Estrogen receptor β-dependent Notch1 activation protects vascular endothelium against tumor necrosis factor α (TNFα)-induced apoptosis - PubMed (original) (raw)
Estrogen receptor β-dependent Notch1 activation protects vascular endothelium against tumor necrosis factor α (TNFα)-induced apoptosis
Francesca Fortini et al. J Biol Chem. 2017.
Abstract
Unlike age-matched men, premenopausal women benefit from cardiovascular protection. Estrogens protect against apoptosis of endothelial cells (ECs), one of the hallmarks of endothelial dysfunction leading to cardiovascular disorders, but the underlying molecular mechanisms remain poorly understood. The inflammatory cytokine TNFα causes EC apoptosis while dysregulating the Notch pathway, a major contributor to EC survival. We have previously reported that 17β-estradiol (E2) treatment activates Notch signaling in ECs. Here, we sought to assess whether in TNFα-induced inflammation Notch is involved in E2-mediated protection of the endothelium. We treated human umbilical vein endothelial cells (HUVECs) with E2, TNFα, or both and found that E2 counteracts TNFα-induced apoptosis. When Notch1 was inhibited, this E2-mediated protection was not observed, whereas ectopic overexpression of Notch1 diminished TNFα-induced apoptosis. Moreover, TNFα reduced the levels of active Notch1 protein, which were partially restored by E2 treatment. Moreover, siRNA-mediated knockdown of estrogen receptor β (ERβ), but not ERα, abolished the effect of E2 on apoptosis. Additionally, the E2-mediated regulation of the levels of active Notch1 was abrogated after silencing ERβ. In summary, our results indicate that E2 requires active Notch1 through a mechanism involving ERβ to protect the endothelium in TNFα-induced inflammation. These findings could be relevant for assessing the efficacy and applicability of menopausal hormone treatment, because they may indicate that in women with impaired Notch signaling, hormone therapy might not effectively protect the endothelium.
Keywords: Akt; ERβ; Notch pathway; Notch1; apoptosis; endothelial dysfunction; estrogen; estrogen receptor; tumor necrosis factor (TNF).
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Conflict of interest statement
The authors declare that they have no conflicts of interest with the contents of this article.
Figures
Figure 1.
Effect of E2, TNFα, and DAPT treatments on Notch receptors and ligands in HUVECs. A, HUVECs were treated for 24 h with DMSO (CTRL), E2 (1 n
m
), TNFα (10 ng/ml), or DAPT (5 μ
m
), alone and in combination. Cell lysates were electrophoresed and immunoblotted with antibodies for total Notch1 (C20), cleaved Notch1 (Val-1744), active Notch2 (clone C651.6DbHN), active Notch4 (H-225), Jagged1, and Dll4 to detect the precursor (Notch1PC), the transmembrane (Notch1TM) and the active form of Notch1 (Notch1ICD), the active form of Notch2 (Notch2ICD), the active form of Notch4 (Notch4ICD), the ligands Jagged1 and Dll4. β-Actin antibody was used to ensure equal loading. Densitometry analyses are shown in
supplemental Fig. S1
. B, HUVECs were treated for 24 h with DMSO (CTRL), TNFα (10 ng/ml), or E2 (1 n
m
) alone and in combination. Total RNA was extracted and qRT-PCR analysis of Notch1, Notch2, Notch4, Jagged1, and Dll4 genes expression was performed. Relative changes in mRNA expression levels were calculated according to the 2−ΔΔ_Ct_ method using RPL13A as reference gene. Results are expressed as mean ± S.D. of three independent experiments, each performed in triplicate. **, p < 0.01; ***, p < 0.001 (pairwise comparison between plus or minus TNFα).
Figure 2.
Effect of E2 and DAPT treatments on TNFα-induced HUVECs apoptosis. A, HUVECs treated for 24 h with DMSO (CTRL), E2 (1 n
m
), TNFα (10 ng/ml), or DAPT (5 μ
m
), alone and in combination, were stained with Annexin V and PI and cytometric analysis was performed. Representative Annexin V-PI plots are shown for each treatment. B, percentage of apoptotic cells (ratio of Annexin V-positive cells/total cells) is shown. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; ***, p < 0.001.
Figure 3.
TNFα-induced apoptosis in Notch1-silenced HUVECs. A, HUVECs were treated for 48 h with siRNA against Notch1. Cell lysates were electrophoresed and immunoblotted with antibody for total Notch1 (C20). β-Actin was used to ensure equal loading. qRT-PCR analyses were performed to detect reduction of Notch1 mRNA levels in HUVECs after siRNA against Notch1 treatment for 48 h. Scrambled siRNA was used as control. Relative changes in mRNA expression levels were calculated according to the 2−ΔΔ_Ct_ method using RPL13A as reference gene. Results are expressed as mean ± S.D. of three independent experiments, each performed in triplicate. ***, p < 0.001. B, HUVECs were transfected with siRNA against Notch1 and, 24 h later, they were treated with TNFα (10 ng/ml) and E2 (1 n
m
), alone, and in combination, for 24 h. Cells were stained with Annexin V and PI, then cytometric analysis was performed. Representative Annexin V-PI plots are shown for each treatment. C, percentage of apoptotic cells (ratio of Annexin V-positive cells/total cells) is shown. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01.
Figure 4.
TNFα-induced apoptosis in Notch1 overexpressing HUVECs. A, HUVECs were transfected with a plasmid encoding the Notch1 intracellular domain (Notch1ICD) and, 24 h later, were treated with TNFα (10 ng/ml) for 24 h. The empty vector (pcDNA3) was used as a control. Lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val-1744) antibody. β-Actin antibody was used to ensure equal loading. B, HUVECs transfected for 24 h were treated for 24 h with TNFα (10 ng/ml), stained with Annexin V and PI, then cytometric analysis was performed. Representative Annexin V-PI plots are shown for each treatment. C, the histogram shows the percentage of apoptotic cells, represented as ratio of Annexin V-positive cells/total cells. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05.
Figure 5.
Role of E2 and Notch1 on TNFα regulation of Akt phosphorylation in HUVECs. A and B, Western blotting and densitometry of HUVECs treated with TNFα (10 ng/ml) for 1, 2, or 24 h in the presence of E2 (1 n
m
). Cells treated with DMSO were used as control (CTRL). Lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val-1744), pAkt (Ser-473), and total Akt antibodies. β-Actin antibody was used to ensure equal loading. B, graphs show protein levels after the indicated treatments normalized to untreated control levels, after signal comparison to β-actin expression. Phosphorylated Akt was normalized to the total Akt level. Results are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (comparison between TNFα treatment and corresponding control); °, p < 0.05; °°, p < 0.01; °°°, p < 0.001 (pairwise comparison between plus or minus E2). C and D, Western blotting and densitometry of HUVECs treated with TNFα (10 ng/ml) for 1, 2, or 24 h in the presence of DAPT (5 μ
m
). Cells treated with DMSO were used as control (CTRL). Lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val-1744), pAkt (Ser-473), and total Akt antibodies. β-Actin antibody was used to ensure equal loading. Graphs show protein levels after the indicated treatments normalized to untreated control levels, after signal comparison to β-actin expression. Phosphorylated Akt was normalized to the total Akt level. Results are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; ***, p < 0.001 (comparison between TNFα treatment and corresponding control); °, p < 0.05; °°°, p < 0.001 (pairwise comparison between plus or minus DAPT). E and F, Western blotting and densitometry of HUVECs treated with TNFα (10 ng/ml) for 1, 2, or 24 h in the presence of E2 (1 n
m
) or E2 (1 n
m
)/DAPT (5 μ
m
). Cells treated with DMSO were used as control. Lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val-1744), pAkt (Ser-473), and total Akt antibodies. β-Actin was used to ensure equal loading. Graphs show protein levels after the indicated treatments were normalized to untreated control levels, after signal comparison to β-actin expression. Phosphorylated Akt was normalized to the total Akt level. Results are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (comparison between TNFα treatment and corresponding control); °, p < 0.05; °°°, p < 0.001 (pairwise comparison between E2 and E2 plus DAPT treatment).
Figure 6.
Effect of wortmannin on TNFα-induced apoptosis in HUVECs. A, HUVECs were treated for 24 h with wortmannin (100 n
m
). Lysates were electrophoresed and immunoblotted with pAkt (Ser-473) and total Akt antibodies. β-Actin antibody was used to ensure equal loading. B, HUVECs treated for 24 h with DMSO (CTRL), 17β-estradiol (E2, 1 n
m
), and TNFα (10 ng/ml) in the presence or absence of wortmannin (100 n
m
), were stained with Annexin V and PI and then cytometric analysis was performed. Representative Annexin V-PI plots are shown for each treatment. C, percentage of apoptotic cells (ratio of Annexin V-positive cells/total cells) is shown. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; ***, p < 0.001.
Figure 7.
Role of estrogen receptor β on Notch1 activation and protection against TNFα-mediated apoptosis in HUVECs. A, HUVECs were transfected with siRNA against ERα and ERβ and, 24 h later, they were treated with E2 (1 n
m
) and TNFα (10 ng/ml), alone and in combination, stained with Annexin V and PI and then cytometric analysis was performed. Representative Annexin V-PI plots are shown for each treatment. B, the histogram depicts the percentage of apoptotic cells as ratio of Annexin V-positive cells/total cells. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01. C, HUVECs were transfected with siRNA against ERα or ERβ for 48 h. Lysates were electrophoresed and immunoblotted with ERα and ERβ antibodies, respectively. β-Actin antibody was used to ensure equal loading. D, Western blot analysis for cleaved Notch1 (Val-1744) in HUVECs after the transfection with siRNA against ERβ and TNFα- (10 ng/ml) and 17β-estradiol (E2, 1 n
m
) treatments for 24 h. β-Actin antibody was used to ensure equal loading. Densitometric analyses are shown in
supplemental Fig. S2_A_
. E, HUVECs were treated for 24 h with E2 (1 n
m
), TNFα (10 ng/ml), and DAPT (5 μ
m
), alone and in combination. Lysates were electrophoresed and immunoblotted with ERβ or ERα antibodies. β-Actin antibody was used to ensure equal loading. Densitometric analyses are shown in
supplemental Fig. S2_B_
.
Figure 8.
Effect of DPN and PPT on TNFα-induced apoptosis in HUVECs. A, HUVECs were treated for 24 h with 17β-estradiol (E2, 1 n
m
), DPN (ERβ agonist, 10 n
m
), and PPT (ERα agonist, 10 n
m
). Lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val-1744) antibody. β-Actin antibody was used to ensure equal loading. B, densitometric analysis. Graphs show protein levels after the indicated treatments normalized to untreated control levels, after signal comparison to β-actin expression. Results are expressed as mean ± S.D. of three independent experiments. ***, p < 0.001 (comparison between control and agonist treatment). C and D, HUVECs treated for 24 h with DMSO (CTRL), DPN (10 n
m
), TNFα (10 ng/ml), DAPT (5 μ
m
), alone and in combination, were stained with Annexin V and PI and then cytometric analysis was performed. Representative Annexin V-PI plots are shown for each treatment. The histogram depicts the percentage of apoptotic cells (ratio of Annexin V-positive cells/total cells) following treatment as shown. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; ***, p < 0.001. E and F, HUVECs treated for 24 h with DMSO (CTRL), PPT (10 n
m
), TNFα (10 ng/ml), DAPT (5 μ
m
), alone and in combination, were stained with Annexin V and PI and then cytometric analysis was performed. Representative Annexin V-PI plots are shown for each treatment. The histogram depicting the percentage of apoptotic cells (ratio of Annexin V-positive cells/total cells) following treatment is shown. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05.
Figure 9.
Effect of PHTPP on TNFα-induced apoptosis, Notch1 activation, and Akt phosphorylation in HUVECs. A, HUVECs treated for 24 h with DMSO (CTRL), E2 (1 n
m
), PHTPP (1 μ
m
), and TNFα (10 ng/ml) alone and in combination, were stained with Annexin V and PI and then cytometric analysis was performed. Representative Annexin V-PI plots are shown for each treatment. B, percentage of apoptotic cells (ratio of Annexin V-positive cells/total cells) following treatment is shown. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; ***, p < 0.001. C, Western blotting analysis of HUVECs treated with DMSO (CTRL), 17β-estradiol (E2,1 n
m
), PHTPP (1 μ
m
), and TNFα (10 ng/ml). Lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val-1744), pAkt (Ser-473), and total Akt antibodies. β-Actin antibody was used to ensure equal loading. D, densitometric analysis. Graphs show protein levels after the indicated treatments were normalized to untreated control levels, after signal comparison to β-actin expression. Phosphorylated Akt was normalized to the total Akt level. Results are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01 (comparison between treatments and corresponding control); °°, p < 0.01; °°°, p < 0.001 (pairwise comparison between plus or minus TNFα treatment).
References
- Nichols M., Townsend N., Scarborough P., and Rayner M. (2014) Cardiovascular disease in Europe 2014: epidemiological update. Eur. Heart J. 35, 2929. - PubMed
- Jensen H. A., and Mehta J. L. (2016) Endothelial cell dysfunction as a novel therapeutic target in atherosclerosis. Expert. Rev. Cardiovasc. Ther. 14, 1021–1033 - PubMed
- Sitia S., Tomasoni L., Atzeni F., Ambrosio G., Cordiano C., Catapano A., Tramontana S., Perticone F., Naccarato P., Camici P., Picano E., Cortigiani L., Bevilacqua M., Milazzo L., Cusi D., et al. (2010) From endothelial dysfunction to atherosclerosis. Autoimmun. Rev. 9, 830–834 - PubMed
- Florian M., and Magder S. (2008) Estrogen decreases TNF-alpha and oxidized LDL induced apoptosis in endothelial cells. Steroids 73, 47–58 - PubMed
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