Estrogen modulates NFκB signaling by enhancing IκBα levels and blocking p65 binding at the promoters of inflammatory genes via estrogen receptor-β

Abstract

Background: NFκB signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17β-estradiol, E2) inhibits expression of these genes in an estrogen receptor (ER)-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF)-α treated rat aortic smooth muscle cells (RASMCs). This study tested whether E2 inhibits NFκB signaling in RASMCs and defined the mechanisms.

Methodology/principal findings: TNF-α treated RASMCs demonstrated rapid degradation of IκBα (10-30 min), followed by dramatic increases in IκBα mRNA and protein synthesis (40-60 min). E2 enhanced TNF-α induced IκBα synthesis without affecting IκBα degradation. Chromatin immunoprecipitation (ChIP) assays revealed that E2 pretreatment both enhanced TNF-α induced binding of NFκB p65 to the IκBα promoter and suppressed TNF-α induced binding of NFκB p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP)-1 and cytokine-induced neutrophil chemoattractant (CINC)-2β genes. ChIP analyses also demonstrated that ERβ can be recruited to the promoters of MCP-1 and CINC-2β during co-treatment with TNF-α and E2.

Conclusions: These data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of IκBα, thus accelerating a negative feedback loop in NFκB signaling, and directly inhibiting binding of NFκB to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NFκB signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury.

Figure 1. Representative Western blots of phospho-IκBα and IκBα in E2±TNF-α treated RASMCs.

Cells were pretreated with/without E2 (10⁻7 M) for 24 hrs then stimulated with TNF-α (1 ng/mL) for the times shown (A). Line graph shows the ratio of IκBα to β-actin in E2±TNF-α treated RASMCs (B). Results are mean±SE from 3 samples/group. #p<0.05 vs. TNF-α-treated RASMCs.

Figure 2. IκBα mRNA expression measured by real-time RT-PCR and normalized using 18 S rRNA.

Cells were pretreated with/without E2 (10⁻⁷ M) for 24 hrs then stimulated with TNF-α (1 ng/mL) for the times shown. Results are mean±SEM from 6 wells/group. *p<0.05 vs. Vehicle-treated RASMCs; #p<0.05 vs. TNF-α-treated RASMCs.

Figure 3. Role of ER isoforms on IκBα protein level.

A. Pretreatment of ERβ agonist DPN (10⁻⁷ M) or E2 enhanced IκBα protein level in response to TNF-α treatment compared to TNF-α alone; ERα antagonist MMP (10⁻⁶ M) did not block the effect of E2 in TNF-α-treated cells. B. Pretreatment of ERα agonist PPT (10⁻⁷ M) did not affect IκBα protein level in response to TNF-α treatment compared to TNF-α alone; C. ERβ antagonist R,R- THC (10⁻⁶ M) blocked the effect of E2 in TNF-α-treated cells. Cells were pretreated with E2, DPN, PPT or vehicle for 24 h, then treated with TNF-α (1 ng/ml) for an additional 45 min. In some experiment groups, cells were pretreated with THC or MPP for 1 hr prior of E2. Bar graph shows the densitometric analysis of relative IκBα expression normalized to to β-actin Level. Results are mean±SE from 6 samples/group. *p<0.05 vs. Vehicle-treated RASMCs; #p<0.05 vs. TNF-α-treated RASMCs.

Figure 4. IκBα mRNA expression measured by real-time RT-PCR and normalized using 18 S rRNA.

Cells were pretreated with E2 (10⁻⁷ M), DPN (10⁻⁷ M), PPT (10⁻⁷ M) or vehicle for 24 hr, then treated with TNF-α (1 ng/ml) for an additional 1 hr. MPP (10⁻⁶ M) or THC (10⁻⁶ M) was given to cells at 1 h before E2 treatment in some experiments. Results are mean±SEM from 6–9 wells/group. *p<0.05 vs. Vehicle-treated RASMCs; #p<0.05 vs. TNF-α-treated RASMCs.

Figure 5. ChIP assays of the binding of NFκB p65 (A), ERβ (B) and AcH4 (C) to the IκBα promoter.

Cells were pretreated with/without E2 (10⁻⁷ M) or DPN (10⁻⁷ M) for 24 hrs and then stimulated with TNF-α (1 ng/mL) for 1 hr. THC (10⁻⁶ M) was given to cells at 1 h before E2 treatment in some experiments. ChIP samples were prepared as described in the text and analyzed using antibodies specific for p65, ERβ or AcH4. The immunoprecipitated DNA fragments and input DNA were analyzed by real-time PCR. The y axis shows values were normalized to input DNA with values for vehicle treatment defined as 1. The numbers represent the mean±SEM from three experiments repeated in duplicate. *p<0.05 vs. Vehicle-treated RASMCs; #p<0.05 vs. TNF-α-treated RASMCs.

Figure 6. ChIP assays of binding of NFκB p65, ERβ and AcH3 to the MCP-1 and CINC-2β promoters.

Cells were pretreated without or with E2 for 24 hrs, then stimulated with TNF-α (1 ng/mL) for 1 hr. ChIP samples were prepared as described in the text and analyzed using antibodies specific for p65, ERβ or AcH3. The immunoprecipitated DNA fragments and input DNA were analyzed by by real-time PCR. The y axis shows values were normalized to input DNA with values for vehicle treatment defined as 1. The numbers represent result from three experiments repeated in duplicate. *p<0.05 vs. Vehicle-treated RASMCs; #p<0.05 vs. TNF-α-treated RASMCs.

Figure 7. E2 inhibited TNF-α-induced MCP-1 and CINC-2β mRNA expression in RASMCs through ERβ.

Cells were grown to subconfluence (≈95%) in 6-well plates, deprived of serum for 24 hrs, pretreated with E2 (10⁻⁷ M), DPN (10⁻⁷ M) or vehicle for 24 h, and then treated with TNF-α (1 ng/ml) for an additional 1 hr. MPP (10⁻⁶ M), or R, R-THC (10^–6 M) was given to cells at 1h before E2 treatment in some experiments. Data, expressed as means±SEM, are from real-time quantitative RT-PCR assays and are normalized by 18 S RNA. Data for MCP-1 and CINC-2β are standardized to the mean mRNA level of the TNF-α-treated RASMCs. *p<0.05 vs. respective vehicle-treated RASMCs; #p<0.05 vs. respective TNF-α-treated RASMCs.