Soluble Frizzled-Related Protein 1 Is Estrogen Inducible in Bone Marrow Stromal Cells and Suppresses the Earliest Events in Lymphopoiesis This information is current as of October 1, 2021. Takafumi Yokota, Kenji Oritani, Karla P. Garrett, Taku Kouro, Makoto Nishida, Isao Takahashi, Michiko Ichii, Yusuke Satoh, Paul W. Kincade and Yuzuru Kanakura J Immunol 2008; 181:6061-6072; ; doi: 10.4049/jimmunol.181.9.6061 Downloaded from http://www.jimmunol.org/content/181/9/6061 References This article cites 67 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/181/9/6061.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Soluble Frizzled-Related Protein 1 Is Estrogen Inducible in Bone Marrow Stromal Cells and Suppresses the Earliest Events in Lymphopoiesis1 Takafumi Yokota,2* Kenji Oritani,* Karla P. Garrett,‡ Taku Kouro,§ Makoto Nishida,† Isao Takahashi,* Michiko Ichii,* Yusuke Satoh,* Paul W. Kincade,‡ and Yuzuru Kanakura* It has long been known that lymphopoiesis is transiently suppressed during pregnancy, which can be experimentally sim- ulated by estrogen treatment. We now confirm with Rag1/GFP reporter mice that early lymphoid progenitors in the lineage marker؊ c-kithigh ScaI؉, hematopoietic stem cell-enriched fraction of bone marrow are particularly depressed in these circumstances. Hematopoietic and environmental cells are both potential hormone targets and, because of this complexity, Downloaded from very little is known regarding mechanisms. We have now identified soluble Frizzled-related protein (sFRP)1 as an estrogen- inducible gene in stromal cells, whose expression corresponded to inability to support lymphopoiesis. Bone-lining stromal cells express sFRP1, and the transcripts were elevated by pregnancy or estrogen injection. Estrogen receptor-␣ was essential for both lymphoid suppression and induction of the sFRP family. SFRP1 has been mainly described as an antagonist for complex Wnt signals. However, we found that sFRP1, like Wnt3a, stabilized ␤-catenin and blocked early lymphoid pro- gression. Myeloerythroid progenitors were less affected by sFRP1 in culture, which was similar to estrogen with respect to http://www.jimmunol.org/ lineage specificity. Hematopoietic stem cells expressed various Frizzled receptors, which markedly declined as they differ- entiated to lymphoid lineage. Thus, hormonal control of early lymphopoiesis in adults might partly relate to sFRP1 levels. The Journal of Immunology, 2008, 181: 6061–6072. ymphocytes are produced throughout life from self-re- negative (LinϪ) ScaIϩ c-kithigh CD150Ϫ CD27ϩ Flk-2ϩ subset of newing hematopoietic stem cells (HSC)3 and are indis- bone marrow (BM) cells, and some fraction of them replenish all L pensable elements of the immune system. Much has been of the lymphocyte populations, including B cells (4). Knowl- learned about molecular mechanisms that regulate lymphocyte dif- edge about extracellular cues that direct early steps in lympho- ferentiation (1, 2). For example, it is now clear that lymphoid gene cyte formation is incomplete, but cytokines such as stem cell by guest on October 1, 2021 expression in progenitor cells is determined by levels and combi- factor and Flt-3 ligand are certainly important. Cell adhesion nations of key transcription factors cooperating or cross-competing molecules allow stem and progenitor cells to be in proximity to with each other in a hierarchy (3). HSC give rise to multipotent stromal cells that produce those factors, as well as ligands in the progenitors (MPP) that lack extensive self-renewal ability and extremely complex Notch and Wnt families that control many eventually segregate into the various blood cell lineages. Lym- differentiation events (5). phoid-primed MPP in the mouse represent a rare lineage marker- There is evidence that sex steroids play important regulatory roles in lymphocyte production. All cells in the lymphoid differ- entiation series decline dramatically in the BM of pregnant mice, whereas they are abnormally elevated in castrated male, ovariec- *Department of Hematology and Oncology and †Department of Cardiovascular Med- icine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; ‡Immu- tomized female, hypogonadal, and androgen receptor-deficient an- nobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma imals (6). Treatment with estrogen does not precisely simulate all City, OK 73104; and §Department of Immunobiology and Pharmacological Genetics, Graduate School of Medicine and Pharmaceutical Science for Research, University of of the pregnancy-related changes, but it restores B lymphopoiesis Toyama, Toyama City, Toyama, Japan in hypogonadal mice to the normal range and selectively depletes Received for publication March 5, 2008. Accepted for publication August 19, 2008. lymphoid progenitors in normal animals (7, 8). In fact, sensitivity The costs of publication of this article were defrayed in part by the payment of page to estrogen made it possible to identify primitive cells in the MPP charges. This article must therefore be hereby marked advertisement in accordance fraction that can generate T and B lymphocytes (9). These early with 18 U.S.C. Section 1734 solely to indicate this fact. lymphoid progenitors (ELP) are not homogenous, but express dif- 1 This work was partly supported by Grants AI20069 and AI058162 from the National Institutes of Health. ferent combinations of lymphoid genes, such as TdT and Rag1 (10). 2 Address correspondence and reprint requests to Dr. Takafumi Yokota, Department of Hematology and Oncology, Osaka University Graduate School of Medicine, C9, BM contains multiple cell types that could be hormone targets, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail address: yokotat@ accounting in part for the modest progress made in determining bldon.med.osaka-u.ac.jp exactly how sex steroids regulate lymphopoiesis. Lymphoid pro- 3 Abbreviations used in this paper: HSC, hematopoietic stem cell; BIO, 6-bro- moindirubin-37-oxime; BM, bone marrow; CLP, common lymphoid progenitor; genitors express estrogen and androgen receptors in a develop- CRD, cysteine-rich domain; ELP, early lymphoid progenitor; ER, estrogen recep- mental age-dependent manner (11). Furthermore, they are directly Ϫ ϩ tor; LSK, Lin ScaI c-kithigh; MC, mesenchymal cell; MPP, multipotent pro- sensitive to estrogen in stromal cell-free, serum-free cultures (12). genitor; MSC, mesenchymal stem cell; sFRP, soluble Frizzled-related protein. Transplantation studies with estrogen receptor (ER)-deficient mice Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 also implicated hematopoietic cells in this process (13). However, www.jimmunol.org 6062 ESTROGEN-INDUCIBLE sFRP1 INHIBITS LYMPHOPOIESIS other findings suggest that stromal cells can make suppressive fac- maceuticals. Recombinant sFRP1–4 and Wnt3a were obtained from R&D tors for lymphopoiesis when exposed to hormone (14). We have Systems. now identified one such substance as soluble Frizzled-related pro- Antibodies tein (sFRP)1. sFRPs are among several types of extracellular regulators for FITC anti-Mac1 (M1/70), Gr-1 (Ly-6G; RB6-8C5), erythroid (TER-119), CD8a (53-6.7), CD19 (1D3), CD45R/B220 (RA3/6B2), and CD34 Wnt signaling (15). The five members of this family contain a (RAM34) Abs; PE-anti-Mac1, CD45R/B220, CD19, c-kit (2B8), ScaI characteristic cysteine-rich domain (CRD) that shares 30–50% ho- (Ly6A/E; D7), and IL-7R␣ (SB/199.1) Abs; biotin anti-Mac1, Gr-1, mology with Frizzled for Wnt. Several studies have shown that CD45R/B220, erythroid, and CD3 (145-2C11) Abs were obtained from BD sFRPs can antagonize Wnt signaling by interrupting the binding of Biosciences. Allophycocyanin anti-CD45R/B220 and c-kit Abs were pur- Wnt to Frizzled (16, 17). However, it is not clear whether all chased from eBioscience. Purified anti-Mac1, Gr-1, erythroid, CD3 (145- 2C11), CD45RA (14.8), and CD19 Abs from BD Bioscience were used for sFRPs down-regulate Wnt signaling or how specific these interac- depleting Linϩ cells, followed by incubation with goat anti-rat IgG-coated tions are. There are at least 19 Wnt proteins that bind to 9 Frizzled magnetic beads (Miltenyi Biotec). A rabbit polyclonal Ab to sFRP1 or other receptors and exhibit their activities depending on receptor (ab4193) and an anti-␤-catenin Ab (14) were from Abcam and BD Bio- context (18). Furthermore, recent studies demonstrated that sFRPs sciences, respectively. can even function through Wnt-independent mechanisms (19, 20). Flow cytometry and cell sorting These complex observations make it extremely difficult to define the physiological role