Nodal and ALK7 Inhibit Proliferation and Induce Apoptosis in Human Trophoblast Cells
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m400641200
ISSN1083-351X
AutoresSadia Munir, Guoxiong Xu, Yaojiong Wu, Burton B. Yang, Peeyush K. Lala, Chun Peng,
Tópico(s)Kruppel-like factors research
ResumoNodal, a member of the transforming growth factor-β superfamily, is known to play critical roles in early vertebrate development, but its functions in extraembryonic tissues are unclear. ALK7 is a type I receptor for Nodal. Recently, we demonstrated that Nodal mRNA and several ALK7 transcripts are expressed in human placenta throughout pregnancy (Roberts, H. J., Hu, S., Qiu, Q., Leung, P. C. K., Cannigia, I., Gruslin, A., Tsang, B., and Peng, C. (2003) Biol. Reprod. 68, 1719–1726). In this study, we determined the role of Nodal and ALK7 in trophoblast cell proliferation and apoptosis. Overexpression of Nodal in normal trophoblast cells (HTR8/SVneo) and several choriocarcinoma cell lines resulted in a significant decrease in the number of metabolically active cells. The effect of Nodal could be mimicked by constitutively active ALK7 (ALK7-ca), but was blocked by kinase-deficient ALK7. The growth inhibitory effect of Nodal was also blocked by dominant-negative Smad2/3. Overexpression of Nodal and ALK7-ca induced apoptosis in trophoblast cells as determined by Hoechst staining, flow cytometry, and caspase-3 Western blotting. In addition, Nodal and ALK7-ca decreased the number of proliferating cells as measured by bromodeoxyuridine assays. Furthermore, overexpression of Nodal or ALK7-ca increased p27 expression, but reduced the levels of Cdk2 and cyclin D1. Taken together, this study demonstrates for the first time that Nodal, acting through ALK7 and Smad2/3, inhibits proliferation and induces apoptosis in human trophoblast cells. Our findings also suggest that the Nodal-ALK7 pathway inhibits cell proliferation by inducing G1 cell cycle arrest and that this effect is mediated in part by the p27-cyclin E/Cdk2 pathway. Nodal, a member of the transforming growth factor-β superfamily, is known to play critical roles in early vertebrate development, but its functions in extraembryonic tissues are unclear. ALK7 is a type I receptor for Nodal. Recently, we demonstrated that Nodal mRNA and several ALK7 transcripts are expressed in human placenta throughout pregnancy (Roberts, H. J., Hu, S., Qiu, Q., Leung, P. C. K., Cannigia, I., Gruslin, A., Tsang, B., and Peng, C. (2003) Biol. Reprod. 68, 1719–1726). In this study, we determined the role of Nodal and ALK7 in trophoblast cell proliferation and apoptosis. Overexpression of Nodal in normal trophoblast cells (HTR8/SVneo) and several choriocarcinoma cell lines resulted in a significant decrease in the number of metabolically active cells. The effect of Nodal could be mimicked by constitutively active ALK7 (ALK7-ca), but was blocked by kinase-deficient ALK7. The growth inhibitory effect of Nodal was also blocked by dominant-negative Smad2/3. Overexpression of Nodal and ALK7-ca induced apoptosis in trophoblast cells as determined by Hoechst staining, flow cytometry, and caspase-3 Western blotting. In addition, Nodal and ALK7-ca decreased the number of proliferating cells as measured by bromodeoxyuridine assays. Furthermore, overexpression of Nodal or ALK7-ca increased p27 expression, but reduced the levels of Cdk2 and cyclin D1. Taken together, this study demonstrates for the first time that Nodal, acting through ALK7 and Smad2/3, inhibits proliferation and induces apoptosis in human trophoblast cells. Our findings also suggest that the Nodal-ALK7 pathway inhibits cell proliferation by inducing G1 cell cycle arrest and that this effect is mediated in part by the p27-cyclin E/Cdk2 pathway. Members of the transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; RT, reverse transcription; ALK7-ca, constitutively active ALK7; ALK7-kd, kinase-deficient ALK7; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ALK7-wt, wild-type ALK7; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. superfamily regulate a variety of cellular functions and play critical roles in many developmental and physiological processes (1Massagué J. Chen Y.-G. Genes Dev. 2000; 6: 627-644Google Scholar, 2Zimmerman C.M. Padgett R.W. Gene (Amst.). 2000; 249: 17-30Google Scholar, 3Chang H. Brown C.W. Matzuk M.M. Endocr. Rev. 2002; 23: 787-823Google Scholar), including placental development (4Graham C.H. Lala P.K. J. Cell. Physiol. 1991; 148: 228-234Google Scholar, 5Graham C.H. Lysiak J.J. McCrae K.R. Lala P.K. Biol. Reprod. 1992; 46: 561-572Google Scholar, 6Peng C. J. Obstet. Gynaecol. Can. 2003; 25: 834-845Google Scholar). This family consists of a large group of peptide growth factors/hormones, including TGF-β, activins, bone morphogenetic proteins, growth and differentiation factors, as well as Nodal and its related proteins (1Massagué J. Chen Y.-G. Genes Dev. 2000; 6: 627-644Google Scholar, 2Zimmerman C.M. Padgett R.W. Gene (Amst.). 2000; 249: 17-30Google Scholar, 3Chang H. Brown C.W. Matzuk M.M. Endocr. Rev. 2002; 23: 787-823Google Scholar). The role of Nodal has been extensively studied in mouse, Xenopus, and zebrafish, in which Nodal and its related proteins have been found to be critical for mesoderm formation and left-right axis patterning during early development (7Brennan J. Norris D.P. Robertson E.J. Genes Dev. 2002; 16: 2339-2344Google Scholar, 8Eimon P.M. Harland R.M. Development (Camb.). 2002; 129: 3089-3103Google Scholar, 9Nonaka S. Shiratori H. Saijoh Y. Hamada H. Nature. 2002; 418: 96-99Google Scholar). The role of Nodal in adult physiological processes is unknown. Studies in rodents have shown that Nodal may inhibit the differentiation of stem trophoblast cells into giant cells. Homozygous mutations of the nodal gene in mice result in excessive numbers of trophoblast giant cells (10Iannaccone P.M. Chou X. Khokha M. Boucher D. Kuehn M.R. Dev. Dyn. 1992; 194: 198-208Google Scholar). On the other hand, overexpression of Nodal in the rat choriocarcinoma cell line Rcho-1 decreases giant cell numbers (11Ma G.T. Soloveva V. Tzeng S.-J. Lowe L.A. Pfendler K.C. Iannacone P.M. Kuehn M.R. Linzer D.H. Dev. Biol. 2001; 236: 124-131Google Scholar). We have recently found that Nodal mRNA is expressed in human placenta from early to late gestation and in JEG-3 choriocarcinoma cells (12Roberts H.J. Hu S. Qiu Q. Leung P.C.K. Cannigia I. Gruslin A. Tsang B. Peng C. Biol. Reprod. 2003; 68: 1719-1726Google Scholar), suggesting that Nodal may regulate human placental development and functions. ALK7 (activin receptor-like kinase-7) is a type I receptor belonging to the serine/threonine kinase receptor family. The serine/threonine kinase receptor family consists of two related groups of receptors termed type I and type II receptors. The type I and type II receptors form functional complexes to mediate signaling by members of the TGF-β superfamily (1Massagué J. Chen Y.-G. Genes Dev. 2000; 6: 627-644Google Scholar, 2Zimmerman C.M. Padgett R.W. Gene (Amst.). 2000; 249: 17-30Google Scholar, 3Chang H. Brown C.W. Matzuk M.M. Endocr. Rev. 2002; 23: 787-823Google Scholar). In mammals, seven type I (known as ALK1–7) and five type II receptors have been characterized. Both type I and type II receptors have similar structural features, including an extracellular ligand-binding domain, a transmembrane domain, and an intracellular serine/threonine kinase domain (1Massagué J. Chen Y.-G. Genes Dev. 2000; 6: 627-644Google Scholar). In addition, type I receptors also have a GS box, which serves as an activation domain for their type II receptor partners (13Wrana J.L. Attisano L. Wieser R. Ventura F. Massagué J. Nature. 1994; 370: 341-347Google Scholar). Upon activation, type I receptors phosphorylate two distinct sets of receptor-regulated Smad proteins, Smad1/5/8 (activated by ALK1/2/3/6) and Smad2/3 (activated by ALK4/5/7) (14Miyazawa K. Shinozaki M. Hara T. Furuya T. Miyazono K. Genes Cells. 2002; 7: 1191-1204Google Scholar, 15Liu F. Front. Biosci. 2003; 8: S1280-S1303Google Scholar, 16Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Google Scholar). In turn, these activated receptor-regulated Smad proteins form complexes with Smad4 and, through their interaction with other transcription factors, regulate target gene expression (1Massagué J. Chen Y.-G. Genes Dev. 2000; 6: 627-644Google Scholar, 2Zimmerman C.M. Padgett R.W. Gene (Amst.). 2000; 249: 17-30Google Scholar, 3Chang H. Brown C.W. Matzuk M.M. Endocr. Rev. 2002; 23: 787-823Google Scholar, 14Miyazawa K. Shinozaki M. Hara T. Furuya T. Miyazono K. Genes Cells. 2002; 7: 1191-1204Google Scholar, 15Liu F. Front. Biosci. 2003; 8: S1280-S1303Google Scholar, 16Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Google Scholar). ALK7 was initially cloned from the rat and found to be predominantly expressed in the central nervous system (17Tsuchida K. Sawchenko P.E. Nishikawa S.I. Vale W.W. Mol. Cell. Neurosci. 1996; 7: 467-478Google Scholar, 18Rydén M. Imamura T. Jörnvall H. Belluardo N. Neveu I. Trupp M. Okadome T. ten Dijke P. Ibanez C.F. J. Biol. Chem. 1996; 271: 30603-30609Google Scholar). Nodal has been recently identified to be the physiological ligand of ALK7 (19Reissmann E. Jornvall H. Blokzijl A. Anderson O. Chang C. Minchiotti G. Persico M.G. Ibanex C. Brivanlou A.H. Genes Dev. 2001; 15: 2010-2022Google Scholar). The kinase domain of ALK7 is closely related to that of ALK4 and ALK5 (12Roberts H.J. Hu S. Qiu Q. Leung P.C.K. Cannigia I. Gruslin A. Tsang B. Peng C. Biol. Reprod. 2003; 68: 1719-1726Google Scholar, 17Tsuchida K. Sawchenko P.E. Nishikawa S.I. Vale W.W. Mol. Cell. Neurosci. 1996; 7: 467-478Google Scholar, 18Rydén M. Imamura T. Jörnvall H. Belluardo N. Neveu I. Trupp M. Okadome T. ten Dijke P. Ibanez C.F. J. Biol. Chem. 1996; 271: 30603-30609Google Scholar, 20Bondestam J. Huotari M.A. Moren A. Ustinov J. Kaivo-Oja N. Kallio J. Horelli-Kuitunen N. Aaltonen J. Fujii M. Moustakas A. ten Dijke P. Otonkoski T. Ritvos O. Cytogenet. Cell Genet. 2001; 95: 157-162Google Scholar). Similar to ALK4 and ALK5, ALK7 also activates the Smad2/3 signaling pathway (20Bondestam J. Huotari M.A. Moren A. Ustinov J. Kaivo-Oja N. Kallio J. Horelli-Kuitunen N. Aaltonen J. Fujii M. Moustakas A. ten Dijke P. Otonkoski T. Ritvos O. Cytogenet. Cell Genet. 2001; 95: 157-162Google Scholar, 21Jörnvall H. Blokzijl A. ten Dijke P. Ibanez C.F. J. Biol. Chem. 2001; 276: 5140-5146Google Scholar, 22Watanabe R. Yamada Y. Ihara Y. Someya Y. Kubota A. Kagimoto S. Kuroe A. Iwakura T. Shen Z.P. Inada A. Adachi T. Ban N. Miyawaki K. Sunaga Y. Tsuda K. Seino Y. Biochem. Biophys. Res. Commun. 1999; 254: 707-712Google Scholar). The human ALK7 cDNA has been recently cloned by us (12Roberts H.J. Hu S. Qiu Q. Leung P.C.K. Cannigia I. Gruslin A. Tsang B. Peng C. Biol. Reprod. 2003; 68: 1719-1726Google Scholar) and another group (20Bondestam J. Huotari M.A. Moren A. Ustinov J. Kaivo-Oja N. Kallio J. Horelli-Kuitunen N. Aaltonen J. Fujii M. Moustakas A. ten Dijke P. Otonkoski T. Ritvos O. Cytogenet. Cell Genet. 2001; 95: 157-162Google Scholar). We found that alternative splicing of the ALK7 gene generates four transcripts, designated ALK7-1, -2, -3, and –4 (12Roberts H.J. Hu S. Qiu Q. Leung P.C.K. Cannigia I. Gruslin A. Tsang B. Peng C. Biol. Reprod. 2003; 68: 1719-1726Google Scholar). ALK7-1 encodes full-length ALK7, whereas the other transcripts encode three novel ALK7 isoforms (12Roberts H.J. Hu S. Qiu Q. Leung P.C.K. Cannigia I. Gruslin A. Tsang B. Peng C. Biol. Reprod. 2003; 68: 1719-1726Google Scholar). These isoforms include a truncated receptor missing the ligand-binding domain and two soluble proteins that have the ligand-binding domain, but no transmembrane domain (12Roberts H.J. Hu S. Qiu Q. Leung P.C.K. Cannigia I. Gruslin A. Tsang B. Peng C. Biol. Reprod. 2003; 68: 1719-1726Google Scholar). Unlike rat ALK7, human ALK7 is widely distributed in various tissues, including brain, pancreas, kidney, ovary, and placenta (12Roberts H.J. Hu S. Qiu Q. Leung P.C.K. Cannigia I. Gruslin A. Tsang B. Peng C. Biol. Reprod. 2003; 68: 1719-1726Google Scholar). Using reverse transcription (RT)-PCR and Western blot analysis, we have found that ALK7 and its isoforms are expressed in placenta from early to late gestation (12Roberts H.J. Hu S. Qiu Q. Leung P.C.K. Cannigia I. Gruslin A. Tsang B. Peng C. Biol. Reprod. 2003; 68: 1719-1726Google Scholar), suggesting a role for ALK7 during pregnancy. The role of ALK7 is largely unknown. In the rat neuronal cell line PC12, ALK7 has been found to arrest cell proliferation and to induce morphological differentiation (21Jörnvall H. Blokzijl A. ten Dijke P. Ibanez C.F. J. Biol. Chem. 2001; 276: 5140-5146Google Scholar). Nodal has been shown to interact with two types of receptor complexes. First, Nodal can bind to the activin receptor complex containing activin receptors IIB and IB (ALK4) (19Reissmann E. Jornvall H. Blokzijl A. Anderson O. Chang C. Minchiotti G. Persico M.G. Ibanex C. Brivanlou A.H. Genes Dev. 2001; 15: 2010-2022Google Scholar, 23Yeo C. Whitman M. Mol. Cell. 2001; 7: 949-957Google Scholar, 24Yan Y.T. Liu J.J. Luo Y. Chaosu E. Haltiwanger R.S. Abate-Shen C. Shen M.M. Mol. Cell. Biol. 2002; 22: 4439-4449Google Scholar). Signaling of Nodal through this receptor complex requires Cripto since, in the absence of Cripto, Nodal loses its signaling activity through the activin receptor complex (19Reissmann E. Jornvall H. Blokzijl A. Anderson O. Chang C. Minchiotti G. Persico M.G. Ibanex C. Brivanlou A.H. Genes Dev. 2001; 15: 2010-2022Google Scholar, 23Yeo C. Whitman M. Mol. Cell. 2001; 7: 949-957Google Scholar, 24Yan Y.T. Liu J.J. Luo Y. Chaosu E. Haltiwanger R.S. Abate-Shen C. Shen M.M. Mol. Cell. Biol. 2002; 22: 4439-4449Google Scholar, 25Kumar A. Novoselov V. Celeste A.J. Wolfman N.M. ten Dijke P. Kuehn M.R. J. Biol. Chem. 2001; 276: 656-661Google Scholar). The other receptor complex that mediates Nodal action is activin receptor IIB-ALK7 (19Reissmann E. Jornvall H. Blokzijl A. Anderson O. Chang C. Minchiotti G. Persico M.G. Ibanex C. Brivanlou A.H. Genes Dev. 2001; 15: 2010-2022Google Scholar). Interaction of Nodal with activin receptor IIB-ALK7 is not dependent on Cripto, although overexpression of Cripto enhances the signaling activity of Nodal through ALK7 (19Reissmann E. Jornvall H. Blokzijl A. Anderson O. Chang C. Minchiotti G. Persico M.G. Ibanex C. Brivanlou A.H. Genes Dev. 2001; 15: 2010-2022Google Scholar). Progression of cells through the cell cycle requires sequential activation of cyclin-dependent kinases and their regulatory subunits, cyclins (26Schafer K.A. Vet. Pathol. 1998; 35: 461-478Google Scholar, 27Vidal A. Koff A. Gene (Amst.). 2000; 247: 1-15Google Scholar, 28Morgan D.O. Ann. Rev. Cell Dev. Biol. 1997; 13: 261-291Google Scholar). During progression through G1 phase, expression of D-type cyclins (D1, D2, and D3) increases, and they then associate with and activate Cdk4/6 (27Vidal A. Koff A. Gene (Amst.). 2000; 247: 1-15Google Scholar). On the other hand, an increase in cyclin E/Cdk2 activity is required for transition from G1 to S phase (27Vidal A. Koff A. Gene (Amst.). 2000; 247: 1-15Google Scholar, 28Morgan D.O. Ann. Rev. Cell Dev. Biol. 1997; 13: 261-291Google Scholar). The Cdk4/6 and Cdk2 activities are controlled by two groups of cell cycle inhibitors, the Ink4 and Cip/Kip families, respectively (26Schafer K.A. Vet. Pathol. 1998; 35: 461-478Google Scholar, 27Vidal A. Koff A. Gene (Amst.). 2000; 247: 1-15Google Scholar). The Ink4 family is composed of several proteins, including p15, p16, p18, and p19 whereas the Cip/Kip family has three members, p21, p27, and p57 (28Morgan D.O. Ann. Rev. Cell Dev. Biol. 1997; 13: 261-291Google Scholar). Many of these cell cycle inhibitors, such as p21, p27, and p15, have been shown to be regulated by TGF-β and are involved in TGF-β-induced cell growth arrest (29Donovan J. Slingerland J. Breast Cancer Res. 2000; 2: 116-124Google Scholar). In this study, we investigated the role and signaling of Nodal and ALK7 in normal and tumor trophoblast cell lines. We demonstrated that overexpression of Nodal and constitutively active ALK7 inhibited proliferation and induced apoptosis. The effect of Nodal is mediated by ALK7 and Smad2/3, as the dominant-negative forms of these molecules blocked the effect of Nodal on trophoblast cell growth. Furthermore, we identified several genes that are involved in cell cycle progression as targets of Nodal and ALK7. Cell Lines and Cell Culture—The human choriocarcinoma cell lines JEG-3, JAR, and BeWo were obtained from American Type Culture Collection (Manassas, VA). The immortalized first trimester trophoblast cell line HTR8/SVneo was established from normal human trophoblast cells as described previously (30Graham C.H. Hawley T.S. Hawley R.G. MacDougall J.R. Kerbel R.S. Khoo N. Lala P.K. Exp. Cell Res. 1993; 206: 204-211Google Scholar). A stable JAR cell line overexpressing Smad3 (JAR-Smad3/c) was developed as described previously (31Xu G. Chakraborty C. Lala P.K. Biochem. Biophys. Res. Commun. 2002; 294: 1079-1086Google Scholar). JEG-3, JAR, JAR-Smad3/c, and HTR8/SVneo cells were cultured in RPMI 1640 medium (Invitrogen), whereas BeWo cells were cultured in Ham's F-12 medium and Dulbecco's modified Eagle's medium (1:1; Hyclone Laboratories, Logan, UT) supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen) in the presence of 10% fetal bovine serum (Sigma). RNA Extraction and RT-PCR—Total RNA was extracted using TRIzol reagent (Invitrogen) following the manufacturer's protocols and stored at –80 °C until RT-PCR analysis. Five micrograms of total RNA was reverse-transcribed into cDNA in a total volume of 50 μl using 0.5 μg of oligo(dT) primer (Amersham Biosciences) and 500 units of Moloney murine leukemia virus reverse transcriptase (New England Biolabs Inc., Mississauga, Ontario, Canada). The reaction was carried out at 37 °C for 2 h in 1× reaction buffer (50 mm Tris-HCl (pH 8.3), 75 mm KCl, 3 mm MgCl2, and 10 mm dithiothreitol) containing 0.5 mm dNTPs, 10 mm dithiothreitol, and 50 units of RNase inhibitor (RNAguard, Amersham Biosciences) and terminated by heating the mixture at 95 °C for 5 min. An aliquot of the cDNA sample (2 μl) was subjected to PCR, which was performed in the presence of 10 mm Tris-HCl (pH 8.3), 2.0 mm MgCl2, 50 μm deoxynucleotide triphosphate, 1 unit of Hotstar Taq (QIAGEN Inc., Mississauga), and 10 pmol of primers for 25–40 cycles depending on the cDNA target to be amplified. Primers for ALK7 transcripts, Nodal, p15, p21, p27, and the internal control GAPDH are listed in Table I. Primers for Smad2, -3, and -4 have been reported previously (32Xu G. Chakraborty C. Lala P.K. Biochem. Biophys. Res. Commun. 2001; 287: 47-55Google Scholar). The annealing temperature for PCR ranged from 50 to 65 °C depending on the primer sets used. Levels of p15, p21, and p27 mRNAs were quantified by normalizing the spot densities of these molecules to their respective GAPDH control. Data from individual experiments were converted to -fold of control (empty vector-transfected cells) before being pooled.Table IPrimer sequencesTarget mRNASequence (5′ → 3′)ALK7-1, -3, -4 (AY127050)Forward primerGCACTTCAAAAGGGTGTCGReverse primerGATCATTCCAGCCATGGTCALK7-2 (AF525679)Forward primerCGAATTCACTGCGCCAAGGTCReverse primerGTGCAGTGTTGTGTTGTTGCAALK7-1 (AY127050)Forward primerATGACCCGGGCGCTCTGCTCAReverse primerATACTGTCAGCATCGCAGCTANodal (AB067630)Forward primerAGACATCATCCGCAGCCTACAReverse primerGTCCATCTGAAACCGCTCTAAGp15 (BC014469)Forward primerTGGGAAAGAAGGGAAGAGTGReverse primerGAACCTGGCGTCAGTCCCCCGTGGCTp21 (U03106)Forward primerTGAGCGATGGAACTTCGACTReverse primerTTAGGGCTTCCTCTTCGACTp27 (U10906)Forward primerTGCCCGAGTTCTACTACAGAReverse primerTTTGGGGAACCGTCTGAAACGAPDH (M33197)Forward primerAAGGTCATCCCTGAGCTGAACReverse primerACGCCTGCTTCACCACCTTCTALK7 expression construct (AY127050)Forward primerCGGAATTCCGCGATGACCCGGGCGCTCTGCTReverse primerTCCCCGCGGGGCTTTGCAGTCTTCTTTGACACANodal expression construct (AB067630)Forward primerCGGAATTCCACCATGCACGCCCACTGCReverse primerCGGAATTCCGGAGGCACCCACATTCTTCCAC Open table in a new tab Generation of Recombinant Constructs—Expression constructs containing the coding regions of ALK7-1 and Nodal were cloned into pcDNA4/TO/Myc-His (Invitrogen). ALK7-1 and Nodal cDNAs were amplified from normal placental tissues by RT-PCR using primers with EcoRI and SacII linkers for ALK7-1 and two EcoRI linkers for Nodal (Table I). Constitutively active ALK7 (ALK7-ca) was generated by replacing Thr194 with aspartic acid, and kinase-deficient ALK7 (ALK7-kd) was generated by replacing Lys222 with arginine using site-directed mutagenesis. Expression constructs of dominant-negative Smad2 and Smad3 in the pRK5F vector were kindly provided by Dr. R. Derynck (University of California, San Francisco) and were subcloned into pcDNA3.1 (Invitrogen) before being used in the experiments. All constructs were fully sequenced. Transient Transfection—Transient transfection was carried out using 25-kDa polyethyleneimine (Sigma) as described previously (33Wu D. Luo S. Wang Y. Zhuang L. Chen Y. Peng C. Mol. Cell. Endocrinol. 2001; 175: 111-121Google Scholar). Briefly, cells were seeded at 50% cell density on tissue culture dishes (Sarstedt, Inc., Montreal, Quebec, Canada) and allowed to adhere and grow overnight. The cells were washed, and the culture medium was replaced with reduced serum Opti-MEM (Invitrogen) 2 h prior to transfection. Plasmid DNA and 0.18 mm polyethyleneimine (0.28 μl/1 μg of DNA) were diluted into 150 mm NaCl. After incubation at room temperature for 10 min, these two solutions were mixed and further incubated at room temperature for 20 min. The polyethyleneimine/DNA mixture was then diluted into Opti-MEM and added to the cells. After overnight incubation at 37 °C, the culture medium was changed to RPMI 1640 medium supplemented with 10% fetal bovine serum. Transfection efficiency, estimated by transfecting the cells with pEGFP, was >60% for 100-mm dishes, 40–50% for cells in 24-well plates, and 30% for cells cultured in 96-well plates. Determination of Cell Growth—Cell growth was determined either by direct cell counting or by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell counting experiments, JEG-3, JAR, and JAR-Smad3/c cells were cultured in 24-well culture plates at a cell density of 2 × 105 cells/well. Cells were transiently transfected with 1 μg of pcDNA4, wild-type ALK7 (ALK7-wt), and ALK7-ca plasmid DNA (n = 6). Cells were trypsinized 48 h after transfection, and cell number was counted using a hemocytometer. Each experiment was repeated three times. For MTT assays, JEG-3, JAR, JAR-Smad3/c, and HTR8/SVneo cells were seeded on 96-well plates at a density of 104 cells/well. After 16–18 h of incubation, cells were transfected with different constructs (0.3 μg/well) as indicated in the figures using the above-mentioned procedure (n = six to eight wells). Forty-eight hours after transfection, the number of metabolically active cells was measured using an MTT cell proliferation kit I (Roche Diagnostics) following the manufacturer's instructions. Briefly, MTT labeling solution was added to the culture medium at the end of each experiment, and cells were further incubated for 3 h. Mitochondrial dehydrogenase activity reduced the yellow MTT dye to a purple formazan, which was then solubilized by overnight incubation with the MTT-solubilizing reagent (10% SDS in 0.01 HCl), and absorbance was read at 595 nm on an enzyme-linked immunosorbent assay plate reader (Bio-Tek Instruments, Inc.). Flow Cytometry—JEG-3 cells were plated on 100-mm dishes and transfected with 15 μg of pcDNA4, ALK7-wt, ALK7-ca, or Nodal constructs. Forty-eight hours after transfection, proliferation and apoptosis assays were performed. Proliferation was determined by measuring incorporation of bromodeoxyuridine (BrdUrd) into DNA. Cells were incubated with 10 μm BrdUrd (Sigma) for 1 h, trypsinized, washed twice with phosphate-buffered saline (PBS), and fixed in ethanol. The cell suspension was then treated with 2 n HCl at 37 °C for 30 min, washed with 0.1 m NaB4O7, and resuspended in PBS containing 0.5% Tween 20 and 1% bovine serum albumin to a density of 106 cells/100 μl. Cells were further incubated with 10 μl of fluorescein isothiocyanate-conjugated mouse anti-BrdUrd monoclonal antibody (Dako Corp., Mississauga) for 30 min. After washing, the cells were resuspended in PBS containing 5 μg/ml propidium iodide and immediately analyzed using a FACScan (BD Biosciences). Apoptosis was assayed using an annexin V apoptosis detection kit (Santa Cruz Biotechnology Inc., Santa Cruz, CA) following the instructions of the manufacturer. Briefly, cells were trypsinized and then pelleted by centrifugation. The cells were washed twice with cold PBS and resuspended in the assay buffer to obtain a density of 106 cells/ml. Annexin V-fluorescein isothiocyanate was added to cell aliquots (2 × 105 cells), followed by incubation at room temperature in the dark for 15 min. The cells were then washed with cold PBS and subjected immediately to FACScan analysis. Hoechst Staining—JEG-3 cells were seeded on 100-mm culture dishes and transfected with pcDNA4, ALK7-wt, ALK7-ca, or Nodal plasmid DNA (15 μg/dish). Forty-eight hours after transfection, cells were subjected to trypsin treatment at 37 °C for 3 min, fixed with 4% formalin in PBS, and washed with PBS. Cells were then incubated with 0.1 μg/ml Hoechst 33248 (bisbenzimide, Sigma) and spotted on slides for microscopy. Nuclear morphology was observed and photographed using a Zeiss Axiovert 35 fluorescent microscope. Protein Extraction and Western Blot Analyses—HTR8/SVneo and JEG-3 cells cultured in 100-mm dishes were transfected with 15 μg/dish plasmid DNA (pcDNA4, ALK7-ca, ALK7-kd, Nodal, or Nodal plus ALK7-kd). Forty-eight hours after transfection, cells were washed twice with ice-cold PBS and lysed with radioimmune precipitation assay buffer (50 mm Tris-HCl, 150 mm NaCl, 1% Triton X-100, 0.5% deoxycholate, and 1% SDS) containing 1 mm dithiothreitol, 1 mm Na3VO4, 5 mm NaF, 100 mm EDTA, 10 mg/ml aprotinin, and 100 mm phenylmethylsulfonyl fluoride. Protein samples were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was blocked with TBST (10 mm Tris-Cl (pH 8.0), 150 mm NaCl, and 0.05% Tween 20) containing 10% nonfat dry milk powder (TBSTM) at room temperature for 30 min. The membrane was then incubated for 2 h at room temperature with a primary antibody (rabbit anti-cleaved caspase-3 (Asp175) polyclonal antibody (1:2000) (Cell signaling Technology); goat anti-Cdk2 polyclonal antibody (1:500), mouse anti-cyclin D1 monoclonal antibody (1:2000), mouse anti-cyclin E monoclonal antibody (1:2000) (all from Santa Cruz Biotechnology Inc.); or goat anti-p27 polyclonal antibody (1:2000) (BD Signal Transduction)) prepared in TBSTM. The membranes were washed three times with TBST for 15 min each and then incubated for 2 h with a horseradish peroxidase-conjugated secondary antibody (donkey anti-rabbit IgG, goat anti-mouse IgG, or rabbit anti-goat IgG antibody (1:5000)) in TBSTM. After washing as described above, signals were detected using an ECL kit (Amersham Biosciences) according to the instructions of the manufacturer. Statistical Analysis—Differences among several groups were determined by one-way analysis of variance, followed by Student-Newman-Keul's test using GraphPad InStat software. For comparison between two groups, Student's t test was used. p < 0.05 was considered significant. Expression of ALK7, Nodal, and Smad mRNAs in Trophoblast Cell Lines—The mRNA expression of ALK7, Nodal, Smad2, and Smad3 was determined in several trophoblast cell lines, including JEG-3, JAR, and BeWo choriocarcinoma cells; a stable JAR cell line overexpressing Smad3; and an immortalized normal trophoblast cell line, HTR8/SVneo. Using a pair of primers spanning exons III and IV of the ALK7 gene, which are known to be alternatively spliced to generate different transcripts (12Roberts H.J. Hu S. Qiu Q. Leung P.C.K. Cannigia I. Gruslin A. Tsang B. Peng C. Biol. Reprod. 2003; 68: 1719-1726Google Scholar), we detected three DNA fragments corresponding to the expected sizes of ALK7-1, -3, and -4 in all trophoblast cell lines tested (Fig. 1A). Similarly, when a pair of primers specific for ALK7-2 was used in PCR, a DNA fragment of the expected size was obtained (Fig. 1A). Since ALK7 has been shown to mediate the effect of Nodal in mesoderm induction (19Reissmann E. Jornvall H. Blokzijl A. Anderson O. Chang C. Minchiotti G. Persico M.G. Ibanex C. Brivanlou A.H. Genes Dev. 2001; 15: 2010-2022Google Scholar) and to activate Smad2 and Smad3 (23Yeo C. Whitman M. Mol. Cell. 2001; 7: 949-957Google Scholar, 25Kumar A. Novoselov V. Celeste A.J. Wolfman N.M. ten Dijke P. Kuehn M.R. J. Biol. Chem. 2001; 276: 656-661Google Scholar), we also examined mRNA expression of Nodal, Smad2, and Smad3. Similar to our previous studies that showed that Nodal mRNA is expressed in JEG-3 cells and in normal placenta (12Roberts H.J. Hu S. Qiu Q. Leung P.C.K. Cannigia I. Gruslin A. Tsang B. Peng C. Biol. Reprod. 2003; 68: 1719-1726Google Scholar), RT-PCR detected Nodal mRNA in JEG-3, JAR, JAR-Smad3/c, and HTR8/SVneo cells (Fig. 1A). Both Smad2 and Smad3 mRNAs were detected in the trophoblast cell lines (Fig. 1B). Whereas Smad2 mRNA levels were similar in all cell lines tested, Smad3 mRNA levels were much lower in JEG-3 and JAR cells than in the other cell lines, confirming our previous findings (33Wu D. Luo S. Wang Y. Zhuang L. Chen Y. Peng C. Mol. Cell. Endocrinol. 2001; 175: 111-121Google Scholar). Nodal and ALK7 Inhibit Trophoblast Cell Growth via Smad2/3—To determine the function of ALK7 in trophoblast cells, we first evaluated its effect on cell growth. JEG-3, JAR, and JAR-Smad3/c cells were transiently transfected with ALK7-wt, ALK7-ca, or the empty vector as a negative control, and cell
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