Implication of microRNAs in atrial natriuretic peptide and nitric oxide signaling in vascular smooth muscle cells

Abstract

MicroRNAs (miRs) are endogenous small RNA molecules that suppress gene expression by binding to complementary sequences in the 3' untranslated regions of their target genes. miRs have been implicated in many diseases, including heart failure, ischemic heart disease, hypertension, cardiac hypertrophy, and cancers. Nitric oxide (NO) and atrial natriuretic peptide (ANP) are potent vasorelaxants whose actions are mediated through receptor guanylyl cyclases and cGMP-dependent protein kinase. The present study examines miRs in signaling by ANP and NO in vascular smooth muscle cells. miR microarray analysis was performed on human vascular smooth muscle cells (HVSMC) treated with ANP (10 nM, 4 h) and S-nitroso-N-acetylpenicillamine (SNAP) (100 μM, 4 h), a NO donor. Twenty-two shared miRs were upregulated, and 21 shared miRs were downregulated, by both ANP and SNAP (P < 0.05). Expression levels of four miRs (miRs-21, -26b, -98, and -1826), which had the greatest change in expression, as determined by microarray analysis, were confirmed by quantitative RT-PCR. Rp-8-Br-PET-cGMPS, a cGMP-dependent protein kinase-specific inhibitor, blocked the regulation of these miRs by ANP and SNAP. 8-bromo-cGMP mimicked the effect of ANP and SNAP on their expression. miR-21 was shown to inhibit HVSMC contraction in collagen gel lattice contraction assays. We also identified by computational algorithms and confirmed by Western blot analysis new intracellular targets of miR-21, i.e., cofilin-2 and myosin phosphatase and Rho interacting protein. Transfection with pre-miR-21 contracted cells and ANP and SNAP blocked miR-21-induced HVSMC contraction. Transfection with anti-miR-21 inhibitor reduced contractility of HVSMC (P < 0.05). The present results implicate miRs in NO and ANP signaling in general and miR-21 in particular in cGMP signaling and vascular smooth muscle cell relaxation.

Fig. 1.

Atrial natriuretic peptide (ANP) and S-nitroso-N-acetylpenicillamine (SNAP) dose-response curves for intracellular cGMP. Human vascular smooth muscle cells (HVSMCs) were pretreated with isobutyl methyl xanthine (IBMX; 100 μM, 30 min) and incubated with ANP (10⁻⁷-10⁻¹⁰ M, 4 h; top) and SNAP (10⁻²-10⁻⁵ M, 4 h; bottom). Cells were harvested, and cell lysates are subjected to cGMP assay, as described in materials and methods. Values are means ± SD; n = 4.

Fig. 2.

microRNA (miR) microarray analysis of control and ANP- and SNAP-treated HVSMC. miRs were extracted from HVSMC cells treated with either ANP (10 nM, 4 h) or SNAP (100 μM, 4 h). Microarray analysis was performed (materials and methods). The color bar on the top conveys changes in miR expression, with green and red indicating minimal and maximal expression of miRs, respectively. The samples from left to right are control lanes-3, ANP-3 lanes, and SNAP-3 lanes. The identifications of miRs are shown on the right. n = 3 for each treatment. P < 0.05.

Fig. 3.

Validation of SNAP- and ANP-mediated changes and the role of cGMP-dependent kinase (cGK) in the expression of miR-21 in HVSMCs by quantitative RT (qRT)-PCR. Total RNA isolated from control and ANP- and SNAP-treated cGMP (top) analog 8-bromo-cGMP (8-Br-cGMP)-treated (bottom) and cGK inhibitor β-phenyl-1,N²-etheno-8-bromo-cGMP-treated HVSMC were subjected to qRT-PCR using TaqMan miR assays (materials and methods). For cGK inhibitor studies, cells were pretreated with Rp-8-bromo-β-phenyl-1,N²-ethenoguanosine 3′,5′-cyclic monophosphothioate (Rp-8-Br-PET-CGMPS) (25 μM × 30 min), followed by treatment with 8-Br-cGMP. The fold change in the expression of the miR-21, as indicated on the X-axis, is relative to that of control cells. Values are means ± SD; n = 4. P < 0.01 compared with control.

Fig. 4.

miR-21-mediated contraction of HVSMCs. HVSMC were transfected with pre-miR-21, anti-miR-21 inhibitor, or controls (pre-miR, anti-miR inhibitor), as indicated, and then embedded in the collagen gels and subjected to a contraction assay (see materials and methods). Representative images (A) and summarized data (B) are as follows. A and B, top: the effect of pre-miR-21 and anti-mir-21 inhibitor on HVSMC transfected with pre-miR-21 and anti-miR-21 inhibitor. A, middle, and B, bottom: the effect of ANP and SNAP on pre-miR-21-induced contraction of HVSMC transfected with pre-miR-21 and treated with ANP and SNAP. A, bottom: the effect of ANP and SNAP on anti-miR-21 inhibitor-induced relaxation of HVSMC transfected with anti-miR-21 inhibitor and treated with ANP and SNAP (arrow: lattice border). n = 4. P < 0.05. C: miR-21 promotes proliferation of HVSMC. HVSMCs transfected with pre-miR control and pre-miR-21 (as described above) are subjected to proliferation assay using a kit from Promega. Values are means ± SD; n = 4. P < 0.05.

Fig. 5.

Pre-miR21 and miR-21 inhibitor decreased and increased myosin phosphatase and Rho interacting protein (M-RIP), cofilin-2 (CFL-2), and cGK-1 expression, respectively. Cells were transfected with pre-miR control, pre-miR-21, miR inhibitor control, and miR-21 inhibitor (50 nM each). Forty-eight hours after transfection, cell lysates were analyzed for expression of M-RIP, cGK-1, and CFL2 by Western blot analysis with polyclonal antibody to M-RIP and monoclonal antibodies to CFL-2 and cGK-1 (materials and methods). A representative Western blot is shown. Values are means ± SD; n = 3. *P < 0.05, **P < 0.01 compared with control.

Fig. 6.

Molecular pathway by which miR-21 regulates HVSMC contraction. ANP and SNAP via cGMP/cGK signaling reduce the expression of miR-21, which has a feedback loop inhibitory effect on cGK-1. miR-21 targets (CFL-2, M-RIP, and cGK-1) and their effects on HVSMC contraction/relaxation are depicted. NO, nitric oxide.