Abstract
cGMP-dependent protein kinase 1 (PKG1) plays an important role in nitric oxide (NO)/cGMP-mediated maintenance of vascular smooth muscle cell (VSMC) phenotype and vasorelaxation. Inflammatory cytokines, including tumor necrosis factor-α (TNFα), have long been understood to mediate several inflammatory vascular diseases. However, the underlying mechanism of TNFα-dependent inflammatory vascular disease is unclear. Here, we found that TNFα treatment decreased PKG1 expression in cultured VSMCs, which correlated with NF-κB-dependent biogenesis of miR-155-5p that targeted the 3'-UTR of PKG1 mRNA. TNFα induced VSMC phenotypic switching from a contractile to a synthetic state through the down-regulation of VSMC marker genes, suppression of actin polymerization, alteration of cell morphology, and elevation of cell proliferation and migration. All of these events were blocked by treatment with an inhibitor of miR-155-5p or PKG1, whereas transfection with miR-155-5p mimic or PKG1 siRNA promoted phenotypic modulation, similar to the response to TNFα. In addition, TNFα-induced miR-155-5p inhibited the vasorelaxant response of de-endothelialized mouse aortic vessels to 8-Br-cGMP by suppressing phosphorylation of myosin phosphatase and myosin light chain, both of which are downstream signal modulators of PKG1. Moreover, TNFα-induced VSMC phenotypic alteration and vasodilatory dysfunction were blocked by NF-κB inhibition. These results suggest that TNFα impairs NO/cGMP-mediated maintenance of the VSMC contractile phenotype and vascular relaxation by down-regulating PKG1 through NF-κB-dependent biogenesis of miR-155-5p. Thus, the NF-κB/miR-155-5p/PKG1 axis may be crucial in the pathogenesis of inflammatory vascular diseases, such as atherosclerotic intimal hyperplasia and preeclamptic hypertension.
Highlights
We found that TNF␣ treatment decreased PKG1 expression in cultured vascular smooth muscle cell (VSMC), which correlated with NF-B– dependent biogenesis of miR-155-5p that targeted the 3-UTR of PKG1 mRNA
These results suggest that TNF␣ impairs nitric oxide (NO)/cGMP-mediated maintenance of the VSMC contractile phenotype and vascular relaxation by down-regulating PKG1 through NF-B– dependent biogenesis of miR-155-5p
We found that TNF␣-induced biogenesis of NF-B–responsive miR-155-5p promotes VSMC phenotypic switching and vasodilatory dysfunction by inhibiting PKG1 expression in VSMCs
Summary
As pro-inflammatory cytokines, including TNF␣, are crucially involved in the pathogenesis of various inflammatory vascular diseases [14], we investigated whether TNF␣ regulates PKG1 expression in human aortic smooth muscle cells (HASMCs) and isolated primary mouse aortic smooth muscle cells (MASMCs). Knockdown or inhibition of PKG1 in 8-Br-cGMP– exposed HASMCs by treatment with PKG1 siRNA, KT 5823, or Rp-8-pCPTcGMP promoted cell cycle progression from the G0/G1 to S phase, with subsequent entry to the G2/M phase (Fig. 5A) These PKG1 inhibitors effectively suppressed mRNA and protein levels of VSMC-specific genes, such as ␣-SMA, SM22␣, SM-MHC11, and calponin (Fig. 5, B and C). 8-Br-cGMP treatment maintained low phosphorylation of myosin light-chain phosphatase 1 (MYPT1) at Thr-696 and myosin light chain (MLC), which are signal mediators downstream of PKG1 [17]; their phosphorylation levels were elevated by co-treatment with either TNF␣ or miR-155-5p mimic, and TNF␣-induced phosphorylation of MYPT1 and MLC was blocked by an miR-155-5p inhibitor (Fig. 6A) Based on these results, we further examined the effect of the miR-155-5p/PKG1 axis on the ex vivo vasorelaxant responses of de-endothelialized mouse aortic vessels to NO and cGMP.
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