Conceived and designed the experiments: WX. Performed the experiments: WP XG LS. Analyzed the data: WP XG. Contributed reagents/materials/analysis tools: WX. Wrote the paper: WP XG.
The authors have declared that no competing interests exist.
To investigate the role of bone morphogenetic protein 2 (BMP-2) in regulation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and apoptosis of pulmonary artery smooth muscle cells (PASMCs) under hypoxia.
Normal human PASMCs were cultured in growth medium (GM) and treated with BMP-2 from 5–80 ng/ml under hypoxia (5% CO2+94% N2+1% O2) for 72 hours. Gene expression of PTEN, AKT-1 and AKT-2 were determined by quantitative RT-PCR (QRT-PCR). Protein expression levels of PTEN, AKT and phosph-AKT (pAKT) were determined. Apoptosis of PASMCs were determined by measuring activities of caspases-3, -8 and -9. siRNA-smad-4, bpV(HOpic) (PTEN inhibitor) and GW9662 (PPARγ antagonist) were used to determine the signalling pathways.
Proliferation of PASMCs showed dose dependence of BMP-2, the lowest proliferation rate was achieved at 60 ng/ml concentration under hypoxia (82.2±2.8%). BMP-2 increased PTEN gene expression level, while AKT-1 and AKT-2 did not change. Consistently, the PTEN protein expression also showed dose dependence of BMP-2. AKT activity significantly reduced in BMP-2 treated PASMCs. Increased activities of caspase-3, -8 and -9 of PASMCs were found after cultured with BMP-2. PTEN expression remained unchanged when Smad-4 expression was inhibited by siRNA-Smad-4. bpV(HOpic) and GW9662 (PPARγ inhibitor) inhibited PTEN protein expression and recovered PASMCs proliferation rate.
BMP-2 increased PTEN expression under hypoxia in a dose dependent pattern. BMP-2 reduced AKT activity and increased caspase activity of PASMCs under hypoxia. The increased PTEN expression may be mediated through PPARγ signalling pathway, instead of BMP/Smad signalling pathway.
Hypoxic pulmonary hypertension, one of the most common pulmonary arterial hypertension, is characterized by increased proliferation and reduced apoptosis of smooth muscle cells
Bone morphogenetic protein 2 (BMP-2), which belongs to the transforming growth factor beta super-family, is a negative regulator of PASMC growth
It has been shown that PPARγ agonist up-regulated phosphatase and tensin homologue deleted on chromosome ten (PTEN) expression in allergen-induced asthmatic lungs
Though BMP-2 activates PPARγ to achieve anti-proliferative effects on SMCs, its role in regulation of PTEN expression in SMC proliferation is yet established. Here we report that BMP-2 increased PTEN expression of PASMCs under hypoxia in a dose dependent pattern. BMP-2 reduced AKT activity and increased caspase activity of PASMCs under hypoxia. The increased PTEN expression may be mediated through PPARγ signalling pathway, instead of BMP/Smad signalling pathway.
Human primary PASMC was purchased from ScienCell Research Laboratories (CA, USA). PASMC was cultured and expanded in SMC growth medium (GM): SMC basal medium (BM) (ScienCell Research Laboratories, CA USA) supplemented with SMC growth supplement (ScienCell Research Laboratories), 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin. PASMCs were regularly passaged every 4–5 days. BMP-2 (Miltenyi Biotec, Singapore) was supplemented in the cell culture medium to determine its effect on PASMC proliferation.
bpV(HOpic) (Merck, Germany), a PTEN inhibitor, and GW9662 (Sigma Aldrich, USA), an antagonist of PPARγ, were used to determine the possible signalling pathways mediated by BMP-2.
PASMC were cultured in hypoxic condition to determine the anti-proliferative effect of BMP-2 on PASMCs. Hypoxia was created in an incubator: 5% CO2+94% N2+1% O2, 37°C.
PASMCs cultured in GM supplemented with BMP-2 were cultured in hypoxia incubator for 72 hours. The supernatant and cells were collected for cytotoxic, gene and protein expression studies.
PASMCs were analyzed by QRT-PCR to determine gene expression after treated with BMP-2. Total RNA was isolated using RNA Isolation Kit (QIAGEN, USA) according to the manufacturer’s instructions
GAPDH60°C, 90 bp, | forward | 5' |
reverse | 5' |
|
PTEN60°C, 155 bp | forward | 5' |
reverse | 5' |
|
AKT-158°C, 125 bp | forward | 5' |
reverse | 5' |
|
AKT-258°C, 127 bp | forward | 5' |
reverse | 5' |
|
Smad-460°C, 128 bp | forward | 5' |
reverse | 5' |
Cell proliferation rate of PASMCs was determined by CyQUANT® Cell Proliferation Assay Kit (Invitrogen, USA). Briefly, 1×104 PASMC/well were seeded into 24-well plate and cultured with BM for 24 hours. The cell culture medium was changed to GM supplemented with BMP-2 for 72 hours in incubator under hypoxia. After that, cell supernatant was removed. Cells were washed with PBS and frozen at −80°C freezer for at least 1 hour. Then each well was incubated with 200 µl CyQUANT® cell-lysis buffer containing DNase-free RNase (1.35 U/ml) to eliminate the RNA component of the fluorescent signal for 1 hour at room temperature. After that, 200 µl cell lysis buffer containing 2X solution of CyQUANT® GR dye was added into each sample for 10 min. The fluorescence intensity was measured using a Tecan fluorescence microplate reader (Tecan Infinite M200, LabX Canada) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.
The cyto-toxicity of BMP-2 towards PASMCs was determined using CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega, USA). Briefly, 5×104 PASMCs/well were seeded into 12-well plate in GM supplemented with BMP-2 for 72 hours in incubator at 37°C. After that, cell culture supernatant was collected and mixed with CytoTox-ONE™ Reagent for at least 10 min. After addition of 50 µl stop solution, the fluorescence signal was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm.
Protein expression levels from treated and non-treated PASMCs were determined by western blot analysis
Proteins from treated and non-treated PASMC were used to determine the apoptosis of PASMCs by determining the activities of caspase -3, -8 and -9.
Caspase -3 and -8 activities were determined by Caspase -3 and -8 Assay Kits, Fluorimetric (Sigma Aldrich, CASP3F and CASP8F). The fluorescence intensity of caspase-3 was recorded at wavelength of 360 nm for excitation, and at wavelength of 460 nm for emission, while it was 360 nm of excitation, and 440 nm of emission for caspase-8. The activity of caspase was calculated as Fluorescence intensity (FI)/min/ml = ΔFlt/(t x v), where ΔFlt = difference in fluorescence intensity between time zero and time t minutes, t = reaction time in min, and v = volume of sample in ml.
Similarly, Caspase-9 activity was determined by Caspase 9 Assay Kit, Fluorimetric (EMD4Biosciences, QIA72). The fluorescence intensity was recorded at wavelength 400 nm of excitation, and wavelength 505 nm of emission. The same formula as the one used for calculation of caspase-3 activity was used to calculate caspase-9 activity.
Trypsinized PASMCs were seeded at a density of 1×105cells/well in 12-well plates and cultured with SMC growth medium without antibiotics. A plasmid carrying enhanced green fluorescent protein (pEGFP) was used to transfect PASMCs to optimize the transfection condition
The lipoplexes were manufactured by mixing lipofectamine-2000 (Lipo) (Invitrogen, USA) with pEGFP (2 ug) from 1∶1 to 4∶1 (volume/weight: ul/ug). Lipofectamine and plasmid DNA were diluted in 50 µl Opti-MEM® I Reduced Serum Medium (Invitrogen, USA). The pEGFP lipoplexes were developed by mixing the respective solutions. After mixing, the mixture was vortexed for 10 seconds followed by centrifuge at lowest speed for 10 minutes. Then lipoplex mixture was sedated for 10 min at room temperature and added into cell culture medium (SMC growth medium without antibiotics) to transfect PASMCs for 24 hours at 37°C in incubator.
siRNA-Smad-4 or siRNA-control lipoplexes were developed by replacing pEGFP with plasmid siRNA-Smad-4 or siRNA-control. The volume of lipofectamine used would be the one that resulted in the highest EGFP gene transfection efficiency. Next, lipofectamine volume was fixed and siRNA concentration was adjusted fom 40 nM up to 200 nM to identify the optimal ratio between lipofectamine and siRNA-Smad-4 to inhibit Smad-4 gene expression.
All statistical analyses were performed using SPSS (version 10.0). The data were presented as mean± standard error means (SEM) and analyzed by the method of analysis of variance (ANOVA) using Bonferroni test. All tests were performed with a significance level of 5%.
The proliferative rate of PASMCs showed dose dependence of BMP-2 when PASMCs was cultured under hypoxia (
PASMCs cultured in GM supplemented with 0–80 ng/ml BMP-2. BMP-2 at the concentrations of 40 and 60 ng/ml significantly inhibited PASMC proliferation as compared with GM without BMP-2. Typical pictures of PASMCs cultured in GM only (
The toxicity of BMP-2 towards PASMCs was determined by measuring LDH in the cell culture supernatant. No significant cell injury was found when BMP-2 was increased up to 60 ng/ml (
It appears that only at 80 ng/ml concentration of BMP-2 resulted in significantly increased LDH leakage as compared with GM with 0 ng/ml BMP-2. The percentage of LDH leakage was normalized to fresh GM (consider as 0%). (*: vs 0 ng/ml, p<0.05).
The percentage of LDH in supernatant was 2.3±0.6% when PASMCs were cultured in GM without BMP-2. They were 2.7±0.4% with 5 ng/ml BMP-2, 2±0.4% with 10 ng/ml, 1±0.3% with 20 ng/ml, 1±0.2% with 40 ng/ml, 2±0.8% with 60 ng/ml. However, the leakage of LDH increased to 4.2±0.6% when BMP-2 was 80 ng/ml concentration, which was significantly increased as compared with that cultured in GM medium only (
BMP-2 significantly increased PTEN gene expression at 8 hours (2.4±0.2 folds, p<0.05) compared to any other time point (
BMP-2 significantly increased PTEN expression at 8 and 24 hours after treatment. (&: vs any other time point, p<0.05; *: vs 0, 1, and 4 hours, p<0.05).
Generally, no significant reduction or increment of AKT-1 gene expression was found after addition of BMP-2 (
No significant reduction or increment of AKT-1 gene expression was found when PASMCs were cultured in GM supplemented with BMP-2 (
Western blot analysis suggested that PTEN protein expression level was BMP-2 dose dependent (
(
Next, PASMCs were cultured in GM supplemented with 40 ng/ml BMP-2 for a serial time (
To determine whether PTEN inhibitor or PPARγ antagonist will inhibit or block the effect of BMP-2 on PTEN production, PASMCs were pre-treated with 2.5 uM bpV (HOpic) (PTEN inhibitor) or 1 uM GW9662 (PPARγ antagonist) for 1 hour before addition of BMP-2. It was found that bpV (HOpic) and GW9662 reduced PTEN protein expression (
To determine whether BMP-2 mediated PTEN up-regulation was mediated by Smad signalling pathway, a plasmid carrying siRNA-Smad-4 was used to inhibit Smad-4 gene expression. Lipofectamine-2000 was used as a transfection vehicle to encapsulate siRNA-Smad-4 plasmid (Lipo-siRNA-Smad-4).
The transfection condition was first optimized using a plasmid carrying enhance green fluorescent protein (pEGFP). Lipo-pEGFP was added into cell culture medium for 24 hours. Then gene transfection efficiency was determined (
(Magnification = 40×).
Based on the optimized transfection condition, PASMCs were transfected with lipo-siRNA-Smad-4. QRT-PCR for Smad-4 gene expression demonstrated that Smad-4 gene expression was successfully reduced to 25% of non-transfected cells when 6 µl Lipofectamine-2000 was used to carry 120 nM siRNA-Smad-4 (
(
It was found that BMP-2 induced up-regulation of PTEN protein expression was not abolished even when Smad-4 protein expression was significantly reduced (
To determine whether PTEN inhibitor could reverse the anti-proliferative effect of BMP-2 on PASMC, bpV (HOpic) was included in cell culture medium for 72 hours under hypoxia (
PPARγ antagonist and PTEN inhibitor were added into respective cell culture medium 1 hour before adding BMP-2 (40 ng/ml). (The number of PASMCs after cultured in GM only was considered as 100%). (*: vs GM only) (Lipo = Lipofetamine −2000).
GW9662 was also included in cell culture medium to investigate whether the up-regulated PTEN was through PPARγ signalling pathway (
Significantly increased caspase-3 activity was found when PASMCs were cultured in medium (6.5±0.18 FI/min/ml) supplemented with BMP-2 for 8 hours (
BMP-2 (40 ng/ml) significantly increased caspases 3 (
Significantly increased caspase-9 activity was found when PASMCs were cultured in medium (5.9±0.17 FI/min/ml) supplemented with BMP-2 for 8 hours (
These suggested that BMP-2 increased caspase activity and may promote apoptosis of PASMC under hypoxia.
The present study demonstrated that BMP-2 up-regulated PTEN gene and protein expression levels of PASMCs under hypoxia. BMP-2 increased caspase activities of PASMCs under hypoxia. The increased PTEN expression was mediated through BMP-2/PPARγ signalling pathway.
The pulmonary vascular remodelling in pulmonary arterial hypertension is characterized by changes in pulmonary vascular structure
BMP-2 has been shown to inhibit human aortic SMCs
BMP-2 can activate Smad signalling system through BMP receptor-II (BMP-RII)
In the current study, BMP-2 up-regulated PTEN gene and protein expression levels. The up-regulated PTEN expression can be inhibited by PPARγ antagonist suggesting the up-regulated PTEN gene and protein expressions were mediated by PPARγ signalling. We propose here that BMP-2 mediated up-regulation of PTEN of PASMC under hypoxia was through PPARγ signalling. Hassman et al.,
Current study also suggested that BMP-2 could increase caspase activity of PASMC under hypoxia. It is known that chronic hypoxia can prolong the growth of human vascular SMC by inducing telomerase activity and telomere stabilization
In summary, the study highlights that BMP-2 can increase PTEN expression under hypoxia in a dose dependent pattern. BMP-2 can increase caspase activities of PASMC under hypoxia. The increased PTEN expression may be mediated through PPARγ signalling pathway, instead of BMP/Smad signalling pathway.