CaMKIα regulates AMP kinase-dependent, TORC-1-independent autophagy during lipopolysaccharide-induced acute lung neutrophilic inflammation - PubMed (original) (raw)

CaMKIα regulates AMP kinase-dependent, TORC-1-independent autophagy during lipopolysaccharide-induced acute lung neutrophilic inflammation

Lanping Guo et al. J Immunol. 2013.

Abstract

Autophagy is an evolutionarily conserved cytoplasmic process regulated by the energy rheostats mammalian target of rapamycin and AMP kinase (AMPK) that recycles damaged or unused proteins and organelles. It has been described as an important effector arm of immune cells. We have shown that the cytoplasmically oriented calcium/calmodulin-dependent protein kinase (CaMK)Iα regulates the inflammatory phenotype of the macrophage (M). In this study, we hypothesize that CaMKIα mediates M autophagy. LPS induced autophagy in RAW 264.7 cells and murine peritoneal M that was attenuated with biochemical CaMK inhibition or CaMKIα small interfering RNA (siRNA). Inhibition of CaMKIα reduced LPS-induced p-Thr(172)AMPK and target of rapamycin complex-1 activity, and expression of a constitutively active CaMKIα but not a kinase-deficient mutant induced p-Thr(172)AMPK and autophagy that was attenuated by the AMPK inhibitor compound C. Coimmunoprecipitation and in vitro kinase assays demonstrated that CaMKIα activates AMPK, thereby inducing ATG7, which also localizes to this CaMKIα/AMPK complex. During LPS-induced lung inflammation, C57BL/6 mice receiving CaMKIα(siRNA) displayed reduced lung and bronchoalveolar immune cell autophagy that correlated with reduced neutrophil recruitment, myeloperoxidase activity, and air space cytokine concentration. Independently inhibiting autophagy, using siRNA targeting the PI3K VPS34, yielded similar reductions in lung autophagy and neutrophil recruitment. Thus, a novel CaMKIα/AMPK pathway is rapidly activated in M exposed to LPS and regulates an early autophagic response, independent of target of rapamycin complex-1 inhibition. These mechanisms appear to be operant in vivo in orchestrating LPS-induced lung neutrophil recruitment and inflammation.

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Figures

Figure 1

Figure 1. CaMK cascade mediates LPS-induced autophagy in macrophages

RAW 264.7 cells were incubated with the broad CaMK inhibitor KN62 (20uM) for 30 minutes and then exposed to LPS (100ng/ml) for 30 minutes. Total cellular lysate (20μg per lane) was isolated and the induction of ATG7, LC3B, and ATG5–12 assessed by immunoblot (n=4 independent experiments).

Figure 2

Figure 2. CaMKI regulates LPS-induced autophagy in macrophages

A, Primary peritoneal macrophages were incubated with either media or the highly selective CaMKK inhibitor STO609 (10μM) for 60 minutes and then exposed to LPS (100ng/ml) for the durations indicated. Total cellular lysate (20μg per lane) was isolated and the induction of active p-Thr177/200-CaMKI assessed by immunoblot (n=3 independent experiments). B, RAW 264.7 cells and primary peritoneal macrophages were subjected to RNAi using Non-target (NT) or CaMKIα siRNA. After 72 h, cells were exposed to LPS (100ng/mL), total cellular lysate (20ug per lane) was isolated and the induction of ATG7, LC3B, and ATG5–12 assessed by immunoblot (n=4 independent experiments).

Figure 3

Figure 3. CaMKIα is sufficient to induce autophagy in macrophages

RAW 264.7 cells were transfected with either a constitutively active CaMKIα(CaMKI293) or a kinase-deficient mutant (CaMKI293K49A) for the durations indicated. Total cellular lysate (20μg per lane) was isolated and analyzed for the induction of ATG7, LC3B, ATG5–12, CaMKIα, and actin by immunblot (representative blot of 3 independent experiments). Lipo=lipofectamine.

Figure 4

Figure 4. CaMKIα is an AMPK kinase

RAW 264.7 cells were subjected to RNAi using Non-target (NT) or CaMKIα siRNA. After 72 h, cells were exposed to LPS (100ng/mL) for 30 minutes. Total cellular lysate (20μg per lane) was isolated and analyzed for p-AMPK, AMPK, p-mTOR, mTOR, p-p70 S6 kinase, p-S6, and CaMKIα by immunblot (representative blot of 3 independent experiments).

Figure 5

Figure 5. CaMKIα induces autophagy through an AMPK-dependent mechanism

RAW 264.7 cells were incubated with the AMPK inhibitor Compound C for 30 minutes and then transfected with either a constitutively active CaMKIα (CaMKI293) or a kinase-deficient mutant (CaMKI293K49A) for 8 hours. Total cellular lysate (20μg per lane) was then isolated and analyzed for the expression of ATG7, LC3B, ATG5–12, p-AMPK, AMPK, p-mTOR, mTOR, p-p70 S6 kinase, p-S6, CaMKIα**,** and actin by immunblot (representative blot of 3 independent experiments).

Figure 6

Figure 6. LPS induces a CaMKIα-AMPK-ATG7 complex

A, RAW 264.7 macrophages were exposed to LPS (100ng/ml) for the durations indicated at which point total cell lysate was harvested, immunoprecipitated for p-AMPK, subjected to immunoblot analysis (20μg per lane), and probed for CaMKIα, AMPK, or ATG7 (n=3 independent experiments). B, AMPK was isolated by immunoprecipitation from RAW 264.7 cells and then incubated in the presence or absence of active p-CaMKI (25ng) 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 immunoblot, and probed with anti-p-Thr172 AMPK or AMPK antibody (representative blot of 3 independent experiments).

Figure 7

Figure 7. ATG7 siRNA inhibits LPS-induced LC3B

RAW 264.7 macrophages were subject to RNAi of ATG7 or LC3. After 72 hours cells were exposed to LPS (100ng/ml) for 30 minutes, at which point total cell lysate was harvested and subjected to immunoblot analysis (20μg per lane) and probed for ATG7, LC3B, and actin (n=3 independent experiments).

Figure 8

Figure 8. CaMKIα siRNA inhibits pulmonary CaMKIα expression and autophagy in vivo

C57Bl/6 mice were administered either CaMKIα (CaMKIαsiRNA) or Non-target (NTsiRNA) siRNA (6mg/kg) by hydrodynamic tail vein injection. After 72 hours, CaMKIαsiRNA and NTsiRNA mice were subjected to intratracheal (IT) administration of LPS (1.5mg/kg). After 12 hours, mice were euthanized, the lungs were harvested, and pulmonary CaMKIα, ATG7, LC3B and actin expression assessed by immunoblot (20μg per lane) (representative blot of 3 independent experiments where 5 mice per experimental condition is shown; N=12 mice total per experimental condition for all 3 experiments combined). Densitometry of CaMKIα was performed, adjusted for densitometry of actin control, and then tested by Mann-Whitney.

Figure 9

Figure 9. CaMKIα regulates autophagy in bronchoalveolar immune cells

CaMKIαsiRNA and NTsiRNA mice were subjected to intratracheal (IT) administration of LPS (1.5mg/kg). After 6 and12 hours, mice were euthanized, bronchoalveolar lavage (BAL) was performed, and BAL immune cells were plated by cytospin. The expression of ATG7 (green, Alexa 488), LC3B (red, Cy3), and Hoechst (blue) staining was determined by immunofluorescence (n=3 independent experiments). 40X magnification.

Figure 10

Figure 10. CaMKIα regulates bronchoalveolar cytokine and chemokine concentrations during LPS-induced lung inflammation

CaMKIαsiRNA and NTsiRNA mice were subjected to intratracheal (IT) administration of LPS (1.5mg/kg). After 6 hours, mice were euthanized, the lungs were lavaged (BAL), and BAL fluid was analyzed for cytokine and chemokines by ELISA. Data represent mean ± SEM for each group. Statistical significance was determined by Mann-Whitney non-parametric analysis (3 independent experiments with 4 mice per experimental condition; N=12 mice total per experimental condition for all 3 experiments combined).

Figure 11

Figure 11. CaMKIα regulates neutrophil recruitment in LPS-induced lung inflammation

CaMKIαsiRNA and NTsiRNA mice were subjected to intratracheal (IT) administration of LPS (1.5mg/kg). After 6 and12 hours, mice were euthanized, the lungs were lavaged (BAL), and BAL fluid was analyzed for neutrophil count, myeloperoxidase (MPO) activity, and protein concentration (3 independent experiments with 4 mice per experimental condition; N=12 mice total per experimental condition for all 3 experiments combined).

Figure 12

Figure 12. Inhibition of VPS34 attenuates autophagy and neutrophil recruitment in LPS-induced lung inflammation

A, VPS34siRNA and NTsiRNA mice were subjected to intratracheal (IT) administration of LPS (1.5mg/kg). After 12 hours, mice were euthanized, the lungs were harvested, total cell lysate was isolated, subjected to immunoblot (20μg per lane), and probed for ATG7, LC3B, VPS34, and actin (representative blot of 2 independent experiments where 6 mice per experimental condition is shown; N=11 mice total per experimental condition for all 2 experiments combined). B, VPS34siRNA and NTsiRNA mice were subjected to intratracheal (IT) administration of LPS (1.5mg/kg). After 12 hours mice were euthanized, the lungs were lavaged (BAL), and BAL fluid was analyzed for neutrophil count, myeloperoxidase (MPO) activity, and protein concentration. Data represent mean ± SEM for each group. Statistical significance was determined by Mann-Whitney non-parametric analysis (2 independent experiments with 4 mice per experimental conditions; N=8 mice total per experimental condition for all 2 experiments combined).

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