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Ca2+/calmodulin-dependent protein kinase kinase β phosphorylation of Sirtuin 1 in endothelium is atheroprotective

  1. John Y.-J. Shyyb,f,1
  1. aDepartment of Physiology and Pathophysiology, Peking University Health Sciences Center, Beijing 100191, China;
  2. bDivision of Biomedical Sciences and
  3. cDepartment of Chemistry, University of California, Riverside, CA 92521;
  4. dDepartment of Kinesiology and Health Sciences, California State University, San Bernardino, CA 92407; and
  5. Departments of eBioengineering and
  6. fMedicine, University of California, San Diego, La Jolla, CA 92093

  1. Contributed by Shu Chien, May 16, 2013 (sent for review March 3, 2013)

  1. Fig. 1.
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    Fig. 1.

    CaMKKβ is required for SIRT1 induction in ECs under PS. (A–C) Immunoblots of HUVECs subjected to PS for the indicated time periods (A), pretreated with STO-609 (2.5 μg/mL) or DMSO for 30 min and then subjected to PS for 8 h (B), and transfected with control siRNA or CaMKKβ siRNA and then exposed to PS for 8 h or kept under static conditions for the same time (C). (D) Immunoblots of CaMKKβ+/+ and CaMKKβ−/− MEFs exposed to static condition or PS for 8 h. The antibodies against targeted proteins are indicated for each immunoblot. Bar graphs below immunoblots summarize the means ± SEM of three independent experiments. *P < 0.05.

  2. Fig. 2.
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    Fig. 2.

    PS increases SIRT1 stability via CaMKKβ phosphorylation at Ser-27 and Ser-47. Immunoblots of HUVECs pretreated with CHX (0.1 mg/mL) for 30 min before PS or kept under static conditions for the indicated time (A and B), transfected with or without control siRNA or CaMKKβ siRNA and then PS for 8 h (B and C), and transfected with control siRNA or CaMKKβ siRNA and then PS or static conditions for 8 h (D). (E) Immunoblots of lysed SIRT1−/− MEFs transfected with plasmid encoding SIRT1-S27AS47A or SIRT1-S27DS47D and then treated with CHX for the indicated times. The antibodies against targeted proteins are indicated. The plots below the immunoblots summarize the mean ± SEM results from three independent experiments. *P < 0.05.

  3. Fig. 3.
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    Fig. 3.

    CaMKKβ phosphorylates SIRT1 at Ser-27 and Ser-47. (A) Alignment of peptide sequences flanking SIRT1 Ser-27, SIRT1 Ser-47, AMPK Thr-172, CaMKI Thr-177, CaMKIV Thr-196, PKB Thr-308, and BRSK1 Thr-189. (B and C) Immunoblots of reaction products of mixtures of recombinant GST-CaMKKβ (50 ng) incubated with recombinant human SIRT1 (200 ng) (B) or AMPK (200 ng) (C) with 2 mM Ca2+ and recombinant calmodulin for 12 h (B) or for 30 min (C) at 30 °C. The phosphorylation of SIRT1 and AMPK was determined with the indicated antibodies. (D and E) MS/MS of phosphorylated SIRT1 tryptic peptides corresponding to residues 23–34 (EAASSPAGEPLR) (D) and 47–58 (SPGEPGGAAPER) (E) obtained from the kinase reaction mixtures described in B and analyzed by LC-MS/MS. The asterisk indicates that an ion bears a phosphate group, and neutral loss of an H3PO4 is represented by Δ.

  4. Fig. 4.
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    Fig. 4.

    Phosphorylation of SIRT1 Ser-27 and Ser-47 increases SIRT1 activity and expression of SIRT1 target genes. (A and B) Levels of SOD1, SOD2, catalase, NRF1, Nrf2, KLF2, HO-1, and Trx1 mRNA (relative to GAPDH) in HUVECs transfected with control siRNA or CaMKKβ siRNA (A) and in SIRT1−/− MEFs transfected with pcDNA, SIRT1-S27AS47A, or SIRT1-S27DS47D (B) and then exposed to PS or static conditions for 8 h. (C and D) SIRT1−/− MEFs were transfected with expression plasmids as indicated. Whole-cell lysates were collected for SIRT1 activity assays (C) and NO bioavailability expressed as NOx (D). *P < 0.05.

  5. Fig. 5.
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    Fig. 5.

    CaMKKβ and SIRT1 are involved in the regulation of antioxidative and anti-inflammatory genes in mouse aorta. (A) Immunoblots of tissue lysates from the aortic arch (AA) and the thoracic aorta (TA) isolated from CaMKKβ+/+ mice and their CaMKKβ−/− littermates performed with indicated antibodies. Bar graphs to the right summarize the means ± SEM from six mice in each group. (B) Levels of SOD1, SOD2, catalase, NRF1, Nrf2, KLF2, PGC1α, eNOS, ICAM-1, VCAM-1, E-selectin, and MCP-1 mRNA (relative to GADPH) in TA from CaMKKβ−/− and their CaMKKβ+/+ littermates. The results summarize the means ± SEM from 15 mice in each group. *P < 0.05.

  6. Fig. 6.
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    Fig. 6.

    CaMKKβ and SIRT1 ablation enhances atherogenesis in mouse aorta. Macrophotographs of oil red O-stained aorta from CaMKKβ+/+ApoE−/− and CaMKKβ−/−ApoE−/− mice (A) and EC-SIRT1+/+/ApoE−/− and EC-SIRT1−/−/ApoE−/− mice (C) fed a Paigen diet for 9 wk and killed. (Scale bar: 0.5 cm.) (B and D) Quantification of percentage of lesion areas in the whole aorta (Left) and aortic arch (AA) and thoracic aorta (TA) (Right). *P < 0.05; n denotes the number of animals used.

  7. Fig. 7.
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    Fig. 7.

    Graphic summary for the antioxidative and anti-inflammatory effects of CaMKKβ phosphorylation of SIRT1 in ECs responding to atheroprotective flow. Together, AMPK and SIRT1 activate PGC1α in the nucleus, leading to up-regulation of antioxidant enzymes such as SOD and catalase. AMPK and SIRT1 also act in concert in the cytoplasm to activate eNOS and augment eNOS-derived NO to exert an anti-inflammatory effects by repressing MCP-1, VCAM-1, ICAM-1, and E-selectin. Collectively, the coregulation of AMPK and SIRT1 by CaMKKβ contributes to endothelial homeostasis and an atheroprotective phenotype of ECs.

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