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Transforming Growth Factor β (TGFβ) Signaling via Differential Activation of Activin Receptor-like Kinases 2 and 5 during Cardiac Development

2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês

10.1074/jbc.m209668200

1083-351X

Simone M. Ward, Jay S. Desgrosellier, Xiaoli Zhuang, Joey V. Barnett, Jonas Galper,

Cardiac Fibrosis and Remodeling

Little is known regarding factors that induce parasympathetic responsiveness during cardiac development. We demonstrated previously that in atrial cells cultured from chicks 14 days in ovo, transforming growth factor β (TGFβ) decreased parasympathetic inhibition of beat rate by the muscarinic agonist, carbamylcholine, by 5-fold and decreased expression of Gαi2. Here in atrial cells 5 days in ovo,TGFβ increased carbamylcholine inhibition of beat rate 2.5-fold and increased expression of Gαi2. TGFβ also stimulated Gαi2 mRNA expression and promoter activity at day 5 while inhibiting them at day 14 in ovo. Over the same time course expression of type I TGFβ receptors, chick activin receptor-like kinase 2 and 5 increased with a 2.3-fold higher increase in activin receptor-like kinase 2. Constitutively active activin receptor-like kinase 2 inhibited Gαi2 promoter activity, whereas constitutively active activin receptor-like kinase 5 stimulated Gαi2 promoter activity independent of embryonic age. In 5-day atrial cells, TGFβ stimulated the p3TP-lux reporter, which is downstream of activin receptor-like kinase 5 and had no effect on the activity of the pVent reporter, which is downstream of activin receptor-like kinase 2. In 14-day cells, TGFβ stimulated both pVent and p3TP-lux. Thus TGFβ exerts opposing effects on parasympathetic response and Gαi2 expression by activating different type I TGFβ receptors at distinct stages during cardiac development. Little is known regarding factors that induce parasympathetic responsiveness during cardiac development. We demonstrated previously that in atrial cells cultured from chicks 14 days in ovo, transforming growth factor β (TGFβ) decreased parasympathetic inhibition of beat rate by the muscarinic agonist, carbamylcholine, by 5-fold and decreased expression of Gαi2. Here in atrial cells 5 days in ovo,TGFβ increased carbamylcholine inhibition of beat rate 2.5-fold and increased expression of Gαi2. TGFβ also stimulated Gαi2 mRNA expression and promoter activity at day 5 while inhibiting them at day 14 in ovo. Over the same time course expression of type I TGFβ receptors, chick activin receptor-like kinase 2 and 5 increased with a 2.3-fold higher increase in activin receptor-like kinase 2. Constitutively active activin receptor-like kinase 2 inhibited Gαi2 promoter activity, whereas constitutively active activin receptor-like kinase 5 stimulated Gαi2 promoter activity independent of embryonic age. In 5-day atrial cells, TGFβ stimulated the p3TP-lux reporter, which is downstream of activin receptor-like kinase 5 and had no effect on the activity of the pVent reporter, which is downstream of activin receptor-like kinase 2. In 14-day cells, TGFβ stimulated both pVent and p3TP-lux. Thus TGFβ exerts opposing effects on parasympathetic response and Gαi2 expression by activating different type I TGFβ receptors at distinct stages during cardiac development. A decrease in heart rate in response to parasympathetic stimulation (negative chronotropic response) involves the binding of acetylcholine to M2 muscarinic receptors and the dissociation of the heterotrimeric G-protein, Gi2, into αi2 and βγ subunits. The latter activates the inward rectifying K+ channel, GIRK1, increasing diastolic depolarization and decreasing heart rate (1Logothetis D.E. Kurachi Y. Galper J. Neer E.J. Clapham D.E. Nature. 1987; 325: 321-326Crossref PubMed Scopus (869) Google Scholar). A decrease in the force of contraction in response to muscarinic stimulation (negative inotropic effect) involves the binding of the αi2 subunit to adenylate cyclase followed by a decrease in cAMP production. Several studies support the conclusion that control of Gαi2expression regulates the response of the heart to parasympathetic stimulation. The development of parasympathetic responsiveness in the embryonic chick heart is associated with an increase in Gαi2 expression (2Liang B.T. Hellmich M.R. Neer E.J. Galper J.B. J. Biol. Chem. 1986; 261: 9011-9021Abstract Full Text PDF PubMed Google Scholar). Furthermore, growth of chick atrial cells in the absence of lipoproteins, which has been shown to result in an increased response to parasympathetic stimulation, is associated with an increase in the expression of Gαi2 (3Haigh L.S. Leatherman G.F. O'Hara D.S. Smith T.W. Galper J.B. J. Biol. Chem. 1988; 263: 15608-15618Abstract Full Text PDF PubMed Google Scholar, 4Barnett J.V. Haigh L.S. Marsh J.D. Galper J.B. J. Biol. Chem. 1989; 264: 10779-10786Abstract Full Text PDF PubMed Google Scholar). Finally, expression of Gαi2 in the porcine atrioventricular node resulted in an increase in parasympathetic tone (5Donahue J.K. Heldman A.W. Fraser H. McDonald A.D. Miller J.M. Rade J.J. Eschenhagen T. Marban E. Nat. Med. 2000; 6: 1395-1398Crossref PubMed Scopus (160) Google Scholar). A role for TGFβ 1The abbreviations used are: TGFβ, transforming growth factor β; TBRI, type I TGFβ receptor; TBRII, type II TGFβ receptor; ActRII, type II activin receptor; BMP, bone morphogenic protein; BMPRII, type II BMP receptor; GAPDH, glyceraldehyhyde phosphate dehydrogenase; ALK, activin receptor-like kinase; chALK, chick ALK; chALK+, constitutively active chALK; PAP, placental alkaline phosphatase; dio, days in ovo; Luc, luciferase; PAI-1, plasminogen activator inhibitor; pBS, pBluescript1The abbreviations used are: TGFβ, transforming growth factor β; TBRI, type I TGFβ receptor; TBRII, type II TGFβ receptor; ActRII, type II activin receptor; BMP, bone morphogenic protein; BMPRII, type II BMP receptor; GAPDH, glyceraldehyhyde phosphate dehydrogenase; ALK, activin receptor-like kinase; chALK, chick ALK; chALK+, constitutively active chALK; PAP, placental alkaline phosphatase; dio, days in ovo; Luc, luciferase; PAI-1, plasminogen activator inhibitor; pBS, pBluescript in the development of the parasympathetic response of the heart was suggested by studies in which medium conditioned by co-culture of chick heart cells and ciliary ganglia induced a negative chronotropic response to carbamylcholine in chick heart cells 3.5 days in ovo (dio). This induction of a parasympathetic response was accompanied by an increase in Gαi2 expression (6Barnett J.V. Taniuchi M. Yang M.B. Galper J.B. Biochem. J. 1993; 292: 395-399Crossref PubMed Scopus (6) Google Scholar) and was reversed by addition of a neutralizing antibody to TGFβ1 to the medium. 2J. V. Barnett and J. B. Galper, unpublished observation.2J. V. Barnett and J. B. Galper, unpublished observation. In contrast, we recently demonstrated that in atrial cells from hearts 14 dio, TGFβ1 decreased the expression of Gαi2 and decreased the negative chronotropic response to carbamylcholine (7Ward S. Gadbut A. Tang D. Papageorge A. Wu L. Li G. Barnett J. Galper J. J. Mol. Cell. Cardiol. 2002; 34: 1217-1226Abstract Full Text PDF PubMed Google Scholar). These data suggest that TGFβ exerts opposing effects on parasympathetic responsiveness at different stages of cardiac development. The TGFβ family is composed of at least three 25-kDa homodimeric proteins, TGFβ1, TGFβ2, and TGFβ3. TGFβ signaling involves the binding of TGFβ ligand to two transmembrane serine threonine kinases, the type I TGFβ receptor I (TBRI) and the type II TGFβ receptor (TBRII). TBRII has a constitutively active cytoplasmic kinase domain and an extracellular domain that binds TGFβ1 and TGFβ3. TGFβ binding results in the phosphorylation of TBRI by TBRII. TBRI then activates a signaling cascade, which may include a series of transcription factors known as Smads (8Zhu H.J. Burgess A.W. Mol. Cell. Biol. Res. Commun. 2001; 4: 321-330Crossref PubMed Scopus (95) Google Scholar). Other TGFβ family members such as the activins and bone morphogenic proteins (BMPs) also signal through a type I receptor by binding to specific type II receptors for activin (ActRII and ActRIIB) and BMP (BMPRII) (9Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3975) Google Scholar). To date, seven type I receptors have been identified and designated activin receptor-like kinases (ALKs) 1–7. The ligand specificity of these ALKs has been determined by their ability to bind to a given ligand and to activate downstream signals in the presence of a specific type II receptor subtype. ALK1 and ALK5 are activated by TGFβ via TBRII (9Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3975) Google Scholar, 10ten Dijke P. Yamashita H. Ichijo H. Franzen P. Laiho M. Miyazono K. Heldin C.H. Science. 1994; 264: 101-104Crossref PubMed Scopus (509) Google Scholar). ALK5 in association with TBRII specifically stimulates the plasminogen activator inhibitor (PAI-1) promoter. ALK5 mediates growth arrest in mink lung epithelial cells following the formation of the ALK5/TRBII complex and the phosphorylation of ALK5 (11Wrana J.L. Attisano L. Wieser R. Ventura F. Massague J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2098) Google Scholar). ALK2 interacts with TBRII as well as ActRII and BMPRII type II receptors (12Attisano L. Carcamo J. Ventura F. Weis F.M. Massague J. Wrana J.L. Cell. 1993; 75: 671-680Abstract Full Text PDF PubMed Scopus (601) Google Scholar). ALK2 does not mediate TGFβ signaling in mink lung epithelial cells but has been implicated in the TGFβ-stimulated epithelial-mesenchymal transformation in the mammary gland of the mouse (13Miettinen P.J. Ebner R. Lopez A.R. Derynck R. J. Cell Biol. 1994; 127: 2021-2036Crossref PubMed Scopus (790) Google Scholar). The regulation of TGFβ receptor signaling by selective interactions with different type I receptors is an intriguing mechanism that might help explain the pleiotropic effects of TGFβ. Here we demonstrate that TGFβ mediates opposing effects on Gαi2 expression and the response of the heart to parasympathetic stimulation at different stages of chick heart development and that these pleiotropic effects are due to differential activation of ALK2 and ALK5 by TGFβ. Embryonic chick atrial myocyte cultures were prepared by a modification of the method of DeHaan (14DeHaan R.L. Circulation. 1967; 35: 821-833Crossref PubMed Scopus (33) Google Scholar) as described previously (15Gadbut A.P. Toupin D.K. Kilbourne E.J. Galper J.B. J. Biol. Chem. 1994; 269: 30707-30712Abstract Full Text PDF PubMed Google Scholar). Eggs were staged according to the method of Hamberger and Hamilton (16Hamberger U. Hamilton H. J. Morphol. 1945; 88: 49-92Crossref Scopus (9975) Google Scholar). The embryos 5 dio corresponded to stage 27, and the 14-day embryo corresponded to stage 40. A Gαi2 RNase protection probe was generated from a PstI fragment derived from the chick Gαi2 cDNA subcloned into pBluescript and linearized with BamHI (17Kilbourne E.J. Galper J.B. Gene (Amst.). 1994; 150: 341-344Crossref PubMed Scopus (6) Google Scholar). Using T7 RNA polymerase (Roche Molecular Biochemicals) in the presence of [32P]UTP (800 Ci/mmol, PerkinElmer Life Sciences), this template gave a 307-nucleotide antisense riboprobe. The glyceraldehyde phosphate dehydrogenase (GAPDH) RNase protection probe, used as a control, was generated from a cDNA template (gift of R. Runyan), which was linearized with HindIII. Using T3 RNA polymerase, this template gave a 250-nucleotide antisense riboprobe. Probes were purified by PAGE on a 6% gel, and the major band corresponding to the predicted molecular weight for the riboprobe was excised and eluted overnight. Total RNA was isolated from cultures of embryonic chick atrial cells 14 dio using guanidinium CsC12 centrifugation as described (18Sambrook J. Fritsh E.F. Maniatis T. Press C.S.H.L. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar). RNase protection was carried out as described previously (15Gadbut A.P. Toupin D.K. Kilbourne E.J. Galper J.B. J. Biol. Chem. 1994; 269: 30707-30712Abstract Full Text PDF PubMed Google Scholar). Riboprobes were hybridized to 15 μg of total RNA prepared from cells treated with either vehicle or 5 ng/ml TGFβ1. The samples were treated with RNase and analyzed by PAGE on 6% gels containing urea followed by autoradiography. Radiographic exposure was 6 h for Gαi2 and 2 h for GAPDH. The relative intensity of the bands was determined by densitometry scanning using NIH Image Pro. Embryonic chick atrial cells from hearts of embryos 5 dio cultured on coverslips at 5 × 105cells/cm2 were treated either with vehicle (4 mm HCl and 0.5 mg/ml bovine serum albumin) or with 5 ng/ml TGFβ1 and placed in a perfusion chamber as described (15Gadbut A.P. Toupin D.K. Kilbourne E.J. Galper J.B. J. Biol. Chem. 1994; 269: 30707-30712Abstract Full Text PDF PubMed Google Scholar), on the stage of a Zeiss inverted phase contrast microscope enclosed in a Lucite box maintained at 37 °C. The inlet side of the chamber was connected via polyethylene tubing to two syringe pumps allowing the cells to be sequentially perfused by two different solutions. Perfusion at 0.98 ml/min did not disturb cell adhesion to the coverslip. Cells were perfused with an HEPES-buffered salt solution containing 1% fetal calf serum, 11 mmglucose, 0.6 mm HEPES, 0.6 mmCaCl2, 4.0 mm KCl, 140 mm NaCl, and 5 mm MgCl2. In this study, each cell served as its own control with the spontaneous beat rate determined before and after exposure to 0.1 mm carbamylcholine. Beating was determined by monitoring the movement of the border of a single cell with a video-motion detector and recording the output with a physiologic recorder (Hewlett-Packard Co., Palo Alto, CA) as described (3Haigh L.S. Leatherman G.F. O'Hara D.S. Smith T.W. Galper J.B. J. Biol. Chem. 1988; 263: 15608-15618Abstract Full Text PDF PubMed Google Scholar). Polyclonal (rabbit) antiserum raised to the carboxyl-terminal decapeptide from rat Gαi2 was a gift of David Manning. TBRII, ALK2, and ALK5 antibodies were prepared as described (8Zhu H.J. Burgess A.W. Mol. Cell. Biol. Res. Commun. 2001; 4: 321-330Crossref PubMed Scopus (95) Google Scholar, 19Lai Y.T. Beason K.B. Brames G.P. Desgrosellier J.S. Cleggett M.C. Shaw M.V. Brown C.B. Barnett J.V. Dev. Biol. 2000; 222: 1-11Crossref PubMed Scopus (56) Google Scholar). Cultured chick atrial cells 5 and 14 dio were grown for 3 days in fetal calf serum, homogenates were prepared and Western blot analysis was carried out as described (15Gadbut A.P. Toupin D.K. Kilbourne E.J. Galper J.B. J. Biol. Chem. 1994; 269: 30707-30712Abstract Full Text PDF PubMed Google Scholar). Equal amounts of protein were loaded as determined by a DC protein assay (Bio-Rad). Equal loading was determined by Coomassie staining. Embryonic chick atrial cells 5 and 14 dio were cultured in medium supplemented with fetal calf serum. On the second culture day, 1 μg of Gαi2-Luc consisting of 1.5 kb of the 5′ upstream region of the chick Gαi2 promoter ligated to a luciferase reporter (7Ward S. Gadbut A. Tang D. Papageorge A. Wu L. Li G. Barnett J. Galper J. J. Mol. Cell. Cardiol. 2002; 34: 1217-1226Abstract Full Text PDF PubMed Google Scholar) and 0.2 μg of a human placental alkaline phosphatase under the control of an SV40 promoter (pSV2Apap, a gift of L. Ercolani) were transfected into heart cells cultured on 35-mm plates by the use of FuGENE 6 transfection reagent (Roche Molecular Biochemicals) as recommended by the manufacturer. Total DNA was maintained at 2.1 μg by addition of pBluescript (pBS) DNA. At 16 h prior to harvesting, cells were incubated as indicated. At 72 h after transfection, cells were washed in phosphate-buffered saline and solubilized in lysis buffer at 425 μl/plate (24 mm glycyl-glycine, 15 mm MgSO4, 4 mm EGTA, 1% Triton X-100, and 1 mm dithiothreitol). The extract was sonicated three times for 10 s and then centrifuged at 13,000 ×g for 3 min at 4 °C, and the supernatant was assayed for luciferase and alkaline phosphatase activity as described (20Holtzman E.J. Soper B.W. Stow J.L. Ausiello D.A. Ercolani L. J. Biol. Chem. 1991; 266: 1763-1771Abstract Full Text PDF PubMed Google Scholar). In other experiments, cells were transfected with the pVent promoter luciferase reporter construct, Xvent2-luc, containing ∼250 bp of Xvent2 promoter sequences, which was a gift from Christof Niehrs, or p3TP-lux containing the putative TGFβ-responsive region of the human PAI-1 (plasminogen activator inhibitor) promoter, which was a gift of Joan Massague. In some experiments, cells were co-transfected with pVent-Luc, p3TP-lux, or Gαi2-Luc and constitutively active TBRIs (constitutively active chicken ALK2 (chALK2+) and chicken ALK5 (chALK5+)). Constitutively active TBRIs were generated as described (21Charng M.J. Zhang D. Kinnunen P. Schneider M.D. J. Biol. Chem. 1998; 273: 9365-9368Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 22Suzuki A. Kaneko E. Ueno N. Hemmati-Brivanlou A. Dev. Biol. 1997; 189: 112-122Crossref PubMed Scopus (85) Google Scholar). Briefly, chALK2 and chALK5 (19Lai Y.T. Beason K.B. Brames G.P. Desgrosellier J.S. Cleggett M.C. Shaw M.V. Brown C.B. Barnett J.V. Dev. Biol. 2000; 222: 1-11Crossref PubMed Scopus (56) Google Scholar) cDNAs were altered in the GS box (chALK2 Q202D; chALK5T204D) (23Wieser R. Wrana J.L. Massague J. EMBO J. 1995; 14: 2199-2208Crossref PubMed Scopus (596) Google Scholar,24Macias-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 (399) Google Scholar). The specificity of these constitutively active mutants of chALK2 and chALK5 was determined by cotransfection of chick atrial cells with p3TP-lux, which is specifically activated by chALK5 or cotransfection with pVent, which is specifically activated by chALK2. chALK5+-stimulated p3TP-lux 2.8 ± 0.4-fold (± S.E.,n = 6, p < 0.01) while having no significant effect on pVent promoter activity. chALK2+stimulated pVent promoter activity 2.0 ± 0.3-fold (± S.E.,n = 6, p < 0.01) while having only a minimal effect on p3TP-lux promoter activity. Statistical analysis was by Student'st test. During embryonic development, the negative chronotropic response of the chick heart to muscarinic stimulation developed between 2 and 7 dio (25Galper J.B. Klein W. Catterall W.A. J. Biol. Chem. 1977; 252: 8692-8699Abstract Full Text PDF PubMed Google Scholar). To determine the effect of TGFβ on the development of the parasympathetic response, embryonic chick atrial cells from 5 dio hearts were incubated for 16 h with either 5 ng/ml TGFβ1 or vehicle, and beat rate was determined in the presence of carbamylcholine. In the absence of TGFβ1, 0.1 mm carbamylcholine decreased beat rate by 30 ± 1% (± S.E., n = 21, p < 0.001, Fig.1). However, after incubation with TGFβ1, carbamylcholine decreased beat rate by 76 ± 1% (± S.E., n = 21, p < 0.01, TableI). These effects on beat rate were reversible within 5 min after reperfusion of cells with carbamylcholine-free medium. Thus TGFβ1 increases the chronotropic response to carbamylcholine by more than 2.5-fold in atrial myocytes from hearts 5 dio. This result is opposite to the effect of TGFβ1 in cells from atria of hearts 14 dio in which we demonstrated that TGFβ1 decreased the chronotropic response to carbamylcholine by more than 5-fold (Table I) (7Ward S. Gadbut A. Tang D. Papageorge A. Wu L. Li G. Barnett J. Galper J. J. Mol. Cell. Cardiol. 2002; 34: 1217-1226Abstract Full Text PDF PubMed Google Scholar).Table IDevelopmental changes in TGFβ1 regulation of the parasympathetic response in embryonic chick atrial cells5 dio1-aData derived from Fig. 1.14 dio1-bData derived from Ref.7.VehicleTGFβ1VehicleTGFβ131.1 ± 1.4%75.4 ± 1.5%94.8 ± 2.1%18.3 ± 1.8%Percent inhibition of beat rate by carbamylcholine in atrial cells 5 and 14 dio treated with vehicle or 5 ng/ml TGFβ1. Data are the means ± S.E.1-a Data derived from Fig. 1.1-b Data derived from Ref.7Ward S. Gadbut A. Tang D. Papageorge A. Wu L. Li G. Barnett J. Galper J. J. Mol. Cell. Cardiol. 2002; 34: 1217-1226Abstract Full Text PDF PubMed Google Scholar. Open table in a new tab Percent inhibition of beat rate by carbamylcholine in atrial cells 5 and 14 dio treated with vehicle or 5 ng/ml TGFβ1. Data are the means ± S.E. The expression of Gαi2 increases in parallel with the development of parasympathetic responsiveness in the embryonic chick heart (2Liang B.T. Hellmich M.R. Neer E.J. Galper J.B. J. Biol. Chem. 1986; 261: 9011-9021Abstract Full Text PDF PubMed Google Scholar). Hence the opposing effects of TGFβ on the negative chronotropic response of the heart to muscarinic stimulation might be associated with alterations in Gαi2 expression. To test this hypothesis, we determined whether TGFβ1 altered Gαi2 expression in atrial myocytes cultured from hearts between 5 and 14 dio. Incubation of cells from hearts 5 dio with TGFβ1 increased the level of Gαi2 mRNA, whereas in cells derived from hearts 7, 9, and 14 dio, TGFβ1 decreased levels of Gαi2 mRNA (Fig.2 A). The mean of five experiments similar to that in Fig. 2 A demonstrated that when compared with vehicle, TGFβ1 stimulated Gαi2 mRNA 2.10 ± 0.16-fold (± S.E.,n = 5, p < 0.002) at day 5 in ovo while decreasing Gαi2 mRNA at days 7, 9, and 14 in ovo by 0.44 ± 0.06-fold (± S.E., n = 4,p < 0.003); 0.52 ± 0.06-fold (± S.E.,n = 5, p < 0.002); and 0.60 ± 0.02-fold (± S.E., n = 5, p < 0.001) respectively. Similarly, TGFβ1 stimulated expression of Gαi2 protein in cells from hearts 5 dio (Fig.2 B) by 2.30 ± 0.10-fold (± S.E., n = 3, p < 0.002, Fig. 2 C) but decreased Gαi2 protein in extracts of cells from hearts 14dio (Fig. 2 B) by 0.42 ± 0.04-fold (± S.E., n = 4, p < 0.001, Fig.2 C). Finally, TGFβ1 stimulated Gαi2 promoter activity in chick atrial cells from hearts 5 dio by 2.40 ± 0.40-fold (± S.E., n = 5, Fig.3 A) and decreased Gαi2 promoter activity by 54 ± 6% (± S.E.,n = 4) in atrial cells from hearts 14 dio (Fig.3 B). These data demonstrate that the opposing effects of TGFβ on the negative chronotropic response to muscarinic stimulation are accompanied by similar alterations in Gαi2expression.Figure 3Effect of TGFβ1 on Gαi2 promoter activity. Chick atrial cells 5 (A) and 14 (B) dio cultured in medium supplemented with fetal calf serum were co-transfected with a 2-kb fragment from the 5′-flanking region of Gαi2ligated to a promoterless luciferase reporter (Gαi2-Luc) and a human placental alkaline phosphatase (PAP). Following transfection, cells were incubated for 16 h with either 5 ng/ml TGFβ or an equal volume of vehicle. Cells were harvested, and luciferase activity and PAP activity were determined as described previously. Data are normalized to the ratio of luciferase to PAP activity in cells cultured with vehicle adjusted to 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These opposing effects in the response of chick atrial cells to TGFβ might reflect changes in the expression of TGFβ receptors involved in signaling at different stages of cardiac development. Western blot analysis demonstrated that embryonic chick atrial cells expressed TBRII, ALK2, and ALK5 (Fig.4, A and C). ALK2 and ALK5 have been reported to mediate distinct responses to TGFβ signaling (9Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3975) Google Scholar, 10ten Dijke P. Yamashita H. Ichijo H. Franzen P. Laiho M. Miyazono K. Heldin C.H. Science. 1994; 264: 101-104Crossref PubMed Scopus (509) Google Scholar, 11Wrana J.L. Attisano L. Wieser R. Ventura F. Massague J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2098) Google Scholar, 13Miettinen P.J. Ebner R. Lopez A.R. Derynck R. J. Cell Biol. 1994; 127: 2021-2036Crossref PubMed Scopus (790) Google Scholar). For this reason, we studied developmental changes in these two TGFβ receptors. chALK2 and chALK5 were initially expressed at low levels at day 5 in ovo but increased markedly between days 5 and 14 in ovo (Fig.4 A). Comparison of the fold increase in ALK2 and ALK5 expression between 5 and 14 dio demonstrated that chALK2 increased 2.30 ± 0.20-fold (± S.E., n = 4,p < 0.01) more than chALK5 (Fig. 4 B). TBRII levels increased 4.40 ± 0.20-fold (± S.E., n = 3) between 5 and 14 dio (Fig. 4, C and D). Thus each receptor increased between 5 and 14 dio with the largest increase in chALK2. To determine whether TGFβ signaling might preferentially activate chALK2 or chALK5 at different stages of cardiac development, we compared the effect of TGFβ1 on p3TP-lux and pVent reporter activity in atrial cells from hearts 5 and 14dio. chALK5 specifically activates the p3TP-lux reporter (12Attisano L. Carcamo J. Ventura F. Weis F.M. Massague J. Wrana J.L. Cell. 1993; 75: 671-680Abstract Full Text PDF PubMed Scopus (601) Google Scholar). pVent is known to be activated by BMP, not by TGFβ, and is one of the best known reporters of ALK2 activation (26Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massague J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (558) Google Scholar). To determine whether ALK2 might be mediating a TGFβ response, in our system, pVent was used as a reporter of ALK2 activation. In atrial cells from hearts 5 dio, 5 ng/ml TGFβ1 stimulated p3TP-lux activity 5.20 ± 0.30-fold (± S.E., n = 5,p < 0.002, Fig.5 A), whereas in atrial cells from hearts 14 dio, TGFβ1 stimulated p3TP-lux by 2.5 ± .01-fold (± S.E., n = 6, p< 0.003, Fig. 5 B). In atrial cells from hearts 5 dio, TGFβ1 had no effect on pVent reporter activity (Fig.5 C). However, in atrial cells 14 dio, we observed an unexpected 2.2 ± 0.2-fold (± S.E., n = 7,p < 003, Fig. 5 D) increase in pVent reporter activity in response to TGFβ1. These data demonstrate that in chick atrial cells, pVent is activated by TGFβ and that this activation is specific for cells 14 dio. These data also suggest that in chick atrial cells 5 dio TGFβ signals via chALK5 and not chALK2. The ability of chALK2 to mediate a TGFβ response at 14 dio but not at 5 dio suggests a potential mechanism for the opposing effects of TGFβ at these ages. Specifically, chALK2 might act to decrease Gαi2 expression, whereas chALK5 might act to increase Gαi2. To test this hypothesis, atrial cells from hearts 14 dio were transfected with chALK5+. chALK5+ stimulated Gαi2promoter activity 2.5 ± 0.20-fold (± S.E., n = 4) (Fig. 6 A, column 3). Furthermore, chALK5+ not only reversed TGFβ1 inhibition of Gαi2 promoter activity but also stimulated Gαi2 promoter activity 2.4 ± 0.1-fold (± S.E., n = 4) above basal (Fig.6 A, column 4). In contrast, transfection of cells from chick atria 14 dio with chALK2+ not only mimicked the effect of TGFβ1 but completely inhibited Gαi2 promoter activity (Fig. 6 B, column 3). If chALK2 mediates the inhibition of the Gαi2 promoter by TGFβ signaling, then overexpression of chALK2 in atrial cells from chicks 5 dio should inhibit TGFβ-stimulated Gαi2promoter activity. In experiments summarized in Fig.7, TGFβ1 stimulated Gαi2 promoter activity 2.10 ± 0.10-fold above basal (± S.E., n = 4). Cotransfection of these cells with chALK2+ followed by incubation with TGFβ1 not only reversed TGFβ stimulation of Gαi2 promoter activity but also decreased Gαi2 promoter activity by 9-fold to 0.40 ± 0.06 (± S.E., n = 4)-fold below basal. As expected, chALK5+ alone stimulated Gαi2 promoter activity. These data demonstrate that chALK2 inhibits Gαi2 promoter activity, whereas chALK5 stimulates Gαi2 promoter activity, and that these effects are independent of the developmental stage of the atrial myocytes. The data presented here provide novel insight into TGFβ signaling and the regulation of parasympathetic responsiveness in the heart. TGFβ stimulates the negative chronotropic response of chick atrial cells 5 dio to carbamylcholine, whereas it decreases the inhibition of beat rate by carbamylcholine in atrial cells 14 dio (7Ward S. Gadbut A. Tang D. Papageorge A. Wu L. Li G. Barnett J. Galper J. J. Mol. Cell. Cardiol. 2002; 34: 1217-1226Abstract Full Text PDF PubMed Google Scholar). These effects of TGFβ correlate with alterations in Gαi2 expression. At 5 dio, TGFβ stimulates Gαi2 expression, and at 14 dio, TGFβ inhibits Gαi2 expression. Examination of two TBRIs reported to play a role in TGFβ signaling reveals that chALK5 increases Gαi2 expression, whereas chALK2 decreases Gαi2 expression independent of the embryonic age of the cells. Further, TGFβ stimulates pVent, a reporter of ALK2 activation, in 14 dio, but not in 5 dio, atrial cells. These data, taken together with the finding that chALK2 expression increases markedly between 5 and 14 dio, suggests that at 5 dio, TGFβ activates only chALK5, but at 14 dio, TGFβ activates both chALK5 and chALK2. These findings offer a potential mechanism to explain the change in TGFβ regulation of Gαi2 expression and parasympathetic response during cardiac development. The induction of a parasympathetic response is a critical step in the physiological development of the mammalian heart. The regulation of the parasympathetic responsiveness of the heart not only controls the rate and force of contraction but also may play a role in the development of cardiac arrhythmias (27Schwartz P.J. La Rovere M.T. Vanoli E. Circulation. 1992; 85: I77-I91PubMed Google Scholar, 28Eschenhagen T. Mende U. Diederich M. Hertle B. Memmesheimer C. Pohl A. Schmitz W. Scholz H. Steinfath M. Bohm M. Michel M.C. Brodde O.E. Raap A. Circulation. 1996; 93: 763-771Crossref PubMed Scopus (35) Google Scholar). We have demonstrated previously that during embryonic development of the chick heart, the negative chronotropic response to carbamylcholine increased markedly between 5 and 7 dio, reaching a plateau at 7 dio (25Galper J.B. Klein W. Catterall W.A. J. Biol. Chem. 1977; 252: 8692-8699Abstract Full Text PDF PubMed Google Scholar). The development of the parasympathetic response in the embryonic chick heart was associated with an increase in Gαi2 expression (2Liang B.T. Hellmich M.R. Neer E.J. Galper J.B. J. Biol. Chem. 1986; 261: 9011-9021Abstract Full Text PDF PubMed Google Scholar). Regulation of Gαi2 expression has been associated with the control of parasympathetic responsiveness in the adult heart. A recent study demonstrated that overexpression of Gαi2 in the porcine atrioventricular node resulted in a decrease in atrioventricular conduction and a decreased response to sympathetic stimulation consistent with an increase in parasympathetic tone (5Donahue J.K. Heldman A.W. Fraser H. McDonald A.D. Miller J.M. Rade J.J. Eschenhagen T. Marban E. Nat. Med. 2000; 6: 1395-1398Crossref PubMed Scopus (160) Google Scholar). Here we demonstrate a striking parallel between developmental changes in TGFβ regulation of the response of the heart to parasympathetic stimulation and TGFβ regulation of Gαi2 expression. These data emphasize the importance of the regulation of Gαi2 expression on parasympathetic responsiveness and cardiac function. Our data support the notion that the transition of TGFβ signaling in atrial cells from a stimulatory effect on Gαi2 expression and parasympathetic response to an inhibitory effect during embryonic development reflects differential activation of the TGFβ type I receptors, chALK2 and chALK5. Complexes of ALK5 and TBRII bind TGFβ1 to mediate TGFβ effects such as growth arrest in mink lung epithelial cells (11Wrana J.L. Attisano L. Wieser R. Ventura F. Massague J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2098) Google Scholar). Although ALK2 binds TGFβ when co-expressed with TBRII, it does not mediate growth arrest in mink lung epithelial cells. A role for ALK2 has been described during the TGFβ-dependent epithelial-mesenchymal transformation of mouse mammary epithelial cells (13Miettinen P.J. Ebner R. Lopez A.R. Derynck R. J. Cell Biol. 1994; 127: 2021-2036Crossref PubMed Scopus (790) Google Scholar). A similar TGFβ-stimulated epithelial-mesenchymal transformation occurs in the atrioventricular cushion during valvulogenesis. Studies using an in vitroculture system demonstrated that anti-chALK2 antisera blocked transformation, whereas anti-chALK5 antisera was without effect (19Lai Y.T. Beason K.B. Brames G.P. Desgrosellier J.S. Cleggett M.C. Shaw M.V. Brown C.B. Barnett J.V. Dev. Biol. 2000; 222: 1-11Crossref PubMed Scopus (56) Google Scholar). The finding that specific TGFβ effects may be attributed to ALK2 or ALK5 suggested that the specificity of the downstream response to TGFβ signaling is dependent on the identity of the TBRI activated in a given cell type. In support of this conclusion, chALK2 and chALK5 were shown to exert opposing effects on Gαi2 promoter activity. Constitutively active chALK2 inhibited Gαi2promoter activity, and constitutively active chALK5 stimulated Gαi2 promoter activity independent of the embryonic age of the cell in which they were expressed. Hence differential activation of chALK2 and chALK5 by TGFβ at 5 and 14 dio might result in opposing effects of TGFβ on Gαi2 expression during cardiac development. To test this hypothesis, we compared the effect of TGFβ1 on pVent and p3TP-lux reporter activity in cells from atria 5 and 14 dio. The pVent reporter is activated by BMP signaling via ALK2 (22Suzuki A. Kaneko E. Ueno N. Hemmati-Brivanlou A. Dev. Biol. 1997; 189: 112-122Crossref PubMed Scopus (85) Google Scholar, 26Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massague J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (558) Google Scholar, 29Schuler-Metz A. Knochel S. Kaufmann E. Knochel W. J. Biol. Chem. 2000; 275: 34365-34374Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), whereas the p3TP-lux reporter is activated by ALK5 signaling (11Wrana J.L. Attisano L. Wieser R. Ventura F. Massague J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2098) Google Scholar). TGFβ1 stimulated p3TP-lux reporter activity in atrial cells from hearts 5 dio but had no effect on pVent reporter activity in these cells. Furthermore, although TGFβ stimulated both p3TP-lux and the pVent reporter in cells 14 dio, the stimulation of pVent was significantly higher than p3TP-lux in these cells. These data support the conclusion that TGFβ signaling at 5 dio occurs via chALK5 and that signaling at 14 dio occurs via both chALK2 and chALK5, with chALK2 predominating. Although it is not possible to directly compare the level of expression of chALK2 and chALK5 at 5 or 14 dio, we noted a larger increase in ALK2 expression than ALK5 expression, consistent with the conclusion that the increase in ALK2 signaling at 14 dio was due at least in part to an increase in expression levels. Taken together with the data which demonstrate that ALK5 stimulates Gαi2 promoter activity and ALK2 inhibits Gαi2 promoter activity, the finding of differential activation of ALK2 and ALK5 would account for the opposing effects of TGFβ on Gαi2 expression at 5 and 14 dio. The unexpected observation that TGFβ stimulates pVent expression in chick atrial cells 14 dio is the first report of activation of a BMP-like signal by TGFβ. TGFβ signaling via ALK5 has been shown to involve Smads 2/3 (30Kretzschmar M. Massague J. Curr. Opin. Genet. Dev. 1998; 8: 103-111Crossref PubMed Scopus (429) Google Scholar). We demonstrated that constitutively active chALK5 did not stimulate pVent promoter activity, which indicates that Smads 2/3 cannot activate pVent in these cells. Furthermore, studies of pVent have demonstrated stimulation by the BMP-specific Smads 1/5/8 (26Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massague J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (558) Google Scholar). This would suggest that TGFβ stimulation of pVent might be mediated by a BMP-specific pathway in these cells. The significance of these developmental changes in TGFβ signaling may be related to a dual role of TGFβ signaling in cardiac physiology and development. In an in vitro model for parasympathetic innervation of the heart, we have demonstrated that induction of a negative chronotropic response to carbamylcholine and the expression of Gαi2 were dependent on the release of a soluble factor (6Barnett J.V. Taniuchi M. Yang M.B. Galper J.B. Biochem. J. 1993; 292: 395-399Crossref PubMed Scopus (6) Google Scholar) whose effect was inhibited by a neutralizing antibody to TGFβ.2These findings implicate TGFβ in the development of the parasympathetic response. Studies in explanted, intact chick heart have previously demonstrated a marked increase in the response of the heart to parasympathetic stimulation between days 2 and 7 in ovo(31Galper J.B. Barnett J.V. Kilbourne E. Gadbut A.P. Speralakis N. Physiology and Pathophysiology of the Heart. Kluwer Academic Publishers, Boston1995: 431-455Google Scholar). Here TGFβ stimulates a significant increase in both Gαi2 expression and parasympathetic response in atrial cells 5 dio. These data support the conclusion that TGFβ plays a role in the development of a parasympathetic response in the heart. At 14 dio, functional parasympathetic innervation of the chick heart is complete (31Galper J.B. Barnett J.V. Kilbourne E. Gadbut A.P. Speralakis N. Physiology and Pathophysiology of the Heart. Kluwer Academic Publishers, Boston1995: 431-455Google Scholar). The significance of TGFβ inhibition of Gαi2 expression and parasympathetic responsiveness at this developmental stage is unclear. However, TGFβ has been shown to play a role in a number of processes important to cardiac function such as angiogenesis, cardiac hypertrophy, inflammation, and the response of the heart to myocardial infarction (32Hanatani A. Yoshiyama M. Kim S. Omura T. Ikuno Y. Takeuchi K. Iwao H. Yoshikawa J. Jpn. Heart J. 1998; 39: 375-388Crossref PubMed Scopus (10) Google Scholar, 33Lijnen P.J. Petrov V.V. Fagard R.H. Mol. Genet. Metab. 2000; 71: 418-435Crossref PubMed Scopus (394) Google Scholar). The relationship between TGFβ inhibition of parasympathetic responsiveness and Gαi2 expression to these processes remains to be determined. These data suggest that TGFβ is an important regulator of parasympathetic responsiveness during cardiac development and may regulate the parasympathetic response at least in part by modulating Gαi2 expression. Further, we suggest that TGFβ signaling may involve the activation of both ALK5 and ALK2 in atrial cells and that the relative contribution of each of these receptors determines the level of Gαi2 expression and parasympathetic responsiveness. Our observations suggesting differential activation of two different type I receptors are an attractive mechanism to explain the pleiotropic effects of TGFβ. We thank R. Runyan for GAPDH cDNA and L. Ercolani for the pSV2-Apap construct.