Dehydroepiandrosterone inhibits the Src/STAT3 constitutive activation in pulmonary arterial hypertension

Roxane Paulin, Jolyane Meloche, Maria Helena Jacob, Malik Bisserier, Audrey Courboulin, Sébastien Bonnet

Abstract

Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. This phenotype is sustained by the activation of the Src/signal transducer and activator of transcription 3 (STAT3) axis, maintained by a positive feedback loop involving miR-204 and followed by an aberrant expression/activation of its downstream targets such as Pim1 and nuclear factor of activated T-cells (NFATc2). Dehydroepiandrosterone (DHEA) is a steroid hormone shown to reverse vascular remodeling in systemic vessels. Since STAT3 has been described as modulated by DHEA, we hypothesized that DHEA reverses human pulmonary hypertension by inhibiting Src/STAT3 constitutive activation. Using PASMCs isolated from patients with PAH (n = 3), we demonstrated that DHEA decreases both Src and STAT3 activation (Western blot and nuclear translocation assay), resulting in a significant reduction of Pim1, NFATc2 expression/activation (quantitative RT-PCR and Western blot), as well as Survivin and upregulation of bone morphogenetic protein receptor 2 (BMPR2) and miR-204. Src/STAT3 axis inhibition by DHEA is associated with 1) mitochondrial membrane potential (tetramethylrhodamine methyl-ester perchlorate; n = 150; P < 0.05) depolarization increasing apoptosis by 25% (terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling; n = 150; P < 0.05); and 2) decreased intracellular Ca2+ concentration (fluo-3 AM; n = 150; P < 0.05) and proliferation by 30% (PCNA). Finally, in vivo similarly to STAT3 inhibition DHEA improves experimental PAH (monocrotaline rats) by decreasing mean PA pressure and right ventricle hypertrophy. These effects were associated with the inhibition of Src, STAT3, Pim1, NFATc2, and Survivin and the upregulation of BMPR2 and miR-204. We demonstrated that DHEA reverses pulmonary hypertension in part by inhibiting the Src/STAT3.

  • proliferation
  • apoptosis
  • remodeling
  • therapies
  • signal transducer and activator of transcription 3

pulmonary arterial hypertension (PAH; WHO Group 1) is a devastating disease of the pulmonary vascular network defined by an increase in pulmonary arterial pressure due to a marked and sustained elevation of pulmonary vascular resistance. Progressively, patients develop a compensatory right ventricular hypertrophy, which becomes insufficient and leads to dilatation and failure (4).

At the cellular level, PAH is characterized by enhanced inflammation (29), proliferation, and resistance to apoptosis (4) of pulmonary artery smooth muscle cells (PASMCs). The sustainability of this phenotype is due in part to the activation of neoplasic pathways involving the transcription factor signal transducer and activator of transcription 3 (STAT3; Ref. 46). The mechanism accounting for STAT3 activation in PAH-PASMCs exclusively relies on the Src pathway and is independent of the regular STAT3 activator Janus-activated kinase (JAK2; Ref. 12). This suggests that Src/STAT3 inhibition could be of a great therapeutic interest for PAH. Similarly to cancer, Src/STAT3 activation in PAH has been associated with the upregulation of the oncogene provirus integration site for Moloney murine leukemia virus (Pim-1) promoting the activation of the transcription factor nuclear factor of activated T-cells (NFATc2). NFATc2 activation has been shown to account for both proliferation and resistance to apoptosis in cancer and PAH. Indeed, by downregulating K+ channels like Kv1.5, NFATc2 leads to cell depolarization, increasing intracellular Ca2+ concentration ([Ca2+]i), and promoting cell proliferation (7, 9, 10); while by upregulating Bcl-2, NFATc2 activation leads to mitochondrial hyperpolarization and apoptosis resistance (7, 9, 10). Moreover, the Src/STAT3 axis is implicated in tumoral upregulation of Survivin, described as an important protein in the pathogenesis of PAH (39). Finally, Src/STAT3 has been also associated with bone morphogenetic protein receptor 2 (BMPR2) downregulation (11), further promoting the proproliferative, antiapoptotic PAH phenotype (54, 58).

The Src-dependant activation of STAT3 is mediated (i.e., phosphorylated P-STAT3 and translocated to the nucleus) by cytokines (such as IL-6; Ref. 14), growth factors (such as PDGF; Ref. 14), or agonists [such as endothelin-1 (ET-1) and ANG II; Ref. 6], factors that all appear to be implicated in PAH (13, 21, 50, 51). Additionally, we (12) provided evidence showing that STAT3 regulates a positive feedback loop sustaining its proper activation. By downregulating miR-204 in PAH, STAT3 activation leads to a constitutive c-Src activation maintaining STAT3 activated. For all these reasons, STAT3 can be considered as a major signaling hub of PAH and associated distal PA remodeling processes.

Dehydroepiandrosterone (DHEA) is a naturally and abundantly produced hormone, known to improve PAH through its vasodilator properties. Indeed, by opening K+ channel-like Kv and BKCa (8, 20, 47) and increasing cGMP generation and responsiveness to nitric oxide (45), DHEA enhances VSMC relaxation (25). Nonetheless, the effects of DHEA on vascular remodeling processes seen in PAH remain unknown. Several literature evidences have demonstrated the implication of the Src/STAT3 axis in both K+ channel modulation (44) (enhancing vasocontriction) along with NFATc2 activation (12, 46) and BMPR2 downregulation (11, 57) (promoting PASMCs proliferation and resistance to apoptosis). Since DHEA has been associated with decreased STAT3 activation in liver cells (34), we hypothesized that DHEA could reverse PAH through the inhibition of the Src-dependent activation of STAT3. DHEA could potentiate its vasodilating effects by decreasing the NFAT-dependant proproliferative and antiapoptotic phenotype (10).

MATERIALS AND METHODS

All the experiments were performed with the approval of the Laval University Ethic and Biosafety Committee. The investigation conforms the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and the principles outlined in the Declaration of Helsinki (1).

Cell culture.

PAH-PASMCs were isolated from <1,500-μm-diameter small PAs from two males with idiopathic PAH (31 and 48 yr old) and one female with PAH Group 1 (lupus; 54 yr old) from lung explants (38). All patients had right catheterization that confirmed pulmonary hypertension (mean pulmonary arterial pressure >25 mmHg at rest). Age- and sex-matched control-PASMCs (3 males 45, 21, and 64 yr old and 2 females 17 and 35 yr old) were used.

Control-PASMCs from five patients were purchased (Cell Application Group). PASMCs (less than passage 6) were grown in high-glucose DMEM media supplemented with 10% FBS (GIBCO, Invitrogen, Burlington, ON, Canada) and 1% antibiotic/antimytotic (GIBCO, Invitrogen; Ref. 7). All cells were used until the fifth passage.

Cell treatments.

DHEA was dissolved in methanol and used at a final concentration of 100 μmol/l (methanol final concentration ≤0.1%). IL-6 stimulations (100 μmol/l) were applied for 48 h (9). Small interfering (si)RNA (Ambion, Austin, TX) were transfected at a final concentration of 20 nmol/l with CaCl2 (46). After 24 h, medium was changed and experiments were performed 48 h after the beginning of the transfection.

Measurement of the mitochondrial membrane potential and [Ca]i.

Measurement of the mitochondrial membrane potential and [Ca]i in live PASMCs (37°C) was performed using a FV1000 confocal microscope equipped with a live cell apparatus (Olympus, Center Valley, PA).

Mitochondrial membrane potential was determined using 10 nM tetramethylrhodamine methyl-ester perchlorate (TMRM; Invitrogen, Branchburg, NJ). [Ca2+]i was determined using 5 μmol/l fluo-3 AM (Invitrogen; Refs. 9, 10). Nuclei were stained using 50 nM of Hoechst 33342 (Invitrogen). Between 100 and 150 cells were measured by experiment in 3 experiments by cell line in 5 control and 3 PAH-PASMCs cell lines.

Proliferation and apoptosis measurements.

PASMCs were exposed to 10% FBS (a condition known to promote proliferation; Refs. 7, 10) or 0.1% FBS (a “starvation” condition that promotes apoptosis; Refs. 7, 10). Apoptosis rates were measured using Apoptag apoptosis detection kit [terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL); Millipore, Temecula, CA]. Proliferation rates were measured by the PCNA antibody from (1/400; Dako, Carpinteria, CA) and Ki67 antibody (1/400; Millipore) according to the manufacturer's instructions (9, 10). The percentage of positive PASMCs for TUNEL or Ki67 was determined by the number of cell showing the presence of a nuclear staining over the total number of cell. The percentage of PCNA positive cell was determined using a threshold based on the intensity obtained in control PASMCs. This threshold was applied in all the other groups, and cells showing a nuclear fluorescent intensity superior to the threshold were considered as positive. Between 100 to 150 cells were measured by experiment in 3 experiments by cell line in 5 control and 3 PA-PASMCs cell lines.

Nuclear translocation assay.

In PASMCs, STAT3 and NFATc2 activation were measured in ≥100 to 150 cells were measured by experiment in 3 experiment by cell lines in 5 control and 3 PA-PASMCs cell lines.

Briefly, PY705-STAT3 (1/250; Cell Signaling) and NFATc2 (1/250; ABCam) staining were performed as previously described (10). Secondary antibodies used were Alexa Fluor 488 or 594 (1/1,000; Invitrogen). The percentage of positive cells was determined using a threshold based on the intensity obtained in control PASMCs. This threshold was applied in all the other groups, and cells showing a nuclear fluorescent intensity superior to the threshold were considered as positive.

The same analysis was performed in rats in 10 distal arteries (<100 μm)/rat in 5 rats per groups. Colocalization between the targeted protein stained in green and the nucleus stained in blue with DAPI was further assessed using Volocity software from Perkin Elmer.

Quantitative RT-PCR and immunoblots.

Quantitative RT-PCR and immunoblots were performed as previously described (9, 10). Quantitative RT-PCR 2ΔCt was calculated with 18S as housekeeping gene (Taqman Gene Expression Assay; Applied Biosystems, Foster, CA). For immunoblots, protein expression of PY705-STAT3, STAT3, PY419-Src (1/1,000; Cell Signaling), and c-Src (1/1,000; Santa Cruz) was quantified. PY705-STAT3/STAT3 and PS419-Src/c-Src ratio evaluations were obtained from the same membrane after a 30-min stripping at 50°. The phosphorylated forms were rapported on the total form of the protein, and normalized by the amido black as previously described (10).

In vivo experiments.

Sprague-Dawley rats (250–350 g) were injected subcutaneously with 60 mg/kg of monocrotaline (MCT; n = 20). Intratracheal nebulization (50 μl) of siSCR (Ambion; 1 nmol in n = 5 rats) and siSTAT3 (Ambion; 1 nmol in n = 5 rats) was performed under anesthesia on day 18, once pulmonary hypertension was established. Freshly prepared DHEA (Sigma-Aldrich, St. Louis, MO) was administered by gavage (n = 5 rats) once a day at the concentration of 10 mg·kg−1·day−1 (30) during 2 wk. Experiments were performed with the approval of the Laval University Animal Ethics Committee.

Hemodynamic measurements.

Rats were initially anesthetized with 3–4% isoflurane and maintained with 2% during procedures. All rats underwent hemodynamic and echocardiography studies as previously described (10, 53). Right catheterizations (closed chest) were performed using SciScence catheters to directly measure PA pressures. Longitudinal and noninvasive evaluations of PAH were performed by echocardiography (using Vevo 2100 VisualSonics equipment) as previously described (10, 53). Euthanasia was performed by exsanguinations.

Histology measurements.

Percent media wall thickness was assessed as previously described (10). Briefly, lungs slides were colored with hematoxylin-eosin, and distal PA <100 μm wall thickness was measured using image Proplus software (Media Cybernetics). Two measurements per artery in 5 to 10 distal arteries per rat in 5 rats per groups were performed.

Statistics.

We tested if the data sets we are using for statistical assessment are coming from a normal distribution using the Shapiro-Wilk test. As all our data sets passed the test significantly (all P > 0.05), we were able to apply parametric analysis to our study and to present our results as means ± SE. For comparison between two means, we used a Student's t-test. For comparison between more than two means, we used a one-way ANOVA followed by a Tukey-Kramer tests. Probability values <0.001, 0.01, and 0.05 were considered as statistically significant.

RESULTS

DHEA decreases Src/STAT3 activation in PAH-PASMCs.

Using Western blot, we showed an increase in the amount of phosphorylated STAT3 on tyrosine 705 (i.e., active form of STAT3) over total STAT3. STAT3 activation is increased by 3.4-fold in PAH-PASMCs compared with control (Fig. 1A). By immunofluorescence, we evaluated the level of P-STAT3 translocated to the nucleus, which is associated with its transcriptional activity. The percentage of P-STAT3-positive cells (presenting an increased nuclear P-STAT3 intensity compared with control cells) is increased by seven times in PAH-PASMCs compared with control-PASMCs (Fig. 1A).

Fig. 1.

Dehydroepiandrosterone (DHEA) decreases Src/STAT3 activation in pulmonary arterial hypertension (PAH)-pulmonary artery smooth muscle cells (PASMCs). A: DHEA decreases STAT3 activation in PAH-PASMCs. Signal transducer and activator of transcription 3 (STAT3) activation was evaluated by immunoblots in triplicates (ratio PY705-STAT3/STAT3 normalized to amido black) and nuclear translocation assay measuring the amount of P-STAT3 (%P-STAT3-positive cells) in green in the nucleus in blue (DAPI) and the colocalization in yellow. Note that the greater the yellow intensity, the more P-STAT3 is activated. DHEA was dissolved in methanol and used at final concentration of 100 μmol/l (methanol final concentration ≤0.1%) for 48 h (n = 100–150 cells by experiment in 3 experiments by cell line in 5 controls and 3 PAH-PASMCs cell lines, P < 0.05). B: DHEA increases miR-204 level, disrupting the positive feedback loop. Src activation was evaluated under STAT3 inhibition by silencer RNA. Representative gels and mean data show that an effective STAT3 inhibition in PAH-PASMCs by siRNA is associated with decreased activation of Src (P-Src/Src ratio normalized to amido black) compared with PAH-PASMCs treated with siSCR. miR-204 mRNA level was measured by quantitative (q)RT-PCR and normalized to U6 in control, PAH, PAH + DHEA, PAH + siSCR, and PAH + siSTAT3 groups. All the experiments were performed in triplicates using PASMCs from 5 controls and 3 PAH-patients (P < 0.05.) C: DHEA decreases Src activation. Src activation was evaluated by immunoblots. Representative gels showing the amount of Src phosphorylated on tyrosine 419 compared with the total amount of Src. All the experiments were performed in triplicates using PASMCs from 5 controls and 3 PAH-patients (P < 0.05). *P < 0.05, **P < 0.01, and ***P < 0.001.

PAH-PASMCs treated with DHEA (100 μmol/l) for 48 h showed a significant decrease in both P-STAT3/STAT3 ratio and nuclear translocation of P-STAT3 (Fig. 1A), suggesting that DHEA inhibits the transcriptional activity of this protein.

DHEA and STAT3 inhibition reverses miR-204 downregulation in human PAH-PASMCs.

In addition to the circulating cytokines and growth factors accounting for STAT3 activation in PAH-PASMCs, we (12) recently demonstrated that the sustained STAT3 activation in these cells also depends on a STAT3-dependent downregulation of the microRNA miR-204. This positive feedback loop mechanism relies on the binding of STAT3 on the host gene of miR-204 leading to its downregulation. This downregulation accounts for a sustained c-Src activation, which is responsible of the constitutive STAT3 activation (12). Since DHEA has the ability to decrease STAT3 activation (34), we speculated that this positive feedback loop should be disrupted by DHEA. To this end, we first studied the effect of STAT3 inhibition on Src activation. PAH-PASMCs were transfected by either scramble RNA or RNA directed against STAT3. As expected, STAT3 inhibition is associated with decreased Src activation in PAH-PASMCs compared with scramble sample (Fig. 1B). As miR-204 is one of the major players of this loop, miR-204 expression was measured in control-PASMCs, PAH-PASMCs, and in PAH-PASMCs treated with STAT3 siRNA, scramble siRNA, or DHEA. STAT3-dependent regulation of miR-204 was confirmed since STAT3 inhibition by siSTAT3 in PAH-PASMCs increases miR-204 expression to a level similar as the one seen in control PASMCs (Fig. 1B). Similarly, DHEA significantly increase miR-204 expression in PAH-PASMCs. Finally, to confirm our previous findings we studied whether c-Src activation (induced by miR-204 downregulation) is decreased by DHEA. Using Western Blot, we showed that DHEA decreases c-Src activation (i.e., phosphorylated Src on tyrosine 418/total c-Src ratio) in PAH-PASMCs (Fig. 1C), disrupting the STAT3-dependant positive feedback loop sustaining its proper activation.

DHEA-dependant inhibition of the Src/STAT3 decreases the oncogenic pim-1/NFATc2/survivin axis and promotes BMPR2 expression.

To determine whether DHEA-dependent STAT3 inhibition is transduced, we studied the effects of DHEA on the STAT3 downstream targets implicated in PAH including Pim-1, NFATc2, BMPR2, and Survivin (12, 46, 54, 58). Pim1 and NFATc2 mRNA levels were measured in PAH-PASMCs in presence or not of DHEA. As STAT3 molecular inhibition (siRNA), DHEA decreases Pim1 and NFATc2 mRNA expression in PAH-PASMCs (Fig. 2A). Since Pim1 accounts for NFATc2 activation in PAH (46), we measured the effects of DHEA on NFATc2 activation by a nuclear translocation assay/immunostaining. We previously described that >60% of PAH-PASMCs show NFATc2 activation, whereas only 20% are positive for activated NFATc3 and <5% for NFATc4. For these reasons, we consider that NFATc2 is the predominant NFAT isoform in PAH-PASMCs (10). DHEA, as well as STAT3 siRNA, decreases NFATc2 activation by sevenfold compared with untreated or scrambled treated PAH-PASMCs (Fig. 2A). These results show that through inhibition of STAT3 phosphorylation, DHEA has an inhibitory effect on the Pim1/NFATc2 axis.

Fig. 2.

A: DHEA restores expression/activity of STAT3 downstream targets aberrantly expressed/activated in PAH. Pim1, nuclear factor of activated T-cells (NFATc2), bone morphogenetic protein receptor 2 (BMPR2), and Survivin mRNA levels were measured by qRT-PCR in triplicate and normalized to 18S in PASMCs from 5 control, 3 PAH, and 3 PAH treated with DHEA, scramble silencer RNA (siSCR; 20 μmol/l), or STAT3 silencer RNA (siSTAT3; 20 μmol/l). Similarly to STAT3 inhibition by silencer RNA, DHEA impairs STAT3 signal transduction to downstream targets, resulting in Pim1, NFATc2, and Survivin decreased mRNA level and increased BMPR2 mRNA level. NFATc2 activation measured by nuclear translocation assay and expressed as the percentage of nuclear NFATc2-positive cells (n = 100 to 150 cells by experiment in ≥3 experiments by cell line in 5 controls and 3 PAH-patient PASMCs; P < 0.05). B: DHEA restores the proliferation/apoptosis balance. PAH-PASMCs had increased intracellular Ca2+ concentrations ([Ca2+]i) and proliferation rates (%PCNA) compared with healthy PASMCs. Both STAT3 siRNA and DHEA significantly decrease PAH-PASMCs proliferation. DHEA in PAH-PASMCs reverses mitochondrial membrane potential (ΔΨm) hyperpolarization [tetramethylrhodamine methyl-ester perchlorate (TMRM)] and decreases apoptosis [%terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL); n = 100–150 cells by experiment in ≥3 experiments by cell line in 5 controls and 3 PAH-patient PASMCs; P < 0.05]. FU, fluorescence units. *P < 0.05, **P < 0.01, and ***P < 0.001.

BMPR2 downregulation is recognized as an important marker of the proproliferative, antiapoptotic PAH phenotype (54, 58). It has been recently suggested that STAT3 activation in PASMCs could be associated with repression of BMPR2 (11). We thus speculated that DHEA, by inhibiting STAT3, could rescue BMPR2 levels. Indeed, both STAT3 inhibition by siRNA and DHEA enhance BMPR2 mRNA levels in PAH-PASMCs (Fig. 2A).

STAT3 activation was also associated in cancer (22, 35) and systemic vascular remodeling (40) with an upregulation of Survivin mRNA levels. Survivin, a member of the inhibitor of apoptosis family of proteins, was described as a major player in PAH pathogenesis (39). For these reasons, we studied whether STAT3 inhibition by siRNA or DHEA can decrease Survivin expression in PAH. We observed a fivefold increase of Survivin expression in PAH-PASMCs compared with control PASMCs. Both siSTAT3 and DHEA normalized Survivin mRNA level in PAH compared with siSCR and untreated PAH-PASMCs respectively (Fig. 2A), showing for the first time that Survivin expression in PAH is dependent at least in part on STAT3 activation. All these results offer for the first time a putative mechanism explaining at the same time the activation of Pim-1/NFATc2/Survivin and the downregulation of BMPR2. The fact that STAT3 inhibition by DHEA reverses all these abnormalities and the fact that DHEA can also restore miR-204 expression place DHEA as an ideal candidate therapy for PAH (18).

DHEA and STAT3 inhibition decreases proliferation and resistance to mitochondria-dependent apoptosis in PAH-PASMCs.

In PAH, NFATc2 activation downregulates important K+ channels, like Kv1.5, leading to PASMCs depolarization, increasing [Ca2+]i and promoting PASMCs proliferation (7, 9, 10). Using fluo-3 AM, we measured the effects of STAT3 inhibition in PASMCs. PAH-PASMCs had greater [Ca2+]i compared with control-PASMCs. STAT3 inhibition by siRNA or DHEA in PAH-PASMCs decreases [Ca2+]i to a similar level as in control-PASMCs (n = 200; P < 0.05; Fig. 2B). PAH-PASMCs had increased proliferation rates (increased %PCNA-positive cells), and STAT3 inhibition by siRNA and DHEA in PAH-PASMCs (10% FBS) decreases proliferation by 2.3-fold (Fig. 2B).

The mitochondria transition pore is voltage-dependent (59), and the mitochondrial membrane potential (ΔΨm) depolarization is an index of the threshold for mitochondrial-dependent apoptosis. In this way, apoptosis is associated with decreased ΔΨm. Since STAT3 (23) and its downstream targets Pim1 (43, 52), NFATc2 (7, 9, 10), and Survivin (39) are implicated in (ΔΨm) regulation, we measured ΔΨm in PAH-PASMCs transfected with STAT3 siRNA, siSCR, or treated with DHEA, using TMRM. STAT3 inhibition by siRNA or DHEA treatment caused a significant ΔΨm depolarization, compared with scrambled-treated PAH-PASMCs and PAH-PASMCs, respectively (Fig. 2B). These data confirmed the role of STAT3 in ΔΨm regulation and in mitochondrial-dependant apoptosis. Apoptosis rates were studied using TUNEL assay. PAH-PASMCs appeared to be resistant to starvation (0.1% FBS)-induced apoptosis (decreased %TUNEL-positive cells compared with control-PASMCs; Fig. 2B). Similarly to STAT3 inhibition, DHEA in “starved” PAH-PASMCs increased apoptosis by fivefold.

DHEA decreases IL6-dependant Src/STAT3 activation and downstream target expression, reduces proliferation, and increases apoptosis in control-PASMCs stimulated by IL-6.

Since IL-6 is one of the major STAT3 activator (60) and plays a critical role in PAH (11, 49, 51), we studied the DHEA effect on control-PASMCs stimulated by IL-6 (100 μmol/l, 48 h). DHEA decreases IL-6-dependant Src/STAT3 activation measured by Western blot, as it can be seen in PAH-PASMCs (Fig. 3A). This result is confirmed by nuclear translocation assay, showing that DHEA reduces the percentage of cell presenting nuclear P-STAT3 (Fig. 3A). Moreover, DHEA decreases expression of STAT3 downstream targets Pim1, NFATc2, BMPR2, and Survivin in control-PASMCs stimulated by Il-6 (Fig. 3B). Finally, in this model, DHEA treatments were associated with decreased [Ca2+]i and proliferation rates, ΔΨm depolarization, and increased apoptosis rates (Fig. 3B).

Fig. 3.

A: DHEA decreases IL-6-dependant Scr/STAT3 activation in control PASMCs. Stat3 activation was studied in control-PASMCs, stimulated by IL-6, and treated with DHEA. Amount of STAT3 phosphorylated on tyrosine 705 is increased by IL-6 stimulation and reversed by DHEA treatment. This was confirmed by immunostaining, showing the nuclear localization of PY705-STAT3 after IL-6 stimulation, which is reversed by DHEA. B: Src activation was evaluated by immunoblots with the ratio PY419-Src/c-Src in control-PASMCs stimulated by IL-6. DHEA decreased significantly IL6-dependant Src activation in control PASMCs. B: DHEA decreases IL-6-dependent STAT3 downstream target expression. Pim1, NFATc2, BMPR2, and Survivin mRNA levels were measured by qRT-PCR and normalized to 18S in PASMCs control stimulated by IL-6. C: DHEA restores the proliferation/apoptosis balance [Ca2+]I and proliferation rates (%PCNA) as well as ΔΨm hyperpolarization (TMRM) and decreases in apoptosis (%TUNEL) were measured in healthy PASMCs stimulated with IL-6 (n = 100–150 cells by experiment in ≥3 experiments by cell line in 5 controls and 3 PAH-patient PASMCs; P < 0.05). *P < 0.05, **P < 0.01, and ***P < 0.001.

DHEA and STAT3 inhibition reverse MCT-PAH.

To validate our in vitro findings, we performed in vivo studies using the MCT rat model of PAH to determine whether DHEA reverses PAH through the inhibition of STAT3. Therapeutical interventions were assessed with DHEA as well as STAT3 silencer RNA.

Rats subcutaneously injected with MCT-PAH (60 mg/kg) and control rats were followed using noninvasive measurements (Doppler and echocardiography) and exercise capacity on treadmill to evaluate disease development. After established PAH (decreased PA acceleration time at day 18), treatments were started. Freshly prepared DHEA (10 mg·kg−1·day−1) was administered by gavage once a day, and STAT3 siRNA (1 nmol) was selectively delivered into the lung by intratracheal nebulization (10). MCT-PAH nontreated rats and nebulized scrambled siRNA rats were used as PAH control. We showed that DHEA and STAT3 inhibition decreases PA pressure, right ventricular (RV) hypertrophy (RV/LVS; Fig. 4A), general cardiac functions (exercise capacity on treadmill; Fig. 4B), and PA wall remodeling (Fig. 4C).

Fig. 4.

DHEA and silencer RNA directed against STAT3 reverse monocrotaline (MCT)-PAH. DHEA (10 mg·kg−1·day−1) and STAT3 inhibition (1 nebulization of 1 nmol) 18 days after MCT injection decreased mean PA pressure (PAP; n = 5 rats/group) and right ventricular hypertrophy (RV/LVS; n = 5 rats/group) (A), improved exercise capacity (B; n = 5 rats/group), and diminished distal PA wall thickness (C; n = 5 to 10 PA/rat with 5 rats/group). H&E, hematoxylin-eosin. *P < 0.05 and ***P < 0.001.

These findings were associated with significant decrease in Src/STAT3 activation in the lung (Western blot; Fig. 5A) and distal PA (decreased yellow staining; Fig. 5B) in rats treated with DHEA as those treated with siSTAT3. Since STAT3 transcriptional activity is decreased, STAT3 downstream target expressions were studied. We showed that miR-204 expression is increased in lung of rats treated with DHEA and siSTAT3 (Fig. 6A). Pim1 and NFATc2 mRNA levels are decreased in lungs, and NFATc2 activation is inhibited within distal PAs (Fig. 6, B and C). Survivin and BMPR2 mRNA levels are restored as well in lungs (Fig. 6, D and E).

Fig. 5.

DHEA decreases Src/STAT3 activation in lung and distal PA in vivo. A: Src and STAT3 activation were evaluated by immunoblots performed in triplicate on rats lungs with the ratios PY419-Src/c-Src and PY705-STAT3/STAT3 normalized to amido black. As in vitro, DHEA effects in vivo were mediated by the inhibition of Src/STAT3 activation (n = 5 rats in each group). B: DHEA-dependent STAT3 inhibition was confirmed in vivo by nuclear translocation assay, showing a decrease in PY705-STAT3/nuclei colocalization (yellow; n = 5 to 10 arteries per rats in 5 rats in each group). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6.

DHEA restores expression/activity of STAT3 downstream targets aberrantly expressed/activated in experimental PAH. A: miR-204 mRNA level was measured by qRT-PCR and normalized to U6 in distal PAs of rats in each group. Like STAT3 inhibition, DHEA enhances miR-204 expression (n = 5 rats in each group; P < 0.005). Pim1 and NFATc2 (B), BMPR2 (D), and Survivin (E) mRNA levels were measured by qRT-PCR and normalized to 18S in distal PAs. Similarly to MCT-PAH siSTAT3 rats, DHEA-treated rats have impaired STAT3 signal transduction to downstream targets, resulting in Pim1, NFATc2, and Survivin decreased mRNA level, and increased BMPR2 mRNA level. C: NFATc2 activation measured by nuclear translocation assay and expressed as the percentage of NFATc2 positive cells in distal PAs (<300 μm) is decreased by DHEA treatment (n = 10 arteries per rats in 5 rats per group; P < 0.005). *P < 0.05, **P < 0.01, and ***P < 0.001.

To validate our therapeutical intervention, we studied proliferation and apoptosis rates within distal PAs. PASMCs proliferation (%PCNA- and Ki67-positive cells) is inhibited by 50% within PAs in rats treated with DHEA and siSTAT3. Resistance to apoptosis is decreased as well, with treatments increasing apoptosis in the vascular wall by 20% (%TUNEL-positive cells; Fig. 7A).

Fig. 7.

DHEA decreases proliferation and increases apoptosis in MCT-PAH distal PAs. Proliferation and apoptosis rates in distal PAs were measured by PCNA, Ki67, and TUNEL assays respectively in 5 to 10 arteries/rat in 5 rats/group (P < 0.005). Schematic representation of STAT3 signaling in PAH and DHEA effects. Increased circulating growth factors, agonists, and cytokines trigger STAT3 activation. DHEA, previously shown to decrease [Ca2+]i, by inhibiting STAT3 leads to 1) decreased NFATc2, Pim1, and Survivin expression; 2) decreased [Ca2+]i dependent PASMC proliferation and enhancing mitochondrial dependent apoptosis; and 3) enhanced BMPR2 and miR-204 levels leading to c-Src inhibition and disruption of the positive feedback loop sustaining STAT3 activated. *P < 0.05, **P < 0.01, and ***P < 0.001.

DISCUSSION

Here we provide, for the first time, evidence showing that DHEA decreases the Src/STAT3 constitutive activation seen in PAH. We showed that DHEA acts as a pharmacological STAT3 inhibitor since its effects are similar to those obtained by molecular inhibition of STAT3 with siRNA. DHEA-dependant Src/STAT3 inhibition reverses PAH phenotype both in vitro and in vivo. Indeed, DHEA restores most of the molecular and cellular abnormalities seen in PAH-PASMCs, including decreased expression of the onco-proteins Pim1 and Survivin, decreased activation of NFATc2, and upregulation of BMPR2 and miR-204 (Fig. 2A). All these findings contribute to the decrease in PAH-PASMC proliferation and resistance to apoptosis seen in our human PASMC and to the decrease in distal PA remodeling seen in our in vivo model. These findings are supportive of our comprehensive mechanism proposed in Fig. 7.

These findings provide a better understanding of the mechanism by which DHEA reverses PAH. In the past, several groups including us have demonstrated the beneficial effects of DHEA in experimental model of PAH. Indeed, by opening BKCa DHEA can decrease [Ca2+]i (8, 20, 47) and vasodilating PASMCs. Since Src and STAT3 are known as K+ channel inhibitors/Ca2+ channel openers (3, 17, 28, 44, 55), their inhibition could explain how DHEA upregulates Kv and BKCa channels (8). Our present findings provide a comprehensive mechanism linking all the previous findings to the Src/STAT3 axis. Indeed, several studies (5, 36) have demonstrated the implication of the Src/STAT3 axis in the upregulation of the RhoA/ROCK pathway; thus the Src/STAT3 axis inhibition by DHEA could be an explanation of how RhoA/ROCK are modulated by DHEA (26). Although STAT3/Pim1 are recognized as the regulators for both NFATc2 expression and activation in PAH (46), several new findings (15, 16) have proposed that RhoA/ROCK could also be implicated in NFAT activation in the vasculature. Thus whether the inhibition of RhoA (26) by DHEA is independent of the Src/STAT3 axis, we propose it will similarly affect the NFAT pathway. Further studies are thus necessary for a better understanding of how DHEA affect RhoA.

Our findings are not restricted to the PASMCs but could be extend to the PA endothelial cells (PAECs) as well. Indeed, STAT3-induced proliferation and resistance to apoptosis in PAH is also present in proliferative PAH-PAECs (Refs. 37, 41) that constitute plexiform lesions. Thus STAT3 inhibitory drugs like DHEA represent a putative ideal treatment for PAH (Fig. 7B).

To this end, we performed preclinical studies showing that indeed STAT3 inhibition reverses PAH in MCT-induced PAH in rats. As in our human PAH-PASMCs, STAT3 inhibition was associated with decreased PAH-PASMCs proliferation and resistance to apoptosis. Although the MCT model has limitations, this model has largely contributed to the development of new therapeutics for PAH over the last decade. A variety of therapeutic strategies have been tested in MCT-based models and were later shown to be effective in PAH patients; conversely, all clinically proven treatments also work in the MCT model. Nonetheless, STAT3 is activated in PAH-PAECs, it will be of a great clinical interest to determine whether or not STAT3 inhibition can reverse plexiform lesions. Thus, in a further study, we will measure the effects STAT3 inhibition in the hypoxia/Sugen model of PAH, which has been recently described (2).

Although we believe our findings are very promising, the translation to the clinic of STAT3 inhibition using nebulized STAT3 siRNA will be very challenging. Thus utilization of an already clinically approved drug like DHEA is of a great interest. DHEA supplementation is an efficient way in reducing carotid stenosis in response to angioplasty (9). In fact, we previously published that by inhibiting Akt DHEA increases glycogen synthase kinase 3 activation pushing NFAT out of the nucleus and thus limiting its transcriptional activity (9). We (46) recently showed that Akt is not activated in PAH-PASMCs excluding the possibility that the present DHEA effects seen in PAH are Akt mediated. Several studies have demonstrated the critical role of STAT3 in regulating Pim-1/NFAT axis in both cancer (27) and cardiovascular diseases (42, 43); thus inhibition of STAT3 could account for the DHEA effects seen in both our in vitro and in vivo models.

We provide evidence for the first time in vascular tissue that DHEA is an efficient STAT3 inhibitor confirming previous findings in the liver (34). The fact that DHEA can disrupt the positive feedback loop involving miR-204 (which sustained Src/STAT3 activated independently of the activation of the tyrosine kinase receptor; Ref. 12) and that DHEA inhibitory effects on STAT3 are sustained over long period of time (18) is clinically appealing.

Limitations.

Although we believe that our study provides for the first time evidence that DHEA is a good and efficient STAT3 inhibitor in human and rodent PASMCs, we have not elucidated the exact molecular mechanism by which DHEA decreases Src/STAT3 activation. This remain to be established and will constitute a further study. Nonetheless, we have evidence that the DHEA effects in vascular tissue are likely mediated through a plasma membrane receptor coupled to G protein (9). Indeed, we previously showed in carotid smooth muscle cells that through a plasma membrane receptor DHEA could decrease NFAT activation and expression. As STAT3 regulates both expression and activation (through Pim-1) of NFAT, we believe that the DHEA inhibitory effect on Src/STAT3 is also mediated by the plasma membrane DHEA receptor. Nonetheless, other mechanisms cannot be ruled out. For example, it has been demonstrated that activation of the peroxisome proliferator-activated receptor-γ (PPAR-γ), a member of the nuclear receptor family that is downregulated in PAH (24), has an inhibitory effect on STAT3 (31, 33, 56). A direct physical protein-protein interaction has been shown between PPAR-γ and activated STAT3, resulting in decreased transcriptional activity of STAT3. Moreover, treatment with PPAR-γ agonist ciglitazone in glioblastoma cell lines showed a decreased expression of phosphorylated form of STAT3 associated with an increased expression of STAT3 inhibitors like the Suppressor of cytokine signaling (SOCS) 3 and the protein inhibitor of activated STAT3 (PIAS3; Ref. 19). As DHEA has been shown to increase PPAR-γ mRNA levels (48), PPAR-γ could be implicated in the DHEA-dependant STAT3 inhibition. Considering that PPAR-γ agonists used in the treatment of PAH patients (rosiglitazone and pioglitazone) could be associated with adverse cardiovascular events (32), DHEA could offers an alternative therapeutic approach. Although the inhibitory effects of DHEA on the Src/STAT3 axis might link together all the previously published findings on DHEA in pulmonary hypertension, other uncovered pathways might also be affected including FAK, Rac1, and other pathways implicated in cell migration that are also implicated in vascular lesion formation seen in pulmonary and are currently understudied. Finally, enlarging our findings to other pulmonary hypertension isoforms including PA secondary to chronic obstructive pulmonary disease or left heart diseases could be beneficial to greater population.

Conclusion/Perspectives

In conclusion, our findings demonstrated for the first time that DHEA is an efficient inhibitor of the Src/STAT3 axis in human PASMCs. As described in our comprehensive schematic in Fig. 7, we showed that in both humans and rodents this newly characterized effect of DHEA links together all the previously published findings on the therapeutic effects of DHEA seen in rodent pulmonary hypertension including K+ channels and RhoA/ROCK abnormalities. Moreover, we provide evidence for the first time in PAH that DHEA a nontoxic, safe, and already available over the counter drug can reverse vascular remodeling processes seen in PAH. Thus, with a single drug we could affect many features associated with pulmonary hypertension including vasoconstriction and vascular remodeling. Moreover, as Src/STAT3 are activated by many different signals including inflammation, growth factor; extracellular matrix, hypoxia (all of which can trigger pulmonary hypertension), we believe that DHEA might be efficient in many different PH isoforms, which is not the case of the presently used medicine.

This finding is of a great clinical interest as it offers us the perspective to enlarge the ongoing clinical trial NCT01273259 actually restricted to hypoxic pulmonary hypertension patients to PAH patients. Finally, our findings also offer new therapeutic perspective to clinician to treat diseases in which STAT3 is implicated including cancer.

GRANTS

This work was supported by the Heart and Stroke Foundation of Canada, Canadian Institute for Health Research, and Canadian Research Chair (to S. Bonnet).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

R. P. and S. B. conception and design of research; R. P., J. M., M. H. J., M. B., and A. C. performed experiments; R. P., J. M., M. H. J., M. B., and A. C. analyzed data; R. P. prepared figures; R. P. and S. B. drafted manuscript; R. P., J. M., M. H. J., M. B., A. C., and S. B. approved final version of manuscript; J. M. interpreted results of experiments; S. B. edited and revised manuscript.

ACKNOWLEDGMENTS

We thank the “Societé Québecoise d'Hypertension Artérielle” (scholarships to J. Meloche and R. Paulin). M. H. Jacob was supported by CAPES (Brazilian Research Agency).

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View Abstract