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Vascular endothelial growth factor (VEGF), 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 (MSCs) produce greater VEGF and provide better cardiac protection compared to male MSCs. The purpose of this study was to determine the mechanisms by which estrogen affects MSC stem cell function as a potential therapeutic measure during ex vivo expansion, prior to therapeutic use.
A single-step purification method using adhesion to cell culture plastic was adopted to isolate MSCs from wild type, estrogen receptor alpha (ERα) knockout (KO), ERβKO and signal transducer and activator of transcription (STAT)3 KO mice. MSCs were treated with or without 17β-estradiol (E2), ERα agonist (propyl pyrazoletriol) and ERβ agonist (diarylpropionitrile), respectively. E2 significantly increased MSC VEGF production in a dose-dependant manner. Both ERα and ERβ were expressed in MSCs. Administration of E2 or ERα agonist (not ERβ agonist) elevated MSC VEGF, hypoxia inducible factor (HIF)-1α expression and STAT3 activation. However these effects were neutralized in ERαKO MSC, not ERβKO. STAT3KO abolished ERα-induced HIF-1α and subsequent VEGF production.
E2-induced VEGF production from MSCs appears to be mediated through ERα-activated STAT3 mediated HIF-1α expression.
A rapidly growing body of evidence indicates that stem cells may exert their beneficial effects on the injured heart in part by paracrine actions (1–3). A number of in vitro and in vivo experiments have suggested that vascular endothelial growth factor (VEGF), a growth factor with potent angiogenic, chemotactic and vasodilatory effects, is one important mediator of these cardioprotective paracrine actions of stem cells (4–7). Additional studies have established that bone marrow cells genetically modified to overexpress VEGF appear to be even more effective for cardiac repair (6). Furthermore, our recent study has shown that decreasing VEGF production by MSCs with the use of siRNA abolishes their ability to protect myocardial function following acute ischemia/reperfusion (I/R) injury (2). Taken together, these data highlight the need for understanding mechanisms of MSC VEGF production so that its production may be increased and the therapeutic effectiveness of stem cells may thereby be enhanced.
Sex differences exist in MSC paracrine growth factor production. Female MSCs produce greater levels of VEGF compared to males in response to toxic stimuli in vitro, and may also provide better cardioprotection (8). In addition, 17β-estradiol (E2) pretreatment of MSCs has been reported to increase MSC VEGF production and may also improve MSC-mediated cardiac protection (9). These results indicate that estrogen may have a favorable effect on MSC function. Additionally, estrogen has been shown to regulate cell function in a variety of stem/progenitor cells, including mesenchymal stem cell-derived osteoblasts, human endothelial progenitor cells and embryonic cortical progenitor cells (10–13). Furthermore, estrogen treatment also upregulates VEGF production by differentiated cells (14, 15). However, the mechanisms by which E2 mediates VEGF production in MSCs remain unknown.
Accumulating evidence demonstrates that hypoxia-inducible factor 1α (HIF-1α) plays a critical role in mediating VEGF expression in a wide range of cell types in vitro (16–18). A number of growth factors, cytokines, and hormones have been reported to activate HIF-1α-induced VEGF production (14, 16). More importantly, HIF-1 mediates prostaglandin E2-induced VEGF gene expression in cancer cells, suggesting that estrogen may mediate HIF-1 induced VEGF expression (17). It is also known that estrogen initiates its biological activity by binding to estrogen receptor alpha (ERα) and/or estrogen receptor beta (ERβ). Activated ER has been reported to induce HIF-1α activation and thereby, stimulates VEGF-mediated angiogenesis in osteoblasts (19). In addition, estrogen induces VEGF production in epithelial cells through activation of HIF-1α and its recruitment to the VEGF promotor by ERα (14). Surprisingly, no information exists regarding the effects of estrogen or its associated receptors on HIF-1α-mediated VEGF production in MSCs. It is known, however, that activation of HIF-1α by hypoxia may be linked to the signal transducer and activator of transcription-3 (STAT3) pathway (20, 21). Estrogen-activated STAT3 has also been noted to mediate physiological and pathological processes in several cell types (22–24). Therefore, an investigation into whether estrogen induces HIF-1α-mediated VEGF production through activation of STAT3 in MSCs is worthwhile.
In the present study, we examined the effects and mechanisms of action of E2 on VEGF production by MSCs. We hypothesized that E2 would increase the stem cell release of VEGF by ERα-induced STAT3 activation and subsequent HIF-1α expression.
C57BL/6J mice with and without targeted deletion or mutation of estrogen receptor alpha (ERαKO: The Jackson Laboratory, Bar Harbor, ME) or estrogen receptor beta (ERβKO: Taconic Farms, Inc., Hudson, NY) and the STAT3 deficiency mouse line (25) (a gift from Dr. Fu’s group) were maintained in a quiet quarantine room for two weeks before the experiments. The animal protocol was reviewed and approved by the Indiana Animal Care and Use Committee of Indiana University. All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication No. 85–23, revised 1985).
A single-step purification method using adhesion to cell culture plastic is employed as previously described (26). Breifly, mouse bone marrow mesenchymal stromal cells were harvested from male bilateral femurs and tibias by removing the epiphyses and flushing the shaft with complete media [Iscove’s Modified Dulbecco’s Medium with 10% fetal bovine serum and 1% pen-strep (GIBCO Invitrogen, Carlsbad, CA)]. Cells were washed by adding complete media, centrifuging for 5 min at 300 rpm @ 24°C and removing supernatant. The cell pellet was then resuspended and cultured with complete media at 37°C, 5% CO2 and 90% humidity. MSCs preferentially attached to the polystyrene surface; after 48 h, nonadherent cells in suspension were discarded. Fresh complete medium was added and replaced every three days thereafter. MSCs were measured for CD markers at passage 3 by flow cytometry and they were double negative for the hematopoietic markers CD34 and CD45 (>90%), negative for the macrophage marker CD11b (>95%), and positive for the mesenchymal stem cell marker CD44 (>95%) (2). MSCs were used for experiments until passage five.
After five passages, MSCs were plated in 12 well plates at a concentration of 1 × 105 cells/well/ml. First, a dose response experiment was performed to determine the concentration of 17β-estradiol (E2) that would cause maximum VEGF production in WT stem cells. MSCs were treated with E2 (Sigma, Saint Louis, Missouri) at 0, 1, 10, 100, 1000 and 10000 nM, respectively for 24 hours. Supernatants were then collected for VEGF assay (ELISA). The experiment was repeated on three separate occasions.
Wild type (WT), ERαKO, ERβKO and STAT3KO MSCs were divided into 4 experimental groups (triplicate wells per group: 1) control; 2) 100nM of E2; 3) 100nM of ERα agonist (propyl pyrazoletriol-PPT: Tocris Cookson Inc., Ellisville, MO); 4) 100nM of ERβ agonist (diarylpropiolnitrile-DPN: Tocris Cookson Inc., Ellisville, MO). To distinguish acute or chronic effects of E2 on VEGF production, supernatants were collected for VEGF assay (ELISA) after 6-hour or 24-hour incubation. The experiment was repeated on three separate occasions (n=6–11 wells/group). In addition, after different time incubation (15-minute, 30-minute, 1-hour, 2-hour and 4-hour), cell lysates were harvested for the assay of STAT3 activation by western blot. Furthermore, activation of mitogen activated protein kinases (MAPKs: p38 MAPK, JNK and ERK1/2) and PI3K/Akt was also analyzed. Expression of nuclear HIF-1α, ERα and ERβ was examined by using nuclear extracts.
VEGF in the MSC supernatant was determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available ELISA set (R&D Systems Inc., Minneapolis, MN). ELISA was performed according to the manufacturer’s instructions. All samples and standards were measured in duplicate.
Total RNA was extracted from 4-hour incubated WT, ERαKO, ERβKO and STAT3KO MSCs with or without E2, ERα agonist and ERβ agonist by using RNA STAT-60 (TEL-TEST, Friendswood, TX). 0.5 μg of total RNA was subjected to cDNA synthesis using cloned AMV first-strand cDNA synthesis kit (Invitrogen life technologies, Carlsbad, CA). cDNA from each sample was analyzed for 18S rRNA (assay ID# Hs99999901_s1) and VEGFa (assay ID# Mm00545913_s1) by using TaqMan gene expression assay (Real-time PCR) (Applied Biosystems, Foster City, CA). Each sample was assayed in duplicate. Values were normalized to corresponding 18S rRNA real-time RT-PCR values and expressed as the fold increase relative to corresponding control.
Western blot analysis was performed to measure MAPKs (p38 MAPK, JNK and ERK1/2), STAT3 activation, PI3K/Akt signaling and HIF-1α. Cells were lysed in cold RIPA buffer (Product No. R 0278: Sigma, Saint Louis, MO) and centrifuged at 12000 rpm for 10 minutes to get whole cell extracts. Nuclear extracts were isolated by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL). The protein extracts (5 μg/lane) were subjected to electrophoresis on a 4–12% precise protein gel (Pierce, Rockford, IL) and transferred to a nitrocellulose membrane. The membranes were incubated in 5% dry milk for 1 hour and then incubated with the following primary antibodies: STAT3, phosphor-STAT3 (Tyr705), p38 MAPK, phosphor-p38 MAPK, JNK, phosphor-JNK (Thr183/Tyr185), ERK1/2, phosphor-ERK1/2 (Thr180/Tyr182), PI3K, phosphor-PI3K, Akt, phosphor-Akt (1:1000 dilution, Cell Signaling Technology, Beverly, MA), HIF-1α, ERα (MC-20), ERβ (H-150) (1:200 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and GAPDH (1:5000 dilution, Biodesign International, Saco, Maine), or TATA binding protein-TBP (1:2000 dilution, Abcam Inc., Cambridge, MA). Membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (Pierce, Rockford, IL) and detection using supersignal west pico stable peroxide solution (Pierce, Rockford, IL). Films were scanned using an Epson Perfection 3200 Scanner (Epson America, Long Beach, CA) and band density was analyzed using ImageJ software (NIH).
All reported values are mean ± SEM. Data was compared using one-way analysis of variance (ANOVA) with post-hoc Tukey test or student’s t-test. A two-tailed probability value of less than 0.05 was considered statistically significant.
Sex differences were noted in MSC production of VEGF in response to injury, with higher levels of VEGF observed in female MSCs (27). To determine what concentration of E2 would cause maximum VEGF production in WT stem cells, different doses of E2 at 1, 10, 100, 1000 nM and 10 μM were adopted. Our data demonstrated that E2 increased MSC VEGF production in a dose-dependant manner in WT (figure 1A). Pharmacologic concentrations of E2 at 100 nM yielded maximal VEGF release in WT MSCs.
To examine which estrogen receptor mediated the effects of E2 on MSC VEGF production, the expression of ERα and ERβ in MSCs was first assessed. Our results indicated that both ERα and ERβ were present in nuclear extracts of MSCs as shown by western blot analysis (figure 1B). Additionally, PPT, a selective ERα agonist, and DPN, a selective ERβ agonist were used to investigate this interesting unknown. 100 nM of E2 and ERα agonist (PPT) significantly increased VEGF production in WT MSCs by more than 200 pg/ml/105 cells (table 1). In addition, elevated VEGF mRNA levels, ranging from 1.5- to 2- fold, were also noted in WT MSCs treated with E2 or the ERα agonist (table 1). However, the ERβ agonist (DPN) did not affect stem cell VEGF production (mRNA and protein). To further confirm this observation, ERαKO and ERβKO MSCs were adopted. In line with these results, we found that ablation of ERα abolished E2- or ERα-induced MSC VEGF expression (table 1). However, application of E2 or the ERα agonist maintained increased levels of VEGF mRNA and protein expression in ERβKO MSCs (table 1).
HIF-1α has been shown to mediate VEGF expression in a variety of cell types by estrogen and other hormones. To understand the molecular mechanisms of E2-increased VEGF expression, we analyzed the expression of VEGF regulator-HIF-1α by western blot. As seen in figure 2, HIF-1α expression was increased by more than 2 times in WT MSCs 30 minutes and 1 hour after administration of E2. In addition, the ERα agonist also elevated HIF-1α expression by 3 times at 30 minutes and 1 hour. However, the selective ERβ agonist did not affect HIF-1α expression. These data suggested that E2 induced HIF-1α expression via the ERα.
As seen in figure 3A, western blot analysis showed that there was a 2-fold increase in STAT3 activation (exhibited as phosphorylated STAT3) 15 minutes after E2 or ERα agonist treatment. This elevated activation of STAT3 was also noted within 30 minutes of treatments, whereas total STAT3 levels were not changed. However, DPN, the selective ERβ agonist, did not increase STAT3 activation in MSCs. In addition, the trend of increased p-STAT3 was noted to be maintained until 2 hours after administration of E2 or ERα agonist in WT MSCs. After 4 hours of treatment, the ERα agonist-induced STAT3 activation normalized.
Interestingly, basal levels of p-STAT3 were significantly higher in WT MSCs than in ERαKO (7-fold) or ERβKO MSCs (2-fold) (figure 3B). However, ablation of ERα or ERβ did not change total STAT3 expression. Notably, deficiency of ERα neutralized E2- or ERα-induced STAT3 activation in MSCs (figure 3C).
STAT3 activation has been linked to upregulation of HIF-1α expression and VEGF production. We already found that E2-induced STAT3 activation and HIF-1α expression was mediated through ERα. However, it was unclear whether ERα-induced STAT3 activation would stimulate HIF-1α expression in MSCs. Therefore, STAT3KO MSCs were used to examine this mechanism. As shown in figure 4, deficiency of STAT3KO abolished E2- or ERα agonist-induced HIF-1α expression.
STAT3KO MSCs were also adopted to further determine the effects of STAT3 on ERα-mediated VEGF production. Significantly increased VEGF production was observed in WT MSCs following 6-hour and 24-hour incubation periods after treatment with E2 or the ERα agonist (figure 5A and C). However, these elevated VEGF protein levels were neutralized in STAT3KO MSCs (figure 5B and D). In addition, deficiency of STAT3 also abolished E2- or ERα-increased VEGF mRNA levels (figure 5E and F).
Given that estrogen stimulates the MAPK pathway and activates PI3K/Akt signaling, and that both the MAPK pathway and PI3K/Akt have been linked to HIF-1α expression (14, 17, 28), we determined the effects of E2, the ERα agonist and the ERβ agonist on activation of MAPKs and PI3K/Akt in MSCs. The results from western blot indicated that administration of E2, PPT or DPN did not change activation of p38 MAPK or ERK1/2 in MSCs within 30 minutes, 1 hour or 2 hours after administration (supplemental figure 1A and C). However, a transient decrease in JNK activation was observed 30 minutes after treatment with E2, ERα agonist or ERβ agonist (supplemental figure 1B). This decreased pattern in JNK activation was also noted in STAT3KO MSCs (data not shown). This suggests that changes in JNK activation are not involved in STAT3 pathway. Additionally, E2, the ERα agonist and the ERβ agonist did not affect activation of PI3K and Akt (supplemental figure 2).
VEGF is a critical factor in angiogenesis and has been shown to facilitate stem cell paracrine protection in the ischemic myocardium (3, 6, 7). Accumulating evidence has demonstrated that estrogen plays a role in modulating stem cell function and in mediating VEGF production (9–13). Our previous study has further suggested that sex differences exist in MSC VEGF production, with relatively higher levels of VEGF being produced by female MSCs (27). These sex disparities in stem cell VEGF production correlate to biologically relevant sex differences in stem cell-mediated cardioprotection, as MSCs from females confer greater cardioprotection compared to male MSCs (8). In addition, E2 pretreated MSCs have exhibited increased VEGF production and better cardiac protection (9). These studies have led to the important appreciation that estrogen may facilitate the induction of stem cell VEGF production, which may enhance the therapeutic effectiveness of MSCs.
Estrogen initiates its biological effects by interacting with multiple estrogen receptors. Two ER subtypes have been identified: ERα and ERβ, both of which belong to the nuclear receptor gene family of transcription factors. In this study, we provide the evidence that E2 can increase MSC production of VEGF in a dose-dependant manner. Our results also indicate that both ERα and ERβ are expressed in MSCs and that E2-induced VEGF expression (both mRNA and protein levels) is mediated through ERα, but not ERβ.
However, the downstream signaling cascades associated with ERα-induced VEGF production in MSCs have not been fully elucidated. HIF-1 is the primary mediator of the physiological responses associated with reduction in tissue oxygen tension (hypoxia), and plays an important role in VEGF transcriptional activation (29, 30). Transcription factor HIF-1 consists of two subunits: HIF-1α, the hypoxia-induced component, and HIF-1β, which is expressed constitutively. HIF-1α is rapidly degraded in normoxic cells. However, under conditions of low oxygen tension, HIF-1α stabilizes due to hypoxia-prevented degradation. Recently, HIF-1α has been reported to regulate the induction of VEGF expression in several cell types under normoxic conditions (9, 14, 15). In vascular smooth muscle cells without hypoxia, angiotensin II upregulates HIF-1α-induced VEGF expression (18). Treatment with insulin-like growth factor 1 has also been shown to elevate HIF-1α-induced VEGF mRNA and protein levels in cancer cells (14). Therefore, the activation of HIF-1α may be required for estrogen-induced-VEGF expression via one or more estrogen receptors (17, 19). Indeed, ERα-activated HIF-1α has been shown to mediate estrogen-induced VEGF production in epithelial cells (14). In the current study, our data regarding E2- or ERα- increased HIF-1α expression in MSCs are in line with results from previous studies, and further confirm that non-hypoxic stimuli can induce HIF-1α. In addition, we observed that E2-elevated HIF-1α is likely mediated by ERα and not ERβ. These results suggest that E2-increased VEGF mRNA and protein levels are likely mediated through ERα-induced HIF-1α in MSCs.
STAT3 has been implicated in a variety of cellular functions, including cell survival/apoptosis, proliferation, inflammation and angiogenesis (26, 31, 32). Activation of STAT3 has been found in a wide range of tissues and organs, and appears to be elevated by stress, growth factors, and cytokines. Of interest, STAT3 has been reported to be a direct target gene for estradiol. Not only has chronic estradiol treatment been shown to induce STAT3 activation, but acute treatment with the ERα agonist PPT has also been shown to upregulate STAT3 expression in the mouse liver (22, 33). Additionally, E2-acitvated STAT3 plays a role in conveying non-genomic effects of estrogen in endothelial cells (24). Moreover, active STAT3 signaling by raloxifene plus 17β-estradiol has been shown to modulate proliferation of vascular smooth muscle cells and human mammary endothelial cells (23). Herein, our results indicate that administration of E2 or the ERα agonist PPT increases activation of STAT3 in MSCs. In addition, significantly decreased basal levels of active STAT3 have been observed in ER knockout MSCs with the lowest levels being noted in ERαKOs. Furthermore, deficiency of ERα abolished E2- or ERα agonist-increased STAT3 activation. Taken together, these data suggest that cross-talk exists between STAT3 and estrogen signaling, and that ERα is required for E2-mediated STAT3 activation in MSCs. However, we recognized that STAT3KO was employed in this study and ablation of a complete signaling moiety might cause some non-specific effects. Therefore, further investigation by using siRNA of STAT3 or transfection of dominant negative STAT3 is required to determine the detailed mechanisms by which STAT3 mediates MSC function.
Recent evidence has demonstrated that STAT3 is a direct transcriptional activator of VEGF expression in a variety of cell types (21, 26, 31, 32). Our previous study has indicated that STAT3 is required for both basal and stress-induced VEGF production in MSCs (26). Over-expression of active STAT3 in cardiomyocytes increased myocardial VEGF production (34), and active STAT3-mediated HIF-1α has been reported to up-regulated transcription of the VEGF promoter in human renal carcinoma cells (20). Blockade of STAT3 down-regulates HIF-1α, and thereby decreases VEGF expression (35). Moreover, active STAT3 has been shown to stabilize and increase HIF-1α protein levels by inhibition of HIF-1α degradation and acceleration of its synthesis (20). Therefore, it can be proposed that STAT3 signaling may mediate MSC production of VEGF through regulation of HIF-1α. In the current study, we found that ablation of STAT3 neutralized E2- or ERα-induced HIF-1α expression, and thus, abolished E2- or ERα-increased VEGF mRNA and protein expression in MSCs. Although Gray MJ et al. have reported that maximum transcription of VEGF expression requires the binding of both STAT3 and HIF-1α to the VEGF promoter in carcinomas (36), the detailed mechanistic interactions of STAT3 and HIF-1α in the regulation of VEGF promoter requires further investigation.
Based on these findings, ERα-activated STAT3 increases stem cell HIF-1α-mediated VEGF expression induced by the administration of estrogen (figure 6). A better understanding of the effects of gender, estrogen and the estrogen receptor may allow for the modification of stem cells in vitro in order to maximize stem cell paracrine protective properties such as the expression of VEGF.
Supplemental figure 1. Activation of p38 MAPK, JNK and ERK1/2 in WT MSCs 30 minute, 1 hour and 2 hour after administration of E2, ERα agonist and ERβ agonist. (A): p-p38 MAPK and total-p38 MAPK. (B): phosphor- and total-JNK. (C): p-ERK1/2 and total-ERK1/2. Two lanes/group are Shown.
Supplemental figure 2. Activation of PI3K and Akt in WT MSCs 30 minute, 1 hour and 2 hour after treatment with E2, the ERα agonist and the ERβ agonist. (A): p-PI3K and total-PI3K. (B): phosphor- and total-Akt. Shown are 2 lanes/group.
this work was supported in part by NIH R01GM070628 (DRM), NIH K99/R00 HL0876077 (MW), and NIH R01HL085595 (DRM).
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