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. 2022 Mar 12;8(1):112.
doi: 10.1038/s41420-022-00906-9.

HDAC11 promotes both NLRP3/caspase-1/GSDMD and caspase-3/GSDME pathways causing pyroptosis via ERG in vascular endothelial cells

Feng Yao  1 , Zhen Jin  1 , Zihan Zheng  1 , Xiaohan Lv  2 , Lingxuan Ren  1 , Jianjun Yang  1 , Danli Chen  1 , Bo Wang  1 , Wei Yang  3 , Lifang Chen  4 , Weirong Wang  4 , Jianli Gu #  5 , Rong Lin #  6
Affiliations

HDAC11 promotes both NLRP3/caspase-1/GSDMD and caspase-3/GSDME pathways causing pyroptosis via ERG in vascular endothelial cells

Feng Yao et al. Cell Death Discov. .

Abstract

Histone deacetylase 11 (HDAC11), a sole member of the class IV HDAC subfamily, participates in various cardiovascular diseases. Recent evidence showed that pyroptosis was a form of inflammatory programmed cell death and is critical for atherosclerosis (AS). However, little is known about the effect of HDAC11 on endothelial cell pyroptosis in AS. Thus, this study aims to investigate the role of HDAC11 in vascular endothelial cell pyroptosis and its molecular mechanism. Firstly, we found that HDAC11 expression was up-regulated and pyroptosis occurred in the aorta of ApoE-/- mice fed with a high-fat diet (HFD) for 8 or 12 weeks. Then, in vitro study found the treatment of human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor-α (TNF-α) resulted in pyroptosis, as evidenced by activation of caspase-1 and caspase-3 activation, cleavage of downstream gasdermin D (GSDMD) and gasdermin E (GSDME/DFNA5), the release of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and IL-18, as well as elevation of LDH activity and increase of propidium iodide (PI)-positive cells. Besides, TNF-α increased HDAC11 expression and induced pyroptosis via TNFR1 in HUVECs. HDAC11 knockdown mitigated pyroptosis by suppressing both NLRP3/caspase-1/GSDMD and caspase-3/GSDME pathways in TNF-α-induced HUVECs. Moreover, GSDME knockdown by siRNA significantly decreased pyroptosis and inflammatory response, while treatment with disulfiram or necrosulfonamide (NSA) further augmented the inhibitory effects of GSDME siRNA on pyroptosis and inflammatory response. Further studies found HDAC11 formed a complex with ERG and decreased the acetylation levels of ERG. More importantly, ERG knockdown augmented vascular endothelial cell pyroptosis in TNF-α-induced HUVECs. Taken together, our study suggests that HDAC11 might promote both NLRP3/caspase-1/GSDMD and caspase-3/GSDME pathways leading to pyroptosis via regulation of ERG acetylation in HUVECs. Modulation of HDAC11 may serve as a potential target for therapeutic strategies of AS.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. HDAC11 expression is upregulated and pyroptosis is activated in the aorta of HFD-fed ApoE−/− mice.
ApoE−/− mice were fed an HFD for 0, 4, 8, or 12 weeks. A Representative images showing the atherosclerotic lesions of aortic root sections with H&E staining and the lipid deposition of aortic root sections with Oil red O staining, ×100. Scale bar indicates 200 μm. B The atherosclerotic lesion area of aortic root sections was analyzed (n = 3). C GSDMD, GSDMD-N, GSDME, and GSDME-N protein expressions in the aorta were determined by Western blotting (n = 3). D The levels of TNF-α, IL-1β, and IL-18 in the serum were determined by ELISA (n = 10). E, F HDAC11 protein and mRNA expressions in the aorta were determined by Western blotting and quantitative real-time PCR (n = 3). G The expression of HDAC11 in aortic intima by immunofluorescent double staining of the aortic sinus of HDAC11 and CD31. The nuclei were stained blue with DAPI. Scale bar indicates 50 μm. *P < 0.05, **P < 0.01 vs. ND group. Results are expressed as mean ± SD.
Fig. 2
Fig. 2. TNF-α induces pyroptosis and upregulates HDAC11 expression in HUVECs.
A HUVECs were treated with TNF-α (10, 20, 40, 60, or 80 ng/mL) for 12 h. GSDMD, GSDMD-N, GSDME, and GSDME-N protein expressions were determined by Western blotting. B HUVECs were treated with 40 ng/mL TNF-α for 2, 4, 8, 12, or 24 h. GSDMD, GSDMD-N, GSDME, and GSDME-N protein expressions were determined by Western blotting. C The levels of IL-1β, IL-6, and IL-18 in the cellular supernatant were detected by ELISA assay. D The cellular supernatant LDH level was evaluated with a cytotoxicity detection LDH kit. E The representative photographs of double-fluorescent staining with PI (red) and Hoechst 33342 (blue), and the quantification of PI-positive cells, ×200. Scale bar indicates 100 μm. F Immunofluorescence staining was performed to determine the expression and localization of HDAC11, ×200. Scale bar indicates 50 μm. G, H The protein and mRNA expressions of HDAC11 were determined by Western blotting and quantitative real-time PCR. *P < 0.05, **P < 0.01 vs. control group. Results are expressed as mean ± SD (n = 3).
Fig. 3
Fig. 3. Blockade of TNFR1 inhibits pyroptosis and downregulates HDAC11 expression in TNF-α-induced HUVECs.
HUVECs were treated with TNF-α (20, 40, or 80 ng/mL) for 12 h. A, B The expressions of TNFR1 and TNFR2 protein and mRNA were determined by Western blotting and quantitative real-time PCR, respectively. The HUVECs were pretreated with a TNFR1 neutralizing antibody (5 µg/mL) for 30 min and then stimulated with TNF-α (40 ng/mL) for 12 h. C The protein expressions of GSDMD, GSDMD-N, GSDME, and GSDME-N were determined by Western blotting. D The levels of IL-1β, IL-6, and IL-18 in the cellular supernatant were detected by ELISA assay. E The cellular supernatant LDH level was evaluated with a cytotoxicity detection LDH kit. F The representative photographs of double-fluorescent staining with PI (red) and Hoechst 33342 (blue), and the quantification of PI-positive cells, ×100. Scale bar indicates 200 μm. G Immunofluorescence staining was performed to determine the expression and localization of HDAC11, ×200. Scale bar indicates 50 μm. H, I The protein and mRNA expressions of HDAC11 were determined by Western blotting and quantitative real-time PCR. **P < 0.01, vs. NC siRNA group; #P < 0.05, ##P < 0.01 vs. NC siRNA + TNF-α group. Results are expressed as mean ± SD (n = 3).
Fig. 4
Fig. 4. HDAC11 knockdown inhibits pyroptosis in TNF-α-induced HUVECs.
A, B HUVECs were transfected with HDAC11 siRNA or NC siRNA for 48 h, and then the protein and mRNA expressions of HDAC11 were determined by western blotting and quantitative real-time PCR. HUVECs were stimulated with 40 ng/mL TNF-α for 12 h after their transfection with HDAC11 siRNA or NC siRNA. C The protein expressions of GSDMD, GSDMD-N, GSDME, and GSDME-N were determined by Western blotting. D The levels of IL-1β, IL-6, and IL-18 in the cellular supernatant were detected by ELISA assay. E The cellular supernatant LDH level was evaluated with a cytotoxicity detection LDH kit. F The representative photographs of double-fluorescent staining with PI (red) and Hoechst 33342 (blue), and the quantification of PI-positive cells, ×200. Scale bar indicates 100 μm. **P < 0.01, vs. NC siRNA group; #P < 0.05, ##P < 0.01 vs. NC siRNA + TNF-α group. Results are expressed as mean ± SD (n = 3).
Fig. 5
Fig. 5. HDAC11 knockdown suppresses both NLRP3/caspase-1/GSDMD and caspase-3/GSDME pathways in TNF-α-induced HUVECs.
HUVECs were treated with TNF-α (20, 40, or 80 ng/mL) for 12 h. A The expressions of NLRP3, ASC, pro-caspase-1, cleaved caspase-1, pro-caspase-3, and cleaved caspase-3 were determined by Western blotting. B NLRP3 mRNA expression was assayed by quantitative real-time PCR. C Caspase-1 and caspase-3 activity were determined using the Caspase-1 activity assay kit and the Caspase-3 activity assay kit. *P < 0.05, **P < 0.01 vs. control group. D HUVECs were stimulated with 40 ng/mL TNF-α for 12 h after their transfection with HDAC11 siRNA or NC siRNA for 48 h. The expressions of NLRP3, ASC, pro-caspase-1, cleaved caspase-1, pro-caspase-3, and cleaved caspase-3 were determined by Western blotting. E NLRP3 mRNA expression was assayed by quantitative real-time PCR. F Caspase-1 and caspase-3 activity were determined using the Caspase-1 activity assay kit and the Caspase-3 activity assay kit. **P < 0.01 vs. NC siRNA group; #P < 0.05, ##P < 0.01 vs. NC siRNA + TNF-α group. Results are expressed as mean ± SD (n = 3).
Fig. 6
Fig. 6. GSDMD inhibitors and GSDME knockdown inhibit pyroptosis and inflammatory response in TNF-α-induced HUVECs.
A HUVECs were treated with different concentrations of disulfiram and NSA (1.25, 2.5, 5, 10, or 20 μmol/L) for 13 h.The cell viability was evaluated by MTT assay (n = 6). B HUVECs were pretreated with disulfiram or NSA (5 μmol/L) for 1 h and then stimulated with TNF-α for 12 h. The representative photographs of double-fluorescent staining with PI (red) and Hoechst 33342 (blue), and the quantification of PI-positive cells, ×100. Scale bar indicates 200 μm. C The cellular supernatant LDH level was evaluated with a cytotoxicity detection LDH kit. D The levels of IL-1β, IL-6, and IL-18 in the cellular supernatant were detected by ELISA assay. E The mRNA expressions of IL-1β, IL-6, and MCP-1 were determined by quantitative real-time PCR. F, G HUVECs were transfected with GSDME siRNA or NC siRNA for 48 h, and then the protein and mRNA expressions of GSDME were determined by western blotting and quantitative real-time PCR. H HUVECs were pretreated with disulfiram or NSA for 1 h and then incubated with TNF-α for 12 h after their transfection with GSDME siRNA or NC siRNA. The representative photographs of double-fluorescent staining with PI (red) and Hoechst 33342 (blue), and the quantification of PI-positive cells, ×100. Scale bar indicates 200 μm. I The cellular supernatant LDH level was evaluated with a cytotoxicity detection LDH kit. J The levels of IL-1β, IL-6, and IL-18 in the cellular supernatant were detected by ELISA assay. K The mRNA expressions of IL-1β, IL-6, and MCP-1 were determined by quantitative real-time PCR. **P < 0.01 vs. control group or NC siRNA group; #P < 0.05, ##P < 0.01 vs. TNF-α group or NC siRNA + TNF-α group; $P < 0.05, $$P < 0.01 vs. GSDME siRNA + TNF-α group. Results are expressed as mean ± SD (n = 3).
Fig. 7
Fig. 7. HDAC11 formes a complex with ERG and decreases ERG acetylation in HUVECs.
A ApoE−/− mice were fed an HFD for 0, 4, 8, or 12 weeks. ERG protein expression was determined by Western blotting (n = 3). **P < 0.01 vs. ND group. B HUVECs were treated with TNF-α (20, 40, or 80 ng/mL) for 12 h. ERG protein expression was determined by Western blotting. HUVECs were stimulated with 40 ng/mL TNF-α for 12 h after their transfection with HDAC11 siRNA or NC siRNA for 48 h. C, D The expression and localization of ERG were determined by Western blotting and Immunofluorescence staining, ×200. Scale bar indicates 50 μm. E HUVECs were transfected with HDAC11 siRNA or NC siRNA for 48 h. The protein lysates were precipitated with ERG or IgG antibody and then were analyzed by Western blotting. F HUVECs were treated with TNF-α for 12 h. The protein lysates were precipitated with acetylated-lysine antibody and then were analyzed by Western blotting. G HUVECs were transfected with HDAC11 siRNA or NC siRNA for 48 h. The protein lysates of HUVECs transfected with HDAC11 siRNA or NC siRNA were precipitated with acetylated-lysine antibody and then were analyzed by Western blotting. *P < 0.05, **P < 0.01 vs. control group or NC siRNA; #P < 0.05 vs. NC siRNA + TNF-α group. Results are expressed as mean ± SD (n = 3).
Fig. 8
Fig. 8. ERG knockdown further augmented pyroptosis in TNF-α-induced HUVECs.
A, B HUVECs were transfected with ERG siRNA or NC siRNA for 48 h, and the protein and mRNA expressions of ERG were determined by western blotting and quantitative real-time PCR. C HUVECs were stimulated with 40 ng/mL TNF-α for 12 h after their transfection with ERG siRNA or NC siRNA. The protein expressions of GSDMD, GSDMD-N, GSDME, and GSDME-N were determined by Western blotting. D The levels of IL-1β, IL-6, and IL-18 in the cellular supernatant were detected by ELISA assay. E The cellular supernatant LDH level was evaluated with a cytotoxicity detection LDH kit. F The representative photographs of double-fluorescent staining with PI (red) and Hoechst 33342 (blue), and the quantification of PI-positive cells, ×200. Scale bar indicates 100 μm. *P < 0.05, **P < 0.01 vs. NC siRNA group; #P < 0.05 vs NC siRNA + TNF-α group. Results are expressed as mean ± SD (n = 3).
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