An antagonism between the AKT and beta-adrenergic signaling pathways mediated through their reciprocal effects on miR-199a-5p

Abstract

We have recently reported that downregulation of miR-199a-5p is necessary and sufficient for inducing upregulation of its targets, including hypoxia-inducible factor-1 alpha (Hif-1 alpha) and Sirt1, during hypoxia preconditioning (HPC). Conversely, others and we have reported that miR-199a-5p is upregulated during cardiac hypertrophy. Thus, the objective of this study was to delineate the signaling pathways that regulate the expression of miR-199a-5p and its targets, and their role in myocyte survival during hypoxia. Since HPC is mediated through activation of the AKT pathway, we questioned if AKT is sufficient for inducing downregulation of miR-199a-5p. Our present study shows that overexpression of a constitutively active AKT (caAKT) induced 70% reduction in miR-199a-5p and was associated with a robust increase in HiF-1 alpha (10+/-2 fold) and Sirt1 (4+/-0.8 fold) that was reversed by overexpression of miR-199a-5p. Similarly, insulin receptor-stimulated activation of the AKT pathway induced downregulation of miR-199a-5p and upregulation of its targets. In contrast, beta-adrenergic receptor (beta AR) activation in vitro and in vivo, induced 1.8-3.5-fold increase in miR-199a-5p. Accordingly, we predicted that beta AR would antagonize AKT-induced, miR-199a-5p-dependent, upregulation of Hif-1 alpha and Sirt1. Indeed, pre-treating the myocytes with isoproterenol before applying HPC, caAKT, or insulin resulted in 87+/-3%, 75+/-15%, and 100% reductions in Hif-1 alpha expression, respectively, and sensitized the cells to hypoxic injury. Thus, activation of beta-adrenergic signaling counteracts the survival effects of the AKT pathway via upregulating miR-199a-5p.

Figure 1. MiR-199a-5p is upregulated during cardiac hypertrophy

Mice were subjected to transverse aortic banding (TAC) or a sham operation. After the indicated time periods RNA was extracted from the left ventricles and analyzed by Northern blotting for miR-199a-5p, alpha skeletal actin (SkAc) as a marker of cardiac hypertrophy, and U6 as an internal control. At each time point cardiac function was assessed by echocardiography and hemodynamic measurements. Any decrease in % ejection fraction, associated with an increase in left ventricular end diastolic pressure, was used as a sign of cardiac failure. MiR-199a-5p signals were quantified by UN-SCAN-IT software and averaged after normalizing each to the corresponding internal control (n=3). The results were graphed as fold increase in miR-199a-5p in TAC vs. Sham hearts. Error bars represent standard deviation. *<0.001 vs. sham.

Figure 2. MiR-199a-5p is reciprocally regulated by βAR stimulation and AKT

a. RNA was extracted from the hearts of 3 month-old β1AR- and β2AR-overexpressing mice and their wild type (WT) littermates, prior to the onset of any cardiac pathology. These were analyzed by Northern blotting for miR-199a-5p and miR-133 as an internal control (n=2). b. Cultured neonatal cardiac myocytes were treated with 10 μM isoproterenol (ISO), Ad.caAKT, or 200 nM insulin, for 16 h (n=3). RNA was then extracted and analyzed by Northern blotting for miR-199a-5p and miR-199a-3p as an internal control. c. MiR-199a-5p signals were quantified, normalized, and averaged. The results were graphed on a log scale as fold change of miR-199a-5p vs. wild type mice or control samples.

Figure 3. cAMP-dependent signaling antagonizes effects of the AKT pathway

a. HPC was applied to cardiac myocytes with or without pretreatment with 10 µM ISO for 16 h, as indicated with the + sign. In parallel, cells were treated with Ad.caAKT with or without pretreatment with Ad.miR-199a-5p or 10 µM ISO for 16 h, where indicated with the + sign (n=5). b. HPC was applied to the myocytes with or without pretreatment of the cells with 100 µM forskolin (forsk), 5 mM wortmannin (wort), or a 4 µM cAMP analogue for 16 h, as indicated with the + sign. At the conclusion of all the treatments listed above, protein was extracted and analyzed by Western blotting for the molecules indicated on the left of each panel (n=3). c. The Hif-1α signals were quantified, averaged, and graphed as fold increase over control. Error bars represent standard deviation. *<0.001 vs HPC (no pretreatment). **<0.001 vs Ad.caAKT (no pretreatment).

Figure 4. Isoproterenol inhibits the protective effect of HPC

Cardiac myocytes were untreated (a, e), stimulated with 10 µM ISO (b, d, f, h), or HPC (c, d, g, h), before exposure to 24 h of hypoxia (e, f, g, h). Following these treatments, the myocytes were loaded with the JC-1 dye and immediately imaged live. The upper panel of each treatment is an image of the cells using the FITC filter, while the lower panel is an image of the same field using the TRITC filter (n=3). i. The red mitochondrial (mito) and the green cytosolic (cyto) signals were quantified using Adobe Photoshop software. The results from 5 fields were averaged and graphed as relative values to the red mitochondrial signal during normoxia adjusted to 1. Error bars represent SEM and *p<0.001 vs control normoxia. ** p<0.01 vs control HPC. # p<0.01 vs control HPC + hypoxia 24 h.

Figure 5. Isoproteronol and miR-199a-5p abrogate insulin-induced upregulation of Hif-1α in cardiac myocytes

a. Cardiac myocytes were treated with insulin using various doses and durations, as indicated. The Hif-1α signal from 4 different experiments were quantified, normalized to GAPDH, averaged, and plotted relative to its control levels vs. insulin dose and stimulation duration. Error bars represent SEM. b. Cardiac myocytes were treated with insulin for various time intervals or Ad. miR.199a-5p for 24 h, following pretreatment with vehicle or 10 µM ISO for 16 h, as indicated (n=3). c. Myocytes were treated with 10 µM ISO for various time intervals before stimulating them with 200 nM insulin for 24 h (n=4). d. Cells were treated with Ad.miR-199a-5p or a control virus for 20 h followed by 200 nM insulin for 6 h, as indicated by the + sign. At the conclusion of all the treatments listed above, protein was extracted and analyzed by Western blotting for the molecules indicated on the left of each panel (n=3). e. Hif-1α signal from experiments shown in c and d were quantified, normalized to GAPDH, averaged, and plotted relative to its control levels after adjusting it to 1. Error bars represent SEM and *p<0.001 vs. control.

Figure 6. Isoproterenol-induced upregulation of miR-199a-5p counteracts its downregulation by HPC or insulin

Cardiac myocytes were subjected to HPC or treated with 200 nM insulin for 6 h following treatment with vehicle or 10 µM ISO for 16 h, as indicated by + signs. RNA was extracted and analyzed for miR-199a-5p expression (n=3). MiR-199a-5p signals were quantified, normalized to 5S, averaged, and plotted relative to its control levels after adjusting it to 1. Error bars represent SEM and *p<0.01 vs. control; #p<0.01 vs. HPC.

Figure 7. Overexpression of ILK, but not inhibition of GSK3β, is sufficient for inducing upregulation of Hif-1α

a. Cardiac myocyte were treated with various doses of Ad.ILK for 20 h or shRNA targeting GSK3β (Ad.GSK3βi) for 24 or 48 h, as indicated (n=2). b. Cardiac myocytes were treated with Ad.miR-199a-5p or a control virus for 16 h before applying Ad.ILK for an additional 20 h. At the conclusion of all the treatments listed above, protein was extracted and analyzed by Western blotting for the molecules indicated on the left of each panel (n=3). Hif-1α signal from experiments shown in a and b were quantified, normalized to GAPDH, averaged, and plotted relative to its control levels after adjusting it to 1. Error bars represent SEM and *p<0.001 vs. control.

Figure 8. An illustration of the pathways that regulate miR-199a-5p and its targets, as suggested by the data

Subjecting cells to HPC or stimulation with insulin results in ILK-AKT-dependent downregulation of miR-199a-5p and upregulation of its targets Hif-1α and Sirt1. Conversely, βAR stimulation induces AC-cAMP-dependent upregulation of miR-199a-5p and, thereby, neutralizes the effects of the AKT pathway. Abbreviations: PIP2, phosphatidylinositol 4,5 bisphosphate; PI3K, phosphatidylinositol-3 kinase; PIP3, phosphatidylinositol 3,4,5 trisphosphate; PKD, protein kinase D; AC adenylyl cyclase; PKA, protein kinase A.