Portal de Periódicos da CAPES

Bone Morphogenetic Protein (BMP) Type II Receptor Deletion Reveals BMP Ligand-specific Gain of Signaling in Pulmonary Artery Smooth Muscle Cells

2005; Elsevier BV; Volume: 280; Issue: 26 Linguagem: Inglês

10.1074/jbc.m502825200

1083-351X

Paul B. Yu, Hideyuki Beppu, Noriko Kawai, En Li, Kenneth D. Bloch,

Myeloproliferative Neoplasms: Diagnosis and Treatment

Bone morphogenetic protein (BMP) ligands signal by binding the BMP type II receptor (BMPR2) or the activin type II receptors (ActRIIa and ActRIIb) in conjunction with type I receptors to activate SMADs 1, 5, and 8, as well as members of the mitogen-activated protein kinase family. Loss-of-function mutations in Bmpr2 have been implicated in tumorigenesis and in the etiology of primary pulmonary hypertension. Because several different type II receptors are known to recognize BMP ligands, the specific contribution of BMPR2 to BMP signaling is not defined. Here we report that the ablation of Bmpr2 in pulmonary artery smooth muscle cells, using an ex vivo conditional knock-out (Cre-lox) approach, as well as small interfering RNA specific for Bmpr2, does not abolish BMP signaling. Disruption of Bmpr2 leads to diminished signaling by BMP2 and BMP4 and augmented signaling by BMP6 and BMP7. Using small interfering RNAs to inhibit the expression of other BMP receptors, we found that wild-type cells transduce BMP signals via BMPR2, whereas BMPR2-deficient cells transduce BMP signals via ActRIIa in conjunction with a set of type I receptors distinct from those utilized by BMPR2. These findings suggest that disruption of Bmpr2 leads to the net gain of signaling by some, but not all, BMP ligands via the activation of ActRIIa. Bone morphogenetic protein (BMP) ligands signal by binding the BMP type II receptor (BMPR2) or the activin type II receptors (ActRIIa and ActRIIb) in conjunction with type I receptors to activate SMADs 1, 5, and 8, as well as members of the mitogen-activated protein kinase family. Loss-of-function mutations in Bmpr2 have been implicated in tumorigenesis and in the etiology of primary pulmonary hypertension. Because several different type II receptors are known to recognize BMP ligands, the specific contribution of BMPR2 to BMP signaling is not defined. Here we report that the ablation of Bmpr2 in pulmonary artery smooth muscle cells, using an ex vivo conditional knock-out (Cre-lox) approach, as well as small interfering RNA specific for Bmpr2, does not abolish BMP signaling. Disruption of Bmpr2 leads to diminished signaling by BMP2 and BMP4 and augmented signaling by BMP6 and BMP7. Using small interfering RNAs to inhibit the expression of other BMP receptors, we found that wild-type cells transduce BMP signals via BMPR2, whereas BMPR2-deficient cells transduce BMP signals via ActRIIa in conjunction with a set of type I receptors distinct from those utilized by BMPR2. These findings suggest that disruption of Bmpr2 leads to the net gain of signaling by some, but not all, BMP ligands via the activation of ActRIIa. Bone morphogenetic protein (BMP) 1The abbreviations used are: BMP, bone morphogenetic protein; BMPR2, BMP type II receptor; Id, inhibitor of differentiation; MAP, mitogen-activated protein; ActRII, activin type II receptor; PASMC, pulmonary arterial smooth muscle cell; siRNA, small interfering RNA; Ad.Cre, adenovirus specifying Cre recombinase; GFP, green fluorescent protein; RT, reverse transcription; p, phosphorylated. signals regulate embryonic tissue patterning and organogenesis, as well as the remodeling of mature tissues (1Goumans M.J. Mummery C. Int. J. Dev. Biol. 2000; 44: 253-265PubMed Google Scholar). BMPs, like other transforming growth factor β superfamily ligands, induce apposition of type I and type II receptors to cause phosphorylation of type I receptors. Activated BMP type I receptors phosphorylate the BMP-responsive SMAD proteins 1, 5, and 8, leading to their nuclear translocation and the regulation of target gene transcription. The Id (inhibitor of differentiation) gene family is an important target of BMP signals (2Hollnagel A. Oehlmann V. Heymer J. Ruther U. Nordheim A. J. Biol. Chem. 1999; 274: 19838-19845Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 3Korchynskyi O. ten Dijke P. J. Biol. Chem. 2002; 277: 4883-4891Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar, 4Lopez-Rovira T. Chalaux E. Massague J. Rosa J.L. Ventura F. J. Biol. Chem. 2002; 277: 3176-3185Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 5Miyazono K. Miyazawa K. Sci. STKE 2002. 2002; : PE40Google Scholar), serving to regulate the differentiation and proliferation of a variety of mature and embryonic cell lineages, including vascular smooth muscle and endothelium (2Hollnagel A. Oehlmann V. Heymer J. Ruther U. Nordheim A. J. Biol. Chem. 1999; 274: 19838-19845Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 6Valdimarsdottir G. Goumans M.J. Rosendahl A. Brugman M. Itoh S. Lebrin F. Sideras P. ten Dijke P. Circulation. 2002; 106: 2263-2270Crossref PubMed Scopus (263) Google Scholar, 7ten Dijke P. Korchynskyi O. Valdimarsdottir G. Goumans M.J. Mol. Cell. Endocrinol. 2003; 211: 105-113Crossref PubMed Scopus (175) Google Scholar, 8Mueller C. Baudler S. Welzel H. Bohm M. Nickenig G. Circulation. 2002; 105: 2423-2428Crossref PubMed Scopus (66) Google Scholar). BMP ligands may also trigger the activation of MAP kinases via SMAD-independent signaling pathways (9Iwasaki S. Iguchi M. Watanabe K. Hoshino R. Tsujimoto M. Kohno M. J. Biol. Chem. 1999; 274: 26503-26510Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 10Hall A.K. Miller R.H. J. Neurosci. Res. 2004; 76: 1-8Crossref PubMed Scopus (62) Google Scholar, 11Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4324) Google Scholar). Three type II receptors, BMPR2 and the activin type II receptors ActRIIa and ActRIIb (12Rosenzweig B.L. Imamura T. Okadome T. Cox G.N. Yamashita H. ten Dijke P. Heldin C.H. Miyazono K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7632-7636Crossref PubMed Scopus (478) Google Scholar, 13Liu F. Ventura F. Doody J. Massague J. Mol. Cell. Biol. 1995; 15: 3479-3486Crossref PubMed Scopus (522) Google Scholar, 14Nohno T. Ishikawa T. Saito T. Hosokawa K. Noji S. Wolsing D.H. Rosenbaum J.S. J. Biol. Chem. 1995; 270: 22522-22526Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 15Yamashita H. ten Dijke P. Huylebroeck D. Sampath T.K. Andries M. Smith J.C. Heldin C.H. Miyazono K. J. Cell Biol. 1995; 130: 217-226Crossref PubMed Scopus (462) Google Scholar, 16Mathews L.S. Vale W.W. Cell. 1991; 65: 973-982Abstract Full Text PDF PubMed Scopus (680) Google Scholar, 17Donaldson C.J. Mathews L.S. Vale W.W. Biochem. Biophys. Res. Commun. 1992; 184: 310-316Crossref PubMed Scopus (79) Google Scholar), can pair with three different type I receptors, ActRIa/ALK2, BMPRIa/ALK3, and BMPRIb/ALK6 (13Liu F. Ventura F. Doody J. Massague J. Mol. Cell. Biol. 1995; 15: 3479-3486Crossref PubMed Scopus (522) Google Scholar, 18Koenig B.B. Cook J.S. Wolsing D.H. Ting J. Tiesman J.P. Correa P.E. Olson C.A. Pecquet A.L. Ventura F. Grant R.A. Chen G.X. Wrana J.L. Massague J. Rosenbaum J.S. Mol. Cell. Biol. 1994; 14: 5961-5974Crossref PubMed Scopus (312) Google Scholar, 19ten Dijke P. Yamashita H. Sampath T.K. Reddi A.H. Estevez M. Riddle D.L. Ichijo H. Heldin C.H. Miyazono K. J. Biol. Chem. 1994; 269: 16985-16988Abstract Full Text PDF PubMed Google Scholar, 20ten Dijke P. Yamashita H. Ichijo H. Franzen P. Laiho M. Miyazono K. Heldin C.H. Science. 1994; 264: 101-104Crossref PubMed Scopus (512) Google Scholar, 21Macias-Silva M. Hoodless P.A. Tang S.J. Buchwald M. Wrana J.L. J. Biol. Chem. 1998; 273: 25628-25636Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 22Ebisawa T. Tada K. Kitajima I. Tojo K. Sampath T.K. Kawabata M. Miyazono K. Imamura T. J. Cell Sci. 1999; 112 (Pt. 20): 3519-3527Crossref PubMed Google Scholar, 23Fujii M. Takeda K. Imamura T. Aoki H. Sampath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (370) Google Scholar), to transduce BMP signals. These various BMP receptors have distinct temporal and spatial expression in tissues and have varying affinities for each of more than 15 known BMP molecules (1Goumans M.J. Mummery C. Int. J. Dev. Biol. 2000; 44: 253-265PubMed Google Scholar, 24Derynck R. Feng X.H. Biochim. Biophys. Acta. 1997; 1333: F105-F150Crossref PubMed Scopus (508) Google Scholar). Although BMPR2, ActRIIa, and ActRIIb can each bind BMP ligands (12Rosenzweig B.L. Imamura T. Okadome T. Cox G.N. Yamashita H. ten Dijke P. Heldin C.H. Miyazono K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7632-7636Crossref PubMed Scopus (478) Google Scholar, 13Liu F. Ventura F. Doody J. Massague J. Mol. Cell. Biol. 1995; 15: 3479-3486Crossref PubMed Scopus (522) Google Scholar, 14Nohno T. Ishikawa T. Saito T. Hosokawa K. Noji S. Wolsing D.H. Rosenbaum J.S. J. Biol. Chem. 1995; 270: 22522-22526Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 15Yamashita H. ten Dijke P. Huylebroeck D. Sampath T.K. Andries M. Smith J.C. Heldin C.H. Miyazono K. J. Cell Biol. 1995; 130: 217-226Crossref PubMed Scopus (462) Google Scholar, 21Macias-Silva M. Hoodless P.A. Tang S.J. Buchwald M. Wrana J.L. J. Biol. Chem. 1998; 273: 25628-25636Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 22Ebisawa T. Tada K. Kitajima I. Tojo K. Sampath T.K. Kawabata M. Miyazono K. Imamura T. J. Cell Sci. 1999; 112 (Pt. 20): 3519-3527Crossref PubMed Google Scholar, 25Greenwald J. Groppe J. Gray P. Wiater E. Kwiatkowski W. Vale W. Choe S. Mol. Cell. 2003; 11: 605-617Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), the preferred ligand-receptor complexes used by cells for BMP signaling are not known. In this study, we examined the signaling mediated by two structurally distinct classes of BMP ligands that are known to be expressed in vascular smooth muscle and endothelial cells and that regulate their function (6Valdimarsdottir G. Goumans M.J. Rosendahl A. Brugman M. Itoh S. Lebrin F. Sideras P. ten Dijke P. Circulation. 2002; 106: 2263-2270Crossref PubMed Scopus (263) Google Scholar, 26Zhang S. Fantozzi I. Tigno D.D. Yi E.S. Platoshyn O. Thistlethwaite P.A. Kriett J.M. Yung G. Rubin L.J. Yuan J.X. Am. J. Physiol. 2003; 285: L740-L754Crossref PubMed Scopus (212) Google Scholar, 27Dorai H. Vukicevic S. Sampath T.K. J. Cell. Physiol. 2000; 184: 37-45Crossref PubMed Scopus (130) Google Scholar, 28Wong G.A. Tang V. El-Sabeawy F. Weiss R.H. Am. J. Physiol. 2003; 284: E972-E979Crossref PubMed Scopus (52) Google Scholar, 29King K.E. Iyemere V.P. Weissberg P.L. Shanahan C.M. J. Biol. Chem. 2003; 278: 11661-11669Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 30Dorai H. Sampath T.K. J. Bone Jt. Surg. Am. 2001; 83-A: S70-S78PubMed Google Scholar). The first class of BMP ligands includes the 92% homologous BMP2 and BMP4, considered prototype BMPs in that they possess greater affinity for type I than type II receptors (19ten Dijke P. Yamashita H. Sampath T.K. Reddi A.H. Estevez M. Riddle D.L. Ichijo H. Heldin C.H. Miyazono K. J. Biol. Chem. 1994; 269: 16985-16988Abstract Full Text PDF PubMed Google Scholar, 31Knaus P. Sebald W. Biol. Chem. 2001; 382: 1189-1195Crossref PubMed Scopus (71) Google Scholar, 32Nohe A. Hassel S. Ehrlich M. Neubauer F. Sebald W. Henis Y.I. Knaus P. J. Biol. Chem. 2002; 277: 5330-5338Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). The second class of BMP ligands we studied consists of the closely related BMP5, BMP6, BMP7, and BMP8, which are ∼60% homologous to BMP2 or BMP4 and share with activin ligands a greater affinity for their corresponding type II receptors (15Yamashita H. ten Dijke P. Huylebroeck D. Sampath T.K. Andries M. Smith J.C. Heldin C.H. Miyazono K. J. Cell Biol. 1995; 130: 217-226Crossref PubMed Scopus (462) Google Scholar, 21Macias-Silva M. Hoodless P.A. Tang S.J. Buchwald M. Wrana J.L. J. Biol. Chem. 1998; 273: 25628-25636Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 22Ebisawa T. Tada K. Kitajima I. Tojo K. Sampath T.K. Kawabata M. Miyazono K. Imamura T. J. Cell Sci. 1999; 112 (Pt. 20): 3519-3527Crossref PubMed Google Scholar, 25Greenwald J. Groppe J. Gray P. Wiater E. Kwiatkowski W. Vale W. Choe S. Mol. Cell. 2003; 11: 605-617Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). To define the contribution of BMPR2 to signaling by these two classes of BMP ligands, we tested the effect of ablating Bmpr2 in pulmonary arterial smooth muscle cells (PASMC). The Bmpr2 gene was disrupted in PASMC isolated from genetically modified mice possessing Bmpr2 alleles with internal loxP sites, using an adenovirus specifying Cre recombinase. In a complimentary approach, BMPR2 expression was inhibited with high efficiency by specific small interfering RNA (siRNA) duplexes. Remarkably, the loss of BMPR2 did not uniformly reduce BMP signal transduction but instead increased the activation of SMAD and p38 by BMP6 and BMP7. In BMPR2-deficient cells, BMP signals were transduced by receptor complexes consisting of the ActRIIa receptor and a set of type I co-receptors distinct from those utilized by BMPR2, resulting in the gain of signaling for certain BMP ligands. Mice with Genetically Modified BMPR2 Alleles—Mice heterozygous for a Bmpr2 mutant allele (Bmpr2+/–) were generated as previously described (33Beppu H. Kawabata M. Hamamoto T. Chytil A. Minowa O. Noda T. Miyazono K. Dev. Biol. 2000; 221: 249-258Crossref PubMed Scopus (336) Google Scholar, 34Beppu H. Ichinose F. Kawai N. Jones R.C. Yu P.B. Zapol W.M. Miyazono K. Li E. Bloch K.D. Am. J. Physiol. 2004; 287: L1241-L1247Crossref PubMed Scopus (174) Google Scholar) and backcrossed for 10 generations to inbred C57BL/6 (wild-type) mice. The generation of mice with a conditionally disrupted Bmpr2 allele was described by Beppu et al. (35Beppu H. Lei H. Bloch K.D. Li E. Genesis. 2005; 41: 133-137Crossref PubMed Scopus (37) Google Scholar). Briefly, a construct containing exons 4 and 5 (which encode the transmembrane and a portion of the kinase domain) of the Bmpr2 gene flanked by two loxP sites and a neomycin selection cassette (pgk-neo) with a third loxP site was used to target Bmpr2 in embryonic stem cells. Mice homozygous for the conditional knock-out allele (Bmpr2flox/flox) on a hybrid C57BL/6 and Sv129 background were viable and appeared normal. Isolation of Pulmonary Artery Smooth Muscle Cells—Explants of murine pulmonary arteries were cultured as described previously (36Takata M. Filippov G. Liu H. Ichinose F. Janssens S. Bloch D.B. Bloch K.D. Am. J. Physiol. 2001; 280: L272-L278Crossref PubMed Google Scholar) to yield PASMCs whose phenotype was confirmed by immunohisto-chemical staining for α-smooth muscle actin. Three separate isolates of PASMCs were obtained, each derived from an individual Bmpr2flox/flox mouse. PASMCs were also isolated from wild-type and Bmpr2+/– mice. Disruption of the BMPR2 Gene in PASMC—To disrupt the Bmpr2 gene, PASMCs isolated from Bmpr2flox/flox mice were infected with an adenovirus specifying Cre recombinase (Ad.Cre) or an adenovirus specifying green fluorescent protein (Ad.GFP) as a control, each at a multiplicity of infection of 150. Cultured cells were allowed to recover for 7 days after infection and then passaged twice to ensure depletion of BMPR2 protein. Measurement of BMPR2 Protein Expression—Proteins were extracted from PASMC in SDS-lysis buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. BMPR2 was detected using a monoclonal antibody directed against the C-terminal domain (BD Biosciences). Bound antibody was detected with an horseradish peroxidase-linked antibody directed against mouse IgG and was visualized using chemiluminescence with ECL Plus (Amersham Biosciences). Measurement of Gene Expression—Total RNA was extracted from PASMCs using TRIzol reagent (Invitrogen), and cDNA was synthesized by the reverse transcriptase reaction. Gene sequences were amplified from cDNA by PCR and quantitated using an ABI Prism 7000 (Applied Biosystems, Inc., Foster City, CA) with the following primers. To detect Bmpr2 cDNA, the forward primer 5′-GAAACGATAATCATTGTTTGGC-3′ corresponding to a sequence in exon 4 and reverse primer 5′-CCCTGTTTCCGGTCTCCTGT-3′ corresponding to a sequence in exon 5 were used. To detect Alk2, Alk3, Alk6, ActRIIa, ActRIIb, Id1, Smad6, Smad7, and 18 S ribosomal cDNA sequences, TaqMan primer sets supplied by Applied Biosystems were used. Changes in the relative gene expression normalized to 18 S rRNA levels were determined using the relative CT method (37Heid C.A. Stevens J. Livak K.J. Williams P.M. Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (5040) Google Scholar, 38Gibson U.E. Heid C.A. Williams P.M. Genome Res. 1996; 6: 995-1001Crossref PubMed Scopus (1781) Google Scholar). Measurement of SMAD1/5/8 and p38 Phosphorylation—After serum starvation in RPMI medium (Invitrogen) for 24 h, PASMCs were incubated with BMPs 2, 4, 6, or 7 (R&D Systems, Inc., Minneapolis, MN). Cultured cell lysates (15 μg/lane) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Amersham Biosciences), and probed with antibodies specific for phosphorylated (p-) SMAD1/5/8 and p-p38 (Cell Signaling Technology, Inc., Beverly, MA). Replicate immunoblots were probed with antibodies directed to total SMAD1 (Upstate Biotechnology Inc., Waltham, MA) and p38 (Cell Signaling). Blots were incubated with horseradish peroxidase-linked antibodies specific for rabbit Ig. Bound complexes were visualized by chemiluminescence (ECL Plus) and quantitated using a Storm imager reading blue fluorescence (Amersham Biosciences). All chemifluorescence measurements obtained were within the dynamic range of the Storm imager. Measurement of BMP-induced Id1 Gene Transcription—Subconfluent cells in 12-well tissue culture plates were transfected using Fu-GENE6 (Roche Applied Science) with 0.5 μg of a plasmid encoding firefly luciferase under the control of the Id1 gene promoter (kindly provided by Dr. Peter ten Dijke, Netherlands Cancer Institute) and 0.1 μg of plasmid encoding the Renilla luciferase gene under the control of the thymidine kinase promoter (Promega Corp., Madison, WI) as a control for transfection efficiency. After 24 h, medium was replaced with serum-free medium, and cells were incubated for 12 h. Cells were then incubated with BMP ligands for 12 h and harvested. Firefly and Renilla luciferase activity was measured using a dual luciferase reagent system (Promega), and relative Id1 transcription was reflected by the firefly luciferase activity divided by the Renilla luciferase activity. siRNA Targeting Constructs and Transfection—siRNA duplexes in annealed and purified form were obtained from Ambion, Inc. (Austin, TX). Sense sequences of duplexes used for gene targeting were (5′ to 3′): for Alk2, GGUCAACCAGAAACUUUACtt; Alk3, GGGCAGAAUCUAGAUAGUAtt; Alk6, GGGUCAGAUUUUCAAUGUCtt; Bmpr2, GGGAGCACGUGUUAUGGUCtt; ActRIIa, GGAGUGUCUUUUCUUUAAUtt; and ActRIIb, GGCUCAGCUCAUGAACGACtt. siRNA duplexes were added at 120 nm to subconfluent PASMCs in 12-well plates in a mixture of Oligofectamine and OptiMEM culture medium (Invitrogen) in the absence of serum or antibiotics. After incubation for 6 h, fetal calf serum was added to cultures at a final concentration of 10%. Assays to measure target mRNA levels were performed 48 h after transfection. Assays to measure BMP-mediated SMAD1/5/8 activation were performed after an additional 12 h of serum starvation. Disruption of the BMPR2 Gene in PASMCs—PASMCs isolated from Bmpr2flox/flox mice were infected with Ad.Cre to excise the genomic fragment containing exons 4 and 5 of the Bmpr2 gene, yielding Bmpr2del/del cells. Cre-mediated recombination was confirmed first by PCR analysis of genomic DNA using primers specific for sequences in introns 3 and 5, which yielded a product consistent with excision of exons 4 and 5 (35Beppu H. Lei H. Bloch K.D. Li E. Genesis. 2005; 41: 133-137Crossref PubMed Scopus (37) Google Scholar). Second, RNA extracted from Bmpr2del/del cells was amplified by RT-PCR with primers specific for sequences in exons 3 and 8, and the product was sequenced to reveal direct splicing of exon 3 to exon 6, with stop codons in exon 6 resulting from a frame-shift (35Beppu H. Lei H. Bloch K.D. Li E. Genesis. 2005; 41: 133-137Crossref PubMed Scopus (37) Google Scholar). Third, quantitative RT-PCR performed on RNA from each isolate of Bmpr2del/del cells using primers specific for exons 4 and 5 demonstrated levels of wild-type Bmpr2 mRNA that were 0.5 to 2% of that found in Bmpr2flox/flox cells infected with Ad.GFP (Fig. 1A). In comparison, PASMC from Bmpr2 heterozygote (Bmpr2+/–) mice expressed Bmpr2 mRNA at levels half of those found in wild-type (Fig. 1B). Bmpr2del/del cells did not express detectable wild-type BMPR2 protein, in contrast to Bmpr2flox/flox cells infected with Ad.GFP (Fig. 1C). SMAD1/5/8 Activation Is Attenuated in BMPR2+/– PASMCs—We previously observed that the ability of Bmpr2+/– cells to activate SMAD1/5/8 in response to BMP2 was diminished compared with wild-type cells (34Beppu H. Ichinose F. Kawai N. Jones R.C. Yu P.B. Zapol W.M. Miyazono K. Li E. Bloch K.D. Am. J. Physiol. 2004; 287: L1241-L1247Crossref PubMed Scopus (174) Google Scholar). To determine whether SMAD signaling in Bmpr2+/– cells was diminished in response to other BMP ligands, cells were stimulated with BMPs 4, 6, or 7 for 30 min. For each of these BMP ligands, the phosphorylation of SMAD1/5/8 was decreased in Bmpr2+/– cells to levels 40–60% of that observed in wild-type cells (results for BMP7 shown in Fig. 2, A and B). SMAD1/5/8 Activation Is Altered in BMPR2-deficient PASMCs—SMAD1/5/8 activation was measured in Bmpr2del/del cells to determine whether the ablation of Bmpr2 would further attenuate BMP signaling in comparison to the reduction in signaling observed in Bmpr2+/– cells. Bmpr2del/del cells retained the ability to phosphorylate SMAD1/5/8 in response to BMP4, at levels that were 30–50% of those in Bmpr2flox/flox cells (Fig. 2, C and D). In response to BMP7, however, Bmpr2del/del cells phosphorylated SMAD1/5/8 at levels 4–5-fold greater than in Bmpr2flox/flox cells. The signaling mediated by other BMP ligands was tested to determine whether the signaling changes observed in BMPR2-deficient cells could be generalized to structurally similar BMPs. Bmpr2del/del cells exhibited attenuated SMAD1/5/8 activation in response to both BMP2 and BMP4 compared with Bmpr2flox/flox cells (Fig. 2E). Bmpr2del/del cells exhibited increased SMAD1/5/8 activation in response to both BMP6 and BMP7 compared with Bmpr2flox/flox cells. The increased ability of Bmpr2del/del cells to activate SMAD1/5/8 was more marked for BMP7 than for BMP6. These changes in the sensitivity to different BMP ligands were consistently observed in Ad.Cre-infected PASMC isolated from three individual Bmpr2flox/flox mice. p38 MAP Kinase Activation Is Altered in BMPR2-deficient PASMC—It has been previously reported that BMPs activate p38 MAP kinase in several cell types independently of SMAD activation (9Iwasaki S. Iguchi M. Watanabe K. Hoshino R. Tsujimoto M. Kohno M. J. Biol. Chem. 1999; 274: 26503-26510Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 10Hall A.K. Miller R.H. J. Neurosci. Res. 2004; 76: 1-8Crossref PubMed Scopus (62) Google Scholar, 11Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4324) Google Scholar). To determine whether the BMP signaling changes observed in Bmpr2del/del cells might be reflected in the activation of effectors other than SMADs, the BMP-induced phosphorylation of p38 was examined in Bmpr2flox/flox and Bmpr2del/del PASMCs (Fig. 2E). In Bmpr2flox/flox cells, stimulation with all BMP ligands induced the activation of p38 by 60 min, which was delayed compared with the activation of SMAD1/5/8. The activation of p38 by BMP2 and BMP4 was decreased in Bmpr2del/del cells compared with Bmpr2flox/flox cells, concordant with decreases in SMAD1/5/8 activation for those ligands. The activation of p38 by BMP6 and BMP7 was increased in Bmpr2del/del cells compared with Bmpr2flox/flox cells, also concordant with increases in SMAD1/5/8 activation for those ligands. BMP-induced Id1 Gene Transcription in BMPR2-deficient Cells—The BMP-induced transcription of Id family genes is a primary means by which BMP ligands achieve effects in target cells (2Hollnagel A. Oehlmann V. Heymer J. Ruther U. Nordheim A. J. Biol. Chem. 1999; 274: 19838-19845Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 3Korchynskyi O. ten Dijke P. J. Biol. Chem. 2002; 277: 4883-4891Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar, 4Lopez-Rovira T. Chalaux E. Massague J. Rosa J.L. Ventura F. J. Biol. Chem. 2002; 277: 3176-3185Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 5Miyazono K. Miyazawa K. Sci. STKE 2002. 2002; : PE40Google Scholar). To determine whether alterations in the ability of BMPR2-deficient cells to activate SMAD1/5/8 were reflected in the ability to transcribe BMP-responsive genes, Id1 gene transcriptional activity was measured using a luciferase reporter gene under the control of the BMP-responsive Id1 gene promoter (Fig. 3, left panel). At baseline, Bmpr2flox/flox and Bmpr2del/del PASMCs had equivalent Id1 transcriptional activity. After stimulation with BMP4, Bmpr2flox/flox cells induced Id1 promoter activity >20-fold. BMP4 also induced Id1 promoter activity in Bmpr2del/del cells but 70% less than that observed in Bmpr2flox/flox cells. After stimulation with BMP7, Bmpr2flox/flox cells had only modest induction of Id1 promoter activity, whereas Bmpr2del/del cells had 5-fold greater induction of Id1 promoter activity in response to this ligand than did Bmpr2flox/flox cells. Consistent with the promoter assay, quantitative RT-PCR measurements revealed that incubation with BMP7 for 1 h increased Id1 mRNA levels to a greater extent in Bmpr2del/del PASMC than in Bmpr2flox/flox cells (Fig. 3, right panel). These findings suggest that the ability of Bmpr2flox/flox and Bmpr2del/del cells to activate SMAD1/5/8 correlated closely with the transcriptional activity of a key BMP-responsive gene. BMPR2 Gene Disruption Does Not Alter the Expression of Other BMP Receptors or Inhibitory SMADs—ActRIIa, ActRIIb, Alk2, and Alk3 receptor sequences were readily detected by quantitative RT-PCR of mRNA from each of the PASMC isolates examined (≤26 cycles of PCR amplification from 200 ng of RNA). The expression levels of ActRIIa, ActRIIb, Alk2, and Alk3 receptors were not altered in Bmpr2del/del cells as compared with Bmpr2flox/flox (shown) or wild-type (data not shown) cells (Fig. 1A). Very little Alk6 cDNA was detected in the PASMC isolates tested (requiring >35 cycles of amplification), consistent with previous reports of low levels of Alk6 mRNA in normal human PASMCs (39Takeda M. Otsuka F. Nakamura K. Inagaki K. Suzuki J. Miura D. Fujio H. Matsubara H. Date H. Ohe T. Makino H. Endocrinology. 2004; 145: 4344-4354Crossref PubMed Scopus (45) Google Scholar). When measured by quantitative RT-PCR, the expression levels of the inhibitory SMADs, Smad6 and Smad7, were not significantly different in Bmpr2del/del and Bmpr2flox/flox cells (data not shown). Inhibition of BMPR2 Expression by Specific siRNA Attenuates BMP4 and Enhances BMP7 Responses—BMPR2 expression was inhibited by transfecting wild-type PASMCs with a gene-specific siRNA duplex for 48 h. This siRNA reduced BMPR2 protein expression by >90% in wild-type PASMCs, whereas transfection with siRNAs specific for Alk6 did not alter BMPR2 protein levels (Fig. 4). Treatment with siRNAs did not affect the expression of total SMAD1 protein. Treatment with siRNA specific for Alk6 did not alter the phosphorylation of SMAD1/5/8 induced by BMP4 or BMP7, consistent with low levels of Alk6 expression observed in PASMCs. Treatment with siRNA specific for Bmpr2 attenuated the phosphorylation of SMAD1/5/8 induced by BMP4 while increasing that induced by BMP7. Changes in BMP signaling resulting from the efficient RNA interference-mediated inhibition of BMPR2 expression in wild-type PASMCs were thus consistent with the ligand-specific changes in BMP signaling observed in Bmpr2del/del cells. Receptors That Transduce BMP Signals in the Presence or Absence of BMPR2—To identify the type II receptors responsible for BMP signaling in Bmpr2flox/flox and Bmpr2del/del PASMC, siRNA-mediated inhibition of receptor expression was employed. 48 h after transfecting cells with siRNAs specific for the type II receptors ActRIIa or ActRIIb, the expression of the target gene relative to 18 S ribosomal RNA expression was measured by quantitative RT-PCR (Fig. 5A). siRNAs specific for either ActRIIa or ActRIIb inhibited their expression (70 and 45%, respectively) in a specific manner with minimal effect on the expression of the homologous receptor. After pretreatment with siRNAs, cells were exposed to BMP ligands for 30 min and the activation of SMAD1/5/8 was assessed (Fig. 5, B–D). The inhibition of ActRIIa or ActRIIb expression in Bmpr2flox/flox cells did not affect the activation of SMAD1/5/8 induced by BMP4 or BMP7 (Fig. 5, B and C). In contrast, the inhibition of ActRIIa in Bmpr2del/del cells impaired the activation of SMAD1/5/8 by BMP4 or BMP7 (Fig. 5, B and D). The inhibition of ActRIIb had no effect under these conditions. These results suggest that BMPR2 is the principal type II receptor transducing BMP signals when it is expressed and that ActRIIa is the principal type II receptor transducing BMP signals when BMPR2 is not expressed. To identify the type I receptors that transduce BMP signals in Bmpr2flox/flox and Bmpr2del/del PASMCs, siRNA specific for Alk2, Alk3, and Alk6 were used. siRNAs for Alk2 and Alk3 inhibited the expression of these type I receptors (60 and 75%, respectively) in a specific manner with minimal effects on the expression of other type I receptors (Fig. 6A). In Bmpr2flox/flox cells, BMP4-mediated SMAD1/5/8 signaling was i