Interleukin-1β mediates the effect of macrophage androgen receptor on hyperphosphate-induced vascular smooth muscle cell calcification
10.3760/cma.j.cn441217-20210408-00060
- VernacularTitle:巨噬细胞雄激素受体通过调控白细胞介素1β基因表达影响高磷诱发的血管平滑肌细胞钙化
- Author:
Haiyan PANG
1
;
Zhi LU
;
Longfei XIAO
;
Haiyan CHEN
;
Zhiqun SHANG
;
Ning JIANG
;
Xiaojuan WANG
;
Fang WEI
;
Aili JIANG
;
Lin WANG
;
Yuanjie NIU
Author Information
1. 天津医科大学第二医院肾脏病血液净化科，天津 300211
- Keywords:
Receptors, androgen;
Macrophages;
Myocytes, smooth muscle;
Interleukin 1β;
Inflammation
- From:
Chinese Journal of Nephrology
2022;38(5):420-427
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate whether it is by regulating interleukin 1β ( IL-1β) gene expression that androgen receptor (AR) in macrophages affects hyperphosphate-induced vascular smooth muscle cell calcification. Methods:The chromatin immunoprecipitation (ChIP) experiment was used to determine whether AR was bound to the androgen receptor element (ARE) sequence of IL-1β promoter in THP-1 cells. Whether the AR regulated IL-1β gene expression was detected by luciferase assay experiments. AR of THP-1 cells was silenced and transfected by lentivirus with vector or shRNA. Flow cytometry was used to select positive transfected cells THP-1ARsc (control) and THP-1ARsi (AR silencing) with fluorescent markers. Western blotting was used to detect AR protein levels of THP-1ARsc (control) and THP-1ARsi cells (AR silencing in monocytes). Macrophages MФARsc (control) or MФARsi (AR silencing) were induced by 50 ng/ml phorbol ester. Enzyme-linked immunosorbent assay was used to detect IL-1β expression levels of MФARsc or MФARsi conditioned medium. The human aortic smooth muscle cells (HASMC) were cultured in MФARsc or MФARsi conditioned medium with phosphate (2.5 mmol/L final concentration of sodium dihydrogen phosphate), and Alizarin red S staining was used to analyze HASMC calcification degree. Western blotting was used to detect the expression levels of RUNX2 (osteoblast marker) and SM22α (HASMC marker), and neutralization assay was performed to test IL-1β-mediating effect of macrophages AR on HASMC calcification. Results:AR was bound to ARE sequence of IL-1β promoter and regulated IL-1β gene expression. The expression level of IL-1β protein in conditioned medium of MФARsi cells decreased significantly compared to MФARsc cells ( P<0.001). Compared with MФARsc conditioned medium group, HASMC calcium deposition in MФARsi conditioned medium group decreased significantly, RUNX2 protein decreased and SM22α protein increased (all P<0.05). The degree of HASMC calcification in the MФARsi conditioned medium+IgG antibody group decreased than that in the MФARsc conditioned medium+IgG antibody group significantly, and the degree of HASMC calcification in the MФARsc conditioned medium+IL-1β antibody group decreased significantly than that in the MФARsc conditioned medium+IgG antibody group; while the degree of HASMC calcification in the MФARsi conditioned medium+IgG antibody group and MФARsi conditioned medium+IL-1β antibody group decreased than that in the MФARsc conditioned medium+IL-1β antibody group (all P<0.05). Conclusions:Macrophage AR regulates IL-1β expression by binding to ARE sequence within IL-1β promoter, and IL-1β mediates the effect of macrophage AR on hyperphosphate-induced HASMC calcification.
