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Insulin-like Growth Factor (IGF)-I Regulates IGF-binding Protein-5 Gene Expression through the Phosphatidylinositol 3-Kinase, Protein Kinase B/Akt, and p70 S6 Kinase Signaling Pathway*

  • Cunming Duan
    Correspondence
    To whom correspondence should be addressed: Dept. of Biology, University of Michigan, Natural Science Bldg., Ann Arbor, MI 48109-1048. Tel.: 734-763-4710; Fax: 734-647-0884;
    Affiliations
    From the Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048
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  • Marya B. Liimatta
    Affiliations
    From the Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048
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  • Olivia L. Bottum
    Affiliations
    From the Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048
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  • Author Footnotes
    * The study is supported in part by National Science Foundation Grant IBN-9728911 and by NIDDK, National Institutes of Health, Grant 5P60DK-20572 through a Pilot/Feasibility grant from the Michigan Diabetes Research and Training Center, University of Michigan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:December 24, 1999DOI:https://doi.org/10.1074/jbc.274.52.37147
      Expression of the insulin-like growth factor-binding protein 5 (IGFBP-5) gene in vascular smooth muscle cells is up-regulated by IGF-I through an IGF-I receptor-mediated mechanism. In this study, we studied the possible involvement of the mitogen-activated protein kinase (MAPK) and PI 3-kinase signaling pathways in mediating IGF-I-regulated IGFBP-5 gene expression. The addition of Des(1–3)IGF-I, an IGF analog with reduced affinity to IGFBPs, resulted in a transient activation of p44 and p42 MAPK. Inhibition of the MAPK activation by PD98059, however, did not affect IGF-I-stimulated IGFBP-5 expression. Des(1–3)IGF-I treatment also strongly activated PI 3-kinase. This activation was probably mediated through IRS-1, because IGF-I stimulation resulted in a significant increase in IRS-1- but not IRS-2-associated PI 3-kinase activity. This activation occurred within 5 min and was sustained at high levels for over 6 h. Likewise, Des(1–3)IGF-I caused a long lasting activation of PKB/Akt and p70s6k. When LY294002 and wortmannin, two specific inhibitors of PI 3-kinase, were added with Des(1–3)IGF-I, the IGF-I-regulated IGFBP-5 expression was negated. The addition of rapamycin, which inhibits IGF-I-induced p70s6k activation, significantly inhibited IGF-I-regulated IGFBP-5 gene expression. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI 3-kinase-PKB/Akt-p70s6k pathway but not the MAPK pathway in vascular smooth muscle cells.
      VSMC
      vascular smooth muscle cell
      ERK
      extracellular signal-regulated kinase
      IGF-1
      insulin-like growth factor-I
      IGFBP
      IGF-binding protein
      IGF-IR
      IGF-I receptor
      IRS
      insulin receptor substrate
      MAPK
      mitogen-activated protein kinase
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PKB/Akt
      protein kinase B
      PKC
      protein kinase C
      p70s6k
      p70 ribosomal protein S6 kinase
      Rb
      retinoblastoma tumor suppressor
      Abnormal vascular smooth muscle cell (VSMC)1 proliferation and directed migration from the media into the intima play major roles in the pathogenesis of atherosclerotic lesions, the formation of restenosis after angioplasty, and the accelerated arteriopathy after cardiac transplantation (
      • Ross R.
      ). These cellular events are regulated by a number of peptide growth factors including insulin-like growth factor-I (IGF-I). Studies have shown that IGF-I is a mitogen and strong chemoattractant for cultured VSMCs (
      • Bornfeldt K.E.
      • Raines E.W.
      • Nakano T.
      • Graves L.M.
      • Krebs E.G.
      • Ross R.
      ,
      • Imai Y.
      • Clemmons D.R.
      ,
      • Jones J.I.
      • Clemmons D.R.
      ). The growth and migration-promoting actions of IGF-I are mediated through the IGF-IR, a tyrosine kinase receptor that is linked to the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathways (
      • Imai Y.
      • Clemmons D.R.
      ). The bioactivity of IGF-I is further modulated by several high affinity binding proteins (IGFBPs) present in the local vasculature. These IGFBPs control the distribution of IGF-I between extracellular environment and cell surface binding sites and can alter IGF bioactivity by modulating its interaction with the receptor (
      • Jones J.I.
      • Clemmons D.R.
      ). Previous studies have shown that VSMCs secrete several IGFBPs, including IGFBP-5 (
      • Giannella-Neto D.
      • Kamyar A.
      • Sharifi B.
      • Pirola C.J.
      • Kupfer J.
      • Rosenfeld R.G.
      • Forrester J.S.
      • Fagin J.A.
      ,
      • Cohick W.S.
      • Gockerman A.
      • Clemmons D.R.
      ,
      • Cohick W.S.
      • Gockerman A.
      • Clemmons D.R.
      ,
      • Ververis J.
      • Li K.
      • Delafontaine P.
      ,
      • Kamyar A.
      • Pirola C.J.
      • Wang H.M.
      • Sharifi B.
      • Mohan S.
      • Forrester J.S.
      • Fagin J.A.
      ,
      • Boes M.
      • Booth B.A.
      • Dake B.L.
      • Moser D.R.
      • Bar R.S.
      ,
      • Duan C.
      • Hawes S.B.
      • Prevette T.
      • Clemmons D.R.
      ). In cultured porcine VSMCs, expression of the IGFBP-5 gene is up-regulated by IGF-I (
      • Duan C.
      • Hawes S.B.
      • Prevette T.
      • Clemmons D.R.
      ). The increased IGFBP-5 protein, in turn, modulates the IGF-I action by a feedback mechanism in these cells (
      • Duan C.
      • Clemmons D.R.
      ).
      The regulatory effect of IGF-I on IGFBP-5 gene expression in VSMCs is mediated through the IGF-IR (
      • Duan C.
      • Hawes S.B.
      • Prevette T.
      • Clemmons D.R.
      ). However, how the activation of the IGF-IR leads to the elevation of IGFBP-5 gene expression is not well understood. In this study, we investigated the effect of IGF-I on MAPK and PI 3-kinase activation and the role of these signaling pathways in mediating IGF-I-stimulated IGFBP-5 gene expression in cultured VSMCs. IGF-I exposure resulted in the activation of both MAPK and PI 3-kinase signaling cascades in these cells. When the activation of PI 3-kinase or p70s6k was suppressed using specific inhibitors, the IGF-I-regulated IGFBP-5 gene expression was negated. The addition of PD98059, which inhibits IGF-I-induced MAPK activation, had no such effect. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI 3-kinase-PKB/Akt-p70s6k pathway but not the MAPK pathway in VSMCs.

      DISCUSSION

      It has been shown that IGF-I stimulates the expression of IGFBP-5 in VSMCs, and this up-regulation of IGFBP-5 by its own ligand is mediated through the IGF-IR (
      • Duan C.
      • Hawes S.B.
      • Prevette T.
      • Clemmons D.R.
      ). How the activation of the IGF-IR leads to the elevation of IGFBP-5 gene expression is poorly defined. In this study, we present evidence that IGF-I stimulation caused a strong and sustained activation of the PI 3-kinase and its downstream signaling molecules, PKB/Akt and p70s6k, in primary porcine VSMCs. We found that inhibition of PI 3-kinase activation by LY294002 or wortmannin abolished the IGF-I-stimulated IGFBP-5 gene expression. Likewise, inhibition of p70s6k also inhibited the IGF-I-stimulated IGFBP-5 gene expression. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI-3-kinase-PKB/Akt and p70s6k pathway in cultured porcine VSMCs.
      Studies using “model” systems (i.e. various tumor or transformed cell lines) have revealed that a complex network of signaling events are activated upon ligand occupancy of the IGF-IR (
      • LeRoith D.
      • Werner H.
      • Beitner-Johnson D.
      • Roberts Jr., C.T.
      ). Two homologous adapter proteins, IRS-1 and IRS-2, have been identified as major substrates of the activated IGF-IR (
      • White M.F.
      ). In this study, we found that IGF-I stimulation resulted in a significant increase in IRS-1- but not IRS-2-associated PI 3-kinase activity. These results strongly suggest (but do not definitively prove) that IGF-I activates the PI 3-kinase signaling pathway primarily through IRS-1. IRS-1 has over 20 potential tyrosine phosphorylation sites, which serve as docking sites for numerous Src homology 2 domain-containing proteins, including the p85 subunit of PI 3-kinase (
      • White M.F.
      ). Like other cell types, activation of IRS-1 in cultured porcine VSMCs results in the activation of the PI 3-kinase pathways. In these primary cells, Des(1–3)IGF-I is a potent activator of PI 3-kinase. This activation is strong and sustained for more than 6 h. In this study, we have used two potent and specific inhibitors of PI 3-kinase, LY294002 and wortmannin, to determine the possible involvement of PI 3-kinase in mediating the effect of IGF-I in regulating IGFBP-5 gene expression. The use of these two structurally distinct compounds has established the important role of PI 3-kinase in transmitting the antiapoptotic and differentiation signals of IGF-I (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Parrizas M.
      • Saltiel A.R.
      • LeRoith D.
      ). Recent studies have shown that PI 3-kinase is required for the effect of insulin on IGFBP-1 gene expression in rat hepatocytes using these compounds (
      • Band C.J.
      • Posner B.I.
      ). In this study, we have found that IGF-I stimulation of IGFBP-5 gene expression was blocked by LY294002 and wortmannin at concentrations that effectively inhibited IGF-I-stimulated PI 3-kinase activity. Thus, activation of PI 3-kinase is required for the action of IGF-I on IGFBP-5 gene expression in VSMCs. This finding is contrary to a recent report indicating that the up-regulation of the IGFBP-5 gene by IGF-I/insulin was rapamycin-sensitive but was not inhibited by LY294002 or wortmannin in mouse C2 myoblast cells (
      • Rousse S.
      • Montarras D.
      • Pinset C.
      • Dubois C.
      ). In that particular study, however, high concentrations of insulin instead of IGF-I were used for the gene expression experiments. In such a setting, one would expect that the insulin receptor would also be activated in addition to the IGF-IR. Therefore, differences in ligands used, experiment conditions, cell type, or species may account for the different findings.
      Treatment of porcine VSMCs with Des(1–3)IGF-I also has a profound and long lasting effect in activating PKB/Akt and p70s6k in cultured porcine VSMCs. PKB/Akt is a serine-threonine kinase immediately downstream of PI 3-kinase (
      • Marte B.M.
      • Downward J.
      ). PKB/Akt has been shown to be a major mediator for IGF-I actions in promoting neuronal survival (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ). A recent study by Cichy et al. (
      • Cichy S.B.
      • Uddin S.
      • Danilkovich A.
      • Guo S.
      • Klippel A.
      • Unterman T.
      ) has shown that PKB/Akt mediates the inhibitory effect of insulin on IGFBP-1 gene expression in hepatocytes. More recent studies from the same group have identified FKHR, a human homologue of the forkhead/winged helix transcription factor initially discovered in Caenorhabditis elegans, as the target of the insulin receptor-PKB/Akt signaling pathway (
      • Guo H.
      • Rena G.
      • Cichy S.
      • He X.
      • Cohen P.
      • Unterman T.
      ). Phosphorylation of FKHR by PKB/Akt at serine 256 disrupts the inhibitory effect of insulin on IGFBP-1 gene transcription (
      • Guo H.
      • Rena G.
      • Cichy S.
      • He X.
      • Cohen P.
      • Unterman T.
      ). Acting further downstream in this pathway is p70s6k. IGF-I-induced cell cycle progression and protein synthesis have been reported to be inhibited by inhibition of p70s6k (
      • Marte B.M.
      • Downward J.
      ). However, other cellular responses mediated by PI 3-kinase, including inhibition of apoptosis, activation of glycogen synthesis, and glucose transport are independent of p70s6k (
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yauch M.
      • Shirouzo M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Yao R.
      • Cooper G.M.
      ). In this study, we have found that rapamycin (20 ng/ml) effectively inhibited Des(1–3)IGF-I-induced p70s6k phosphorylation without affecting MAPK, PI 3-kinase, and PKB/Akt activity or phosphorylation states in porcine VSMCs. Inhibition of p70s6k activation by rapamycin significantly inhibited the IGF-I-stimulated IGFBP-5 gene expression, suggesting that the action of IGF-I in up-regulating IGFBP-5 gene is mediated through the PI 3-kinase, PKB/Akt, and p70s6k signaling pathway in porcine VSMCs. The downstream events of the PI 3-kinase-PKB/Akt-p70s6k pathway that are involved in IGF-I regulation of IGFBP-5 gene expression are not clear at present. p70s6k phosphorylates and activates several nuclear proteins involved in transcriptional regulation of specific genes (
      • Proud G.
      ). One of the nuclear proteins phosphorylated by p70s6k is the transcription factor cAMP-responsive element modulator (
      • De Groot R.F.
      • Ballou L.M.
      • Sassone-Corsi P.
      ), which constitutes a target for mitogenic signaling through p70s6k. It has also been reported that rapamycin treatment leads to a reduction in the phosphorylation of retinoblastoma tumor suppressor (Rb) in human and rat VSMCs (
      • Marx S.O.
      • Jayaraman T.
      • Go L.O.
      • Marks A.R.
      ). The relevance of any of these nuclear proteins to IGFBP-5 gene transcription, however, has yet to be established. It is of interest to note that while inhibition of PI 3-kinase activity by either LY294002 or wortmannin completely abolished IGF-I-induced IGFBP-5 gene expression, rapamycin was only partially effective in inhibiting IGF-I-induced IGFBP-5 expression. Since rapamycin at the concentrations used completely suppressed p70s6k, it is possible that another p70s6k-independent signaling pathway(s) is also involved in this action of IGF-I. Recently we have shown that several PKC isoforms, including PKC-βI, -ε, -η, and -ζ, are expressed in porcine VSMCs and are under the regulation of IGF-I (
      • Yano K.
      • Bauchat J.R.
      • Liimatta M.B.
      • Clemmons D.R.
      • Duan C.
      ). Down-regulation or inhibition of PKC activity by high doses of phorbol 12-myristate 13-acetate or a specific PKC inhibitor (GF109203X) abolished IGF-I-induced IGFBP-5 gene expression in these cells. Several studies indicate that several PKC isoforms, including PKC-ζ and PKC-ε, act downstream of the PI 3-kinase (
      • Liscovitch M.
      • Cantley L.C.
      ,
      • Moriya S.
      • Kazlauskas A.
      • Akimoto K.
      • Hirai S.
      • Mizuno K.
      • Takenawa T.
      • Fukui Y.
      • Watanabe Y.
      • Ozaki S.
      • Ohno S.
      ,
      • Liu Q.
      • Ning W.
      • Dantzer R.
      • Freund G.G.
      • Kelley K.W.
      ). Therefore, some of these PKC isoforms may be essential signaling intermediates for IGF-I acting downstream of PI 3-kinase in the IGF signaling network in VSMCs (
      • Yano K.
      • Bauchat J.R.
      • Liimatta M.B.
      • Clemmons D.R.
      • Duan C.
      ).
      In addition to the PI 3-kinase pathway, we consistently observed a time- and concentration-dependent phosphorylation of ERK1 and -2 induced by Des(1–3)IGF-I treatment in these cells. Although several previous studies reported that IGF-I did not activate MAPK in cultured human and bovine VSMCs (
      • Bornfeldt K.E.
      • Raines E.W.
      • Nakano T.
      • Graves L.M.
      • Krebs E.G.
      • Ross R.
      ,
      • Graves L.M.
      • Bornfeldt K.E.
      • Sidhu J.S.
      • Argast G.M.
      • Raines F.W.
      • Ross R.
      • Leslie C.C.
      • Krebs E.G.
      ,
      • Koyama H.
      • Nishizawa Y.
      • Hosoi M.
      • Kogawa K.
      • Shioi A.
      • Morii H.
      ), other studies reported an increase in MAPK activity after IGF-I treatment in rat and porcine VSMCs (
      • Imai Y.
      • Clemmons D.R.
      ,
      • Thommes K.B.
      • Hoppe J.
      • Vetter H.
      • Sachinidis A.
      ,
      • Takagi Y.
      • Kashiwagi A.
      • Tanaka Y.
      • Maegawa H.
      • Shigeta Y.
      ,
      • Duan C.
      • Bauchat J.R.
      • Hsieh T.
      ). These conflicting data cannot simply be attributed to species differences, because IGF-I is a strong activator of MAPK in many human and bovine cell types (
      • Parrizas M.
      • Saltiel A.R.
      • LeRoith D.
      ,
      • Pavlovic-Surjancev B.
      • Cahill B.L.
      • Perlman R.L.
      ). Our results with porcine VSMCs are not necessarily in contradiction with previous studies using human and bovine VSMCs. Those studies focused on the comparison of PDGF-BB and IGF-I. Indeed, we found that IGF-I is a weaker activator of the MAPK pathway in comparison with PDGF-BB in this cell type; PDGF-BB at a concentration of 5 ng/ml results in a greater response than that of 100 ng/ml IGF-I (data not shown). Likewise, Thommes et al. (
      • Thommes K.B.
      • Hoppe J.
      • Vetter H.
      • Sachinidis A.
      ) also reported that IGF-I was only 30% as effective as PDGF in inducing MAPK activation in rat VSMCs. Furthermore, our results indicated that the IGF-I-induced MAPK activation is a transient event, characterized by a rapid rise and decline within minutes and is only observed when high doses of IGF-I are added. These variations may explain the previous negative findings.
      Activation of the MAPK signaling pathway by IGF-I has been shown in a variety of cell types (
      • LeRoith D.
      • Werner H.
      • Beitner-Johnson D.
      • Roberts Jr., C.T.
      ). The importance of MAPK in cell proliferation and gene expression is generally acknowledged. Several targets of this pathway have been defined, including transcription factors such as Elk-1 or SAP-1 (
      • Hill C.S.
      • Treisman R.
      ). This provides a common route by which signals from various growth factors and hormones converge at a major regulatory element in the promoters of c-fos and other coregulated genes, the serum response element. Several lines of evidence suggested that the up-regulating of the IGFBP-5 gene by IGF-I observed in porcine VSMCs may not act though this pathway. In these primary cells, IGF-I up-regulates IGFBP-5 gene expression in a time frame consistent with that of an intermediate effect rather than an immediate early response. No consensus serum response element is present in the promoter region of the IGFBP-5 gene. In addition, cycloheximide abrogates IGF-I-induced IGFBP-5 gene expression, indicating a requirement for the synthesis of an intermediate protein(s) (
      • Duan C.
      • Hawes S.B.
      • Prevette T.
      • Clemmons D.R.
      ). Furthermore, IGF-I stimulation does not change the c-fos levels in rat VSMCs (
      • Thommes K.B.
      • Hoppe J.
      • Vetter H.
      • Sachinidis A.
      ). In this study, we further demonstrated that specific inhibition of the MAPK activation did not significantly decrease the IGF-I-dependent IGFBP-5 gene expression. Since PD98059 at the given concentration almost completely suppressed IGF-I-induced MAPK activation and inhibited IGF-I-directed VSMC migration, we conclude that MAPK activation is not critical for IGF-I-induced IGFBP-5 gene expression.
      In addition to the IGFBP-5 gene, IGF-I has been shown to regulate transcription of a number of genes in a variety of cell types, but a consensus IGF-responsive element has yet to be established. In VSMCs, IGF-I has been shown to regulate expression of the elastin gene (
      • Wolfe B.L.
      • Rich C.B.
      • Goud H.D.
      • Terpstra A.J.
      • Bashir M.
      • Rosenbloom J.
      • Sonenshein G.E.
      • Foster J.A.
      ,
      • Jensen D.E.
      • Rich C.B.
      • Terpstra A.J.
      • Farmer S.R.
      • Foster J.A.
      ,
      • Conn K.J.
      • Rich C.B.
      • Jensen D.E.
      • Fontanilla M.R.
      • Bashir M.M.
      • Rosenbloom J.
      • Foster J.A.
      ). This regulation has been attributed to several GC-rich sequences in these genes that are capable of binding to IGF-I-regulated nuclear proteins isolated from rat VSMCs (
      • Wolfe B.L.
      • Rich C.B.
      • Goud H.D.
      • Terpstra A.J.
      • Bashir M.
      • Rosenbloom J.
      • Sonenshein G.E.
      • Foster J.A.
      ,
      • Urban R.J.
      • Bodenburg Y.
      ). Two of the proteins have been shown to be transcription factors Sp1 and Sp3 (
      • Jensen D.E.
      • Rich C.B.
      • Terpstra A.J.
      • Farmer S.R.
      • Foster J.A.
      ,
      • Conn K.J.
      • Rich C.B.
      • Jensen D.E.
      • Fontanilla M.R.
      • Bashir M.M.
      • Rosenbloom J.
      • Foster J.A.
      ). The IGFBP-5 promoter contains several GC-rich regions superficially resembling the Sp1 element. In particular, the DNA sequence 5′-CCCCACCCCCACCC-3′ at positions −147 to −134 has this potential. Although this highly conserved sequence contains two overlapping AP-2 elements 5′-CCCCACCC-3′ and is capable of binding to AP-2 in vitro, it does not appear to mediate the AP-2 regulation in vivo (
      • Duan C.
      • Clemmons D.R.
      ). This region contains sequences identical to the Rb control element 5′-CCACCC-3′. The Rb control element motif has been identified as a Sp1-binding sequence responsible for Rb-inducedtrans-activation (
      • Chen L.I.
      • Nishinaka T.
      • Kwan K.
      • Kitabayashi I.
      • Yokoyama K.
      • Fu Y.-H.F.
      • Grunwald S.
      • Chiu R.
      ). Previous studies by Jensen et al. (
      • Jensen D.E.
      • Rich C.B.
      • Terpstra A.J.
      • Farmer S.R.
      • Foster J.A.
      ) suggested that IGF-I may disrupt Sp1 binding to the GC-rich domain of the elastin gene by affecting the phosphorylation state of Rb in rat VSMCs. In preliminary studies, we have found that IGF-I stimulation resulted in Rb phosphorylation in cultured porcine VSMCs.
      C. Duan, M. B. Liimatta, and O. L. Bottum, unpublished observation.
      This regulation of Rb phosphorylation state by IGF-I may require p70s6kactivation, because it has been reported that rapamycin treatment leads to a reduction in the phosphorylation of Rb in human and rat VSMCs (
      • Marx S.O.
      • Jayaraman T.
      • Go L.O.
      • Marks A.R.
      ). Further experiments are needed to determine whether this Rb control element motif and/or another cis sequence(s) is responsible for the IGF-I-stimulated IGFBP-5 gene expression in VSMCs.

      ACKNOWLEDGEMENT

      We thank Dr. Ildiko Szantos-Denes for providing the IRS-1 and IRS-2 antibodies.

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