AMP-activated protein kinase (AMPK) α1 ablation increases atherosclerotic calcification of aortic roots in vivo. ApoE−/−, ApoE−/−/AMPKα1−/−, and ApoE−/−/AMPKα2−/− mice were fed with Western diet for 24 wk. A, Representative images of alizarin red staining of aortic roots in indicated groups. B, ApoE−/−/AMPKα1−/− but not ApoE−/−/AMPKα2−/− mice had increased alizarin red staining in atherosclerotic lesion areas of aortic roots compared with ApoE−/− mice. C, ApoE−/−/AMPKα1−/− mice had increased aortic calcium content compared with ApoE−/− mice. No difference of aortic calcium content between ApoE−/−/AMPKα2−/− and ApoE−/− groups (scale bar, 500 μm. ApoE−/−: n=8; ApoE−/−/AMPKα1−/−: n=8; ApoE−/−/AMPKα2−/−: n=7; *P<0.05; n.s. P>0.05).
AMP-activated protein kinase (AMPK) α1 deletion induces Runx2 expression in atherosclerotic lesions in vivo. ApoE−/−, ApoE−/−/AMPKα1−/−, and ApoE−/−/AMPKα2−/− mice were fed with Western diet for 24 wk. A, Representative immunofluorescence staining of Runx2 (Runt-related transcription factor) in aortic roots. B, ApoE−/−/AMPKα1−/− but not ApoE−/−/AMPKα2−/− mice had increased Runx2 staining in aortic roots compared with ApoE−/− mice. C, Western blot results indicate that ApoE−/−/AMPKα1−/− but not ApoE−/−/AMPKα2−/− had increased Runx2 expression compared with ApoE−/−. D, Representative triple staining of Runx2 (red), VSMC marker α-smooth muscle actin (α-SMA, green), and macrophages marker CD68 (blue) in atherosclerotic lesion areas. Yellow color in merged image indicates colocalization of Runx2 and α-SMA (scale bar, 200 μm in A and 100 μm in B. ApoE−/−: n=8; ApoE−/−/AMPKα1−/−: n=8; ApoE−/−/AMPKα2−/−: n=7; *P<0.05; n.s. P>0.05).
Metformin administration inhibits atherosclerotic calcification and Runx2 (Runt-related transcription factor) expression via AMP-activated protein kinase (AMPK) α1 in vivo. ApoE−/− and ApoE−/−/AMPKα1−/− mice with or without metformin (100 mg/kg per d) were treated with Western diet for 24 wk. A, Representative alizarin red staining of aortic roots of indicated groups. B, ApoE−/−/AMPKα1−/− mice had increased alizarin red staining in atherosclerotic lesion areas of aortic roots compared with ApoE−/− mice. Metformin treatment significantly reduced atherosclerotic calcification in ApoE−/− mice, but had no such effect in ApoE−/−/AMPKα1−/− mice. C, ApoE−/−/AMPKα1−/− mice had increased aortic calcium content compared with ApoE−/− mice. Metformin treatment significantly reduced aortic calcium content in ApoE−/− mice, but had no such effect in ApoE−/−/AMPKα1−/− mice. D, Representative immunofluorescence staining of Runx2 in aortic roots of indicated groups. E, ApoE−/−/AMPKα1−/− had increased Runx2 staining in atherosclerotic lesions compared with ApoE−/− mice. Metformin administration significantly inhibited Runx2 staining in ApoE−/−, but the effect was abolished in ApoE−/−/AMPKα1−/− mice. F, Western blot results show ApoE−/−/AMPKα1−/− had increased aortic Runx2 expression compared with ApoE−/− mice. Metformin administration significantly inhibited Runx2 expression in ApoE−/−, but the effect was abolished in ApoE−/−/AMPKα1−/− mice (scale bar: 500 μmol/L for alizarin red staining images and 200 μmol/L for immunofluorescence staining of Runx2. ApoE−/−: n=8; ApoE−/−+metformin: n=7; ApoE−/−/AMPKα1−/−: n=7; ApoE−/−/AMPKα1−/− plus metformin: n=6; *P<0.05; n.s. P>0.05).
Vascular smooth muscle cell (VSMC)-specific AMP-activated protein kinase (AMPK) α1 deficiency promotes atherosclerotic Runx2 (Runt-related transcription factor) expression and calcification in vivo. ApoE−/−/AMPKα1f/f, ApoE−/−/AMPKα1f/f/SM22Cre, ApoE−/−/AMPKα1f/f/lyzMCre mice were fed with Western diet for 24 wk. A, Representative images of alizarin red staining and immunofluorescence of Runx2 of aortic roots. ApoE−/−/AMPKα1f/f/SM22Cre but not ApoE−/−/AMPKα1f/f/lyzMCre mice had increased alizarin red staining in atherosclerotic lesion areas of aortic roots (B), Runx2 staining (C), and aortic calcium content (D) compared with ApoE−/−/AMPKα1f/f mice. E, Western blot results show that ApoE−/−/AMPKα1f/f/SM22Cre but not ApoE−/−/AMPKα1f/f/lyzMCre had increased aortic Runx2 expression compared with ApoE−/−/AMPKα1f/f mice (scale bar, 500 μmol/L for alizarin red staining images and 200 μmol/L for immunofluorescence staining of Runx2. ApoE−/−/AMPKα1f/f: n=7; ApoE−/−/AMPKα1f/f/SM22Cre: n=7; ApoE−/−/AMPKα1f/f/lyzMCre: n=6. *P<0.05; P>0.05).
AMP-activated protein kinase (AMPK) α1 deficiency promotes osteoblastic differentiation of vascular smooth muscle cell (VSMC) via Runx2 (Runt-related transcription factor). A, Alizarin red staining of calcium nodule formation of murine wild-type (WT), AMPKα1−/−, and AMPKα2−/− VSMC treated with osteogenic media for 14 d. AMPKα1 but not AMPKα2 deficiency significantly induced calcium nodule formation compared with WT VSMC. B, AMPKα1-deficient murine VSMC had significantly increased Runx2 expression compared with WT VSMC. C, ox-LDL (100 μg/mL) significantly induced Runx2 expression, which could be inhibited by metformin (2 mmol/L). AMPKα1 silencing markedly increased Runx2 expression compared with scrambled siRNA-transfected (Scr siRNA) cells. Oxidized low-density lipoprotein (ox-LDL) further induced Runx2 expression, but could not be suppressed by metformin supplementation. D, Representative alizarin red staining of murine WT and AMPKα1−/− VSMC with indicated treatment in osteogenic media for 14 d. AMPKα1 deficiency significantly induced calcium nodule formation compared with WT VSMC. Silencing of Runx2 by lentivirus Runx2 shRNA markedly suppressed the effect (n=3 for each experiment). Lenti-Runx2 shRNA indicates lentivirus Runx2 shRNA; and Lenti-Scr shRNA, lentivirus scramble shRNA. *P<0.05; n.s. P>0.05.
AMP-activated protein kinase (AMPK) α1 enhances Runx2 (Runt-related transcription factor) degradation via proteasome in vascular smooth muscle cell (VSMC). A, AMPKα1 silencing did not alter Runx2 mRNA expression compared with scrambled siRNA-transfected (Scr siRNA). B, MG132 (1 μmol/L) significantly increased Runx2 protein expression in Scr siRNA-transfected VSMC as early as 4 h. The effect of MG132 on Runx2 was mild in AMPKα1-silenced VSMC. C, Metformin treatment markedly reduced Runx2 half-life compared with the control (n=3 for each experiment. CHX indicates cycloheximide. *P<0.05; n.s. P>0.05.
AMP-activated protein kinase (AMPK) α1 phosphorylates protein inhibitor of activated STAT-1 (PIAS1) at serine 510 and promotes Runx2 (Runt-related transcription factor) small ubiquitin-related modifier (SUMO)ylation in vascular smooth muscle cell (VSMC). A, Treatment of metformin (2 mmol/L) for 6 h significantly induced Runx2-SUMO1 expression (≈100-kD band). The effect could be blocked by addition of compound C (10 μmol/L). B, AMPKα1 and PIAS1 in whole-cell lysates were pulled down by the appropriate primary antibody and subjected to Western blot analysis to detect the binding of AMPKα1 and PIAS1. C, Serine phosphorylation of PIAS1 was determined by pull-down PIAS1 antibody and immunoblotting with phosphor-serine antibody. Six hours of metformin treatment induced PIAS1 phosphorylation as indicated by pulling down by PIAS1 and immunoblotting with phosphor-serine antibody (≈80-kD band). The effect could be abolished by compound C. Neither metformin nor compound C altered PIAS1 expression. D, Myc-tagged WT and site-directed mutants of PIAS1 were transfected into human aortic VSMC then treated with metformin for 6 h. Serine phosphorylation of PIAS1 was determined by pull-down with anti-Myc and Western blot analysis with antiphospho-serine antibody (n=3 for each experiment. *P<0.05).
Protein inhibitor of activated STAT-1 (PIAS1) serine 510 phosphorylation is required for AMP-activated protein kinase (AMPK) α1–activated Runx2 (Runt-related transcription factor) small ubiquitin-related modifier (SUMO)ylation and instability. A, Myc-tagged wild-type (WT) and S510A-mutated PIAS1 were transfected into human aortic vascular smooth muscle cell (VSMC). Metformin (2 mmol/L) treatment significantly induced Runx2-SUMO1 expression (≈100-kD band) in WT-PIAS1–transfected cells, but had no such effect in S510A-PIAS1–transfected ones. B, AMPKα1 silencing significantly increased Runx2 expression. The effect could be blocked by transfection with S510E-mutated PIAS1. C, WT and S510A-mutated PIAS1 were transfected into human aortic VSMC. Oxidized low-density lipoprotein (ox-LDL) markedly induced Runx2 expression in WT-PIAS1– and S510A-PIAS1–transfected cells. However, metformin addition could only inhibited ox-LDL–triggered Runx2 expression in WT-PIAS1–transfected cells but not in S510A-PIAS1–transfected ones. (n=3 for each experiment). Scr siRNA indicates scramble siRNA. *P<0.05; n.s. P>0.05.
Rosemeire M Kanashiro-Takeuchi, Lauro M Takeuchi, Raul A Dulce, Darrell Hardin, Marilia Zuttion, Wayne Balkan, Ren Z Cai, Andrew V Schally and Joshua M Hare
