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Published ahead of print on June 28, 2007, doi:10.1165/rcmb.2006-0360OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 598-605, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0360OC

BMP4 Induces HO-1 via a Smad-Independent, p38MAPK-Dependent Pathway in Pulmonary Artery Myocytes

Xudong Yang1, Patty J. Lee2, Lu Long1, Richard C. Trembath3 and Nicholas W. Morrell1

1 Department of Medicine, University of Cambridge, Addenbrooke's and Papworth Hospitals, Cambridge, United Kingdom; 2 Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, New Haven, Connecticut; and 3 Division of Molecular Medicine and Genetics, Kings College London, London, United Kingdom

Correspondence and requests for reprints should be addressed to Dr. Nicholas W. Morrell, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Box 157, Hills Road, Cambridge CB2 2QQ, UK. E-mail: nwm23{at}cam.ac.uk


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Bone morphogenetic proteins (BMPs) are multifunctional cytokines, which play a key role in vascular development and remodeling. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has been shown to be protective against vascular and lung injury. In a microarray study, we identified HO-1 as a major target of BMP4 signaling in human pulmonary artery smooth muscle cells (PASMCs), and confirmed the induction of HO-1 mRNA and protein by RT-PCR and Western blotting, respectively. Immunoblotting demonstrated that incubation of PASMCs with BMP4 rapidly phosphorylated Smad1/5 and activated the mitogen-activated protein kinases, p38MAPK and ERK1/2, in PASMCs, but not JNK. Using pathway selective inhibitors, the induction of HO-1 mRNA and protein was shown to be dependent on activation of p38MAPK. Induction was independent of Smad1/5 signaling, since HO-1 mRNA and protein induction was intact in PASMCs harboring mutations in the kinase domain of BMP type II receptor, with disrupted Smad signaling. In addition, adenoviral transfection of kinase-deficient BMPR-II also failed to inhibit BMP4-induced HO-1 expression. In functional studies, the HO-1 inhibitor, ZnPP-IX, partly reversed the growth-inhibitory effects of BMP4, and overexpression of HO-1 in PASMCs inhibited serum-stimulated [3H]-thymidine incorporation. Taken together, these findings show that HO-1 is an important Smad-independent target of BMP signaling in vascular smooth muscle. Inhibition of HO-1 function or expression will further increase the proproliferative capacity of BMPR-II–deficient PASMCs and may thus represent a potential "second hit" necessary for disease manifestation.

Key Words: pulmonary hypertension • vascular remodelling • bone morphogenetic protein type II receptor • Smads • signal transduction



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We identify heme oxygenase-1 as an important functional target of bone morphogenetic protein signaling in human pulmonary artery smooth muscle cells. These findings have important implications for our understanding of the pathogenesis of familial pulmonary hypertension.

 
Bone morphogenetic proteins (BMPs) are polypeptides, which form a large subgroup within the transforming growth factor-beta (TGF-beta) family. BMPs are multifunctional cytokines that regulate growth and differentiation in many cell types and play a major role both in early embryogenesis and in subsequent organogenesis (1). In common with other members of the TGF-beta family, BMP signaling is mediated through activation of combinations of type I and type II serine/threonine kinase receptors (2). These receptors signal via the activation of the canonical TGF-beta signaling proteins termed Smad proteins, but recent studies have also confirmed that BMPs may also directly activate the mitogen-activated protein kinase (MAPK) pathways (3–5).

The identification of heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II receptor (BMPR-II) in patients with familial pulmonary arterial hypertension (PAH) has highlighted the major role of the BMP/TGF-beta pathway in lung vascular remodeling (6, 7). In the pulmonary circulation, expression of BMPR-II is localized to pulmonary artery endothelial cells and smooth muscle cells. Studies have confirmed that mutations in BMPR-II may decrease BMP-dependent Smad signaling (8, 9). At least in pulmonary artery smooth muscle cells (PASMCs), Smad 1/5 signaling is coupled to growth inhibition (8) and apoptosis (10). Mutations in BMPR-II do not appear to disrupt signaling via the MAPKs, p38MAPK and extracellular signal–regulated kinase (ERK)1/2 in PASMCs (8). Indeed, we have previously reported that overexpression of mutant BMPR-II leads to a gain of function in terms of p38MAPK signaling in an epithelial cell line (8).

As part of an attempt to define the genomic targets of BMP signaling in the pulmonary circulation, we performed microarray studies in normal human PASMCs (our unpublished data). These suggested that heme oxygenase-1 (HO-1) is a major BMP4-inducible gene. HO-1 is the rate-limiting enzyme for the degradation of protoheme-IX, generating equimolar amounts of biliverdin-IX{alpha}, ferrous iron, and carbon monoxide (CO) (11). Of the three isoforms of HO identified to date, HO-2 and HO-3 are constitutively expressed (12, 13). In contrast, HO-1 is strongly induced by a variety of stimuli, including heme, heat shock, hypoxia, heavy metals, and nitric oxide (13, 14). All three HO isoforms are inhibited by various metalloprotoporphyrins, such as zinc (Zn) and tin (Sn) protoporphryin-IX (ZnPP-IX, SnPP-IX) (15).

HO-1 plays an important role in the regulation of vascular tone through the generation of CO, which activates soluble guanylyl cyclase and increases intracellular cyclic GMP (16–18). HO-1 expression has been shown to inhibit the growth of vascular smooth muscle cells in vitro and in vivo (19). For example, serum stimulates HO-1 gene expression, and the HO-1–catalyzed release of CO functions in an autocrine manner to limit vascular SMC proliferation in rats (20). CO has antiapoptotic and antiproliferative effects, some of which may be independent of the guanylyl cyclase/cGMP pathway in vascular cells (21). In addition, HO inhibitors increase blood pressure and peripheral vascular resistance, suggesting a critical vasoregulatory role for HO-1 (17). In vivo rodent models of hypoxia-induced pulmonary hypertension have demonstrated the potential importance of CO (22) in the regulation of pulmonary vascular tone and growth.

Since BMPs and HO-1 are important regulators of pulmonary vascular remodeling, we examined in detail the regulation of HO-1 by BMP4 and the signal transduction pathways involved. Our findings suggest that BMP4 induces HO-1 expression predominantly via a Smad-independent, p38MAPK-dependent pathway in PASMCs. Furthermore, BMP4 is capable of inducing HO-1 in mutant PASMCs harboring a disease-causing mutation in the kinase domain of BMPR-II, suggesting that the protective effects of BMP4-inducible HO-1 are preserved in patients with familial PAH.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from InVitrogen (Paisley, UK). BMP4 was purchased from R&D Systems (Abingdon, UK). The polyclonal antibodies anti-phospho-p38MAPK, total-p38MAPK, phospho-p44/42, total-p44/42, phospho-JNK, total-JNK, and chemiluminescence LumiGLO detection kit were purchased from New England Biolabs (Beverly, MA). Anti–HO-1 (ASO-110) and anti-mouse IgG–horseradish peroxidase (HRP) antibodies were purchased from Stressgen (York, UK). Anti-rabbit IgG-HRP antibody was purchased from DAKO (Ely, UK). Protease inhibitor cocktail tablets were purchased from Roche Molecular Biochemicals (Burgess Hill, UK). The selective MAPK inhibitors, SB203580, PD098059, and JNK Inhibitor l were purchased from Calbiochem (Nottingham, UK). The Access RT-PCR kit was purchased from Promega (Southampton, UK). 3H-thymidine and enhanced chemiluminescence (ECL) reagents were purchased from Amersham Biosciences (Bucks, UK). Human AoSMC Nucleofector Solution was purchased from Amaxa Biosystems (Köln, Germany). MAX efficiency DH5{alpha}-competent cells were purchased from Life Technologies (Paisley, UK). The Endofree Plasmid Maxi Kit was purchased from Qiagen (Crawley, UK). DC Protein Assay was purchased from Bio-Rad (Hemel Hempstead, UK). All other materials were purchased from Sigma (Dorset, UK).

Culture of Human PASMCs
Normal PASMCs were isolated from surgical resection specimens derived from patients undergoing lobectomy or pneumonectomy for suspected lung tumor (n = 5). Only uninvolved tissue was used. PASMCs were explanted from lobar pulmonary arteries and peripheral arteries (< 1–2mm external diameter), as previously described (8, 23). Papworth Hospital ethical review committees approved the study, and subjects gave informed written consent. Cells were maintained in 10% FBS/DMEM and used for experiments between passages 4 and 6. Additional PASMCs were obtained from patients (n = 3) undergoing heart-lung transplantation for familial PAH and known to harbor mutations in the BMPR-II receptor. One isolate was obtained from a patient with a mutation in the kinase domain of BMPR2, comprising a C to T change at position 1471, predicted to substitute arginine for tryptophan at position 491 of the amino acid sequence (R491W), one in which tyrosine is substituted for cysteine at position 347 (C347Y) (24), and one in which a premature stop codon is inserted in place of tryptophan at position 9 of the amino acid sequence (W9X). The smooth muscle phenotype of isolated cells was confirmed by positive immunofluorescence with antibodies to anti–{alpha} smooth muscle actin antibody (IA4) and anti–smooth muscle specific myosin (hsm-v), as described (25).

Transfection Studies
Plasmids Vectors for overexpression of human HO-1 (Psffv-HO-1) and control vector (Psffv-NEO) were employed. Transfection was performed with a Nucleofector electroporation system (Amaxa Bioscience). PASMCs were cultured in 10% FBS/DMEM, and used in passages 4 to 6. One nucleofection sample contained: 1 to 2 x 106 cells, 5 µg plasmid cDNA in less than 5 µl dH2O, and 100 µl Human AoSMC Nucleofector Solution. The electroporation was performed according to the manufacturer's instructions.

Adenoviral transfection AdCMVBMPR2 myc and AdCMVBMPR2(D485G)myc, replication incompetent serotype 5 adenoviral vectors, were created by cloning the full-length human BMPR2 cDNA containing a myc tag at the C-terminus into the plasmid pShuttleCMV, containing the cytomegalovirus promoter. Homologous recombination with plasmid pAdEasy1 resulted in the generation of pAdCMVBMPR2 myc and pAdCMVBMPR2(D485G)myc. Virus was generated by standard techniques involving Pac1 digestion of the plasmid and transfection into HEK293T cells. Large-scale virus preps were generated in HEK293 cells then purified by cesium chloride centrifugation. Viral titer was determined by TCID50 assay and particle titer by OD260. Control viruses contained the CMV promoter driving expression of reporter genes luciferase (Luc) or green fluorescent protein (GFP). Quiescent PASMCs were transfected with wild-type or mutant (D485G) BMPR-II constructs for 48 hours at a dose of 50 plaque-forming units (pfu).

Cell Proliferation Assays
The growth of peripheral PASMCs was determined by 3H-thymidine incorporation, representing DNA synthesis (25). Briefly, PASMCs were seeded in 48-well plates at a density of 1 x 104 per well in 10% FBS/DMEM, and maintained for 3 days. When grown to subconfluence (70–80% confluence), PASMCs were quiesced by incubation with serum-free DMEM for 48 hours. The medium was then replaced with fresh DMEM with 5% FBS in the presence or absence of BMP4 (1–50 ng/ml) for 24 hours. A quantity of 0.25 µCi/well 3H-thymidine was added for the final 6 hours. In some experiments, cells were treated with ZnPP-IX (0.1–5 µM) for 1 hour before adding BMP4.

Western Blot Analysis
PASMCs were plated on 6-cm tissue culture dishes in 10% FBS/DMEM and grown to ~ 90% confluence. After 48 hours of quiescence in serum-free DMEM, the cells were then treated with BMP4 (50 ng/ml) or vehicle for up to 24 hours. In additional studies, cells were pre-treated (30 min) with inhibitors of ERK1/2 (PD098059 10 µM), p38MAPK (SB203580 5 µM), or JNK (JNK inhibitor 1 10 µM). ZnPP-IX (1 µM), a known inducer of HO-1 (26), was added in some experiments. Cells were lysed in 1x sample loading buffer (Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, beta-mercaptoethanol) containing 1x protease inhibitor cocktail on ice. The cell lysates were boiled for 5 minutes and then centrifuged at 18,000 x g for 5 minutes at 4°C. Protein concentrations of the cell lysates were determined by DC Protein Assay. Approximately 100 µg of protein were electrophoresed by 12% SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk with 2% BSA in washing buffer (1x TBS with 0.1% Tween-20, TBS-T) for 1 hour at room temperature, then incubated with anti–HO-1 antibody at 1:1,000 dilution in blocking buffer with gentle agitation overnight at 4°C. After washing, the blots were incubated for 1 hour with a 1:5,000 dilution of HRP-conjugated anti-mouse IgG and visualized using ECL reagents. To detect the expression of phosphorylated p38MAPK, JNK1/2, and ERK1/2 (p44/42), after being transferred to a nitrocellulose membrane, the membranes were blocked for 1 hour at room temperature in blocking buffer (20 mM Tris, 500 mM NaCl, 0.1% Tween-20, 5% non-fat milk), and incubated with specific polyclonal antibodies for anti–phospho-ERK1/2, p38, and JNK1/2 at 1:1,000 dilution in blocking buffer with gentle agitation overnight at 4°C. After washing, the blots were incubated for 1 hour with a 1:2,000 dilution of HRP-conjugated anti-rabbit IgG and visualized using a chemiluminescence LumiGLO detection kit. To verify equivalent sample loading, blots were stripped in stripping solution (2% SDS, 62.5 mM Tris-HCl, pH 6.8, 100 mM beta-mercaptoethanol) at 60°C for 30 minutes, and then re-probed with anti-total ERK1/2, JNK1/2, p38, and anti–beta-actin antibodies, as appropriate.

RT-PCR
Total RNA was extracted from treated human PASMCs using TRIzol reagent. The RNA concentration was determined by spectrophotometry. RT-PCR was performed using the Access RT-PCR System of Promega (Madison, WI). Briefly, 50 ng total RNA was reverse transcribed for 1 hour at 48°C in 12.5 µl containing 2.5 µl 5x RT-PCR buffer, 200 µM dNTP, 1.5 mM MgSO4, 1.25 U AMV reverse transcriptase. Amplification of HO-1 cDNA was performed at temperatures of 62 and 55°C, using 1.25U TfI DNA polymerase. Primers were designed using the computer program Prime3 (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). All primers were synthesized by Sigma-Genosys Ltd, (Cambridgeshire, UK). Primer sequences were as follows: sense, 5'-CTT CTT CAC CTT CCC CAA CA-3'; antisense, 5'-GCT CTG GTC CTT GGT GTC AT-3'. The amount of starting material and the number of cycles were selected so that amplified product signal was quantitatively related to the input of RNA. Samples of the PCR reactions were taken at multiple points throughout the amplification, allowing analysis of the product during the exponential phase of DNA amplification for appropriate semi-quantification. The beta-actin (sense primer, 5'-ATG AAG TGT GAC GTT GAC ATC CG-3'; antisense primer, 5'-GCT TGC TGA TCC ACA TCT GCT G-3') was used as an internal control for PCR. PCR products were visualized by electrophoresis in 2% agarose gels stained with ethidium bromide. Control reactions were run without the addition of reverse transcriptase. The identity of PCR products was confirmed by direct sequencing.

Quantitative Real-Time PCR
Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instruction. DNA from each RNA sample was removed by TURBO DNA-free TURBO DNase treatment and removal reagents (Ambion, Warrington, UK). Reverse transcription was then performed using StrataScript first-strand synthesis system (Stratagene, Cambridge, UK). Synthesized complementary DNA was amplified by a standard PCR protocol using iQ SYBR Green supermix (Bio-Rad). Parallel amplifications with primers for beta-actin were performed. The following cycling conditions were used: 3 minutes preincubation at 95°C, 30 s denaturation at 95°C, 30 seconds annealing at 58°C, and 30 seconds extension at 72°C for 50 cycles using iCycler (Rio-Rad). Product specificity was confirmed by agarose gel electrophoresis and routinely by melting–curve analysis. Real-time PCR data were analyzed by using iCycler software (Bio-Rad). The level of HO-1 gene expression was normalized to the level of beta-actin gene expression in parallel samples. The sequences for the primers were as follows: human HO-1, forward primer, 5'-CAGGCAGAGAATGCTGAG-3', reverse primer, 5'-GCTTCACATAGCGCTGCA-3'; human beta-actin, forward primer, 5'-GCA CCA CAC CTT CTA CAA TGA-3', reverse primer, 5'-GTC ATC TTC TCG CGG TTG GC-3'.

Statistical Analysis
Data were expressed as mean ± SEM and analyzed with GraphPad Prism version 3.0 (GraphPad Software). Comparisons were made by Student's t test. A value of P < 0.05 indicated statistical significance.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
BMP4 Induces Expression of HO-1 mRNA and Protein via p38MAPK
Incubation of normal PASMCs with BMP4 (50 ng/ml) increased HO-1 mRNA by 1 hour (Figures 1A and 1B) and HO-1 protein expression by 2 hours (Figures 1C and 1D). To determine which signal transduction pathways might be involved in BMP4-induced HO-1 induction, we examined the activation of Smad1/5 and ERK1/2 (Figure 2A), p38MAPK and JNK pathways (Figure 2B). Rapid phosphorylation of Smad1/5 was seen after 15 minutes of stimulation with BMP4. BMP4 stimulation led to rapid (15 min) transient activation of ERK1/2, which returned to baseline by 8 hours. In contrast, phosphorylation of p38MAPK was evident at 1 hour and was sustained. There was no activation of JNK apparent.


Figure 1
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  		Figure 1. Semiquantitative RT-PCR gel showing time course of induction of heme oxygenase-1 (HO-1) mRNA over a 24-hour exposure to 10 ng/ml BMP4, compared with the expression of mRNA for the housekeeping gene beta-actin (A). Further real-time PCR analysis of the time course of HO-1 mRNA induction is confirmed in B. (C) Induction of HO-1 protein over a 24-hour exposure to 10 ng/ml BMP4. Membranes were stripped and probed for beta-actin to confirm equally lane loading. (D) Densitometry from three separate experiments confirmed the time course of protein induction. *P < 0.05, **P < 0.01 compared with time 0.

 

Figure 2
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  		Figure 2. Immunoblots demonstrating the time course of activation (phosphorylation) of Smad1/5 and p44/42 extracellular signal–regulated kinase (ERK) in pulmonary artery smooth muscle cells (PASMC) exposed to 10 ng/ml BMP4 (A). Lower panel (B) shows time course of activation of p38MAPK and lack of activation of JNK. Equal loading was confirmed by stripping blots and reprobing with total MAPK or Smad1 antibodies. Results representative of three experiments.

 
The effect of MAPK inhibitors on HO-1 gene transcription was examined at the 4-hour time point. In the presence of MAPK pathway inhibitors, only SB203580, the p38MAPK inhibitor, prevented the induction of HO-1 mRNA by BMP4 (Figures 3A to 3C). The induction of HO-1 mRNA by BMP4 was increased by co-incubation with the known inducer of HO-1 protein expression, ZnPP-IX. At the level of protein expression, SB203580 profoundly inhibited HO-1 expression (Figure 3B), whereas PD980509 and JNKi-1 had no effect. ZnPP-IX increased BMP4-induced expression of HO-1 protein, as expected.


Figure 3
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  		Figure 3. Semiquantitative PCR gel (A) demonstrating that induction of HO-1 mRNA in response to BMP4 (10 ng/ml) is inhibited by the p38MAPK inhibitor SB203580 but not by the ERK inhibitor, PD980509 or the JNK inhibitor JNKi-1. In addition, HO-1 mRNA induction is further increased in the presence of the HO-1 inhibitor ZnPP-IX. Equal RNA concentration was confirmed by amplification of the housekeeping gene, beta-actin. Densitometric analysis of semiquantitative PCR gels from three separate experiments (B) confirmed the effect of SB203580 (*P < 0.05 compared with BMP4 alone). (C) Further real-time PCR analysis confirmed the effect of SB203580 on BMP4-induced HO-1 gene expression (*P < 0.05 compared with BMP4 alone). (D) The effects of the same inhibitors on the induction of HO-1 protein by BMP4 were shown by immunoblotting. ZnPP-IX was again used as a positive regulator of HO-1 expression. (E) Densitometry of immunoblots from three separate experiments confirmed the effect of SB203580 on HO-1 protein expression. *P < 0.05 and ***P < 0.001 compared with control.

 
HO-1 Induction by BMP4 Is Independent of Smad1 Phosphorylation
In concentration–response studies we observed that induction of HO-1 protein expression by BMP4 occurred at a concentration of 0.1 ng/ml. Low-level activation of Smad1/5 and p38MAPK were both observed at this concentration (Figure 4A). To disrupt Smad1/5 signal transduction, normal PASMCs were adenovirally transfected with wild-type (pAdCMVBMPR2 myc) or mutant kinase deficient (pAdCMVBMPR2(D485G)myc) BMPR-II. Adenoviral transfection efficiency was judged as 90% in parallel transfections using Adv-GFP. PASMCs transfected with kinase-deficient BMPR-II showed a markedly attenuated ability to activate Smad1/5 as assessed by immunoblotting (Figure 4B). In contrast, activation of p38MAPK and induction of HO-1 protein by BMP4 was unimpaired in these cells. In addition, we employed PASMCs harboring endogenous germline mutations in BMPR-II. Similar to the results in transfected cells, BMP4 led to preserved activation of p38MAPK and induction of HO-1 protein expression in these cells, despite a marked deficiency of endogenous Smad signaling (Figure 4C).


Figure 4
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  		Figure 4. Immunoblots showing the concentration-response of HO-1 protein induction after 4 hours of exposure to BMP4, and the activation of Smad1/5 and p38MAPK after 1 hour of BMP4 exposure (A). Middle panel (B) shows immunoblots from control PASMCs adenovirally transfected with wild-type or D485G dominant-negative kinase domain mutant BMPR-II. Induction of phospho-p38MAPK and HO-1 by BMP4 was intact despite the almost absent Smad1/5 phosphorylation in D485G cells. Lower panel (C) shows HO-1, phospho-p38MAPK, and Smad1/5 activation in control PASMCs and PASMCs derived from a patient with pulmonary arterial hypertension harboring a kinase domain mutation in BMPR-II (C347Y) showing similar findings. Results representative of three separate experiments in control and mutant cells.

 
BMP4-Induced HO-1 Regulates the Growth of Human PASMCs
Incubation of PASMCs with BMP4 (10 ng/ml) increased 3H-thymidine incorporation into peripheral PASMCs and inhibited proliferation of proximal PASMCs (Figure 5A), as previously described (8). BMP4 induced expression of HO-1 protein to a similar exent in both cell types (Figure 5B). Co-incubation with BMP4 and the HO-1 inhibitor, ZnPP-IX, increased 3H-thymidine incorporation further in peripheral cells and reversed the growth inhibition in proximal cells (Figure 5A). To confirm that HO-1 overexpression inhibits mitogenic responses in human PASMCs, we transiently transfected cells with Psffv–HO-1 or control vector, Psffv-NEO. Overexpression of HO-1 was confirmed by Western blotting (Figure 5C). Control vector had no effect on serum-stimulated 3H-thymidine incorporation, whereas overexpression of HO-1 completely inhibited the serum-induced mitogenesis of PASMCs (Figure 5D).


Figure 5
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  		Figure 5. Bar graphs depicting 3H-thymidine incorporation into peripheral and proximal PASMCs treated with BMP4 (10ng/ml) for 24 hours in the presence or absence of the HO-1 inhibitor, ZnPP-IX (1 µM) (A). Western blotting for HO-1 confirmed that BMP4 induced HO-1 in both peripheral and proximal cells (B) and that overexpression of HO-1 in PASMCs transfected with Psffv–HO-1 increased HO-1 protein levels compared with control vector, Psffv-NEO (C). (D) Transfection of control peripheral PASMCs with Psffv–HO-1 inhibited serum-stimulated 3H-thymidine incorporation. *P < 0.05, **P < 0.01; results representative of three separate experiments.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The BMP pathway is emerging as critical mediator of pulmonary vascular remodeling. In this study we have identified that BMP4, one of the major ligands for the BMPR-II receptor, is a novel and potent regulator of HO-1 expression in human PASMCs. HO-1 is known to exert antiproliferative and cytoprotective effects in vascular cells and may therefore contribute to the beneficial effects of an intact BMP signaling pathway in the normal vasculature. Indeed, we confirmed that induction of HO-1 contributed to the antiproliferative effects of BMPs in PASMCs. Thus, the selective HO-1 inhibitor, ZnPP-IX, partly reversed the growth-inhibitory effects of BMP4 in lobar PASMCs, and heightened the proliferation of peripheral PASMCs to BMP4. We have previously reported the differential effect of BMP4 on PASMC proliferation depending on whether cells are isolated from lobar and peripheral pulmonary arteries (8), but in both cell types, HO-1 induction by BMP4 heightened cell proliferation. Moreover, we demonstrated that overexpression of HO-1 was sufficient to inhibit proliferation of PASMCs exposed to serum. Taken together, these data strongly support a role for BMP-induced HO-1 expression in growth inhibition of PASMCs. The mechanism of growth inhibition by HO-1 is thought to involve production of CO and stimulation of soluble guanylyl cyclase leading to increased intracellular cyclic GMP (27). However, alternative pathways involving activation of MAPKs have also been described (27).

We demonstrated that HO-1 induction by BMP4 was dependent on the activation of p38MAPK, but not ERK or JNK. BMP4 led to rapid phosphorylation of p38MAPK and ERK1/2, but only inhibition of p38MAPK with SB203580 attenuated the induction of HO-1 mRNA and protein expression by BMP4. The induction of HO-1 by p38MAPK seems to be cell- and stimulus-specific. For example, TGF-beta1 has been shown to induce HO-1 via p38MAPK in A549 epithelial cells (28), and the plant polyphenol, quercetin, induces HO-1 via p38MAPK in rat aortic smooth muscle cells (29). However, in rat pulmonary artery endothelial cells, hypoxia activates both p38MAPK and HO-1, but HO-1 induction is partly inhibited by the activated p38MAPK (30). In primary cultured rat hepatocytes, HO-1 induction by arsenic is dependent on activation of JNK, and again inhibited by p38MAPK (31). Our findings represent the first report to our knowledge of HO-1 induction via p38MAPK in PASMCs.

Although activation of Smad1/5 is regarded as the canonical BMP signal transduction pathway, our data strongly suggest that induction of HO-1 by BMP4 is independent of Smad1/5 activation. First, we confirmed that BMP4-induced HO-1 expression was intact in PASMCs derived from patients with familial PAH, with a demonstrable reduction in Smad1/5 phosphorylation. Second, to ensure that this retained activation of HO-1 was not due to a change in cell phenotype as a consequence of the disease state, we also employed normal PASMCs and overexpressed a dominant-negative kinase deficient BMPR-II (9). In both cases HO-1 induction by BMP4 remained intact. In both cases we showed that Smad1/5 signaling was markedly impaired, but p38MAPK signaling was preserved. This is in contrast to the Smad-dependent effect of TGF-beta1 on HO-1 induction in human renal proximal tubule cells (32). In that study, HO-1 induction was not affected by the presence of inhibitors of p38MAPK or ERK1/2, but was inhibited by overexpression of the inhibitory Smad, Smad7. The common partner Smad, Smad 4 is common to both TGF-beta and BMP signal transduction pathways, but the receptor mediated Smads downstream of the TGF-beta receptor, ALK-5, Smads 2 and 3 are distinct from the BMP-regulated Smads 1, 5, and 8 (33). Furthermore, BMP and TGF-beta signaling can exert antagonistic effects in many cells (34, 35).

A number of studies have now shown a role for HO-1 in the control of pulmonary vascular tone and remodeling. Inhibition of HO-1 with tin protoporphyrin exaggerates acute hypoxic pulmonary vasoconstriction (36). In the chronically hypoxic (37, 38) and monocrotaline (39, 40) rat models of pulmonary hypertension, chronic treatment with HO-1 inhibitors has been shown to worsen pulmonary hypertension. In addition, mice overexpressing HO-1 are protected from hypoxia-induced pulmonary hypertension, possibly because of reduced hypoxia-induced expression of proinflammatory cytokines (41). However, not all studies have confirmed these observations. Of note, one study showed that inhibition of HO-1 with tin protoporphyrin during 5 weeks of chronic hypoxia did not elevate pulmonary vascular resistance compared with hypoxia alone (36). A role for HO-1 in idiopathic pulmonary arterial hypertension in humans is supported by the finding that HO-1 expression is lost within the plexiform lesions and precapillary arterioles of these patients (42).

How then might our findings further our understanding of the pathogenesis of pulmonary arterial hypertension? One interpretation of our findings is that HO-1 represents a target for "second hits" within the pulmonary circulation. We know that a mutation in the BMPR-II is an important factor increasing susceptibility to pulmonary hypertension. However, for the majority of mutations, the disease manifests with reduced penetrance (as low as 15–20%), indicating that other factors are necessary to trigger disease development. A lack of intact Smad signaling as a consequence of BMPR-II mutation leads to a proproliferative, prosurvival phenotype in PASMCs, but this may be insufficient to induce disease in vivo. This contention is supported by the observation that mice deficient in BMPR-II require additional stimuli to develop pulmonary hypertension (43). Part of this protection from manifest pulmonary hypertension, despite defective Smad signaling, may reside in the fact that Smad-independent BMP-inducible targets, as exemplified by HO-1 in this study, are preserved. In this model, a second hit that antagonizes the protective effects of HO-1 could further increase the risk of developing pulmonary hypertension (Figure 6). This may occur at any part of the HO-1/CO/cGMP pathway. This effect could be environmental, epigenetic, or result from functional antagonism with other pathways. As such, it would be unlikely that this effect would persist in isolated pulmonary artery smooth muscle cells harboring mutations in BMPR-II. In addition, the second hit could occur at the level of downstream targets of CO action, such as cyclic GMP, potassium channels, or caveolin-1 function (27), all of which have been previously implicated in the pathogenesis of pulmonary hypertension (42, 44).


Figure 6
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  		Figure 6. Proposed mechanism for PASMC proliferation in BMPR-II mutant cells. Disruption of Smad-responsive pathways in mutant cells is not alone sufficient to allow proliferation of PASMCs, because of the preservation of critical antiproliferative Smad-independent pathways, for example HO-1. Disruption of Smad-independent HO-1 expression by environmental or epigenetic factors would further compound the lack of growth inhibitory pathways favoring PASMC proliferation.

 
In summary, we have demonstrated that HO-1 gene and protein expression is an important target of BMP4-regulated transcription in human PASMCs and that this contributes to an antiproliferative effect of BMP4. HO-1 induction by BMP4 is an example of a Smad-independent, p38MAPK-dependent, transcriptional event. In PASMCs harboring mutations in BMPR-II we propose that disruption of HO-1 signaling, in addition to defects in Smad signaling, contributes to the development of pulmonary vascular remodeling and pulmonary hypertension.


   Footnotes
 
This study was supported by a grant from the British Heart Foundation (Programme Grant RG/03/005 to N.W.M. and R.C.T.). This study received financial support from by the European Commission under the 6th Framework Programme (Contract No: LSHM-CT-2005-018725, PULMOTENSION).

This publication reflects only the authors' views and the European Community is in no way liable for any use that may be made of the information contained therein.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0360OC on June 28, 2007

Conflict of Interest Statement: N.W.M acted as a consultant to Novartis plc and received $5,000 in 2005–2006 and received a research grant of $450,000 from Novartis plc in 2007. R.C.T. has participated as a speaker in scientific meetings or courses organized and financed by various pharmaceutical companies (Acctison, Schering, Novartis). None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 24, 2006

Accepted in final form April 16, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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