ULK1 mediates the NO-induced SIRT1 upregulation.

<p>(A) MEF from the <i>Ulk1</i>^−/−, <i>Ulk2</i>^−/−, <i>Ulk1/2</i>^−/−, and <i>WT</i> mice were transfected with GFP or eNOS Adenovirus for 48 h; (B) MEF from the <i>Ulk1</i>^−/−, <i>Ulk2</i>^−/−, <i>Ulk1/2</i>^−/−, and <i>WT</i> mice were treated with NONOate (50 µM) or A23187 (1 µM) for 4 h; (C) HUVECs were transfected with control or <i>ULK1</i> SiRNA for 48 h, then treated with A23187 (1 µM) for 4 h; (D) HUVECs were transfected with control or <i>ULK1</i> SiRNA for 48 h, then treated with NONOate (50 µM) for 4 h. The western blots shown are representative of three independent experiments. *represents <i>p</i><0.05 vs control (<i>n</i> = 3); #represents <i>p</i><0.05 vs A23187 or NONOate alone (<i>n</i> = 3); NS, not significant. GFP, green fluorescent protein; eNOS, endothelial nitric oxide synthase; Ad, Adenovirus; Si-SiRNA.</p

ULK1 regulates SIRT1 protein expression via 26S proteasomes.

<p>(A) HUVECs were transfected with control or <i>ULK1</i> SiRNA for 48 h, then treated with epoxomicin (0.1 µM) for 4 h; (B) HUVECs were transfected with control or <i>ULK1</i> siRNA for 48 h, SIRT1 mRNA levels were determined by RT-PCR; (C) HUVECs were transfected with control or <i>ULK1</i> or β-TrCP1 siRNA for 48 h. The western blots are representative of three independent experiments. *represents <i>p</i><0.05 vs control (<i>n</i> = 3); # represents <i>p</i><0.05 vs <i>si-ULK1</i> alone (<i>n</i> = 3). Si, siRNA.</p

NO stabilizes and upregulates ULK1 protein expression.

<p>(A) HUVECs were transfected with GFP or eNOS adenovirus for 48 h; (B) HUVECs were treated with A23187 (1 µM) for the indicated time; (C) HUVECs were treated with NONOate (50 µM) for the indicated time; (D) HUVECs were treated with CHX (5 µM) for the indicated time, followed by incubation of NONOate (50 µM) for 4 h; (E) HUVECs were treated with CHX (5 µM) for the indicated time, followed by incubation of A23187 (1 µM) for 4 h. The western blots are representative of three independent experiments. *represents <i>p</i><0.05 vs control (<i>n</i> = 3); NS, not significant. GFP, green fluorescent protein; eNOS, endothelial nitric oxide synthase; Ad, Adenovirus; CHX, cycloheximide.</p

ULK1 modulates 26S proteasome functionality.

<p>GFPu-1 cells were transfected with (A) control or <i>ULK1</i> plasmid for 48 h, and (B) the accumulated GFP fluorescence in <i>ULK1</i> plasmid-expressing cells was captured with a fluorescent microscope; (C) HEK293 cells were transfected with control or <i>ULK1</i> plasmid for 48 h and chymotrypsin-like activity was measured; (D) The <i>Ulk1</i>^−/− and <i>WT</i> MEF were transfected with Ub^G76V-GFP plasmid for 48 h; (E) Chymotrypsin-like activity was measured in the <i>Ulk1</i>^−/− and <i>WT</i> MEF; (F) HUVECs were transfected with control or <i>ULK1</i> SiRNA for 48 h and chymotrypsin-like activity was measured. The western blots are representative of three independent experiments. *represents <i>p</i><0.05 vs control (<i>n</i> = 3). Si, siRNA.</p

ULK1 regulates SIRT1 protein expression which is independent of autophagy.

<p>(A) HEK293 cells were transfected with <i>pcDNA</i> or <i>ULK1</i> plasmid for 48 h; (B) HEK293 cells were transfected with <i>pcDNA</i> or <i>ULK1</i> plasmid for 48 h. SIRT1 mRNA levels were determined by RT-PCR; (C) HUVECs were treated with rapamycin (2 µM) for the indicated time; (D) U20S cells which expressed GFP-LC3 reporter were treated with rapamycin (2 µM) or NONOate (50 µM) for 12 h; GFP-LC3 imaging was captured using fluorescent microscope according to instructions of the manufacturer, EMD Millipore; (E) MEF from the <i>Ulk1</i>^−/−, <i>Ulk2</i>^−/−, <i>Ulk1/2</i>^−/−, and <i>WT</i> mice were treated with bafilomycin A1 (10 nM) for 4 h; (F) HUVECs were treated with NONOate (50 µM) for the indicated time; (G) HUVECs were transfected with GFP or eNOS adenovirus for 48 h; (H) HUVECs were treated with A23187 (1 µM) for the indicated time. The western blots are representative of three independent experiments. *represent <i>p</i><0.05 vs control (<i>n</i> = 3). NS, not significant. GFP, green fluorescent protein; eNOS, endothelial nitric oxide synthase; Ad, adenovirus; Baf A1, Bafilomycin A1.</p

Sp1 Mediates a Therapeutic Role of MiR-7a/b in Angiotensin II-Induced Cardiac Fibrosis via Mechanism Involving the TGF-β and MAPKs Pathways in Cardiac Fibroblasts

<div><p>MicroRNA-7a/b (miR-7a/b) protects cardiac myocytes from apoptosis during ischemia/reperfusion injury; however, its role in angiotensin II (ANG II)-stimulated cardiac fibroblasts (CFs) remains unknown. Therefore, the present study investigated the anti-fibrotic mechanism of miR-7a/b in ANG II-treated CFs. ANG II stimulated the expression of specific protein 1 (Sp1) and collagen I in a dose- and time-dependent manner, and the overexpression of miR-7a/b significantly down-regulated the expression of Sp1 and collagen I stimulated by ANG II (100 nM) for 24 h. miR-7a/b overexpression effectively inhibited MMP-2 expression/activity and MMP-9 expression, as well as CF proliferation and migration. In addition, miR-7a/b also repressed the activation of TGF-β, ERK, JNK and p38 by ANG II. The inhibition of Sp1 binding activity by mithramycin prevented collagen I overproduction; however, miR-7a/b down-regulation reversed this effect. Further studies revealed that Sp1 also mediated miR-7a/b-regulated MMP expression and CF migration, as well as TGF-β and ERK activation. In conclusion, miR-7a/b has an anti-fibrotic role in ANG II-treated CFs that is mediated by Sp1 mechanism involving the TGF-β and MAPKs pathways.</p></div

Effect of miR-7a/b mimics on TGF-β and MAPK pathways.

<p>Western blots and analysis of TGF-β (A), p-ERK (B), p-JNK (C), and p-p38 (D) expression. Con: normal untreated CFs; NC: negative control siRNA; *p < 0.05, compared with control; and #p, < 0.05 compared with NC.</p

Effect of miR-7a/b mimics on proliferation, migration, and MMP-2 and MMP-9 expression/activity in CFs.

<p>A: MTT assay. B-C: CFs on the external surface of the Transwell were dyed with Crystal Violet and photographed under a microscope. D-F: Western blot analysis of MMP-2 (D, E) and MMP-9 (D, F) protein levels. G-H: Media were harvested for gelatin zymography analysis of MMP-2 activity; no MMP-9 activity was detected in the media. Con: normal untreated CFs; NC: negative control siRNA; *p, < 0.05 compared with control; and #p, < 0.05 compared with NC.</p

Effect of different concentrations of mithramycin on the regulation of Sp1, collagen Iexpression and signal transduction in CFs.

<p>A-F: Western blots and analysis of mithramycin on the regulation of Sp1 (A), collagen I (B), TGF-β (C), p-ERK (D), p-JNK (E) and p-p38 (F). Con: normal untreated CFs; NC: negative control siRNA; M: mithramycin. *p < 0.05, compared with control; and #p < 0.05, compared with NC.</p

Sp1 mediates miR-7a/b-regulated proliferation migration, and MMP-2 and MMP-9 expression/activity in CFs.

<p>A-C: Western blot analysis of MMP-2 (A, B) and MMP-9 (A, C) protein levels. D-E: Media were harvested for gelatin zymography analysis of MMP-2 activity; no MMP-9 activity was detected in media. F: MTT assay. G-H: CFs on the external surface of the Transwell were dyed with Crystal Violet and photographed under a microscope. Con: normal untreated CFs; NC: negative control siRNA; M: mithramycin. *p < 0.05, compared with control; #p < 0.05, compared with NC; and ◆p < 0.05, compared with mithramycin.</p