Smad-Dependent and Smad-Independent Induction of Id1 by Prostacyclin Analogues Inhibits Proliferation of Pulmonary Artery Smooth Muscle Cells In Vitro and In Vivo
Rationale: Mutations in the bone morphogenetic protein type II receptor (BMPR-II) are responsible for the majority of cases of heritable pulmonary arterial hypertension (PAH). Mutations lead to reduced Smad1/5-driven expression of inhibitor of DNA binding protein 1 (Id1) and loss of the growth suppressive effects of BMPs. The impact of existing PAH therapies on BMP signaling is lacking.
Objective: Because prostacyclin analogues are effective treatments for clinical PAH, we hypothesized that these agents enhance Smad1/Id1 signaling.
Methods and Results: Iloprost alone induced Id1 expression in human pulmonary artery smooth muscle cells (PASMCs), an effect that was independent of Smad1/5 activation but dependent on a cAMP-responsive element in the Id1 promoter. In addition, iloprost and treprostinil enhanced BMP-induced phosphorylation of Smad1/5 and Id1 expression in a cAMP-dependent manner. The mechanism involved suppression of inhibitory Smad, Smad6. Furthermore, iloprost rescued the deficit in Smad1/5 phosphorylation and Id gene expression in PASMCs harboring mutations in BMPR-II and restored growth suppression to BMP4 in mutant PASMCs. We confirmed a critical role for Id1 in PASMC proliferation. Reduced expression of Id1 was observed in concentric intimal lesions of heritable PAH cases. In the monocrotaline rat model of PAH, associated with reduced BMPR-II expression, we confirmed that treprostinil inhibited smooth muscle cell proliferation and prevented progression of PAH while enhancing Smad1/5 phosphorylation and Id1 gene expression.
Conclusions: Prostacyclin analogues enhance Id1 expression in vitro and in vivo and restore deficient BMP signaling in BMPR-II mutant PASMCs.
- cyclic adenosine 5′-monophosphate
- BMP type II receptor
- Smad1 phosphorylation
- inhibitor of DNA binding protein
Pulmonary arterial hypertension (PAH) is characterized by a sustained elevation of pulmonary arterial pressure caused by increased pulmonary vascular resistance. The increase in resistance results from a complex process of vascular obliteration and occlusion involving cellular components of the walls of precapillary pulmonary arteries.1,2 Although the precise sequence of events that initiates the process of remodeling remains unclear, the identification of heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II receptor (BMPR-II) has suggested a central role for the BMP/transforming growth factor-β signaling pathway.3–5 Mutations in BMPR-II have been identified in approximately 80% of cases of heritable PAH.6 Mice deficient in BMPR-II are more susceptible to pulmonary hypertension and vascular remodeling.7 In addition, several studies support a role for dysfunctional BMP signaling in idiopathic PAH8 and in experimental models of PAH-induced by chronic hypoxia, monocrotaline exposure, and high flow.9,10 Restoration of BMPR-II expression by targeted gene delivery to the pulmonary circulation prevents the development of pulmonary hypertension in the chronically hypoxic rat.11
Loss of BMPR-II function, at least in pulmonary artery smooth muscle cells (PASMCs), reduces phosphorylation of downstream Smad1/5 proteins.12 One of the major transcriptional targets of BMP/Smad signaling is the inhibitor of DNA binding (Id) family of proteins, comprising Ids 1 to 4.13 We have previously shown that loss of BMPR-II function or knockdown of Id1 both lead to a loss of the growth suppressive effects of BMPs in PASMCs.14 Thus therapeutic enhancement of the BMPR-II/Smad1/Id1 axis may be a useful intervention in PAH, potentially restoring the growth suppressive effects of BMPs.
To date, little information is available regarding the impact of existing PAH therapies on BMP signaling. One of the most successful therapeutic interventions in PAH is the use of prostacyclin and its analogues.15,16 Prostanoids are thought to exert their beneficial effects by a combination of vasodilatation, inhibition of platelet aggregation, and inhibition of PASMC proliferation.17 Vasodilation of pulmonary arteries by prostanoids and inhibition of PASMC proliferation are largely dependent on the activation of adenylyl cyclase and elevation of intracellular cAMP.18 Recently, it was shown that cAMP can induce Id1 and Id2 gene expression, resulting in the paracrine induction of bone formation in human mesenchymal stem cells.19 Furthermore, the Id1 promoter contains a cAMP response element and elevation of cAMP enhanced BMP4 stimulated Id1 reporter gene activity in C2C12 cells.20 cAMP may also suppress expression of the BMP inhibitory Smad, Smad6, in osteoprogenitor cells.21
In the present study, we investigate the potential interaction between prostanoid stimulation and BMP signaling in PASMCs. We show that prostanoids induce early Id1 gene expression independent of Smad signaling though also enhance BMP4-induced Smad1/5 phosphorylation and Id1 gene expression at later time points via a cAMP/protein kinase A (PKA)-dependent mechanism. Furthermore, prostanoids can partly rescue the defect in Smad1 activation and Id gene expression in PASMCs harboring mutations in BMPR-II and restore BMP4-mediated growth suppression in BMPR-II mutant cells. We further confirm that Id1 gene expression, as a critical target of prostanoid and BMP signaling, regulates the proliferation of human PASMCs. Finally, we show that prostanoids inhibit progression of pulmonary hypertension in the monocrotaline rat model, known to be associated with BMPR-II dysfunction,9 and enhance deficient BMP signaling in this model.
Human PASMC Culture
Control human PASMCs were obtained from patients undergoing lung resection for suspected malignancy (n=5), derived from sites distant to the tumor. Cells were derived from peripheral pulmonary arteries (<2 mm external diameter) as previously described or lobar arteries for growth studies.14 Cells were also derived from the lungs of patients undergoing heart-lung transplantation for PAH (n=3, at Papworth Hospital), known to harbor a mutation in the BMPR2 gene. These included 2 patients with a mutation in the kinase domain of BMPR2 in which arginine or tyrosine is substituted for cysteine at position 347 (C347R and C347Y) and 1 patient with an exon 1 nonsense mutation at amino acid 9, W9X, predicted to lead to haploinsufficiency.22 The local ethics review committee approved the study, and subjects or relatives gave written informed consent.
PASMCs were grown to ≈95% confluence. After 24-hour quiescence, cells were treated with or without BMP4 (0.1 to 10 ng/mL) in 0.1% FBS/Dulbecco modified Eagles medium (DMEM) for up to 24 hours. Treprostinil sodium was kindly provided by Lucie H. Clapp (University College London). Treprostinil sodium (10−7 to 10−6mol/L), iloprost (10−7 to 10−6 mol/L) (Schering), and dibutyryl cAMP (dbcAMP, 1×10−4 to 2 ×10−3 mol/L) were applied in experiments. At specified time points, cells were lysed in buffer as described.18 Samples were electrophoresed by 10% SDS-PAGE and then transferred to nitrocellulose membrane. For studies of Smad1/5 activation, blots were incubated with polyclonal rabbit antiphospho-Smad1/5 antibody as previously described.14 To confirm equal protein loading, blots were reprobed using β-actin. The role of PKA-regulated adenylyl cyclase activity was investigated using H-89, an inhibitor of PKA (10−6 M), for 1 hour before and continuing during incubation with prostacyclin analogues. Anti-Id1 was used followed by anti-rabbit secondary antibody.
Reporter Gene Assays and Constructs
We used a full-length Id1 promoter corresponding to a 2.2 kb 5′ upstream regulatory region of the human Id-1 driving a luciferase reporter gene in a PGL-3 vector.23 Site directed mutagenesis was used to generate additional constructs carrying mutations in the previously described cAMP response binding element (CRE)20 or mutations of the 2 major Smad-binding elements (SBEs) described previously.24,29 These wild-type and mutated promoters were used to determine the specific role of SBE and CRE elements in iloprost-stimulated gene transcription. See the Online Data Supplement at http://circres.ahajournals.org for a description of mutant constructs. All constructs were checked by sequencing. Cotransfection with Renilla luciferase was used as a transfection control (pRL-TK, Promega). Transient transfection of PASMCs was achieved by electroporation using the Amaxa Nucleofector system according to the manufacturer’s instructions.
Quiescent PASMCs were incubated with BMP4 (0.1 to 10 ng/mL) or DMEM alone for 4 or 24 hours. Total RNA was extracted using TRIzol reagent, then reverse transcribed using Superscript III First-strand Synthesis Super Mix. Quantitative-PCR was performed using SYBR1 GreenER qPCR SuperMix, and samples were run on BioRad iCycler iQ. Primers were used to allow amplification of Id1 and normalized to β-actin, which was included in each sample run. See the expanded Methods section, available in the Online Data Supplement at http://circres.ahajournals.org for primer sequences.
Cell Proliferation Assay
The effect of iloprost on PASMC proliferation was determined during stimulation with 10% FBS or 10 ng/mL PDGF. Cells were seeded in 24-well plates (104 cells per well) overnight in DMEM containing 10% FBS, then exposed to iloprost and/or BMP4 in 10% FBS/DMEM or 10% FBS/DMEM alone. Medium was changed on alternate days, and cells were counted on day 4 with a hemocytometer.
SiRNA Knockdown of Id1 and Smad6
PASMCs were plated at 20 000/well in 24-well plates into DMEM containing 10% FBS and left overnight. Cells were then transfected with Id1 siRNA (Smart pool, Dharmacon) or control siRNA (siCP nontargeting pool, Dharmacon) using Dharmafect transfection reagent, following the manufacturer’s instructions. See the Online Data Supplement at http://circres.ahajournals.org for details. On target plus siRNA for Smad6 (Dharmacon) was incubated with cells 48 hours before RNA extraction and protein collection.
Lentiviral Transfection of Id1
Pseudotyped vectors were generated by transfection of plasmid DNA into 293T cells using a calcium phosphate method, as previously described.25 Transfections were performed in 100-mm dishes using optimized ratios of constructs, as outlined in the expanded Methods section, available in the Online Data Supplement at http://circres.ahajournals.org. The efficiency of transfection of PASMCs was assessed by GFP reporter expression 72 hours after transduction. Levels of Id1 were assayed by immunoblotting. Cell proliferation and [3H]-thymidine assays were then conducted in the presence of 10% FBS.
The Monocrotaline Rat Model
Male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) either received a single injection of monocrotaline (MCT, 60 mg/kg, s.c.) or saline. Three weeks after MCT injection, osmotic minipumps containing treprostinil (0.15 mg/mL; to deliver an approximate rate of 45 ng/kg/min) or saline vehicle were implanted for a further 2 weeks. Six animals were used per group.
At the end of the treatment protocol, rats were anesthetized for hemodynamic assessment and then euthanized for measurement of right ventricular hypertrophy and lung tissue collection. Slides of lung tissue were stained by hematoxylin and eosin and elastic van Giessen techniques and examined by light microscopy. Slides were analyzed in a blinded fashion without knowledge of treatment groups. In each rat, 30 to 40 intra-acinar arteries were categorized as muscular, partially muscular, or nonmuscular. All experiments were performed according to institutional guidelines that complied with national and international regulations.
Lung tissue samples were obtained from patients with heritable PAH undergoing lung transplantation (n=3) and controls (lung resection for suspected tumor) (n=3). Fixed sections were dual immunostained with anti-Ki67 and anti-α-smooth muscle actin (SMA) or anti-α-SMA and anti-Id1 overnight. Immunoreactivity was detected by anti-mouse (for SMA) or anti-rabbit (for Id1, Ki67). Nuclei were stained with Hoechst reagent before imaging by confocal microscopy. In rat lungs, anti-Id1, phospho-Smad1, and proliferating cell nuclear antigen (PCNA) were used and visualized with 3, 3′-diaminobenzedine and examined by light microscopy.
Data are presented as means±standard error. Data between groups were compared using a 2-tailed t test or ANOVA, as appropriate. P<0.05 was used to indicate a statistically significant result.
Iloprost Enhances Id1 Gene Expression in PASMCs
Because Id1 is a major target of BMP signaling and the Id1 promoter is reported to have a cAMP-responsive site, we first determined whether iloprost could alter the expression of Id1 in human PASMCs. Iloprost (10−7 mol/L) alone increased the expression of Id1 at least 10-fold (Figure 1A) after 24 hours of exposure. As expected, BMP4 (10 ng/mL) markedly increased the expression of Id1. This effect was further amplified in the presence of iloprost.
To begin to determine the mechanism by which iloprost stimulates Id1 gene expression, we used full-length Id1-Luc,23 SBEmut-Luc, and CREmut-Luc luciferase reporter constructs transfected into PASMCs. Iloprost alone stimulated Id1-Luc reporter activity, as did BMP4 alone. The combination of iloprost and BMP4 further increased Id1-luc reporter activity, similar to the results of Id1 gene expression by real-time PCR (Figure 1B). Iloprost was capable of stimulating activity of the SBEmut-Luc reporter, which contains mutations at the 2 SBE sites (Figure 1C). However, as expected, the SBEmut-Luc reporter demonstrated a minimal response to BMP stimulation. In contrast, iloprost was unable to stimulate activity of the CREmut-Luc reporter, which lacks the cAMP-responsive binding element previously identified in the Id1 promoter20 (Figure 1D). As expected, the CREmut-Luc reporter retained sensitivity to BMP stimulation.
Iloprost Enhances Id1 Protein and BMP-Stimulated Smad1/5 Phosphorylation in PASMCs
The above findings suggest that iloprost may increase Id1 expression by Smad-dependent and Smad-independent pathways. We first confirmed that iloprost alone was unable to activate Smad1/5 phosphorylation at 1 hour or 24 hours (Figure 1E and 1F). Despite this, iloprost alone did increase Id1 protein expression at 24 hours (Figure 1F). In the presence of BMP4, iloprost increased Id1 protein expression without affecting phosphorylation of Smad1/5 at 1 hour and enhanced both Smad1/5 phosphorylation and Id1 protein expression at 24 hours (Figure 1E and 1F). In further experiments, we confirmed that iloprost/BMP4 cotreatment led to a sustained activation of Smad1/5 at later time points (24 to 48 hours) (Figure 1G) and increased Id1 expression at 24 hours (Figure 1H).
Effects of Iloprost on Id1 Expression Are cAMP/PKA-Dependent
The effects of iloprost alone on Id1 gene expression were partly inhibited by the PKA inhibitor H-89 (Figure 2A). The enhanced stimulation of Id1 gene expression seen with BMP4 in the presence of iloprost was completely reversed by H-89 (Figure 2B). To confirm the effect of cAMP/PKA on Id1 protein expression, we show that the cell permeable analogue dibutyryl cAMP alone increased Id1 protein expression without activating Smad1 at 6 hours (Figure 2C). This effect was inhibited in the presence of H-89. In the presence of BMP4 for 24 hours, dbcAMP increased Smad1/5 phosphorylation and Id1 protein expression. Again, this effect was reversed by pretreatment with H89 (Figure 2D).
Treprostinil Also Enhances BMP4-Stimulated Smad1 Activation and Id1 Expression
To confirm that the enhancement of BMP4 simulated Smad1/5 phosphorylation and Id1 protein expression is seen with other prostacyclin analogues, we demonstrated that similar effects are observed with another prostanoid used clinically, treprostinil (Online Figure I, A through C).
Iloprost Reduces Expression of the Inhibitory Smad, Smad6
To explore the potential mechanism of enhanced and prolonged BMP-stimulated Smad1 activation by iloprost, we questioned whether iloprost inhibits expression of the BMP-specific inhibitory Smad, Smad6, as previously reported in osteoprogenitor cells.21 Dibutyryl cAMP (Figure 3A) or iloprost (Figure 3B) significantly reduced BMP4 stimulated Smad6 gene expression in PASMCs. To further confirm the involvement of Smad6 inhibition in the enhancement of BMP signaling by iloprost, we used siRNA to show that knockdown of Smad6 increased the phosphorylation of Smad1/5 and prevented any further enhancement of Smad1/5 activation in the presence of iloprost (Figure 3C).
Iloprost Partly Reverses the Smad1/Id1 Signaling Defect in BMPR-II Mutant Cells
We have previously reported14 that human PASMCs harboring mutations in BMPR-II demonstrate a failure of Smad1/5 activation and a reduced ability to induce Id1 expression in response to BMPs. We questioned whether incubation of mutant PASMCs with iloprost might reverse this defect. We first confirmed that BMPR-II mutant cells were deficient in both Smad1/5 activation and Id1 expression in response to BMP4 (Figure 4A). Iloprost significantly increased both the amount of phosphorylated Smad1/5 and Id1 protein induced by BMP4 in mutant cells at 24 hour (Figure 4B to 4D).
Iloprost Enhances the Antiproliferative Effects of BMP4 on PASMCs Via Id1
To determine whether the interaction between BMPs and iloprost has functional significance, we studied proliferative responses of PASMCs. Iloprost and BMP4 both significantly inhibited the proliferation of proximal PASMCs induced by 10%FBS (Figure 5A) or 10 ng/mL PDGF (Online Figure II) over 4 days. Cotreatment of serum-stimulated PASMCs with BMP4 and iloprost led to a greater reduction in proliferation compared with either treatment alone (Figure 5A).
The involvement of Id1 in iloprost mediated growth suppression was confirmed by siRNA knockdown of Id1 in the presence or absence of iloprost. Interestingly, Id1 knockdown (>90%) alone increased the proliferation of PASMCs maintained in 10%FBS for 4 days (Figure 5B). Iloprost inhibited the proliferation of cells transfected with the control siRNA, but was unable to inhibit proliferation of cells in which Id1 expression was reduced (Figure 5B).
Iloprost Restores the Antiproliferative Effect of BMP4 in BMPR-II Mutant PASMCs
To determine whether iloprost could influence an important functional defect in BMPR-II mutant PASMCs, we first confirmed that mutant cells are resistant to growth suppression by BMP4, as we have previously reported (Figure 5C).12 We also confirmed that BMPR-II mutant cells were growth inhibited by iloprost (Figure 5C). The combination of BMP4 with iloprost led to enhanced growth inhibition of mutant cells compared with either agent alone (Figure 5C). To demonstrate whether induction of Id1 expression could directly modify the proliferation of control and mutant PASMCs, we used lentiviral transfection to overexpress the human Id1 gene. After transfection with empty lentiviral vector, mutant PASMCs proliferated more rapidly than control cells over 4 days (Figure 5D). Overexpression of Id1 significantly reduced the proliferation of control and mutant PASMCs in response to 10%FBS compared with cells transduced with empty vector (Figure 5D), demonstrating that restoration of Id1 expression directly influences the proliferative state of mutant PASMCs. In parallel studies, we confirmed the overexpression of Id1 in transduced PASMCs and confirmed that the transduction efficiency was greater than 90%, using a lentiviral vector driving expression of GFP (Figure 5E).
Lack of Id1 Staining in Concentric Lesions From Familial PAH Patients
In control human lungs, Id1 expression as assessed by immunohistochemistry and confocal microscopy was observed in inflammatory cells and in occasional epithelial smooth muscle and endothelial cells. In small pulmonary arteries from control lungs, numerous vascular wall cells were found that coexpressed Id1 and α-SMA (Figure 6A). In contrast, in concentric intimal and hypertrophied small pulmonary arteries from patients with heritable PAH, no staining for Id1 was observed in cells expressing α-SMA (Figure 6B). In control small pulmonary arteries, expression of the proliferation marker Ki67 was virtually absent in the vascular wall. In contrast, in concentric intimal lesions found in the lungs of patients with heritable PAH, Ki67-positive cells were seen in most lesions (Online Figure III A and III B).
Treprostinil Inhibits Progression of Pulmonary Hypertension and Vascular Remodeling in MCT Rats
Five weeks after exposure to MCT, including 2 weeks of saline infusion, rats exhibited severe elevation of right ventricular systolic pressure, elevated pulmonary vascular resistance, and right ventricular hypertrophy (Figure 7A to D), compared with animals not exposed to MCT. Rats treated with treprostinil for the final 2 weeks demonstrated a reduction in all of these indices compared with saline-treated rats (Figure 7A to 7D). In addition, MCT-exposed rats treated with treprostinil infusion over the final 2 week period exhibited less severe muscularization of small pulmonary arteries compared with saline-exposed animals (Figure 7E and 7F). Chronic treprostinil infusion had no effect on systemic blood pressure in rats (Online Figure IV).
Treprostinil Infusion Restores Lung Smad1/5 Activity and Id1 Expression in MCT-Exposed Rats
We first confirmed that the expression of BMPR-II protein, phospho-Smad1/5, and Id1 gene expression were markedly suppressed in the lungs of MCT-exposed rats treated with saline, consistent with our previous report9 (Figure 8A to 8C). Treatment with treprostinil had no effect on the expression of BMPR-II protein in the lungs of rats exposed to MCT (Figure 8A). In contrast, treprostinil treatment markedly enhanced the levels of phosph-Smad1/5 and Id1 gene expression in the lungs of MCT-exposed rats (Figure 8B and 8C). Immunohistochemistry of rat lung sections also suggested that treprostinil infusion increased pulmonary vascular Id1 expression in MCT-exposed rats and reduced the number of PCNA positive cells, as a marker of cell proliferation, in hypertrophied arteries (Figure 8D).
Prostacyclin and its analogues are effective therapies approved for the treatment of patients with severe PAH.26 Because dysfunction of BMPR-II expression and signaling occur in heritable and idiopathic PAH, we questioned whether prostanoids impact on BMP signaling. We provide evidence that prostanoids, via cAMP, drive the expression of an important BMP target gene, Id1, independent of Smad signaling, via a direct effect on the Id1 promoter. In addition, we report that prostanoids enhance the intensity and duration of Smad1/5 c-terminal phosphorylation in response to BMP4, further enhancing Id1 gene expression, an effect that involves the suppression of the inhibitory Smad, Smad6. To our knowledge, this is the first report that prostacyclin analogues enhance the activity of the Smad/Id axis in response to BMPs in vascular cells. In addition, we found that iloprost partly reversed the defect in BMP-stimulated Smad1/Id1 signaling in PASMCs harboring mutations in BMPR-II. We further show that iloprost enhances the growth suppressive effects of BMPs in human PASMCs and provide direct evidence that overexpression of Id1 leads to growth suppression of PASMCs. Finally, we were able to provide in vivo support for our in vitro findings in the MCT rat model of PAH. In this model, known to be associated with reduced BMPR-II expression and signaling, we show that treprostinil infusion prevents progression of PAH and reduces muscularization of intra-acinar pulmonary arteries while increasing phosphorylation of Smad1/5 and Id1 gene expression in the lung, without altering BMPR-II expression.
We and others have previously identified the Id family of transcription factors as important functional targets of BMP signaling, with relevance to PAH.27,28 Id genes are known to regulate smooth muscle cell phenotype and proliferation, though the precise roles of specific Id genes in these processes are uncertain. Our initial experiments indicated that iloprost alone could induce the expression of Id1 in PASMCs in the absence of Smad1/5 phosphorylation. Further evidence for a Smad-independent effect of iloprost on Id1 transcription was provided by the use of the human Id1-Luc reporter plasmid and reporter plasmids in which the SBE and CRE sites were mutated. The SBEmut-Luc reporter has mutations involving the 2 main Smad binding sites within the Id1 promoter. We demonstrate that iloprost retains the ability to activate this reporter, whereas the response to BMP4 is greatly diminished. In contract, the use of the CREmut-Luc reporter confirmed that mutation within the CRE site completely abolished the induction of Id1 by iloprost.
One of the major pathways downstream of prostanoid receptors is the elevation of intracellular cAMP by Gsα-coupled stimulation of adenylyl cyclase. Elevated cAMP activates protein kinase A (PKA) which phosphorylates among other proteins, the cAMP response element binding (CREB) protein family of transcription factors. The Id1 gene promoter contains a CREB binding site which has previously been shown to enhance BMP stimulated Id1 expression in osteoblasts.20 In human mesenchymal stem cells, cAMP has also been shown to induce expression of Id2 and bone formation.19 In the present study, we found that the early Smad-independent induction of Id1 gene expression by iloprost could be partly inhibited by the PKA inhibitor, H89, indicating the involvement of the cAMP/PKA pathway. Moreover, incubation of PASMCs with dbcAMP was sufficient to induce Id1 protein expression. Taken together, our findings confirm that iloprost, via cAMP but independent of Smad signaling, can regulate the expression of Id1 in PASMCs.
At later time points, coincubation of PASMCs with BMP4 and iloprost led to enhanced and sustained activation of Smad1 and increased expression of Id1, compared with BMP4 treatment alone. We reasoned that sustained activation of Smad1 could be due to iloprost suppressing inhibitory Smad pathways. cAMP has previously been shown to suppress Smad6 expression in bone cells.21 In the present study, we show that BMP4 increases expression of the BMP selective inhibitory Smad, Smad6, but this effect is reduced in the presence of iloprost or dbcAMP in PASMCs. Although siRNA knockdown of Smad6 enhanced phospho-Smad 1/5 activity in response to BMP4, we further showed that Smad6 knockdown prevented any additional enhancement of phospho-Smad1/5 activation by iloprost in response to BMP4. The enhancement of BMP-mediated Id1 induction by iloprost was also partly dependent on cAMP/PKA because it was inhibited by H89 and could be mimicked by dbcAMP. Thus, the potentiation of BMP signaling by iloprost and related compounds appears to be due to direct stimulation of the Id1 promoter as well as enhancement of phospho-Smad1/5 signaling via inhibition of Smad6 expression. A further mechanism we considered was that iloprost may directly affect expression of the BMPR-II receptor. However, although BMP4 induced BMPR-II expression at later time points, iloprost either alone or in combination with BMP4 did not affect BMPR-II transcript levels or protein expression (data not shown). In addition, we found that treprostinil had no effect on BMPR-II protein expression in MCT-exposed rats in vivo.
We have previously shown that a major consequence of BMPR-II mutation in PASMCs is loss of the growth suppressive effects of BMPs.12,14,30 Thus BMPR-II mutation, at least in PASMCs, leads to a proproliferative, apoptosis-resistant cell phenoype30 that may contribute to the process of vascular obliteration observed in the lungs of patients with familial and idiopathic PAH. We have further provided evidence that the antiproliferative effects of BMPs are mediated via the Smad1/Id1 axis.12,14 The present study provides confirmation that Id1 is a critical regulator of PASMC proliferation. First, iloprost inhibited proliferation of serum- or PDGF-stimulated PASMCs, consistent with the observation that Id1 expression is induced or enhanced by iloprost. More direct evidence was provided by the observation that lentiviral overexpression of Id1 suppressed serum-stimulated proliferation of PASMCs. Furthermore, we demonstrated that siRNA knockdown of Id1 caused enhanced serum dependent proliferation of PASMCs and that Id1 knockdown prevented the antiproliferative action of iloprost.
The lack of Id1 expression we observed in concentric intimal lesions from patients with BMPR-II mutations and PAH suggests that loss of Id1 is associated with medial and neointimal hyperplasia. In addition, we observed that thickened pulmonary arteries in MCT-exposed rats demonstrate increased PCNA staining and lack of Id1 expression. In contrast, previous studies have suggested that Id1 is involved in inhibition of smooth muscle differentiation31 and may contribute to restenosis and atherosclerosis.32 Our observations seem to contradict these findings. Intriguingly, in the setting of lung fibrosis, a condition characterized by myofibroblast proliferation, mice lacking Id1 demonstrate increased susceptibility to bleomycin-induced lung fibrosis.33 Clearly, more detailed studies of the roles of specific Id genes in mesenchymal cell plasticity and proliferation are warranted.
To investigate the potential importance of our findings in a disease relevant setting, we used PASMCs derived from patients with mutations in the BMPR2 gene. In these proliferative cells, we confirmed our previous observation that Smad1 activation and Id protein induction in response to BMP4 were deficient compared with control cells.14 We then demonstrated that iloprost enhanced BMP4-stimulated Smad1/5 activation and Id protein expression in BMPR-II mutant cells. Thus, it appears that iloprost can partly rescue the signaling deficit in BMPR-II mutant cells. We further confirmed that BMPR-II mutant PASMCs were resistant to the growth suppressive effects of BMPs but that sensitivity to BMP4 growth inhibition could be restored in the presence of iloprost. This mechanism may contribute to the beneficial effects of iloprost and other prostanoids in PAH.
We provided further evidence for the in vivo relevance of our findings employing the MCT rat model of PAH. This model is associated with a marked reduction in lung BMPR-II protein expression, reduced Smad1/5 activation and reduced Id1 expression.9 Treprostinil infusion was commenced in rats 3 weeks after exposure to MCT and continued for a further 2 weeks. Treprostinil markedly restored lung phospho-Smad1/5 activity and Id1 gene expression, compared with saline infused rats. Consistent with an inhibitory effect on PASMC proliferation in vivo, treprostinil inhibited progression of indices of PAH and prevented muscularization of small pulmonary arteries. PCNA staining of small pulmonary arteries was reduced in treprostinil treated rats compared with those treated with saline.
In summary, the present study describes important cross-talk between the BMP/Smad/Id gene axis and the cAMP/PKA pathway downstream of prostanoid receptors in PASMCs. In addition, we have provided further evidence for the role of Id1 in control of PASMC proliferation. Furthermore, we have demonstrated that prostanoids can partly rescue a major signaling defect in PASMCs harboring BMPR-II mutations and enhance Smad1/5 and Id1 signaling in vivo, when BMPR-II expression is reduced. These findings provide novel insights into the mechanism of action of prostanoids in the treatment of PAH.
Sources of Funding
This study was funded by a Programme Grant from the British Heart Foundation to NWM and partly by the Cambridge NIHR Biomedical Research Centre.
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Novelty and Significance
What Is Known?
Mutations in the bone morphogenetic protein type II receptor (BMPR-II) are a major cause of pulmonary arterial hypertension (PAH).
Loss of BMPR-II signaling leads to a failure of growth suppression of pulmonary artery smooth muscle cells (PASMCs).
Prostanoids inhibit PASMC proliferation in vitro and are effective treatments for patients with PAH.
What New Information Does This Article Contribute?
Prostanoids enhance BMP signaling in normal PASMCs and restore BMP signaling in BMPR-II mutant PASMCs.
Prostanoids inhibit the proliferation of PASMCs in vitro and in vivo via the induction of BMP signaling pathways.
Rescue of specific BMP signaling pathways indicates a novel mechanism of action of prostanoids and identifies further potential therapeutic targets for PAH.
Mutations in the gene encoding BMPR-II underlie the majority of cases of heritable PAH and a proportion of idiopathic cases. Even in patients in whom a BMPR-II mutation is not present, reduced expression and function of BMPR-II is a feature. It is not known whether existing treatments for PAH affect BMP signaling in the pulmonary vasculature. In the present study, we demonstrate for the first time that prostacyclin analogues, used in the treatment of clinical PAH, induce and enhance BMP signaling pathways in PASMCs. In addition, we show that prostacyclin analogues restore deficient BMP signaling in PASMCs harboring mutations in BMPR-II and restore the antiproliferative effects of BMPs in these cells. We identified a critical role for the negative regulator of basic helix-loop-helix transcription factors, Id1 (inhibitor of differentiation 1), in the antiproliferative effects of prostanoids in PASMCs. Furthermore, in the monocrotaline rat model of PAH, which is associated with reduced lung BMPR-II expression, the prostacyclin analogue treprostinil enhanced BMP signaling and Id1 gene expression, inhibited PASMC proliferation, and inhibited the progression of PAH. These findings demonstrate a novel mechanism of action for prostanoids in the therapy of PAH and provide further evidence for Id1 as a key regulator of PASMC proliferation.
In April 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.2 days.
Original received September 24, 2009; revision received May 20, 2010; accepted May 21, 2010.