Zc3h12c inhibits vascular inflammation by repressing NF-κB activation and pro-inflammatory gene expression in endothelial cells

Abstract

Endothelial activation characterized by the expression of multiple chemokines and adhesive molecules is a critical initial step of vascular inflammation, which results in recruitment of leucocytes into the sub-endothelial layer of the vascular wall and triggers vascular inflammatory diseases such as atherosclerosis. Although inhibiting endothelial inflammation has already been well recognized as a therapeutic strategy in vascular inflammatory diseases, the therapeutic targets are still elusive. In the present study we found that Zc3h12c (zinc finger CCCH-type-containing 12C), a recently discovered CCCH zinc finger-containing protein, significantly inhibited the endothelial cell inflammatory response in vitro. Overexpression of Zc3h12c significantly attenuated TNFα (tumour necrosis factor α)-induced expression of chemokines and adhesive molecules, and thus reduced monocyte adherence to HUVECs (human umbilical vein endothelial cells). Conversely, siRNA (small interfering RNA)-mediated knockdown of Zc3h12c increased the TNFα-induced expression of chemokines and adhesive molecules in HUVECs. Furthermore, forced expression of Zc3h12c decreased TNFα-induced IKKα/β [IκB (inhibitor of nuclear factor κB) kinase α/β], IκBα phosphorylation and p65 nuclear translocation, suggesting that Zc3h12c exerted its anti-inflammatory function probably by suppressing the NF-κB (nuclear factor κB) pathway. Thus Zc3h12c is an endogenous inhibitor of TNFα-induced inflammatory signalling in HUVECs and might be a therapeutic target in vascular inflammatory diseases.

Figure 1. TNFα-induced Zc3h12c expression in HUVECs

(A) HUVECs were stimulated with 2.5 ng/ml TNFα and then the RNA was collected at the time points indicated. (B) HUVECs were stimulated with TNFα at different doses as indicated and the RNA was collected 4 h later. The mRNA level of Zc3h12c was detected by Q-PCR. Results are means ± S.D., n =4. *P < 0.05 and **P < 0.01 compared with the untreated group.

Figure 2. Overexpression of Zc3h12c inhibited inflammatory gene expression in HUVECs

(A) HUVECs were infected with 5 pfu/per cells of Ad/Zc3h12c or Ad/GFP and then treated with or without 2.5 ng/ml TNF-α for 4 h. The relative mRNA level of Zc3h12c, VCAM-1, ICAM-1, E-selectin, IL-8 and MCP-1 was detected by Q-PCR. **P < 0.01. (B) HUVECs were treated as above and the protein levels of Zc3h12c, VCAM-1, ICAM-1 and E-selectin were detected by Western blotting. Results are means ± S.D.

Figure 3. Knockdown of Zc3h12c increased inflammatory gene expression in HUVECs

HUVECs were transfected with Zc3h12c siRNA or control siRNA. The transfected cells were treated with or without 2.5 ng/ml TNFα for 4 h. The relative mRNA level of Zc3h12c, VCAM-1, ICAM-1, E-selectin, IL-8 andMCP-1 was detected by Q-PCR. Results are means ± S.D., n =4. *P < 0.05 and **P < 0.01 compared with the control siRNA group.

Figure 4. Zc3h12c inhibited monocyte adhesion to HUVECs

(A) HUVECs were transfected with Ad/Zc3h12c or Ad/GFP and then cultured with Hoechst-labelled THP-1 cells. At 30 min later the floating THP-1 cells were washed away and the adhesive THP-1 cells were detected by fluorescence microscopy. (B) The adhesive THP-1 cells were counted and analysed. Results are means ± S.D from three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5. Zc3h12c inhibited human VCAM-1 promoter activity

(A) HEK-293 cells were transfected with the human VCAM-1 promoter reporter plasmid along with FLAG control or FLAG–Zc3h12c plasmids. At 24 h later, the transfected cells were treated with or without 2.5 ng/ml TNFα. At 24 h after the treatment, the luciferase (Luc.) activity was measured. (B) HEK-293 cells were transfected with the human VCAM-1 promoter reporter and pcDNA3 or pcDNA3-p65 plasmids as indicated. At 24 h later the luciferase activity was measured. Results are means ± S.D., n =4. *P < 0.05 compared with the FLAG control group.

Figure 6. Zc3h12c inhibited the NF-κB signalling pathway

(A) HUVECs were infected with Ad/Zc3h12c or Ad/GFP and then treated with 2.5 ng/ml of TNFα for 0, 5, 15 and 30 min as indicated. The cell lysates were extracted and Western blotting was performed to detect the phosphorylation of IKKα/β and IκBα with specific antibodies as indicated. (B) HUVECs were infected with Ad/Zc3h12c or Ad/GFP and then treated with 2.5 ng/ml TNFα for 0, 15, 30 and 60 min as indicated. The cytosol and nuclear fraction was isolated and Western blotting was performed to detect the p65 protein level in the cytosol and nuclear fractions. Three independent experiments showed similar results.