17β-Estradiol Promotes Apoptosis in Airway Smooth Muscle Cells Through CD38/SIRT1/p53 Pathway

Abstract

17β-Estradiol (E2) is the major estrogen secreted by the premenopausal ovary and shows dual effects on cell apoptosis under pathological conditions. E2 was previously shown to increase CD38 mRNA and protein expression in myometrial smooth muscle, but its function and mechanism remain largely unknown. Here we investigated the role of E2 in hypoxia-induced apoptosis in mouse airway smooth muscle cells (ASMCs) and explored the underlying mechanisms. Results showed that E2 significantly increased CD38 expression at both mRNA and protein levels, accompanied with decreased SIRT1 levels in ASMCs. By using primary ASMCs from the wild type (WT) and the smooth muscle-specific CD38 knockout (CD38 KO) mice, we found that the down-regulation of SIRT1 induced by E2 was abolished in CD38 KO AMSCs. E2 promoted the acetylation of p53 in WT cells, and this effect was also diminished in the absence of CD38. In addition, E2 further activated CD38/SIRT1/p53 signal pathway and promoted cell apoptosis during hypoxia. However, these effects were reversed in CD38 KO ASMCs and by the specific SIRT1 activator Resveratrol. We also found that E2 enhanced CD38 expression through estrogen receptor. The data suggested that CD38 is a direct target for E2 which promotes hypoxia-induced AMSC apoptosis through SIRT1/p53 signal pathway.

Keywords: 17β-estradiol; CD38; SIRT1; apoptosis; hypoxia.

Figure 1

Expression of CD38 and SIRT1 in ASMCs after E2 treatment. ASMCs were pre-treated with the indicated concentrations of E2 for 24 h. (A) CD38 and (B) SIRT1 mRNA levels were detected by real-time PCR. (C) CD38 and SIRT1 protein levels were determined by western blot and quantitative analysis of (D) CD38 and (E) SIRT1 levels was normalized to GAPDH levels. *P < 0.05, **P < 0.01 vs. the control group. N = 3.

Figure 2

Time course of CD38 and SIRT1 expression in ASMCs with E2 treatment. ASMCs were pre-treated with 10 or 100 nM of E2 for 24 h and 48 h respectively. (A) CD38 and (B) SIRT1 mRNA levels were detected by real-time PCR. (C) CD38 and SIRT1 protein levels were determined by western blot and quantitative analysis of (D) CD38 and (E) SIRT1 levels was normalized to GAPDH levels. *P < 0.05, **P < 0.01 vs. the control group. N = 3.

Figure 3

The effects of E2 on SIRT1/p53 signal pathway in WT and CD38 KO ASMCs. (A) CD38, SIRT1, p53, and Ac-p53 levels were determined by western blot. Quantitative analysis of (B) CD38 and (C) SIRT1 levels was normalized to GAPDH levels, and (D) Ac-p53 levels were normalized to total p53 levels. *P < 0.05, **P < 0.01 vs. the WT control group. ^##P < 0.01 vs. the E2-treated WT group. N = 3.

Figure 4

The effects of E2 on SIRT1/p53 signal pathway in WT and CD38 KO ASMCs after hypoxia exposure. (A) CD38 and (B) SIRT1 mRNA levels were detected by real-time PCR. (C) CD38, SIRT1, p53, and Ac-p53 levels were determined by western blot. Quantitative analysis of (D) CD38 and (E) SIRT1 levels was normalized to GAPDH levels and (F) Ac-p53 levels were normalized to total p53 levels. *P < 0.05, **P < 0.01 vs. the WT control group; ^#P < 0.05, ^##P < 0.01 vs. the corresponding WT group. N = 3.

Figure 5

The effects of E2 on apoptosis in WT and CD38 KO ASMCs after hypoxia exposure. (A) Representative images of Hoechst 33258 staining in ASMCs. Scale bar, 50 μm. (B) Quantitative analysis of apoptosis expressed as the percentage of total cell count. (C) Bax and Bcl-2 levels were determined by western blot and (D) the Bax/Bcl-2 ratio were quantitatively analyzed. (E) The activity of caspase-3 was measured by colorimetry. **P < 0.01 vs. the WT control group; ^#P < 0.05, ^##P < 0.01 vs. the WT hypoxia group. N = 3.

Figure 6

The effects of combined treatment with E2 and SIRT1 activator Resveratrol (RSV) on apoptosis after hypoxia exposure. RSV (10 μM) was added to ASMCs for 2 h incubation followed by 24 h of E2 treatment. (A) Bax and Bcl-2 levels were determined by western blot and (B) the Bax/Bcl-2 ratio were quantitatively analyzed. (C) The activity of caspase-3 was measured by colorimetry. *P < 0.05, **P < 0.01 vs. the control group; ^#P < 0.05, ^##P < 0.01 vs. the E2-treated group under hypoxia. N = 3.

Figure 7

The effects of estrogen receptor antagonist on CD38 expression. The estrogen receptor antagonist ICI182,780 (ICI, 10 nM) was added to ASMCs for 2 h incubation followed by 24 h of E2 treatment. (A) CD38 mRNA levels were detected by real-time PCR. (B) CD38 protein levels were determined by western blot and quantitative analysis of CD38 levels was normalized to GAPDH levels. *P < 0.05, **P < 0.01 vs. the control group; ^#P < 0.05, ^##P < 0.01 vs. the E2-treated group. N = 3.