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Am J Physiol Lung Cell Mol Physiol 291: L1059-L1067, 2006; doi:10.1152/ajplung.00180.2006
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BMP-dependent activation of caspase-9 and caspase-8 mediates apoptosis in pulmonary artery smooth muscle cells

Giorgio Lagna,1 Peter H. Nguyen,1 Weihua Ni,1 and Akiko Hata1,2

1Molecular Cardiology Research Institute, Tufts-New England Medical Center, and 2Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts

Submitted 18 May 2006 ; accepted in final form 28 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Germ line mutations in the bone morphogenetic protein (BMP) receptor type II (BMPRII) gene have been found in >50% of familial idiopathic pulmonary arterial hypertension (IPAH) patients and in 30% of sporadic cases of IPAH. Mutations of BMPRII occur in the extracellular ligand-binding domain, in the cytoplasmic serine/threonine kinase domain, or in the long carboxy terminus domain of unknown function. In this study, we demonstrate that BMPs promote apoptotic cell death in normal human pulmonary artery smooth muscle cells (PASMCs) by activation of caspases-3, -8, and -9, cytochrome c release, and downregulation of Bcl-2. Normal PASMCs expressing a kinase domain mutant or a carboxy-terminal domain deletion mutant of BMPRII identified in IPAH patients are resistant to BMP-mediated apoptosis. This dominant-negative effect may act in heterozygous patients and lead to the development of the pulmonary vascular medial hypertrophy found in IPAH patients. Our study also demonstrates an essential role of the carboxy terminus domain of BMPRII in the activation of the apoptotic signaling cascade.

transforming growth factor-beta; Smad; bone morphogenetic protein receptor type II

IDIOPATHIC PULMONARY ARTERIAL hypertension (IPAH) is a progressive disease that is fatal because of right ventricular failure (10, 26, 30). IPAH is characterized by proliferation of vascular cells and obliteration of small pulmonary arteries, leading to sustained elevation of pulmonary arterial pressure. Typical cell morphological changes include increased muscularization of small arteries and thickening or fibrosis of the intima, as well as the presence of plexiform lesions (26, 30). Recently, heterozygous mutations in the gene encoding the type II bone morphogenetic protein (BMP) receptor (BMPRII) were identified in patients with familial and sporadic forms of IPAH (23, 36). Mutations in the BMPRII gene are found not only in the extracellular ligand-binding domain, the transmembrane domain, and the kinase domain, but also in the carboxy-terminal region known as the tail domain, which is unique to BMPRII and has no known function (23, 36). Heterozygous BMPRII-deficient (BMPRII+/–) mice have no apparent developmental phenotype (3) but can develop increased mean pulmonary arterial pressure and pulmonary vascular resistance compared with their wild-type littermates by chronic infusion of serotonin, especially under conditions of chronic hypoxia (22). Histological analyses reveal that the wall thickness of muscularized pulmonary arteries and the number of alveolar capillary units are greater in BMPRII+/– mice than in wild-type mice (3). These results suggest that a mutation in one copy of the BMPRII gene can cause pulmonary hypertension in mice, a scenario similar to that seen in IPAH patients.

Outside its unique tail domain, BMPRII has high homology to other receptors of transforming growth factor (TGF)-beta superfamily ligands and, like other receptors, has an intrinsic serine/threonine kinase activity. In combination with the type I BMP receptor (BMPRIA or BMPRIB), it binds to BMPs and activates downstream signal transducers of the Smad family, which then translocate to the nucleus and regulate transcription of target genes (27). Little is known about the role of BMP signaling in vascular cells. BMP2 and BMP7 have been shown to inhibit proliferation of pulmonary artery smooth muscle cells (PASMCs) in vitro (29). It is also known that BMP2 can induce apoptosis in PASMCs (43). In PASMCs derived from IPAH patients, BMP2-induced apoptosis is significantly decreased compared with PASMCs from secondary PAH patients (43). More recently, it was reported that a mutant form of BMPRII found in IPAH, when expressed in a smooth muscle-restricted fashion in transgenic mice, causes an increase in pulmonary artery pressure and pulmonary arterial muscularization with no increase in systemic arterial pressure (37). These results suggest that loss of BMP signal in smooth muscle cells (SMCs) is sufficient to produce pulmonary hypertension.

In this study, we examined the biological effect of BMP7 and BMP4 in human primary PASMCs and found that both BMP7 and BMP4 promote apoptotic cell death. We also show that BMP-mediated apoptosis is mediated by the activation of caspase-8 and caspase-9 and correlates with the reduced expression of the antiapoptotic factors Bcl-2 and cIAP-2. PASMCs expressing mutant forms of BMPRII identified in IPAH patients are resistant to BMP-mediated proapoptotic effects, suggesting that an alteration of the normal BMP signal might contribute to abnormal pulmonary smooth muscle organization in IPAH patients.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Human PASMCs were purchased from Cambrex and were maintained in Sm-GM2 media (Cambrex) containing 5–10% fetal calf serum (FCS; Hyclone) plus growth factors as indicated. The mouse embryonic carcinoma P19 cell line was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS.

Antibodies and growth factors. BMP receptor-phosphorylated Smad1 and total Smad1 were detected with anti-phospho-Smad1/5/8 rabbit monoclonal antibody (41D10; Cell Signaling Technology) and rabbit polyclonal antisera (Upstate Biotechnology), respectively. Other antibodies used in this study were mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (clone 1D4, Covance), mouse monoclonal anti-flag (M2, Sigma), rabbit monoclonal anti-cleaved caspase-3 (MAB835, R&D Systems), anti-caspase-8 (Ab3, Oncogene Research) and rabbit polyclonal anti-caspase-9 (no. 9502 Cell Signaling Technology, and no. ab2324, Abcam). Human Bcl-2 and Bax were detected with rabbit polyclonal antibodies ab16837 and ab18210 (Abcam), respectively. Recombinant TGF-beta1, BMP2, BMP4, and BMP7 were purchased from R&D Systems. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Roche).

Constructions of recombinant adenoviruses. Recombinant adenoviruses were constructed as described previously (19). Flag epitope-tagged wild-type or mutant BMPRII cDNAs were subcloned into the pShuttle-CMV vector.

Detection of apoptotic cells. Release of lactate dehydrogenase (LDH) was measured by a fluorometric enzyme assay kit (Roche) according to the manufacturer's instructions. PASMCs were cultured in serum-free DMEM for 48 h, followed by stimulation with 10 nM BMP2, BMP4, or BMP7 for 48 h. Cells were then subjected to terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay with a Cell Death Detection kit (Roche). Cytoplasmic release of cytochrome c and mitochondrial permeability were detected with an Apoptosis Detection Kit (MitoSciences) and a Mit-E-{Psi} Apoptosis & Mitochondria Permeability Detection Kit (Biomol), respectively.

Caspase assay. The enzymatic activity of caspases was measured with a caspase colorimetric assay kit (Biomol) according to the manufacturer's instructions. The caspase inhibitors used in the study were Ac-DEVD-CHO for caspase-3, Ac-IETD-CHO for caspase-8, and z-LE(OMe)HD(OMe)-fmk for caspase-9 (Calbiochem). z-VAD-fmk (Calbiochem) was used as a broad-range caspase inhibitor. Cells with activated caspase-9 or caspase-8 were detected by immunoblotting analysis.

RNA interference. Synthetic small interfering RNA (siRNA) corresponding to the sequence of human BMPRII (5'-AAGGAAGCACCGAAGCGAAACTT-3') was purchased from Dharmacon. As controls, siRNA with a nontargeting sequence (Dharmacon) and siRNA against mouse TFII-I (19) were used. The siRNAs were transfected into PASMCs by OligofectAMINE (Invitrogen). Forty-eight hours after transfection, the cells were treated with BMP4 or vehicle and harvested. Under this condition, transfection efficiencies as determined by fluorescent-labeled siRNA were in the range of 90–100%.

Reverse transcriptase-polymerase chain reaction assay. Reverse transcriptase-polymerase chain reaction (RT-PCR) assay was performed as described previously (19). The primers used were 5'-ACGGGAGAGAAGACGAGCCT-3' and 5'-CTAGATCAAGAGAGGGTTCG-3' for human BMPRII, 5'-GACTTCTTCCGCCGCTACCG-3' and 5'-GACAGCCAGGAGAAATGAAAC-3' for human Bcl-2, 5'-TCCTCCTGAGCAGTCAGC-3' and 5'-GGCGATGTCAATAGGACTC-3' for human hypoxanthine-guanine phosphoribosyl transferase (HPRT), and 5'-ACCCGAGCTGGCCCTGGAC-3' and 5'-ATGGTGAGCGAGGCGGTGAG-3' for human Bax. Human GAPDH, BMPRIA, BMPRIB, cIAP-1, cIAP-2, survivin, ALK2, ActRIIA, and ActRIIB primers were described previously (2, 5, 43).

Immunoblot assay. Immunoblot assays were described previously (13). Total cell lysates were prepared in radioimmunoprecipitation assay buffer.

Luciferase assay. After transfection, the cells were reseeded onto 12-well plates and treated with 3 nM BMP4 for 20 h in 0.2% FCS-DMEM. Xvent2-Luc construct and luciferase assays were described previously (13).

Statistical analysis. Statistical significance was determined by ANOVA and Fisher's least significant difference test or by Student's t-test analysis (P < 0.05) as appropriate. Percentages were converted to arcsin values before analysis. All data are plotted as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
BMP induces apoptosis in PASMCs. We investigated a possible proapoptotic effect of BMP treatment on human PASMCs. Real-time PCR analysis indicated that mRNAs encoding both BMPRIs (BMPRIA and BMPRIB) and BMPRII were expressed in human PASMCs (Fig. 1A). BMP7 has also been shown to bind a type I receptor named activin-like receptor kinase 2 (ALK2; also known as AVCRI, ActRI, or ActRIA) in combination with the type II receptors ActRIIA or ActRIIB (25). We found that ALK2, ActRIIA, and ActRIIB mRNAs were also expressed in PASMCs (Fig. 1A). In agreement with a previous report (43), expression of Smad proteins (Smad1–7), BMP ligands (BMP2–7), TGF-beta receptors (TbetaRI and TbetaRII), and TGF-beta ligands (TGFbeta1–3) has been confirmed by RT-PCR in PASMCs (W. Ni and A. Hata, unpublished observation). As both BMP4 and BMP7 signal through BMPRII (15), we studied the effect of both factors in PASMCs. To test whether BMPs can mediate apoptosis in human PASMCs, we first examined the potential damage to the plasma membrane generated by BMP stimulation. For this purpose, we measured the amount of LDH released into the medium by lysed cells in response to BMP stimulation. As shown in Fig. 1B, release of LDH in the medium was observed on both BMP4 and BMP7 treatment of PASMCs (P < 0.001). A similar level of LDH release was observed with treatment with TGF-beta1 or staurosporine (ST; P < 0.001), a known inducer of apoptosis (Fig. 1B).


Figure 1
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  		Fig. 1. Bone morphogenetic proteins (BMPs) promote apoptotic cell death in pulmonary artery smooth muscle cells (PASMCs). A: total RNAs from PASMCs were subjected to RT-PCR analysis using primers to BMP receptor type I [BMPRI; activin-like receptor kinase (ALK)2, ALK3, and ALK6] and type II (BMPRII; ActRIIA and ActRIIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; as a loading control). PCR amplified products are displayed in agarose gel. The cDNA fragments corresponding to each primer set were used as positive controls (Control). RNA samples directly subjected to PCR without reverse transcription reaction were used as negative controls (–RT). B: human PASMCs were serum starved for 48 h and then treated with 10 nM BMP7, 10 nM BMP4, 100 pM transforming growth factor (TGF)-beta1, or 20 nM staurosporine (ST) for 24 h. Lactate dehydrogenase (LDH) activity in cell culture media was measured by a fluorometric enzyme assay. Data are plotted as means ± SE of 3 independent experiments. *P < 0.001 (vs. none) C: PASMCs were serum starved for 48 h and then treated with BMP7 (1 or 10 nM) or ST (20 nM) for 48 h. Cells undergoing apoptosis were stained by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay (green). Phalloidin (red) stains all the cells in the field. D: PASMCs were serum starved for 48 h and treated with BMP7, BMP2, or BMP4 (10 nM) for 48 h. The samples were subjected to TUNEL assay (green). 4',6-Diamidino-2-phenylindole (DAPI) staining (blue) shows all the nuclei in the field.

 
BMP-mediated apoptosis in PASMCs was also detected by TUNEL assay, which measures DNA cleavage in apoptotic cells. In the absence of BMP7, the percentage of TUNEL-positive cells was <0.1%. After treatment with 1 and 10 nM BMP7, 17 ± 1.8% and 85 ± 3.2% of the cells were TUNEL positive, respectively (Fig. 1C). Similar percentages of TUNEL-positive cells were observed after treatment with 10 nM BMP7, BMP4, or BMP2 (Fig. 1D). Morphological changes in the nucleus (chromosomal condensation) could also be detected in all BMP-treated cells (Fig. 1D, DAPI stain). These results confirm the ability of BMPs to induce a typical apoptotic cell death phenotype in PASMCs.

PASMCs undergo caspase-dependent apoptotic cell death in response to BMP7. To assess the components of the apoptotic cascade involved in BMP7-mediated apoptosis, we measured the enzymatic activity of caspase-3, caspase-8, and caspase-9 by an in vitro fluorometric assay using cell lysates of PASMCs treated with BMP7 or ST (positive control) (Fig. 2A). BMP7 treatment activated all three caspases tested (P < 0.001), and a specific inhibitor of each caspase reduced the BMP-induced caspase activity to a level similar to that in untreated cells (Fig. 2A). This result indicates that the caspase cascade was activated in PASMCs in response to BMP7.


Figure 2
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  		Fig. 2. Activation of caspase-3, -8, and -9 by BMP7 treatment. A: enzymatic activity of caspase-3, -8, and -9 was measured by a colorimetric assay after 48-h starvation and stimulation of PASMCs with 10 nM BMP7 or 20 nM ST for 24 h. A specific caspase inhibitor was added during the reaction in some samples. Data are plotted as means ± SE of 3 independent experiments. *P < 0.001 (vs. none); ns, nonsignificant difference (vs. none). B: after incubation with 10 nM BMP7 for the periods of time indicated, PASMC lysate was analyzed by immunoblotting with antibodies to caspase-9 (cas-9), caspase-8 (cas-8), or processed caspase-3 (cas-3). The cell lysate of PASMCs treated with 20 nM ST for 24 h is shown as positive control. C: PASMCs were serum starved for 48 h and treated with 10 nM BMP7 in the presence or absence of caspase inhibitors for 24 h. The cells were then stained by TUNEL assay (green) and DAPI (blue).

 
Caspase activation is accompanied by a proteolytic cleavage of the unprocessed form (procaspase). To test the in vivo activation of caspases in BMP-treated PASMCs, we subjected total cell lysates to immunoblotting using anti-caspase-8 and anti-caspase-9 antibodies, which recognize both the unprocessed and processed forms of these proteins. In unstimulated cells, PASMC lysate contained only the unprocessed, inactive forms of caspase-8 and caspase-9 (Fig. 2B). The processed forms of caspase-8 and caspase-9 were detected in cells stimulated with BMP7 or in ST-treated cells (Fig. 2B). The proteolytic cleavage of caspase-3 was also detected on BMP7 treatment. The anti-caspase-3 antibody specifically detects only the active form of caspase-3 (Fig. 2B). Thus BMP7 mediates the activation of initiator caspases, including caspase-3, -8, and -9.

The requirement for each caspase was examined in vivo. A single inhibitor of caspase-3, -8, or -9 used individually was unable to decrease the percentage of TUNEL-positive cells induced by BMP7 (data not shown). A marginal decrease in the percentage of TUNEL-positive cells was observed with a combination of inhibitors for caspase-3 and -8 or caspase-3 and -9 (Fig. 2C). However, the addition of a combination of inhibitors for caspase-8 and -9 or zVAD-fmk, a broad-range caspase inhibitor, prevented the appearance of TUNEL-positive PASMCs on BMP7 stimulation (Fig. 2C). This result indicates that the activation of either caspase-8 or caspase-9 is sufficient to mediate BMP-induced apoptosis in PASMCs.

BMP-induced apoptosis is mediated by downregulation of Bcl-2. Activation of caspase-9 is generally thought to follow the disruption of the outer mitochondrial membrane, which causes a collapse of membrane potential and a change in permeability (8). Therefore, PASMCs were stained with the cationic dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), which aggregates in negatively charged intact mitochondria in nonapoptotic cells, emitting red fluorescence at 590 nm. On mitochondrial membrane potential collapse in apoptotic cells, the dye can no longer accumulate inside the mitochondria and distributes throughout the cell, assuming a monomeric form that emits green fluorescence at 527 nm. In untreated PASMCs, the detection of red, but not green, fluorescence on JC-1 staining suggested that the mitochondrial outer membrane was intact (Fig. 3A). After treatment with either BMP7 or BMP4, a dramatic increase in green fluorescence was observed, indicating that both treatments reduce mitochondrial membrane potential (Fig. 3A). Next, we examined whether the BMP signal mediates cytochrome c release from mitochondria in PASMCs. BMP4-treated PASMCs were subjected to staining of cytochrome c and complex V{alpha}, a mitochondrial marker whose localization does not change on disruption of the mitochondrial outer membrane (Fig. 3B). Before treatment, cytochrome c and complex V{alpha} colocalized and showed a punctate pattern; however, on BMP4 treatment the cytochrome c stain appeared diffused and failed to colocalize with the mitochondrial marker (Fig. 3B). The results shown in Fig. 3, A and B, indicate that the BMP4 signal mediates the release of cytochrome c from mitochondria as a result of disruption of the mitochondrial outer membrane.


Figure 3
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  		Fig. 3. Release of cytochrome c from mitochondria and decrease of Bcl-2 expression are associated with BMP-mediated apoptotic cell death in PASMCs. A: PASMCs were serum starved for 48 h and treated with 10 nM BMP7, BMP4, or 20 nM ST (as a positive control) for 48 h, followed by staining with the dye JC-1. Cells were analyzed by fluorescence microscopy (emission at 527 and 590 nm). See text for details. B: PASMCs were serum starved for 48 h and treated with 10 nM BMP4. Cells were stained with FITC-conjugated anti-cytochrome c antibody, Texas red-conjugated anti-complex V{alpha} antibody, and DAPI. C: PASMCs were serum starved for 48 h and treated with vehicle or 3 or 10 nM BMP7 for 24 h. Total RNA was extracted from the cells and subjected to RT-PCR analysis using specific primers for Bcl-2, Bax, and HPRT (loading control). The PCR reaction was performed in the presence of [32P]dCTP. PCR products were separated by acrylamide gel electrophoresis and visualized by autoradiography (RT-PCR). Simultaneously, total cell lysates were prepared and subjected to immunoblot using anti-Bcl-2 or anti-Bax antibodies (WB). D: PASMCs were serum starved for 48 h and treated with or without 3 nM BMP4 or BMP7 for 24 h. Total RNA was extracted from the cells and subjected to a real-time PCR reaction. The level of expression of Bcl-2, cIAP-2, survivin, and Bax mRNAs was normalized to GAPDH. mRNA expression of cIAP-2 and survivin was not measured in samples treated with BMP7. Data are means ± SE of triplicates and are normalized to the level of expression of each gene in the absence of BMP. *P < 0.001 (none vs. +BMP4 or +BMP7); ns, nonsignificant difference (none vs. +BMP4 or +BMP7).

 
The release of cytochrome c from mitochondria can be regulated by Bcl-2 family proteins (6). Therefore, we examined the effect of BMPs on the mRNA level of an antiapoptotic molecule (Bcl-2) and a proapoptotic molecule (Bax) by semiquantitative RT-PCR. BMP7 treatment significantly reduced the Bcl-2 mRNA level in PASMCs (Fig. 3C). Unlike Bcl-2, Bax expression was unaffected (Fig. 3C). Consistent with the change of mRNA expression, the amount of Bcl-2 protein, unlike Bax, was dramatically decreased on BMP7 stimulation (Fig. 3C). Change in the mRNA expression of other antiapoptotic factors after BMP4 treatment was examined by RT-PCR analysis. Similar to the result obtained with BMP7 (Fig. 3, C and D), BMP4 stimulation strongly downregulated (30-fold) the Bcl-2 transcript, whereas the Bax mRNA level was unchanged (Fig. 3D). Members of the inhibitor of apoptosis protein (IAP) family, cIAP-2 and survivin, were both downregulated by BMP4 treatment (2.8-fold and 7.5-fold) (Fig. 3D). cIAP-1 was not detectable in PASMCs (data not shown). These results indicate that both BMP4 and BMP7 trigger a change in the balance between proapoptotic and antiapoptotic factors, which might result in the observed increase of active caspases in PASMCs.

BMPRII is essential for BMP-mediated apoptosis in PASMCs. To test the requirement of BMPRII for BMP-mediated apoptosis, a siRNA complementary to the 5'-untranslated region of human BMPRII was transiently transfected into PASMCs. Detection of BMPRII mRNA by PCR indicated that PASMCs transfected with two independent control siRNAs (control siRNA 1 and control siRNA 2) showed no significant reduction of BMPRII expression compared with mock-transfected cells, whereas >90% reduction of the BMPRII mRNA level was observed in anti-BMPRII siRNA-transfected cells (Fig. 4A). Under these conditions, BMP4-induced phosphorylation of the BMP signal transducers Smad1, Smad5, and Smad8 was not detectable, confirming that BMPRII is essential for BMP4 signaling (Fig. 4B). The BMPRII siRNA treatment also dramatically reduced, but did not abolish completely, the phosphorylation of Smad1/5/8 in BMP7-treated cells (Fig. 4B). The residual activity might be due to BMP7 binding to type II receptors other than BMPRII, such as ActRIIA and ActRIIB, which are expressed in PASMCs (see Fig. 1A). In the apoptosis assay, transfection of PASMCs with BMPRII siRNA severely diminished the percentage of TUNEL-positive cells after BMP4 stimulation, demonstrating the essential role of BMPRII in mediating BMP-induced apoptosis in PASMCs (Fig. 4C).


Figure 4
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  		Fig. 4. Downregulation of BMPRII inhibits BMP-mediated apoptosis. A: PASMCs were transfected with 125 or 250 nM small interfering RNA (siRNA) directed against BMPRII, siRNA against the unrelated gene TFII-I (control siRNA 1), or nontargeting siRNA (control siRNA 2) for 48 h. Total RNA was isolated and subjected to a RT reaction, followed by semiquantitative PCR analysis of BMPRII and GAPDH (control). BMPRII expression normalized to GAPDH is shown as % of the expression level in mock-transfected cells (right). B: PASMCs were transfected with nontargeting siRNA (control siRNA 2) or BMPRII siRNA. Forty-eight hours after transfection, cells were stimulated with 10 nM BMP4 or BMP7 for 1 h or left untreated and finally harvested for immunoblot analysis. Expression of phospho-Smad1/5/8 or total Smad1 was analyzed by immunoblotting. B4, BMP4; B7, BMP7. C: PASMCs were transfected with 250 nM control siRNA (control siRNA 1 or 2) or siRNA directed against BMPRII for 48 h. The cells were treated with 10 nM BMP4 for 48 h and then subjected to TUNEL assay. Cells treated with 20 nM ST are shown as positive control. TUNEL-positive cells are shown at top (TUNEL). Propidium iodide stain (PI) displays all the cells in the field. The results are quantified at bottom; the average % of TUNEL-positive cells from 3 independent experiments is plotted on the ordinate axis with SE. Values labeled with the same letter do not differ significantly from one another (P = 0.05).

 
BMPRII mutants found in IPAH patients block BMP-mediated apoptosis. To investigate the potential role of BMP-mediated apoptosis in the etiology of IPAH, we tested the ability of BMPRII mutants found in IPAH patients to mediate BMP responses, such as transcriptional activation of BMP target genes in BMP-responsive cells and apoptosis in PASMCs. We examined two human BMPRII mutant constructs (MutK and MutT) harboring mutations found in familial IPAH patients (Fig. 5A, left). MutK contains a point mutation of cysteine 347 to tyrosine (C347Y) in the serine/threonine kinase domain; MutT contains a point mutation of isoleucine 860 to a stop codon (I860X), which results in a truncation of 178 amino acids in the carboxy terminus tail domain (Fig. 5A, left). We also generated a mutant ({Delta}T) in which the entire carboxy terminus domain is deleted. Adenovirus-mediated expression of these BMPRII mutants in PASMCs was confirmed by immunoblot analysis (Fig. 5A, right). The ability of these mutants to transduce the BMP signal in a Smad-dependent manner was examined by measuring the phosphorylation level of BMP receptor-specific R-Smads (Smad1, Smad5, and Smad8) on BMP stimulation. Overexpression of wild-type BMPRII slightly augmented the level of phosphorylation of BMP-specific Smads, suggesting that BMPRII expression may be a limiting factor in Smad-dependent BMP signaling in PASMCs (Fig. 5B). Overexpression of BMPRII mutants in which the carboxy terminus domain is either truncated (MutT) or completely deleted ({Delta}T) did not significantly interfere with Smad phosphorylation compared with cells infected with control virus (Fig. 5B). Overexpression of the kinase domain mutant MutK completely abolished BMP4-dependent phosphorylation of R-Smads. The degree of the MutK effect is comparable to the result obtained with BMPRII siRNA-treated PASMCs stimulated with BMP4 (see Fig. 4B), indicating that MutK complies with the characteristics of a dominant-negative BMPRII mutant (Fig. 5B). This result also confirms the essential role of BMPRII in the BMP4 signaling pathway.


Figure 5
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  		Fig. 5. Overexpression of BMPRII mutants found in idiopathic pulmonary arterial hypertension (IPAH) patients can block BMP-mediated apoptosis. A: schematic diagram of mutant BMPRII constructs (see text for description). PASMCs were infected with recombinant adenovirus carrying Flag epitope-tagged BMPRII wild type (WT), MutT, MutK, and {Delta}T for 24 h. Expression of each protein was determined by immunoblotting using anti-Flag antibody (M2) and anti-GAPDH (loading control). B: PASMCs were infected with BMPRII adenovirus or a control (LacZ) virus, stimulated with 10 nM BMP4 for 1 h or left untreated, and finally harvested for immunoblot analysis. Expression of phospho-Smad1/5/8 or total Smad1 was analyzed by immunoblotting. C: the BMP-responsive reporter Xvent2-luc was transfected into P19 cells (left) or PASMCs (right), followed by infection with adenovirus expressing WT or mutant (MutK, MutT, and {Delta}T) forms of BMPRII or with a dominant-negative BMPRIB mutant [BMPRIB(DN)] [multiplicity of infection (moi) = 200]. In PASMCs, an OAZ expression vector (300 ng/well) was cotransfected with the reporter construct. The cells were treated with 3 nM BMP4 or vehicle for 20 h, and luciferase activity was measured. D: PASMCs were infected with a control virus (LacZ), wild-type BMPRII (WT), or IPAH mutants (MutK, MutT, and {Delta}T) (moi = 200), followed by treatment with 10 nM BMP7 for 48 h and TUNEL assay (right). The % of TUNEL-positive cells is shown at left. Values labeled with the same letter do not differ significantly from one another (P = 0.05).

 
To test the potential dominant-negative effect of MutK in a BMP-dependent reporter assay, we expressed wild-type or mutant BMPRII proteins in mouse embryonic carcinoma P19 cells and determined their effect on the expression of the BMP reporter construct Xvent2-luc (13). A dominant-negative mutant of the BMP type I receptor IB [BMPRIB(DN)], harboring a mutation in the ATP binding site of the kinase domain (44), was transfected as a control. The kinase domain mutant MutK completely blocked the BMP4-dependent activation of the reporter, as did BMPRIB(DN) (Fig. 5C). Overexpression of the carboxy terminus domain mutants MutT and {Delta}T increased the basal activity of the reporter but had little effect on BMP-mediated induction (Fig. 5C, left). Thus the kinase domain of BMPRII, but not its carboxy terminus domain, is essential for Smad phosphorylation and Smad-dependent gene regulation. Similar results were obtained in PASMCs expressing ectopic OAZ, a Smad1 cofactor required for Xvent2 transcription (13, 18) (Fig. 5C, right).

Finally, we tested the effect of the mutant BMPRII proteins on the BMP7-mediated apoptotic signal in PASMCs. After BMP treatment, the percentage of TUNEL-positive PASMCs in the control (93.3 ± 1.5%) and in the presence of ectopic wild-type BMPRII (94.3 ± 0.9%) was similar; however, a statistically significant reduction of TUNEL-positive cells was observed in the samples expressing MutK (71.3 ± 1.6%) or MutT (68.3 ± 1.4%) (Fig. 5D). This result suggests that BMP-mediated apoptosis in PASMCs is controlled by both the kinase and tail domains of BMPRII. Interestingly, samples expressing the {Delta}T mutant showed no significant difference in TUNEL-positive cells (89.9 ± 1.1%) compared with control (Fig. 5D).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Possible role of BMP-mediated apoptosis in etiology of pulmonary hypertension. Apoptosis is a highly regulated cellular process that maintains tissue homeostasis. Unregulated apoptosis causes developmental defects and various types of disease. In the pulmonary vasculature, it has been speculated that apoptosis may play a role in eliminating cells that have migrated into the vascular lumen and/or hypertrophied cells accumulated in the pulmonary vasculature (7, 32). It has also been demonstrated that increased PASMCs proliferation or inhibition of PASMC apoptosis can contribute to pulmonary vascular medial hypertrophy, a hallmark of pulmonary hypertension (32–34). Therefore, understanding the mechanisms regulating cell growth and apoptosis can help explain the molecular pathogenesis of pulmonary hypertension. Although the homeostasis of PASMCs is regulated by various growth factors and cytokines, the critical role of BMPs and their signaling pathways was illustrated by the discovery of mutations in the BMPRII gene in IPAH patients. In this study we demonstrate an involvement of BMP signaling in the normal physiology of vascular SMCs and evidence that mutations of BMPRII might lead to the pathological condition of the pulmonary arteries of hypertensive patients due to a reduced apoptosis in vivo. It has been reported that treatment of hypertensive rats with a serine elastase inhibitor, which is shown to induce SMC apoptosis, reduces pulmonary artery pressure and medial hypertrophy (7). Therefore, it is plausible that reduced apoptosis in PASMCs may contribute to the development of pulmonary medial hypertrophy. Contrary to our observation in PASMCs, the BMP (BMP2 or BMP7) signaling pathway protects pulmonary artery endothelial cells (PAECs) from apoptosis, and loss-of-function mutations of BMPRII lead to increased apoptosis in PAECs, suggesting that the BMP role is cell type specific (35).

BMP ligands and receptors affecting PASMC proliferation and survival. We found that human PASMCs express all BMP ligands except BMP7 (W. Ni and A. Hata, unpublished observation), as previously reported (43). Although PASMCs do not synthesize BMP7, it may be produced by another type of pulmonary cell, such as the vascular endothelium or the alveolar epithelium, and subsequently secreted into the intercellular space to affect PASMCs (41). In this study, we found that BMP7 and BMP4 show equivalent proapoptotic activities in PASMCs. However, both similar and opposing effects of BMP7 compared with BMP2 or BMP4 on vascular cells have been reported previously. BMP2 and BMP7 inhibit proliferation of human aortic SMCs both in vivo and in vitro (9, 31, 38) and of PASMCs in vitro in a similar manner (29). In vascular SMCs, however, BMP7 stimulates the maintenance of the SMC phenotype (9), whereas BMP2 and BMP4 appear to negatively regulate the expression of SMC markers (17). Such difference in activity between BMP7 and BMP2/4 might be due to the ability of BMP7 to transmit a potentially distinct signal through its binding to ALK2 in combination with ActRIIA or ActRIIB (25). We (see Fig. 4B) and others (42) observed that when BMPRII expression or activity is impaired in PASMCs, BMP7 (but not BMP4) is able to induce a residual level of Smad1/5/8 phosphorylation through an alternative pathway, possibly involving ActRIIA or ActRIIB. Our observation that the dominant-negative BMPRII mutant (MutK) only partially impairs BMP7-induced apoptosis might be due to an alternative BMP7 signal through the ALK2/ActRII receptor complex.

Molecular mechanism of BMP-induced apoptosis in PASMCs. We demonstrate that BMP signaling triggers apoptosis of human PASMCs via a mitochondria-mediated, caspase-dependent mechanism. In primary osteoblasts, BMP2 is shown to induce apoptosis by activating caspase-9 in a Smad-dependent manner (14). We observe not only downregulation of Bcl-2, cIAP-2, and survivin, but also a release of cytochrome c from mitochondria in BMP-treated PASMCs, which could induce activation of caspase-9. Altogether, we conclude that BMP-induced apoptosis in PASMCs is dependent on a caspase-9-mediated signal occurring downstream of mitochondrial dysfunction. The latter could be induced, at least in part, by a relative increase of the proapoptotic protein Bax as a consequence of downregulation of antiapoptotic proteins, such as Bcl-2, cIAP-2, and survivin. Whether the decrease of Bcl-2, cIAP-2, and survivin expression by BMP treatment is regulated at the transcriptional level in a Smad-dependent manner or through a change in mRNA stability is yet to be determined. High levels of survivin expression are considered to be a major pathological mechanism of apoptosis inhibition, in cancer cells as well as in vascular SMCs and endothelial cells (1). For example, vascular injury increases survivin expression in the vessel wall, concomitantly with neointima formation (4). Survivin overexpression also coincides with pulmonary vascular remodeling in rats developing monocrotaline-induced PAH (28). Therefore, we speculate that a loss of BMP-mediated downregulation of survivin might lead to an accumulation of survivin in PASMCs harboring BMPRII mutations; in turn, this event may contribute to an abnormal remodeling of the pulmonary vascular wall and development of pulmonary hypertension as seen in monocrotaline-induced pulmonary hypertensive rats.

Role of BMPRII kinase and tail domain. The kinase domain of the type II receptors is known to be constitutively active, in contrast to the type I receptor kinase domain, which is regulated via phosphorylation of a juxtamembrane domain. Therefore, it is likely that overexpression of the kinase domain mutant of BMPRII (MutK; C347Y) abolishes the endogenous BMP signaling pathway at the level of the activation of the type I receptor kinase, which is essential for phosphorylation of Smad proteins. This interpretation is consistent with a result obtained with PASMCs prepared from a patient harboring the BMPRII C347Y mutation. Our result with the tail domain truncation mutant (MutT) suggests that the tail domain of BMPRII is dispensable for the phosphorylation of Smad1 and transcriptional activation of the BMP reporter but has a specific role in BMP-mediated apoptosis in PASMCs. This observation suggests the existence in PASMCs of a signaling pathway that regulates apoptosis downstream of the BMP receptors in parallel to phosphorylation of Smad1/5/8. It is important to note that PASMCs derived from an IPAH patient with a missense mutation within the tail domain (N903S) reportedly demonstrated a slight reduction in the phosphorylation level of Smad1 in response to a low concentration of BMP4 (30 pM) (40). We speculate that this effect may not appear in our study because of the use of a higher concentration of BMP4 (10 nM) or because of the high protein expression level of the exogenous BMPRII mutant. Unlike cells expressing MutT, cells expressing the BMPRII mutant with a complete deletion of the tail domain ({Delta}T) showed no significant difference in TUNEL assay compared with those expressing the wild-type BMPRII. We found that the {Delta}T mutant is expressed at comparatively very high levels in PASMCs (see Fig. 5A). Therefore, it is possible that the increased level of expression may compensate for the lack of a tail domain.

The tail domain is a unique structure among the TGF-beta family receptors found only in the type II BMP receptor. The tail domain is conserved among the BMPRII homologs of Caenorhabditis elegans (11), Drosophila (21), and Xenopus (16). Because the BMPRII tail domain has recently been shown to bind several potential signal transducers (12, 20, 24, 39), it will be interesting to examine the potential roles of these factors in the apoptotic signaling cascade mediated by BMPs in pulmonary vascular cells.


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This study was supported by grants from the National Institutes of Health and the American Cancer Society (to A. Hata) and from Pfizer and the American Heart Association (to G. Lagna).


   ACKNOWLEDGMENTS
 
We thank Dr. Miyazono for providing the BMPRII mutant constructs and Pamela SooHoo for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Hata and G. Lagna, Molecular Cardiology Research Institute, Tufts-New England Medical Center, 750 Washington St., Box 8486, Boston, MA 02111 (e-mail: akiko.hata{at}tufts.edu and glagna{at}tufts-nemc.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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