Camkiv-Dependent Preservation of Mtor Expression Is Required For

CaMKIV-Dependent Preservation of mTOR Expression Is Required for Autophagy during Lipopolysaccharide-Induced Inflammation and Acute Kidney Injury This information is current as of September 28, 2021. Xianghong Zhang, Gina M. Howell, Lanping Guo, Richard D. Collage, Patricia A. Loughran, Brian S. Zuckerbraun and Matthew R. Rosengart J Immunol published online 28 July 2014 http://www.jimmunol.org/content/early/2014/07/26/jimmun Downloaded from ol.1302798 http://www.jimmunol.org/ Why The JI? Submit online.

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 28, 2014, doi:10.4049/jimmunol.1302798 The Journal of Immunology

CaMKIV-Dependent Preservation of mTOR Expression Is Required for Autophagy during Lipopolysaccharide-Induced Inﬂammation and Acute Kidney Injury

Xianghong Zhang,* Gina M. Howell,* Lanping Guo,* Richard D. Collage,* Patricia A. Loughran,*,† Brian S. Zuckerbraun,* and Matthew R. Rosengart*,‡

Autophagy, an evolutionarily conserved homeostasis process regulating biomass quantity and quality, plays a critical role in the host response to sepsis. Recent studies show its calcium dependence, but the calcium-sensitive regulatory cascades have not been deﬁned. In this study, we describe a novel mechanism in which calcium/calmodulin-dependent protein kinase IV (CaMKIV), through inhibitory serine phosphorylation of GSK-3b and inhibition of FBXW7 recruitment, prevents ubiquitin proteosomal degradation of

mammalian target of rapamycin (mTOR) and thereby augments autophagy in both the macrophage and the kidney. Under the Downloaded from conditions of sepsis studied, mTOR expression and activity were requisite for autophagy, a paradigm countering the current perspective that prototypically, mTOR inhibition induces autophagy. CaMKIV–mTOR-dependent autophagy was fundamentally important for IL-6 production in vitro and in vivo. Similar mechanisms were operant in the kidney during endotoxemia and served a cytoprotective role in mitigating acute kidney injury. Thus, CaMKIV–mTOR-dependent autophagy is conserved in both immune and nonimmune/parenchymal cells and is fundamental for the respective functional and adaptive responses to septic

insult. The Journal of Immunology, 2014, 193: 000–000. http://www.jimmunol.org/

utophagy is an ancient homeostatic process that regulates presentation, and assist pattern recognition receptors by delivering cellular biomass quantity, quality, and distribution through cytosolic pathogen-associated molecular patterns to endosomal A the recycling of cytoplasmic proteins and organelles (1, 2). TLRs (15, 17, 18). Interestingly, nonimmune cells, such as But autophagy may also be acutely induced as an adaptive response hepatocytes and renal tubular cells, also exhibit TLR signaling to stress that enables cells to reuse cytoplasmic components to and respond to the stress of sepsis or LPS by inducing autophagy support vital functions. In the setting of nutrient deprivation, the (19–24). However, a complete understanding of the signaling mechanisms orchestrating this dynamic process involve the main mechanisms regulating autophagy during sepsis and the functional growth rheostat mammalian target of rapamycin (mTOR) and AMP- role of autophagy in cell survival and recovery of organ function by guest on September 28, 2021 activated protein kinase (AMPK), a sensor of cellular energy has yet to be developed. status (2–8). Prototypically, AMPK, sensing reduced cellular energy The calcium/calmodulin-dependent protein kinases (CaMK), (i.e., elevated AMP), inhibits mTOR, a negative regulator of auto- a family of serine/threonine kinases responsive to intracellular Ca2+ phagy, thereby inducing autophagy (3, 8–10). concentration [Ca2+], are integral to the immune response, medi- Though the quintessential function of autophagy is to digest and ating Ca2+-dependent Mf function and regulating septic inflam- recycle portions of the cytoplasm during starvation, recent data mation (25–27). Recently, we and others have defined regulatory support a fundamental role in innate immunity as an antimicrobial roles of this multifunctional family in autophagy. CaMKKb,in effector of TLRs (11–15). In macrophages (Mf), LPS induces response to elevations in cytosolic calcium, functions as an upstream autophagy through mechanisms dependent on TLR4, Toll/IL-1R kinase for AMPK-dependent autophagy (28). In the Mf,LPS domain-containing adapter inducing IFN-b, RIP1, and p38 MAPK induces a CaMKI–AMPK complex that regulates autophagy (29). (14, 16). Biologically, it has been shown to be important in the In this circumstance, however, CaMKI–AMPK-dependent auto- elimination of intracellular microbes, contribute to MHC class II phagy occurs independent of mTOR inhibition. These data suggest that for some cell types, such as the Mf, concomitant autophagy and mTOR activity are necessary for specific cellular function. *Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213; †Department of In this study, we further characterize the regulatory role of the Cell Biology, University of Pittsburgh, Pittsburgh, PA 15213; and ‡Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA 15213 CaMK during autophagy, focusing upon the downstream CaMKIV. Our data suggest that CaMKIV negatively regulates ubiquitin- Received for publication October 17, 2013. Accepted for publication June 20, 2014. mediated degradation of mTOR and titrates basal mTOR expres- This work supported by National Institutes of Health Grant R01 GM082852 (to M.R.R.) and a Surgical Infection Society Resident Research Fellowship grant (to M.R.R.). sion, which serves a permissive role for LPS-induced autophagy. f Address correspondence and reprint requests to Dr. Matthew R. Rosengart, Department This mechanism is not specific to the M , as it is also observed in of Surgery, University of Pittsburgh, 200 Lothrop Street, Suite F1266.1, Pittsburgh, the kidney during endotoxemia and appears to serve a cell-specific PA 15213. E-mail address: [email protected] functional and adaptive response to the stress of sepsis. Abbreviations used in this article: AKI, acute kidney injury; AMPK, AMP-activated protein kinase; CaMK, calcium/calmodulin-dependent protein kinase; Mf, macrophage; mTOR, mammalian target of rapamycin; mTORC1, mammalian target of rapamycin Materials and Methods complex 1; pMf, peritoneal Mf; RNAi, RNA interference; RT, room temperature; Reagents and Abs siRNA, small interfering RNA; TASCC, TOR–autophagy spatial coupling compartment; WT, wild-type. Ultra-pure LPS (Escherichia coli 0111:B4) was obtained from List Bio- logicals (Campbell, CA). STO609 (7-oxo-7H-benzimidazo[2,1-a]benz[de] Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 isoquinoline-3-carboxylic acid–acetic acid) was obtained from Calbiochem

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302798 2 CaMKIV REGULATES AUTOPHAGY DURING ENDOTOXEMIA

(San Diego, CA) and used at concentrations of 10–20 mM. STO609 is highly Technologies, Carlsbad, CA). Following transfection, cells were handled as specific for CaMKK: it has an in vitro IC50 of 0.13–0.38 mM for CaMKK detailed in the figure legends. and 32 mM for CaMK II with little or no inhibition of CaMKI, CaMKIV, protein kinase A, protein kinase C, ERK, or myosin L-chain kinase (30). Cellular protein extraction MG132 was purchased from Calbiochem (San Diego, CA) and used at a Total cellular protein was extracted at 4˚C in 500 ml lysis buffer (29, 33). concentration of 10 mM. Abs for mTOR, p-Ser9–GSK3b,GSK3b,p-Thr389 235/236 Protein concentration was determined using a bicinchoninic acid protein p70 S6 kinase, and p-Ser S6 were obtained from Cell Signaling assay (Pierce, Rockford, IL). Technology (Danvers, MA). Abs for ATG7, ATG5-12, LC3B, ubiquitin, FBXW7, actin, and tubulin were obtained from Abcam (Cambridge, MA). Immunoprecipitation Animal experimentation Five microliters Ab was added to 500 mg isolated cellular protein within We performed all animal experiments in accordance with the National lysis buffer and incubated at 4˚C overnight (29). Thirty microliters 50% Institutes of Health guidelines under protocols approved by the Institutional slurry prewashed Protein G PLUS-Agarose (Santa Cruz Biotechnology, Animal Care and Use Committee of the University of Pittsburgh. We Dallas, TX) were then added to each sample, followed by incubation for an 3 additional 2 h at 4˚C. The samples were spun at 14,000 rpm and washed purchased 6–8-wk-old C57BL/6J and B6.129 1-Camk4tm1Tch/J m (CaMKIV2/2) male mice from The Jackson Laboratory (Bar Harbor, four times in lysis buffer. Samples were then resuspended in 30 l lysis 2/2 buffer for future analysis. ME) (26, 31, 32). The CaMKIV strain is derived from a C57BL/6J background; these mice are viable, fertile,normalinsize,anddonot Immunoblot display any gross physical or behavioral abnormalities. We randomly assigned groups of mice of each strain to a specific experiment. Total cellular lysate was electrophoresed in a 6, 10, or 15% SDS-PAGE gel Mice were anesthetized using a mixture of isoflurane and oxygen bled in (based on protein of interest) and transferred to a Hybond-ECL nitrocel- at 3.5 l/min. LPS (List Biologicals) dissolved in DNase/RNase-free distilled lulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The Downloaded from water (1 mg/ml) was instilled i.p. at a concentration of 1.5 or 5 mg/kg. membrane was blocked for 1 h at room temperature with 5% milk and Animals were sacrificed 18 or 48 h after LPS. Blood was isolated by in- incubated with primary Ab for overnight at 4˚C. Blots were then incubated tracardiac puncture, and the kidneys were harvested. Whole blood was in a HRP-conjugated secondary Ab against the primary Ab at RT for 1 h. stored overnight at 4˚C and the serum isolated by centrifugation at 5000 3 g The blot was developed using the SuperSignal chemiluminescent substrate for 10 min. (Thermo Scientific, Rockford, IL) and exposed on KAR-5 film (Eastman For all studies, one investigator performed the surgical experimentation Kodak, Rochester, NY). All gels were reblotted for total protein to confirm and collected the samples. The data were then analyzed by an investigator equal loading or to assess knockdown after RNA interference (RNAi). blinded to the specific treatment. Densitometry was performed by the National Institutes of Health Image http://www.jimmunol.org/ J64 program (National Institutes of Health, Bethesda, MD). Cell isolation and treatment RNA preparation and RT-PCR The murine Mf cell line RAW 264.7 (American Type Culture Collection, Rockville, MD) was grown in DMEM (BioWhittaker, Walkersville, MD) Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA), supplemented with 10% FCS (Sigma-Aldrich, San Diego, CA), 50 U/ml according to the instructions specified by the manufacturer. The mRNA penicillin, and 50 mg/ml streptomycin (Cellgro Mediatech, Kansas City, level was determined by quantitative RT-PCR. Briefly, 1 mg total RNA was 2 2 MO). Peritoneal Mf (pMf) were isolated from C57BL/6 or CaMKIV / used for the first-strand cDNA synthesis, which was carried out by using a mice by lavaging the peritoneal cavity with five 5-ml (25 ml total) aliquots RT system kit, according to the instructions of the manufacturer (Promega). of 4˚C PBS (26, 33). The lavage was centrifuged at 300 3 g for 5 min, and cDNA (1 ml) was used as a template in PCR reactions, and the PCR reaction the cells were resuspended in RPMI 1640 supplemented with 10% FCS, mixture at a 25 ml volume contained 12.5 ml23 Brilliant II SYBR Green by guest on September 28, 2021 50 U/ml penicillin, and 50 mg/ml streptomycin and plated. Selected cell QPCR Master Mixes (Agilent Technologies, Santa Clara, CA), 1 mlcDNA, populations were treated with STO609 (10 mM) for 1–4 h or MG132 and 1 ml mTOR sense and antisense primer sets (SuperArray Bioscience). (10 mM) for 6 h, as described in the figure legends. DMSO, at concen- After denaturing at 95˚C for 15 min, the amplification protocol consisted of trations similar to comparative experiments, was used as an appropriate 40 cycles of 95˚C for 30 s, followed by 60˚C for 60 s. Expression levels of control and never exceeded 0.2%. Selected cells were then treated with mRNA were normalized by the housekeeping gene GAPDH. LPS (100 ng/ml) for the durations described in the figure legends. In vitro kinase assay Small interfering RNA Recombinant (1 mg) GSK3a/b fusion protein (27 kD) was incubated in RAW 264.7 Mf (2 3 104), murine peritoneal Mf (1 3 105), or HKC-8 the presence or absence of activated p-Thr196–CaMKIV (25 ng) for 10 min 4 (2 3 10 ) were plated in 0.5 ml growth medium (without antibiotics) in at 30˚C with the following additions: 10 mM MgCl2, 0.2 mM ATP, 1 mM each well of a 24-well plate, resulting in 30, 80, or 30% confluence, re- CaCl2,and1mM CaM. Reactions were terminated by boiling in SDS–2-ME spectively. Nontarget and mTOR small interfering RNA (siRNA) were dissociation solution, subjected to 10% SDS-PAGE, and probed with p-Ser9– obtained from Dharmacon (Lafayette, CO). We used the Smartpool siRNA GSK3b and anti-CaMKIV. Total GSK3b was evaluated by Coomassie blue from Dharmacon that incorporates four separate siRNA sequences for each R250 staining. mTOR: The following mTOR sequences were used: mTOR, target sequence 59-GGCAUAUGGCCGAGAUUUA-39;59-UAACAGGGUUCGA- Microscopy GAUAAG-39;59-UAACAGGGUUCGAGAUAAG-39;and59-GCGGAUG- pMf cells were plated on sterilized glass slides and allowed to adhere for GCUCCUGACUAU-39. 2 h at 37˚C. Cells underwent three washes with PBS and were then fixed A total of 3 ml Hiperfect was added in 100 ml Opti-DMEM with 25 nM in 4% paraformaldehyde at 4˚C. For in vivo experiments, kidneys were siRNA and incubated at room temperature (RT) for 10 min. This trans- flushed with PBS and then perfused with 2% paraformaldehyde. After 2-h fection mixture was applied to each well and incubated for 72 h. Inhibition fixation, samples were transferred to 30% sucrose for 24 h with a total of of each targeted protein was determined by immunoblot. All experiments three sucrose changes. The samples were cryopreserved in liquid nitrogen and cell number determinations were performed in triplicate. cooled 2-methylbutane and stored at 280˚C until sectioning to 5 mm Plasmid construction and transfection thickness. Immunostaining for in vivo and in vitro was performed as fol- lows. Samples were permeabilized in 0.1% Triton X-100 (Sigma-Aldrich, Plasmids encoding a constitutively active CaMKIV (CaMKIV-dCT) or a St. Louis, MO) at RT, and then the BSA was removed with three washes of kinase-inactive CaMKIV-dCTK75E mutant were generous gifts from PBS. The samples were then blocked with incubation with 2% BSA in Dr. Douglas Black (Yale University, New Haven, CT) (26). CaMKIV-dCT PBS for 1 h at RT. For experiments with cells, samples were then incubated contains a C-terminally truncated version of the CaMKIV encoding gene, with rabbit anti-FBXW7 (Abcam) or rabbit p-Ser9–GSK3b (Cell Signaling truncated to 317 and an N-terminal FLAG epitope. CaMKIV-dCTK75E Technology) for 60 min at RT. In vivo samples stained with anti-LC3-I/II was constructed by changing lysine 75 to glutamate in CaMKIV, which Ab (Novus; 5 mg/ml) in PBB plus 0.1% Triton X-100 at 4˚C. After negatively affects ATP binding at the catalytic site. For transient trans- washing three times with PBB, cells were incubated with a Cy3 secondary fection, RAW 264.7 cells or pMf were seeded in a 24-well plate at 3 3 Ab (goat anti-rabbit, 1:1000; Jackson ImmunoResearch Laboratories) plus 105 or 13 105 cells/well. After 2 h of adhesion, Mf were transfected with Alexa Fluor 488 phalloidin (1:250; Invitrogen). The slides were rinsed, and 1 mg plasmid CaMKIV-dCT or CaMKIV-dCTK75E using the Lipofectamine HOECHST stain (1 mg/100 ml bisbenzimide) was applied for 30 s. Coverslips 2000 reagent according to the instructions specified by the manufacturer (Life or cover glass were adhered with gelvatol, a water-soluble mounting media, The Journal of Immunology 3 and slides were placed at 4˚C overnight before imaging. Samples were (34). Sample processing and imaging occurred at the Center for Biological viewed at 320 original magnification with a 2-fold digital zoom with a Imaging at the University of Pittsburgh. numeric aperture of 0.8 on an Olympus Fluoview 1000 (Olympus America, Center Valley, PA). Imaging conditions were maintained at identical set- Renal function parameters tings within each Ab-labeling experiment with original gating performed using the negative control. Quantification was performed using Metamorph Renal function was determined by assaying serum for cystatin C, using an (Molecular Devices, Sunnyvale, CA) (34). For transmission electron mi- enzyme immunoassay kit (R&D Systems, Minneapolis, MN). Cystatin C croscopy, kidneys were flushed with PBS and subsequently perfused and has emerged as a more precise marker of glomerular filtration rate and has fixed with 2% glutaraldehyde in 0.1 mol/l phosphate buffer (pH 7.4), been validated in both human and murine studies (35, 36). followed by 1% OsO . The tissue was sectioned into 2-cm cubes and then 4 Cytokine production postfixed with 1% OsO4 and 1% K3Fe(CN)6 for 1 h. Samples were washed three times with PBS and then dehydrated in a graded series of 30–100% IL-10, TNF-a, and IL-6 concentrations were quantified by an enzyme 3 ethanol before incubation in 3 1-h incubations of Polybed 812 epoxy immunoassay kit (R&D Systems) that is based on a coated-well, sandwich resin. Samples were cured at 37˚C for 24 h followed by 48 h at 65˚C. enzyme immunoassay. Ultrathin sections of 60 nm were then cut by a microtome (Leica Ultracut R; Leica Microsystems), collected on 200 mesh grids that were then Statistical analysis poststained with 2% uranyl acetate in 50% methanol for 10 min, followed by 1% lead citrate for 7 min, and analyzed using a JEM 1011CX electron Statistical analyses were performed using Stata 12SE software (Stata, College microscope (JEOL, Peabody, MA). Images were acquired digitally from Station, TX). Values are expressed as means 6 SEM. Groups are compared a randomly selected pool of 10–15 fields for each experimental condition by ANOVA. A p value ,0.05 was considered statistically significant. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Mf LPS-induced autophagy is CaMKIV dependent. (A)PrimarypMf from C57BL/6 or CaMKIV2/2 mice were exposed to LPS (100 ng/ml) for 30 min. Total cellular lysate (10 mg/lane) was isolated and the induction of ATG5-12, ATG7, LC3B, and actin assessed by immunoblot (representative blot of n = 4 independent experiments; n = 4 mice total per experimental condition for all four experiments combined). (B)pMfs were transfected with empty vector, a constitutively active CaMKIV (CaMKIV-dCT), or a kinase-deﬁcient mutant (CaMKIV-dCTK75E) for 8 h. Total cellular lysate (10 mg/lane) was then isolated and analyzed for the expression of ATG5-12, ATG7, LC3B, total CaMKIV, and actin by immunoblot (representative blot of n = 3 independent experiments). (C) WT and CaMKIV2/2 pMf were exposed to LPS (100 ng/ml) for 30 min, and autophagy was assessed by electron microscopy. Top and bottom panels, Scale bars, 500 nm. Black arrowheads indicate double membrane autophagosomes/autophagolysosomes. 4 CaMKIV REGULATES AUTOPHAGY DURING ENDOTOXEMIA Downloaded from FIGURE 2. CaMKIV regulates mTOR expression. (A) Primary pMf from C57BL/6 or CaMKIV2/2 mice were exposed to LPS (100 ng/ml) for 30 min. Total cellular lysate (10 mg/lane) was isolated, and mTOR and actin expression were assessed by immunoblot (representative blot of n = 4 independent experiments in which two mice per group are shown; n = 8 total mice/experimental condition for all four experiments combined): lanes 1 and 2 (WT 1), lanes 3 and 4 (WT 2); lanes 5 and 6 (CaMKIV2/2 1); and lanes 7 and 8 (CaMKIV2/2 2). (B) Primary pMf from C57BL/6 or CaMKIV2/2 mice were exposed to LPS (100 ng/ml) for 30 min. Total cellular lysate (10 mg/lane) was isolated, and p-Thr389p70 S6 kinase, p70 S6 kinase, p-Ser240/244 S6, p-Ser235/236 S6, and actin expression was assessed by immunoblot (representative blot of n = 4 independent experiments; n = 4 mice total/group for all four experiments C f m m combined). ( ) Primary pM were exposed to STO609 (10 M) and LPS (100 ng/ml) for 4 h. Total cellular lysate (10 g/lane) was isolated, and mTOR and http://www.jimmunol.org/ tubulin expression was assessed by immunoblot (representative blot of n = 4 independent experiments; n = 4 mice total for all four experiments combined). (D)pMf were transfected with empty vector, a constitutively active CaMKIV (CaMKIV-dCT), or a kinase-deﬁcient mutant (CaMKIV-dCTK75E) for 8 h. Total cellular lysate (10 mg/lane) was then isolated and analyzed for the expression of mTOR and actin by immunoblot (representative blot of n = 3 independent experiments).

Results LC3B, which was lesser in magnitude with CaMKIV-dCTK75E Mf LPS-induced autophagy is CaMKIV dependent transfection. Electron microscopy shows reduced density of double-membrane autophagosomes in LPS-stimulated CaMKIV2/2 We have previously shown that CaMKIV is activated in Mf ex- pMf, by comparison with similarly treated WT pMf (Fig. 1C). by guest on September 28, 2021 posed to LPS (26). In this study, we show that in Mf responding These data support that active CaMKIV regulates LPS-induced to LPS, autophagy is dependent upon CaMKIV signaling. pMf 2 2 2 2 autophagy in the Mf. isolated from CaMKIV / mice (CaMKIV / pMf) exhibited attenuated ATG5/12, ATG7, and LC3B in response to LPS stim- f ulation, by comparison with wild-type (WT) pMf (Fig. 1A). To CaMKIV regulates M mTOR expression investigate whether CaMKIV is sufﬁcient to induce autophagy, we The preponderance of data suggest that mTOR complex 1 (mTORC1) used a constitutively active CaMKIV plasmid (CaMKIV-dCT) and activity negatively regulates autophagy; and thus mechanisms, like a kinase defective mutant (CaMKIV-dCTK75E) (26). As shown AMPK activity, that inhibit mTORC1, induce autophagy (4, 9, 10, 2 2 in Fig. 1B, CaMKIV-dCT strongly induced ATG5/12, ATG7, and 37). However, we observed that CaMKIV / pMf have reduced

FIGURE 3. mTOR is necessary for Mf autophagy. Primary pMf were subjected to RNAi using nontarget (NT) or mTOR siRNA. After 72 h, cells were exposed to LPS (100 ng/ml) for 30 min. Total cellular lysate (10 mg/lane) was then isolated and analyzed for the expression of mTOR, ATG7, ATG5- 12, LC3B, and tubulin by immunoblot (representative blot of n = 3 independent experiments; n =3 mice total for all three experiments combined). The Journal of Immunology 5 mTOR expression, both at baseline and after LPS stimulation, by and WT pMf expressed similar levels of mTOR mRNA, sug- comparison with WT pMf (Fig. 2A). The reduced mTOR ex- gesting that CaMKIV regulation of mTOR occurs at a posttran- pression in CaMKIV2/2 pMf correlated with attenuated mTORC1 scriptional level (Fig. 4A). Prior work has shown that FBXW7 signaling, as evidenced by reduced p-Thr389p70 S6 kinase and targets mTOR for ubiquitination and proteosomal degradation (38, p-Ser235/236S6 (Fig. 2B). Similarly, exposure to the CaMKK1/2 39). As shown in Fig. 4B, treatment with the proteasome inhibitor biochemical inhibitor STO609 acutely inhibited mTOR protein MG132 increased the expression of mTOR in both WT and expression in pMf (Fig. 2C). Alternatively, elevating CaMKIV CaMKIV2/2 pMf (Fig. 4B). Similarly, MG132 restored the ex- activity through the transfection of active CaMKIV-dCT increased pression of mTOR that had been inhibited with STO609 bio- mTOR protein concentration (Fig. 2D). These data support that chemical CaMKK inhibition (Fig. 4C). The increased mTOR with CaMKIV regulates mTOR expression. MG132 was predominantly the ubiquitinated form of mTOR as shown in Fig. 4D. These data support that CaMKIV inhibits ubiquitin f mTOR is necessary for M LPS-induced autophagy dependent mTOR degradation. These data led us to hypothesize that CaMKIV-dependent regu- Flugel€ et al. (38) described increased ubiquitin-mediated pro- lation of mTOR may play an important role in the induction of tein degradation of hypoxia inducible factor-1 after phosphoryla- autophagy in Mf responding to LPS. As shown in Fig. 3, RNAi of tion by GSK-3b and recruitment of the ubiquitin ligase and tumor mTOR signiﬁcantly inhibited mTOR expression in pMf,bycom- suppressor F-box and WD protein FBXW7. GSK-3b is negatively parison with scrambled, nontargeted, control siRNA. The attenuated regulated by inhibitory phosphorylation of serine 9 (40). Thus, we expression of mTOR correlated with reduced LPS-induced ATG7, explored whether CaMKIV regulated FBXW7 and p-Ser9–GSK3b. 2 2 ATG5/12, and LC3B. Thus, in Mf, mTOR appears to be necessary AsshowninFig.5A,CaMKIV / pMf exhibited increased Downloaded from for autophagy. FBXW7 expression and reduced inhibitory p-Ser9–GSK3b (i.e., increased kinase activity due to a loss of inhibitory serine 9 phos- b CaMKIV negatively regulates FBXW7- and GSK3- – phorylation) by comparison with WT pMf, which appropriately f dependent ubiquitin-mediated degradation of mTOR in M correlated with reduced mTOR expression (Fig. 4B). Similarly, We next explored potential mechanisms by which CaMKIV reg- biochemical CaMKK inhibition with STO609 increased FBXW7 2/2 9 ulates mTOR expression. As shown in Fig. 4A, both CaMKIV and decreased p-Ser –GSK3b (Fig. 5B), which again mirrored the http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. CaMKIV regulates ubiquitin-mediated degradation of mTOR. (A) Primary pMf from C57BL/6 or CaMKIV2/2 mice were isolated, and total mTOR mRNA assayed by real time PCR (n = 4 independent experiments). (B) Primary pMf from C57BL/6 (n = 1) or CaMKIV2/2 (n = 2) mice were isolated and exposed to MG132 (10 mM) for 4 h. Total cellular lysate (10 mg/lane) was then isolated and analyzed for the expression of mTOR and actin by immunoblot (representative blot of n = 3 independent experiments; n = 3 WT and n = 6 CaMKIV2/2 mice total were used for all three experiments combined): lanes 1 and 2 (WT 1), lanes 3 and 4 (CaMKIV2/2 1), and lanes 5 and 6 (CaMKIV2/2 2). (C) RAW 264.7 cells were exposed to STO609 (10 mM) and MG132 (10 mM) for 4 h. Total cellular lysate (10 mg/lane) was isolated, and mTOR and actin expression was assessed by immunoblot (representative blot of n = 3 independent experiments). (D) RAW 264.7 Mf were exposed to STO609 (10 mM) and MG132 (10 mM) for 4 h, at which point total cell lysate was harvested, immunoprecipitated for ubiquitin, subjected to immunoblot analysis, and probed for mTOR (representative blot of n =3 independent experiments). KO, knockout. 6 CaMKIV REGULATES AUTOPHAGY DURING ENDOTOXEMIA Downloaded from

2/2 FIGURE 5. CaMKIV negatively regulates FBXW7 and GSK3b.(A) Primary pMf from C57BL/6 (WT) or CaMKIV mice. FBXW7 (Cy3, red), http://www.jimmunol.org/ p-Ser9–GSK3b (Cy3, red), and Hoechst (blue) staining was determined by immunofluorescence (n = 3 independent experiments; n = 3 mice total/group for three experiments combined). Original magnification 360. Scale bars, 1 mm. Corresponding densitometry blots compare FBXW7 and p-Ser9–GSK3b from WT and CaMKIV2/2 Mf.(B) RAW 264.7 cells were exposed to STO609 (10 mM) for the durations indicated. Total cellular lysate (10 mg/lane) was isolated, and mTOR, FBXW7, p-Ser9–GSK3b, GSK3b, and tubulin expression was assessed by immunoblot (representative blot of n = 4 independent experiments). (C) GSK3ab fusion protein (27 kD) was incubated in the presence or absence of active p-CaMKIV (25 ng) for 10 min at 30˚C with the following additions: 10 mM MgCl2, 0.2 mM ATP, 1 mM CaCl2, and 1 mM CaM. Reactions were terminated by boiling in SDS–2-ME dissociation solution, subjected to immunoblot, and probed with anti–p-Ser9–GSK3b or anti–p-CaMKIV. Total GSK3ab fusion protein was evaluated by Coomassie blue R250 staining (representative blot of n = 3 independent experiments). (D) RAW 264.7 Mf were exposed to STO609 (10 mM) for 2 or 4 h, at which point total cell lysate was harvested, immunoprecipitated (IP) for mTOR, subjected to immunoblot (IB) analysis, and probed for mTOR or FBXW7 (representative blot of n = 3 independent experiments). by guest on September 28, 2021 reduced mTOR expression (Fig. 4C). In vitro kinase assay dem- CaMKIV regulates mTOR and autophagy in the kidney onstrated that active p-CaMKIV directly phosphorylates GSK3b 9 We next explored whether these mechanisms were operant in at its inhibitory Ser residue (Fig. 5C). And finally, an acute re- other organs responding to the stress of sepsis and the functional duction in CaMK activity with STO609 increased the recruitment ramifications. Similar to pMf, kidneys isolated from CaMKIV2/2 of FBXW7 to mTOR as shown in Fig. 5D. Collectively, our data mice exhibited reduced expression of mTOR, which correlated suggest that CaMKIV preserves mTOR protein expression by with reduced p-Ser9–GSK3b (Fig. 7A). However, both WT and negatively regulating GSK3b and FBXW7, which prevents ubiq- 2 2 CaMKIV / kidneys exhibited similar and low expression of uitination and subsequent degradation of mTOR. FBXW7. The loss of renal mTOR was associated with reduced CaMKIV and mTOR regulate Mf IL-6 production renal autophagy during endotoxemia (Figs. 7B). Representative immunoblot shows significantly less LC3B expression in the renal Recently, it has been suggested that colocalization of autophagy 2/2 and mTOR augments the secretory phenotype of certain cell types, cortices of CaMKIV animals than of WT animals 18 h after , 2/2 such as the Mf (41). Thus, we hypothesized that loss of CaMKIV, LPS (p 0.001) (Fig. 7B). CaMKIV kidneys, by contrast to and subsequently mTOR, reduces Mf IL-6 production. In vitro, WT kidneys, exhibited less immunofluorescence and a less pro- RNAi of mTOR reduced LPS-induced IL-6 production: 1594 nounced punctate staining pattern for LC3 (Fig. 7B). Electron versus 1065 pg/ml (p = 0.03) (Fig. 6A). There were no significant microscopy illustrates numerous multimembranous autopha- changes in TNF-a or IL-10 production (data not shown). Similar gosomes and autophagolysosomes in the renal cortex of WT reductions in IL-6 were obtained when mTOR activity was animals after endotoxemia. These structures are notably reduced 2/2 inhibited with rapamycin (1681 versus 372 pg/ml; p , 0.001) or in the kidneys of CaMKIV animals, which exhibit swollen, CaMK activity was biochemically inhibited with KN93 or STO609 damaged mitochondria lacking prototypical architectural features (Fig. 6B). CaMKIV2/2 pMf expressedlessIL-6afterLPSthan (Fig. 7B). We confirmed that mTOR expression is necessary for WT pMf: 3336 versus 1561 pg/ml (p = 0.05) (Fig. 6C). In vivo LPS-induced autophagy using mTOR siRNA and human proximal during endotoxemia, WT mice displayed significant elevations in renal tubular (HKC-8) cells. As shown in Fig. 7C, RNAi of mTOR systemic IL-6 concentrations, which were attenuated in CaMKIV2/2 attenuated LPS-induced autophagy in HKC-8 cells by comparison mice: 88% reduction in IL-6 concentration (1095 versus 381 pg/ml; with NT siRNA. Thus, in the kidney as well, CaMKIV preserves p = 0.02) (Fig. 6D). There were no significant reductions in serum mTOR expression, which is necessary for LPS-induced autophagy. IL-10 concentration (245 versus 383 pg/ml; p = 0.17) or TNF-a Recent literature suggests that autophagic mechanisms directed production (85 versus 57 pg/ml; p = 0.32) (data not shown). at removal of damaged mitochondria, or mitophagy, provide a critical The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. CaMKIV and mTOR regulate macrophage IL-6 production. (A) RAW 264.7 cells were exposed to mTOR siRNA. After 72 h, they were exposed to LPS (100 ng/ml) for 18 h. Supernatant was harvested and assayed for IL-6 by ELISA (n = 3 independent experiments). Data are mean 6 SEM. Total cellular lysate was subjected to immunoblot of mTOR or actin (n = 3 independent experiments). (B) RAW 264.7 cells were exposed to LPS (100 ng/ ml) for 18 h either in the presence or absence of DMSO, KN93 (10 mM), STO690 (10 mM), or rapamycin (50 ng/ml). Supernatant was harvested and assayed for TNF-a, IL-6, and IL-10 by ELISA (n = 3 independent experiments). Data are mean 6 SEM. (C)pMf were harvested from WT and CaMKIV2/2 mice and exposed to LPS (100 ng/ml) for 18 h. Supernatant was harvested and assayed for IL-6 by ELISA (n = 3 independent experiments; n = 8 mice total/ group for all three experiments combined). Data are mean 6 SEM. (D) IL-6 concentrations after endotoxemia comparing WT and CaMKIV2/2 mice (n =3 independent experiments, n = 12 mice total/group for all three experiments combined). Data are mean 6 SEM. 8 CaMKIV REGULATES AUTOPHAGY DURING ENDOTOXEMIA Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. CaMKIV regulates mTOR and autophagy in the kidney. (A) Immunoblot analysis of renal cortex lysate detecting mTOR, p-Ser9–GSK3b, FBXW7, GSK3b, and tubulin from WT and CaMKIV2/2 mice (representative blot of n = 4 independent experiments in which two mice per group are shown; n = 8 mice total/group for all four experiments combined). Corresponding densitometry blots compare mTOR and p-Ser9–GSK3b from WT and CaMKIV2/2 mice. (B) The effect of endotoxemia (LPS 5 mg/kg) on autophagy in the renal cortices of WT and CaMKIV2/2 mice was assessed by immunoblot for LC3B (16 kD). Representative blot is shown at 18-h time point from n = 4 independent experiments in which three mice per group are shown (n = 12 mice total/group for all four experiments combined). Immunoﬂuorescent microscopy (original magniﬁcation 320) of renal cortex in WT and CaMKIV2/2 mice harvested at 18 h after LPS (5 mg/kg). LC3 (Cy3, red), actin (Alexa 488, green), and nucleus (Hoechst, blue). Top panel scale bars, 20 mm. Representative images of n = 3 independent experiments (n = 9 mice total/group for all three experiments combined). Transmission electron microscopy (5 3 104 and 5 3 105) of renal cortex of WT and CaMKIV2/2 mice 18 h after endotoxemia. Middle panel, Scale (Figure legend continues) The Journal of Immunology 9 cytoprotective role in protecting against acute kidney injury (AKI), which primarily affects the mitochondria-rich proximal tubule cells (42). We observed that CaMKIV2/2 mice experienced greater AKI than WT mice. As shown in Fig. 8, endotoxemic CaMKIV2/2 mice, by comparison with their WT counterparts, exhibited significant elevations in cystatin C concentration (992 versus 655 ng/ml; p = 0.002). These data create a paradigm in which CaMKIV maintains mTOR expression which is fundamental for the induction of autophagy and protection against AKI during endotoxemia (Fig. 9).

Discussion Autophagy is an evolutionarily conserved lysosomal mechanism that enables cells to conserve and maintain cellular biomass quality and quantity by targeting damaged or unused proteins and even organelles for degradation (1, 2, 8, 43, 44). Though essential for tissue homeostasis, evidence is accumulating that it may be acutely activated by cells in response to multiple forms of stress. The mechanisms regulating this complex process and the ram- Downloaded from ifications of any defects are just being elucidated. In this study, we describe a novel mechanism in which CaMKIV, by inhibiting GSK3b activity and FBXW7 recruitment, prevents ubiquitin proteosomal FIGURE 8. Loss of CaMKIV-dependent autophagy worsens renal organ degradation of mTOR and thereby directly augments autophagy in dysfunction during endotoxemia. Cystatin C concentrations during endo- both the Mf and the kidney. Under the conditions of sepsis studied, toxemia comparing WT and CaMKIV2/2 mice (three independent experi- mTOR expression and activity are requisite for autophagy, a para- ments, n = 12 mice total mice/group for all three experiments combined). http://www.jimmunol.org/ digm countering the current perspective that prototypically, mTOR Data are mean 6 SEM. inhibition induces autophagy. Observed in both Mf (i.e., immune cells) and the kidney (i.e., parenchymal cells), this CaMKIV– mTOR inhibition (3, 9, 10). Indeed, AMPK-mediated inhibition mTOR-dependent autophagic mechanism is conserved across several of mTOR has been fairly well established as a mechanism that cell types and fundamental for the respective functional (e.g., Mf induces autophagy in a variety of cells and experimental models cytokine production) and adaptive responses (e.g., renal tubular cell (3, 4, 9, 10, 37). Studies have explored the use of pharmacologic cytoprotection) to septic insult. mTOR inhibitors, such as rapamycin, which potently inhibit Our data complement prior investigations that describe mech- downstream mTOR signaling and thereby induce autophagy (47– by guest on September 28, 2021 anisms by which the family of multifunctional CaMK regulate 49). However, in this study, CaMKIV invoked an mTOR-dependent autophagy. Using carcinoma cell lines and NIH3T3 fibroblasts, mechanism to directly augment autophagy. Loss of CaMKIV or Høyer-Hansen et al. (28) observed that calcium-mobilizing agents CaMK activity (i.e., STO609) reduced mTOR expression and (e.g., vitamin D, thapsigargin) inhibited the activity of mTOR, TORC1 activity, which attenuated autophagy. Restoration of active a negative regulator of autophagy, and induced the accumulation CaMKIV induced TORC1 signaling and mTOR-dependent LC3B of autophagosomes in a Beclin-1 and ATG-7 manner. This process expression. Finally, mTOR siRNA inhibited LPS-induced auto- was mediated by CaMKKb and AMPK (28). Other studies have phagy in both Mf and renal tubular cells. This dependence of identified a CaMKKa/b-AMPK pathway in which CaMKKa/b autophagy on mTOR was also observed in renal tubular cells during functions as an indirect AMPK kinase (4, 45). In Mf, LPS induces endotoxemia. In our prior study, CaMKIa regulated AMPK- endoplasmic reticulum stress and calcium mobilization that we dependent autophagy independent of mTOR inhibition (29). Thus, have shown leads to the activation of all members of the CaMK in both immune and nonimmune cells, CaMK-dependent autophagy cascade (26, 29, 33, 46). We have previously shown that LPS- appears to require mTOR, at least in the context of sepsis. induced CaMKI and -IV activity in Mf are CaMKKa/b depen- In addition to being operant in vitro, our data support the in vivo dent (26, 29, 33). Recently, we described a CaMKIa–AMPK importance of these mechanisms in modifying early cell-specific pathway rapidly activated as an early autophagic response in Mf responses to sepsis. In a murine model of endotoxemia CaMKIV– exposed to LPS (29). We now include CaMKIV that like its sibling mTOR autophagy was necessary for IL-6 production, which mirrored CaMKI also regulates autophagy. Thus, we propose a paradigm in our in vitro Mf results. We believe that TORC1 activity, and not which dual CaMKKa/b–CaMKI and CaMKKa/b–CaMKIV trans- merely the structural presence of mTOR, underlies our observa- duction pathways, responding to calcium mobilization, mediate dis- tions as functional inhibition of mTOR with rapamycin similarly tinct aspects of autophagy. Collectively, our data support a functional attenuated IL-6 production. This perspective is supported by other role for all members of the CaMK family during autophagy. independent studies (50). Recently, it has been suggested that Our prior and these new data highlight that CaMKI and -IV autophagy and inflammation are intimately linked (41). In Mf,a augment autophagy independent of mTOR inhibition. The mTOR spatial coupling of mTOR and autophagy into a TOR–autophagy typically serves as a negative regulator of autophagy, and as a con- spatial coupling compartment (TASCC) augments the secretory sequence, initiation of autophagy is largely dependent on release of phenotype and facilitates the mass synthesis of secretory proteins, bars, 2 mm. Bottom panel, Scale bars, 500 nm. Representative images of n = 3 independent experiments (n = 9 mice total/group for all three experiments combined). (C) Human proximal renal tubular epithelial (HKC-8) cells were subjected to RNAi using nontarget (NT) or mTOR siRNA. After 72 h, cells were exposed to LPS (10 ng/ml) for 30 min. Total cellular lysate (10 mg/lane) was then isolated and analyzed for the expression of mTOR, LC3B, and tubulin by immunoblot (representative blot of three independent experiments). 10 CaMKIV REGULATES AUTOPHAGY DURING ENDOTOXEMIA Downloaded from http://www.jimmunol.org/ FIGURE 9. CaMKIV is essential for autophagy during endotoxemia. CaMKIV inhibition of GSK-3b activity and FBXW7 recruitment prevents ubiquitin- mediated proteosomal degradation of mTOR and thereby directly augments autophagy in both the Mf and the kidney. These mechanisms are cytoprotective in the kidney and important for immune cell function. such as cytokines (41). Disrupting the TASCC suppresses the organ physiology and the host response, in attempts to identify synthesis of IL-6 and IL-8 (41). We view this paradigm as explana- the clinical utility of CaMK and autophagic modulation during tory for the mTOR dependency of CaMKIV regulated autophagy. inflammatory states. Though awaiting further investigation, we hypothesize that CaMKIV, by guest on September 28, 2021 by regulating mTOR and thus autophagy, orchestrates construction Disclosures of the TASCC, which is essential for optimal immune cell function. The authors have no financial conflicts of interest. These mechanisms we initially described in Mf were shown to be equally relevant to renal tubular cells. AKI is arguably the most prevalent form of organ dysfunction during severe sepsis. Recent References literature suggests a critical cytoprotective role for autophagy in 1. Klionsky, D. J. 2005. Autophagy. Curr. Biol. 15: R282–R283. both toxin-mediated and ischemia-reperfusion–induced AKI (42). 2. Klionsky, D. J., and S. D. Emr. 2000. Autophagy as a regulated pathway of cellular degradation. Science 290: 1717–1721. Autophagic mechanisms directed at removal of damaged mito- 3. Kim, J., M. Kundu, B. Viollet, and K. L. Guan. 2011. 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