Nuclear factor-κB pathway mediates the effects of CD137 signaling on NFATc1 expression in mice vascular smooth muscle cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe whether CD137 signaling could affect the nuclear factor of activated T cells c1 (NFATc1) expression through nuclear factor-κB (NF-κB) pathway in mice aortic vascular smooth muscle cells (VSMCs).</p><p><b>METHODS</b>Adherence methods for tissues explants were used for primary culture of mouse aortic VSMCs. The mRNA expression of CD137 and NFATc1 was detected by real-time quantitative PCR (RT-qPCR). The VSMCs protein expression of IκB-α, NF-κB p65, phospo-p65 and NFATc1 was determined by Western blot. The level of CD137 was measured by Flow Cytometry (FCM).</p><p><b>RESULTS</b>(1) The mRNA and protein expression of CD137 in VSMCs was significantly upregulated at 24 h after co-culture with TNF-α (10 ng/ml, all P < 0.05). (2) Compared with the control group, the level of p-NF-κB p65 in cytoplasm and nucleus was significantly increased (8.34 ± 0.28 vs. 1, P < 0.05, and 2.64 ± 0.42 vs. 1, P < 0.05) while the level of IκB-α was reduced (1 vs. 2.70 ± 0.28, P < 0.05) after co-treatment with agonist-CD137 mAb, above effects were partly blocked by adding specific NF-κB inhibitor PDTC (30 µmol/L: 1.15 ± 0.14 vs. 8.34 ± 0.28, P < 0.05, and 2.09 ± 0.12 vs. 2.64 ± 0.42, P < 0.05, and 1.78 ± 0.74 vs. 1, P < 0.05). (3) The mRNA (2.07 ± 0.09 vs. 1, P < 0.05) and protein (1.75 ± 0.07 vs. 1, P < 0.05) expression of NFATc1 was significantly upregulated by agonist CD137mAb compared with the control group, and these effects could be reversed by PDTC (1.15 ± 0.07 vs. 2.07 ± 0.09, P < 0.05, and 0.90 ± 0.11 vs. 1.75 ± 0.07, P < 0.05).</p><p><b>CONCLUSION</b>CD137 signaling could affect the NFATc1expression in VSMCs through NF-kappaB pathway.</p>

CD137 signaling regulates the expression of nuclear factor of activated T cells c1 through miR-145a-5p in ApoE(-)/(-) mice / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate if miR-145a-5p participates the modulation process of CD137 signaling on the expression of nuclear factor of activated T cells c1 (NFATc1) in ApoE(-)/(-) mice.</p><p><b>METHODS</b>Atherosclerotic plaque model was produced by perivascular carotid collar placement in ApoE(-)/(-) mice. After surgery, the mice were randomly divided into the following groups: CD137 activated group (CD137 group, n = 6), CD137 inhibited group (anti-CD137 group, n = 6) and control group (n = 6). The mRNA expression of miR-145a-5p in plaque and cells was measured by real-time quantitative PCR (RT-PCR). Immunofluorescence was used to observe the distribution of NFATc1 in plaque and the expression of NFATc1 at mRNA and protein levels were detected by qRT-PCR, Western blot, respectively. The mouse vascular smooth muscle cells (VSMCs) were isolated and transfected with miR-145a-5p mimics or inhibitors by Lipofectamine. The eukaryotic expression vector and luciferase vector including p3xFLAG-NFATc1, p3xFLAG-NFATc1-3'UTR, psicheck2-NFATc1, psicheck2-NFATc1-Mut were constructed through molecular cloning and homologous recombination techniques, 293T cells were transfected with the miR-145a-5p mimics or inhibitors and the protein level and fluorescence intensity were then measured, respectively.</p><p><b>RESULTS</b>In vivo or in vitro, the level of miR-145a-5p was significantly decreased (0.21 ± 0.06 vs. 1.00 ± 0.00, P < 0.05, 0.22 ± 0.07 vs. 0.50 ± 0.12, P < 0.05) while the opposite effects were observed in anti-CD137 group. NFATc1 expression was decreased or increased in VSMCs transfected with miR-145a-5p mimics or inhibitors, respectively (all P < 0.05). miR-145a-5p mimics decreased the expression of p3xFLAG-NFATc1-3'UTR and the fluorescence intensity (0.56 ± 0.08 vs. 1.00 ± 0.00, P < 0.05).</p><p><b>CONCLUSION</b>CD137 signaling participates the regulation process on the expression of NFATc1 through miR-145a-5p in ApoE(-)/(-) mice.</p>