94 and HSP90α)andIRE1α. S1P specifically S1P receptors (S1P1–S1P5), we showed that these pharmacolog- interacts with the N-terminal domain of heat shock proteins. Because ical interventions did not modify the ER stress-induced increase in this ER stress-initiated mechanism is operative in both epithelial cells CAMP expression (7). Although prior study shows that TNFα and macrophages, it appears to be a universal, highly conserved receptor activation can initiate S1P binding to TRAF2 on plasma response, broadly protective against diverse external perturba- membrane, forming a signaling complex (S1P–TRAF2–TRADD– tions that lead to increased ER stress. Finally, these studies further RIP1) that activates NF-κB (11), this mechanism does not appear illuminate how ER stress and S1P orchestrate critical stress-specific to regulate CAMP synthesis. Instead, we identify and delineate a signals that regulate production of one protective response by stim- previously unidentified TNFα receptor- and a S1P receptor- ulating production of the key innate immune element, CAMP. independent mechanism that regulate CAMP production through intracellular assembly of a S1P–TRAF2-stress-responsive protein ER stress | sphingosine-1-phosphate | heat shock protein 90 | cathelicidin | signaling complex that forms in response to ER stress. Elucidation innate immunity Significance ammalian epithelial tissues face hostile external environ- Mments, where they are repeatedly bombarded by external The cathelicidin antimicrobial peptide (CAMP) is an innate im- perturbants, such as UV irradiation, oxidative stress and micro- mune element that promotes antimicrobial defense, but ex- bial pathogens that potentially threaten the integrity of epithelial cessive CAMP can stimulate inflammation and tumorigenesis. and nonepithelial tissues/cells. Antimicrobial peptides (AMPs) We recently discovered that external perturbations that induce represent highly conserved, innate immune elements that protect subtoxic levels of endoplasmic reticulum (ER) stress increase the host from microbial pathogens, while also signaling a variety sphingosine-1-phosphate (S1P) production, in turn activating of downstream responses that further fortify innate and adaptive NF-κB–mediated CAMP synthesis. We report here that S1P in- immunity (1, 2). We have shown that AMP production increases teracts with the heat shock proteins (HSP90α and GRP94) not only in response to microbial challenges, but also in response through a previously unidentified S1P receptor-independent to a wide variety of other external perturbations that converge on intracellular mechanism, followed by the activation of NF-κB the endoplasmic reticulum (ER), where AMP orchestrate a host leading to stimulation of CAMP production. These studies illu- of ER-initiated stress responses (3). Although high doses of ex- minate the critical role of both ER stress and S1P in orchestrating ternal perturbants can cause excessive ER stress, leading to in- stress-specific signals that enhance innate immunity. creased production of the proapoptotic lipid ceramide, threatening Author contributions: K.P. and Y.U. designed research; K.P., H.I., H.S.S., K.-O.S., Y.i.K., and cells with apoptosis (4, 5), subtoxic levels of the same perturbants J.Y.K. performed research; K.P., H.I., K.-O.S., Y.-M.L., T.Y., W.M.H., and Y.U. analyzed data; instead provoke lower levels of ER stress, with incrementally re- K.P., P.E., and Y.U. wrote the paper; Y.U. developed the concept; Y.U. designed experi- duced ceramide production that rescues cells from apoptosis in ments; and Y.U. directed the project. part through metabolic conversion of ceramide to sphingosine- The authors declare no conflict of interest. 1-phosphate (S1P) (6). S1P in turn stimulates production of the key This article is a PNAS Direct Submission. AMP, cathelicidin AMP (CAMP), and its downstream proteolytic 1H.I. and H.S.S. contributed equally to this work. product, LL-37, through transactivation of NF-κB–C/EBPα (7). 2To whom correspondence should be addressed. Email: [email protected]. This mechanism operates independently of the well-known vitamin This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. D receptor-dependent mechanism, which instead regulates CAMP 1073/pnas.1504555113/-/DCSupplemental. E1334–E1342 | PNAS | Published online February 22, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1504555113 Downloaded by guest on September 27, 2021 of this previously unidentified regulatory mechanism could point to supporting the idea that S1P generated by SPHK1 is responsible for PNAS PLUS potentially previously undeveloped therapeutic approaches to ei- stimulation of CAMP production. Moreover, Lys63 (K63)-linked ther enhance innate immunity or to suppress excessive CAMP polyubiquitination of RIP1, which is required for NF-κB activation, production causing inflammation and tumorigenesis. was suppressed by a blockade of SPHK1 using PF-543 or SPHK1 siRNA (Fig. 2 A and D). These results ascertained our prior find- Results ings that S1P signaling generated by SPHK1 is essential for ER S1P Generated by SPHK1 Stimulates CAMP Production Through S1P stress-induced CAMP expression through NF-κB activation. Receptor-Independent Activation of NF-κB in Response to ER Stress. Although previous findings showed that ER stress activates Because mammalian epidermis is continuously threatened by the NF-κB via S1P1, S1P2, and S1P3 receptor activation (9, 10), our external environment, we used cultured human epidermal ker- prior studies revealed that pharmacological interventions using atinocytes (KC) as our primarily model in these studies. specific activators/agonists and inhibitors/antagonists of all five Our prior study characterized that S1P generated by sphingo- identified S1P receptors (S1P1–S1P5) did not modify the ER sine kinase (SPHK) 1 activates NF-κB, leading to stimulation of stress-induced increase in CAMP expression (7). We first con- CAMP production in KC in response to ER stress (7). Consistent firmed whether S1P-mediated stimulation of CAMP expression in with this prior study (7), increased S1P and CAMP production in vivo is S1P receptor-independent. Topical applications of Tg pro- response to ER stress [induced by an ER stressor, thapsigargin (Tg) duced comparable increases in mRNA and protein production of + + that releases Ca2 from ER by inhibition of sarco-ER Ca2 -ATPases; the murine homolog of CAMP (13) in the epidermis of S1P1-, ref. 12] were suppressed by a specific inhibitor of SPHK1, PF-543, S1P2-, and S1P3-null and wild-type mice (SI Appendix,Fig.S3A, which significantly inhibited an isoform of SPHK, SPHK1, but not B, D,andE). Pertinently, topical Tg also comparably increased SPHK2, at concentrations ≥ 1 μMinKC(Fig.1A–C)
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