Signal transducer and activator of transcription 3-stimulated hypoxia inducible factor-1alpha mediates estrogen receptor-alpha-induced mesenchymal stem cell vascular endothelial growth factor production

Abstract

Objective: Vascular endothelial growth factor, a critical factor in angiogenesis, mediates stem cell paracrine protective effects on ischemic myocardium. Studies on the role of sex in stem cell function have demonstrated that female mesenchymal stem cells produce greater vascular endothelial growth factor and provide better cardiac protection compared with male mesenchymal stem cells. The purpose of this study was to determine the mechanisms by which estrogen affects mesenchymal stem cell function as a potential therapeutic measure during ex vivo expansion, before therapeutic use.

Methods: A single-step purification method using adhesion to cell culture plastic was adopted to isolate mesenchymal stem cells from wild-type, estrogen receptor-alpha knockout, estrogen receptor-beta knockout, and signal transducer and activator of transcription 3 knockout mice. Mesenchymal stem cells were treated with or without 17beta-estradiol, estrogen receptor-alpha agonist (propyl pyrazoletriol), and estrogen receptor-beta agonist (diarylpropionitrile).

Results: 17beta-estradiol significantly increased mesenchymal stem cell vascular endothelial growth factor production in a dose-dependent manner. Both estrogen receptor-alpha and estrogen receptor-beta were expressed in mesenchymal stem cells. Administration of 17beta-estradiol or estrogen receptor-alpha agonist (not estrogen receptor-beta agonist) elevated mesenchymal stem cell vascular endothelial growth factor, hypoxia inducible factor-1alpha expression, and signal transducer and activator of transcription 3 activation. However, these effects were neutralized in estrogen receptor-alpha knockout mesenchymal stem cells, not estrogen receptor-beta knockout. Signal transducer and activator of transcription 3 knockout abolished estrogen receptor-alpha-induced hypoxia inducible factor-1alpha and subsequent vascular endothelial growth factor production.

Conclusion: 17beta-estradiol-induced vascular endothelial growth factor production from mesenchymal stem cells appears to be mediated through estrogen receptor-alpha-activated signal transducer and activator of transcription 3-mediated hypoxia inducible factor-1alpha expression.

Figure 1

(A): 17β-estradiol (E2) increases MSC production of VEGF in a dose-dependant manner (1, 10, 100, 1000 nM and 10 μM) (24-hour incubation). (B): Expression of estrogen receptor (ER)α and ERβ in mesenchymal stem cells (MSCs). Nuclear extracts from 6 individual samples are analyzed by western blot. Results are Mean ± SEM (the experiment was repeated on three separate occasions), *p<0.01 vs. control (0), #p<0.01 vs. 1nM of E2, $p<0.01 vs. 10nM of E2.

Figure 2

The nuclear levels of HIF-1α are increased by administration of 17β-estradiol (E2) or the ERα agonist-PPT (ERα), but not the ERβ agonist-DPN (ERβ) in wild type msenchymal stem cells (MSCs). (A): nuclear HIF-1α expression at 30 minute after E2, ERα agonist and ERβ agonist treatment. (B): the nuclear levels of HIF-1α at 1 hour after administration of E2, the ERα agonist and the ERβ agonist. Shown are representative immunoblots. Bar graph indicates relative density (%) of HIF-1α compared to nuclear loading control TATA binding protein (TBP). (Mean ± SEM, *p<0.05 vs. control)

Figure 3

(A): effects of E2, the ERα agonist or the ERβ agonist on STAT3 activation. Increased STAT3 activation is noted within 15 minute and 30 minute after E2 or ERα agonist treatment and this high level of p-STAT3 is maintained until 2 hours. Shown are representative immunoblots. (B): basal levels of STAT3 activation in MSCs from WT, ERαKO and ERβKO mice (2 lanes/group). (C): E2 or the ERα agonist does not induce STAT3 activation in ERαKO MSCs (2 lanes/group). Bar graph indicates relative density of p-STAT3 compared to total STAT3 (%). (Mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 vs. control or WT, #p<0.01 vs. ERβKO)

Figure 3

(A): effects of E2, the ERα agonist or the ERβ agonist on STAT3 activation. Increased STAT3 activation is noted within 15 minute and 30 minute after E2 or ERα agonist treatment and this high level of p-STAT3 is maintained until 2 hours. Shown are representative immunoblots. (B): basal levels of STAT3 activation in MSCs from WT, ERαKO and ERβKO mice (2 lanes/group). (C): E2 or the ERα agonist does not induce STAT3 activation in ERαKO MSCs (2 lanes/group). Bar graph indicates relative density of p-STAT3 compared to total STAT3 (%). (Mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 vs. control or WT, #p<0.01 vs. ERβKO)

Figure 4

Increased nuclear levels of HIF-1α by E2 or the ERα agonist are absent in STAT3KO MSCs. (A): nuclear HIF-1α expression at 30 minute after E2, ERα and ERβ treatment in STAT3KO MSCs. (B): the nuclear levels of HIF-1α at 1 hour after administration of E2, ERα and ERβ agonists in STAT3KO MSCs. Shown are representative immunoblots. Bar graph indicates relative density (%) of HIF-1α compared to nuclear loading control TATA binding protein (TBP). (Mean ± SEM)

Figure 5

Protein and mRNA levels of VEGF in mesenchymal stem cells (MSCs) from wild type (WT) and STAT3KO mice after administration of 17β-estradiol (E2), the ERα agonist-PPT (ERα) and the ERβ agonist-DPN (ERβ). E2 or the ERα agonist increases VEGF secretion at 6-hour (A) and 24-hour treatment (C), as well as VEGF mRNA (E) in WT MSCs. However, STAT3KO abolishes E2- or ERα-induced VEGF production either 6-hour (B) or 24-hour treatment (D), as well as VEGF gene expression (F) (Mean ± SEM, *p<0.05 vs. control)

Figure 6

A model for the mechanism of VEGF production mediated by estrogen receptor (ER) α.