CaMKIV-dependent preservation of mTOR expression is required for autophagy during LPS-induced inflammation and acute kidney injury¹

Reagents and antibodies

Ultra Pure LPS (Escherichia coli 0111:B4) was obtained from List Biologicals (Campbell, CA). STO609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid - acetic acid) was obtained from Calbiochem (San Diego, CA) and used at concentrations of 10-20μM. STO609 is highly specific for CaMKK: it has an in vitro IC₅₀ of 0.13-0.38 μM for CaMKK and 32 μM for CaMK II with little or no inhibition of CaMKI, CaMKIV, PKA, PKC, ERK, or myosin light chain kinase.(30) MG132 was purchased from Calbiochem (San Diego, CA) and used at a concentration of 10μM. Antibodies for mTOR, p-Ser⁹-GSK3β, GSK3β, p-Thr³⁸⁹p70 S6 kinase, p-Ser^235/236S6 were obtained from Cell Signaling Technology (Danvers, MA). Antibodies for ATG7, ATG5-12, LC3B, ubiquitin, FBXW7, actin and tubulin were obtained from Abcam (Cambridge, MA).

Animal experimentation

We performed all animal experiments in accordance with the National Institutes of Health guidelines under protocols approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. We purchased 6-8 week old C57BL/6J and B6.129X1-Camk4tm1Tch/J (CaMKIV^−/−) male mice from the Jackson Laboratory (Bar Harbor, ME).(26, 31, 32) The CaMKIV^−/− strain is derived from a C57BL/6J background; these mice are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. We randomly assigned groups of mice of each strain to a specific experiment.

Mice were anesthetized using a mixture of isoflurane and oxygen bled in at 3.5L/min. LPS (List Biologicals, Inc., Campbell, CA) dissolved in DNase/RNase free distilled water (1mg/ml) was instilled intraperitoneally (IP) at a concentration of 1.5 or 5 mg/kg. Animals were sacrificed 18 or 48 hours after LPS. Blood was isolated by intracardiac puncture, and the kidneys were harvested. Whole blood was stored overnight at 4°C, and the serum isolated by centrifugation at 5,000g for 10min.

For all studies, one investigator performed the surgical experimentation and collected the samples. The data were then analyzed by an investigator blinded to the specific treatment.

Cell isolation and treatment

The murine macrophage cell line RAW 264.7 (American Type Culture Collection, Rockville, MD) was grown in DMEM (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (Sigma, San Diego, CA), 50U/mL penicillin, and 50μg/mL streptomycin (Cellgro Mediatech Inc., Kansas City, MO). Peritoneal Mφ were isolated from C57BL/6 or CaMKIV^−/− mice by lavaging the peritoneal cavity with five 5mL (25mL total) aliquots of 4°C PBS.(26, 33) The lavage was centrifuged at 300g for 5 minutes, and the cells were resuspended in RPMI supplemented with 10% fetal calf serum, 50U/mL penicillin, and 50μg/mL streptomycin and plated. Selected cell populations were treated with STO609 (10μM) for 1-4 hours or MG132 (10μM) for 6 hours, as described in the figure legends. DMSO, at concentrations similar to comparative experiments, was used as an appropriate control and never exceeded 0.2%. Selected cells were then treated with LPS (100ng/mL) for the durations described in the figure legends.

siRNA

RAW 264.7 Mφ (2 X 10⁴), murine peritoneal Mφ (1 X 10⁵), or HKC-8 (2 X 10⁴) were plated in 0.5 ml of growth medium (without antibiotics) in each well of a 24-well plate, resulting in 30%, 80% or 30% confluence, respectively. Non-target and mTOR siRNA were obtained from Dharmacon (Lafayette, Colorado). We utilized the Smartpool siRNA from Dharmacon that incorporates 4 separate siRNA sequences for each mTOR: The following mTOR sequences were used:

3 μl Hiperfect were added in 100 μl Opti-DMEM with 25nM siRNA and incubated at RT for 10 min. This transfection mixture was applied to each well and incubated for 72 h. Inhibition of each targeted protein was determined by immunoblot. All experiments and cell number determinations were performed in triplicate.

Plasmid construction and transfection

Plasmids encoding a constitutively active CaMKIV (CaMKIV-dCT) or a kinase-inactive CaMKIV-dCTK75E mutant were the generous gifts of Dr. Douglas Black (Yale University, New Haven, CT).(26) CaMKIV-dCT contains a C-terminally truncated version of the CaMKIV encoding gene, truncated to 317 and a N-terminal FLAG epitope. CaMKIV-dCTK75E was constructed by changing lysine 75 to glutamate in CaMKIV, which negatively affects ATP binding at the catalytic site. For transient transfection, RAW 264.7 cells or peritoneal Mφ were seeded in a 24-well plate at 3 × 10⁵ or 1× 10⁵ cells/well. After 2 h of adhesion, Mφ were transfected with 1 μg of plasmid CaMKIV-dCT or CaMKIV-dCTK75E using the Lipofectamine 2000 reagent according to the instructions specified by the manufacturer (Life Technologies, Carlsbad, CA). Following transfection, cells were handled as detailed in the figure legends.

Cellular protein extraction

Total cellular protein was extracted at 4° C in 500 μl of lysis buffer.(29, 33) Protein concentration was determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL).

Immunoprecipitation

Five μl of antibody was added to 500 μg of isolated cellular protein within lysis buffer and incubated at 4° C overnight.(29) Thirty μl of 50% slurry of pre-washed protein G-agarose beads (Abcam, San Diego, CA) were then added to each sample, followed by incubation for an additional 2 h at 4° C. The samples were spun at 14,000 rpm and washed 4 times in lysis buffer. Samples were then resuspended in 30 μl of lysis buffer for future analysis.

Immunoblot

Total cellular lysate was electrophoresed in a 6%, 10%, or 15% SDS-PAGE gel (based on protein of interest) and transferred to a Hybond-enhanced chemiluminescence nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was blocked for 1 h at room temperature with 5% milk and incubated with primary antibody for overnight at 4°C. Blots were then incubated in a horseradish peroxidase-conjugated secondary antibody against the primary antibody at room temperature for 1 h. The blot was developed using the SuperSignal chemiluminescent substrate (Themo, Rockford, IL) and was exposed on KAR-5 film (Eastman Kodak, Rochester, NY). All gels were reblotted for total protein to confirm equal loading or to assess knockdown after RNAi. Densitometry was performed by the NIH Image J64 program (National Institutes of Health, Bethesda, MD).

RNA preparation and RT-PCR

Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA), according to the instructions specified by the manufacturer. The mRNA level was determined by quantitative RT-PCR. Briefly, 1 μg total RNA was used for the first-strand cDNA synthesis, which was carried out by using a RT system kit, according to the instructions of the manufacturer (Promega). cDNA (1 μL) was used as a template in PCR reactions, and the PCR reaction mixture at a 25 μL volume contained 12.5 μL 2× Brilliant II SYBR® Green QPCR Master Mixes (Agilent Technologies, Santa Clara, CA, USA), 1 μL cDNA, and 1 μL mTOR sense and antisense primer sets (SuperArray Bioscience). After denaturing at 95°C for 15 min, the amplification protocol consisted of 40 cycles of 95°C for 30 s, followed by 60°C for 60 s. Expression levels of mRNA were normalized by the housekeeping gene GAPDH.

In vitro kinase assay

Recombinant (1 μg) GSK3α/β fusion protein (27KD) was incubated in the presence or absence of activated p-Thr196-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 μM CaM. Reactions were terminated by boiling in SDS-2-ME dissociation solution, subjected to 10% SDS-PAGE, and probed with p-Ser⁹-GSK3β and anti-CaMKIV. Total GSK3β was evaluated by commassi blue R250 staining.

Microscopy

Peritoneal Mφ cells were plated on sterilized glass slides and allowed to adhere for 2 hours at 37°C. Cells underwent 3 washes with PBS and were then fixed in 4% paraformaldehyde at 4°C. For in vivo experiments, kidneys were flushed with PBS and then perfused with 2% paraformaldehyde. After 2h fixation, samples were transferred to 30% sucrose for 24 h with a total of three sucrose changes. The samples were cryopreserved in liquid nitrogen cooled 2-methylbutane and stored at −80°C until sectioning to 5 mm thickness. Immunostaining for in vivo and in vitro was performed as follows. Samples were permeabilized in 0.1% Triton X (Sigma-Aldrich, St. Louis, MO) at room temperature (RT) and than the BSA was removed with 3 washes of PBS. The samples were then blocked with incubation with 2% bovine serum albumin (BSA) in PBS for 1h at RT. For experiments with cells, samples were then incubated with rabbit anti-FBXW7 (Abcam, Cambridge, MA) or rabbit p-Ser⁹-GSK3β (Cell Signaling, Santa Cruz, CA) for 60 minutes at RT. In vivo samples stained with anti-LC3-I/II antibody (Novus, 5ug/ml) in PBB plus 0.1% Triton X-100 at 4°C. After washing 3 times with PBB, cells were incubated with a Cy3 secondary antibody (goat anti-rabbit, 1:1000, Jackson ImmunoResearch Laboratories) plus AlexaFluor-488 phalloidin (1:250, Invitrogen). The slides were rinsed, and HOECHST stain (1 mg/100 ml bisbenzimide) was applied for 30 seconds. Cover slips or cover glass were adhered with gelvatol, a water-soluble mounting media, and slides were placed at 4°C overnight before imaging. Samples were viewed at 20x with a 2 fold digital zoom with a Numeric Aperture (NA) of 0.8 on an Olympus Fluoview 1000 (Olympus America Inc., Center Valley, PA). Imaging conditions were maintained at identical settings within each antibody-labeling experiment with original gating performed using the negative control. Quantification was performed using Metamorph (Molecular Devices, Sunnyvale, CA).(34) For transmission electron microscopy (TEM), kidneys were flushed with PBS and subsequently perfused and fixed with 2% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4), followed by 1% OsO₄. The tissue was sectioned into 2 cm cubes and then postfixed with 1% OsO₄ and 1% K₃Fe(CN)₆ for 1 h. Samples were washed 3× with PBS and then dehydrated in a graded series of 30% to 100% ethanol before incubation in 3× 1 h incubations of Polybed 812 epoxy resin. Samples were cured at 37°C for 24 h followed by 48 h at 65°C. Ultrathin sections of 60 nm were then cut by a microtome (Leica Ultracut R), collected on 200 mesh grids that were then post-stained 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 microscope (JEOL, Peabody, MA). Images were acquired digitally from a randomly selected pool of 10 to 15 fields for each experimental condition.(34) Sample processing and imaging occurred at the Center for Biological Imaging at the University of Pittsburgh.

Renal function parameters

Renal function was determined by assaying serum for cystatin C, using an enzyme immunoassay kit (R&D, Minneapolis, MN). Cystatin C has emerged as a more precise marker of glomerular filtration rate and has been validated in both human and murine studies.(35, 36)

Cytokine production

IL-10, TNFα, IL-6, concentrations were quantified by an enzyme immunoassay kit (R&D Systems, Minneapolis, MN) that is based on a coated-well, sandwich enzyme immunoassay.

Statistical analysis

Statistical analyses were performed using Stata 12SE software (College Station, TX). Values are expressed as means ± SEM. Groups are compared by ANOVA. A p<0.05 was considered statistically significant.

Macrophage LPS-induced autophagy is CaMKIV dependent

We have previously shown that CaMKIV is activated in Mφ exposed to LPS.(26) Here we show that in Mφ responding to LPS, autophagy is dependent upon CaMKIV signaling. Peritoneal macrophages isolated from CaMKIV^−/− mice (CaMKIV^−/− pMφ) exhibited attenuated ATG5/12, ATG7 and LC3B in response to LPS stimulation, by comparison to WT pMφ (Figure 1A). To investigate whether CaMKIV is sufficient to induce autophagy, we utilized a constitutively active CaMKIV plasmid (CaMKIV-dCT) and a kinase defective mutant (CaMKIV-dCTK75E).(26) As shown in Figure 1B, CaMKIV-dCT strongly induced ATG5/12, ATG7 and LC3B, which was lesser in magnitude with CaMKIV-dCTK75E transfection. Electron microscopy shows reduced density of double-membrane autophagosomes in LPS-stimulated CaMKIV^−/− pMφ, by comparison to similarly treated WT pMφ (Figure 1C). These data support that active CaMKIV regulates LPS-induced autophagy in the Mφ.

CaMKIV regulates macrophage mTOR expression

The preponderance of data suggest that mTORC1 activity negatively regulates autophagy; and thus mechanisms, like AMP kinase activity, that inhibit mTORC1, induce autophagy.(4, 9, 10, 37) However, we observed that CaMKIV^−/− pMφ have reduced mTOR expression, both at baseline and after LPS stimulation, by comparison to WT pMφ (Figure 2A). The reduced mTOR expression in CaMKIV^−/− pMφ correlated with attenuated mTORC1 signaling, as evidenced by reduced p-Thr³⁸⁹p70 S6 kinase and p-Ser^235/236S6 (Figure 2B). Similarly, exposure to the CaMKK1/2 biochemical inhibitor STO609 acutely inhibited mTOR protein expression in pMφ (Figure 2C). Alternatively, elevating CaMKIV activity through the transfection of active CaMKIV-dCT increased mTOR protein concentration (Figure 2D). These data support that CaMKIV regulates mTOR expression.

mTOR is necessary for macrophage LPS-induced autophagy

These data led us to hypothesize that CaMKIV-dependent regulation of mTOR may play an important role in the induction of autophagy in Mφ responding to LPS. As shown in Figure 3, RNAi of mTOR significantly inhibited mTOR expression in pMφ, by comparison to scrambled, non-targeted, control siRNA. The attenuated expression of mTOR correlated with reduced LPS-induced ATG7, ATG5/12 and LC3B. Thus, in Mφ, mTOR appears to be necessary for autophagy.

CaMKIV negatively regulates FBXW7 and GSK3-β dependent ubiquitin-mediated degradation of mTOR in macrophages

We next explored potential mechanisms by which CaMKIV regulates mTOR expression. As shown in Figure 4A, both CaMKIV^−/− and WT pMφ expressed similar levels of mTOR mRNA, suggesting that CaMKIV regulation of mTOR occurs at a post-transcriptional level (Figure 4A). Prior work has shown that FBXW7 targets mTOR for ubiquitination and proteosomal degradation.(38, 39) As shown in Figure 4B, treatment with the proteasome inhibitor MG132 increased the expression of mTOR in both WT and CaMKIV^−/− pMφ (Figure 4B). Similarly, MG132 restored the expression of mTOR that had been inhibited with STO609 biochemical CaMKK inhibition (Figure 4C). The increased mTOR with MG132 was predominantly the ubiquitinated form of mTOR as shown in Figure 4D. These data support that CaMKIV inhibits ubiquitin dependent mTOR degradation.

Flugel et al described increased ubiquitin-mediated protein degradation of HIF-1 after phosphorylation by GSK-3β and recruitment of the ubiquitin ligase and tumor suppressor F-box and WD protein FBXW7.(38) GSK-3β is negatively regulated by inhibitory phosphorylation of serine 9.(40) Thus, we explored whether CaMKIV regulated FBXW7 and p-Ser⁹-GSK3β. As shown in Figure 5A, CaMKIV^−/− pMφ exhibited increased FBXW7 expression and reduced inhibitory p-Ser⁹-GSK3β (i.e. increased kinase activity due to a loss of inhibitory serine 9 phosphorylation) by comparison to WT pMφ, which appropriately correlated with reduced mTOR expression (Fig. 4B). Similarly, biochemical CaMKK inhibition with STO609 increased FBXW7 and decreased p-Ser⁹-GSK3β (Figure 5B), which again mirrored the reduced mTOR expression (Fig. 4C). In vitro kinase assay demonstrated that active p-CaMKIV directly phosphorylates GSK3β at its inhibitory Ser9 residue (Figure 5C). And finally, an acute reduction in CaMK activity with STO609 increased the recruitment of FBXW7 to mTOR as shown in Figure 5D. Collectively, our data suggest that CaMKIV preserves mTOR protein expression by negatively regulating GSK3β and FBXW7, which prevents ubiquitination and subsequent degradation of mTOR.

CaMKIV and mTOR regulate macrophage IL-6 production

Recently it has been suggested that colocalization of autophagy and mTOR augments the secretory phenotype of certain cell types, such as the Mφ.(41) Thus, we hypothesized that loss of CaMKIV, and subsequently mTOR, reduces Mφ IL-6 production. In vitro, RNAi of mTOR reduced LPS-induced IL-6 production: 1594 vs. 1065 pg/mL, p=0.03 (Figure 6A). There were no significant changes in TNFα or IL-10 production (data not shown). Similar reductions in IL-6 were obtained when mTOR activity was inhibited with rapamycin (1681 pg/mL vs. 372 pg/mL, p<0.001), or CaMK activity was biochemically inhibited with KN93 or STO609 (Fig. 6B). CaMKIV^−/− pMφ expressed less IL-6 after LPS than WT pMφ: 3336 pg/mL vs. 1561 pg/mL, p=0.05 (Figure 6C). In vivo during endotoxemia, WT mice displayed significant elevations in systemic IL-6 concentrations, which were attenuated in CaMKIV^−/− mice: 88% reduction in IL-6 concentration (1095 vs. 381 pg/mL; p=0.02) (Figure 6D). There were no significant reductions in serum IL-10 concentration (245 vs. 383 pg/mL; p=0.17) or TNF-α production (85 vs. 57 pg/mL; p=0.32) (data not shown).

CaMKIV regulates mTOR and autophagy in the kidney

We next explored whether these mechanisms were operant in other organs responding to the stress of sepsis and the functional ramifications. Similar to pMφ, kidneys isolated from CaMKIV^−/− mice exhibited reduced expression of mTOR, which correlated with reduced p-Ser⁹-GSK3β (Fig. 7A). However, both WT and CaMKIV^−/− kidneys exhibited similar and low expression of FBXW7. The loss of renal mTOR was associated with reduced renal autophagy during endotoxemia (Figs. 7B). Representative immunoblot shows significantly less LC3B expression in the renal cortices of CaMKIV^−/− animals than of WT animals 18 hours after LPS (p<0.001) (Fig. 7B). CaMKIV^−/− kidneys, by contrast to WT kidneys, exhibited less immunofluorescence and a less pronounced punctate staining pattern for LC3 (Fig. 7B). EM illustrates numerous multi-membranous autophagosomes and autophagolysosomes in the renal cortex of WT animals after endotoxemia. These structures are notably reduced in the kidneys of CaMKIV^−/− animals, which exhibit swollen, damaged mitochondria lacking prototypical architectural features (Fig. 7B). We confirmed that mTOR expression is necessary for LPS-induced autophagy using mTOR siRNA and human proximal renal tubular (HKC-8) cells. As shown in Figure 7C, RNAi of mTOR attenuated LPS-induced autophagy in HKC-8 cells by comparison to NT siRNA. Thus, in the kidney as well, CaMKIV preserves mTOR expression, which is necessary for LPS-induced autophagy.

Recent literature suggests that autophagic mechanisms directed at removal of damaged mitochondria, or mitophagy, provide a critical cytoprotective role in protecting against AKI, which primarily affects the mitochondria-rich proximal tubule cells.(42) We observed that CaMKIV^−/− mice experienced greater AKI than WT mice. As shown in Figure 8, endotoxemic CaMKIV^−/− mice, by comparison to their WT counterparts, exhibited significant elevations in Cystatin C concentration (992 vs. 655 ng/mL, p=0.002).