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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gregorio-King, C. C. Articles by Kirkland, M. A. Search for Related Content PubMed PubMed Citation Articles by Gregorio-King, C. C. Articles by Kirkland, M. A. Related Collections Hematopoiesis and Stem Cells Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 October 2001, Vol. 98, No. 7, pp. 2279-2281 BRIEF REPORT ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells Claudia C. Gregorio-King, Gregory R. Collier, Janine S. McMillan, Caryll M. Waugh, Janet L. McLeod, Fiona M. Collier, and Mark A. Kirkland From the Stem Cell Laboratory, Douglas Hocking Research Institute, Barwon Health, The Geelong Hospital, and the Metabolic Research Unit, Deakin University, Geelong Victoria, Australia.     Abstract Top Abstract Introduction Study design Results and discussion References Using differential display polymerase chain reaction, a gene was identified in CD34+-enriched populations that had with low or absent expression in CD34 populations. The full coding sequence of this transcript was obtained, and the predicted protein has a high degree of homology to oxysterol-binding protein. This gene has been designated OSBP-related protein 3 (ORP-3). Expression of ORP-3 was found to be 3- to 4-fold higher in CD34+ cells than in CD34 cells. Additionally, expression of this gene was 2-fold higher in the more primitive subfraction of hematopoietic cells defined by the CD34+38 phenotype and was down-regulated with the proliferation and differentiation of CD34+ cells. The ORP-3 predicted protein contains an oxysterol-binding domain. Well-characterized proteins expressing this domain bind oxysterols in a dose-dependent fashion. Biologic activities of oxysterols include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, among them hematopoietic cells. Characterization and differential expression of ORP-3 implicates a possible role in the mediation of oxysterol effects on hematopoiesis. (Blood. 2001;98:2279-2281) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Molecular processes that maintain the stem cell pool and govern the proliferation and differentiation of hematopoietic stem and progenitor cells are widely investigated but incompletely understood. Identification of these genes will facilitate our understanding of hematopoiesis and may be used to improve clinical outcomes. Using differential display-polymerase chain reaction (dd-PCR), we identified a differentially expressed transcript in CD34-enriched populations, with low or absent expression in CD34-depleted populations. Sequencing of this transcript revealed significant homology to oxysterol-binding protein (OSBP). On the basis of ESTs in the GenBank database, a family of up to 8 human genes that code for OSBP-related proteins is postulated to exist.1 These have been designated OSBP-related proteins (ORPs). A partial sequence for our OSBP-related gene was thus designated ORP-3.1     Study design Top Abstract Introduction Study design Results and discussion References CD34+ purification Umbilical cord blood (UCB) samples were obtained after uncomplicated vaginal or cesarean delivery. Adult bone marrow (ABM) cells were obtained from cell scrapings of discarded rib and femur heads after cardiothoracic or hip-replacement surgery. All samples were donated by volunteers according to approved institutional guidelines. Mononuclear cells were prepared by density-gradient separation through Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden). CD34+ cells were isolated using a MiniMacs bead separation kit (Miltenyi Biotec, Becton Dickinson, Sunnyvale, CA). Flow cytometric analysis of CD34+ populations and fluorescence-activated cell sorting of CD34+ subsets CD34+ cells were labeled with anti-CD38-fluorescein isothiocyanate, anti-CD34-phycoerythrin, and anti-CD45-PECY5 (Coulter Immunotech, Fullerton, CA). CD34+ purity was determined by flow cytometric analysis (FacsCalibur, Becton Dickinson) following modified ISHAGE guidelines2 using CELLQuest software (Becton Dickinson). When required, CD34+38/dull cells were sorted using a FacsCalibur Cell Concentrator Sorting Module. Culture of umbilical cord blood CD34+ cells In selected experiments, isolated CD34+ cells were cultured for 1 week at 37°C in a humidified atmosphere flushed with 5% CO2 in air, at a concentration of 0.5 × 106 cells/mL in  minimum essential medium (Trace, Noble Park, Victoria, Australia) with 20% fetal bovine serum (CSL Biosciences, Parkville, Victoria, Australia), 2 mM L-glutamine, 200 U/mL penicillin-streptomycin (Sigma, St Louis, MO), 20 ng/mL recombinant human stem cell factor (Amgen, Thousand Oaks, CA), 10 ng/mL IL-1B (Endogen, Woburn, MA), 10 ng/mL IL-3, 10 ng/mL IL-6, and 10 ng/mL G-CSF (Amrad, Boronia, Victoria, Australia). dd-PCR RNA was extracted by RNeasy total RNA isolation kit (Qiagen, Clifton Hill, Victoria, Australia) and was DNase treated (Invitrogen, Carlsbad, CA). Reverse transcription and dd-PCR were carried out as described in the RNAimage Differential Display System (GenHunter, Nashville, TN). We used ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA) for all sequencing reactions, and sequences were determined using an ABI PRISM 373 DNA Sequencer (PE Applied Biosystems). mRNA extraction and rapid amplification of cDNA ends mRNA was extracted from UCB mononuclear cells using Oligotex Direct mRNA Kit (Qiagen). 5' and 3' Rapid amplification of cDNA ends (RACE) was carried out using the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA). All primers used in this study are presented in Table 1.                                View this table: [in this window] [in a new window]   Table 1. Primer and probe sequences Northern hybridization A PCR product spanning the ORP-3 coding region was amplified from UCB mononuclear cell cDNA in a standard PCR reaction and subcloned. The ORP-3 insert was isolated and labeled using [-32P]dATP and a Strip-Ez DNA probe synthesis kit (Ambion, Austin, TX). The labeled ORP-3 probe was hybridized to a human multiple tissue Northern blot (Clontech) with ULTRAhyb hybridization buffer (Ambion) and was visualized by autoradiography. Taqman real-time polymerase chain reaction Reverse transcription was carried out using the reverse transcription system (Promega, Madison, WI). Real-time PCR amplification of ORP-3 and -actin was carried out using Taqman Universal PCR Mastermix on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). Bioinformatics Nucleic acid and protein sequences were analyzed using software available from the National Center for Biotechnology Information and Swiss Institute of Bioinformatics databases.     Results and discussion Top Abstract Introduction Study design Results and discussion References Full ORP-3 mRNA of 6.631 kb was obtained using EST database searching and RACE technology (GenBank accession no. AY008372). The size of the complete ORP-3 cDNA has been confirmed by Northern hybridization with the presence of a transcript at approximately 7.0 kb (results not included). Our results also revealed the presence of 2 other transcripts at approximately 4.4 and 3.6 kb. These splice variants were expressed in a variety of normal human tissues and might have been produced as a result of posttranscriptional modification. Further investigation is required to determine their specific functions. We used Taqman real-time PCR to confirm the differential expression of ORP-3. Results indicate that the expression in CD34+ cells from UCB and ABM is 3- to 4-fold higher (respectively) than corresponding CD34 populations (Figure 1A). ORP-3 gene expression (normalized for the expression of -actin24) was also significantly higher in CD34+ cells from ABM than in equivalent cell populations from UCB. View larger version (24K): [in this window] [in a new window]   Figure 1. ORP-3 gene expression as measured by Taqman real-time PCR. All gene expression results are calculated relative to -actin and are expressed in arbitrary units (mean ± SEM).24 Wilcoxon signed-rank test was used to determine the statistical significance of the differences between related groups for nonparametric data. (A) ORP-3 gene expression in CD34+ () and CD34 () hematopoietic cells from UCB (6.3 ± 0.9, 2.0 ± 0.4, n = 21,*P  .0001) and ABM (11.2 ± 2.2, 2.6 ± 1.1, n = 14, #P  .001). Mean purity of the UCB and ABM CD34+-enriched populations were 57.5% ± 2.8% and 55.0% ± 4.8%, respectively. The percentage of CD34+ cells in the CD34 fractions was less than 0.01%. Average viability of the CD34+ and CD34 populations (more than 95%) did not vary significantly. ORP-3 gene expression did not correlate with sample purity (results not shown). (B) ORP-3 gene expression in CD34 (), CD34+ (), and CD34+ cells after 1-week culture () from UCB (6.2 ± 0.9, 2.5 ± 0.6, n = 14, *P  .01) and ABM (10.8 ± 3.5, 3.6 ± 1.1, n = 9, #P  .01). FACS analysis of the CD34+ and CD34+culture samples showed that after 1-week culture under the conditions described, the percentage of CD34+ cells decreased by approximately 33%. Total cell number increased by approximately 10%, and viability of the 2 populations (more than 90%) did not vary significantly. (C) ORP-3 gene expression in UCB CD34 (2.0 ± 0.5), CD34+CD38+ (8.0 ± 2.9), and CD34+CD38 hematopoietic cells (17.6 ± 3.8, n = 5, P  .04). CD34+38/dull cells were sorted using sort gates constructed to include approximately 10% of the total CD34+ population. Viability (greater than 80%) of the 2 populations did not vary significantly. The CD34+ population of cells is heterogeneous and contains multipotential and lineage-restricted cells.3,4 Therefore, we investigated ORP-3 gene expression in this population before and after 7-day culture in media containing growth factors that promote the differentiation and proliferation of hematopoietic progenitors.4,5 Our results indicate that under these culture conditions, ORP-3 gene expression in freshly isolated, uncultured CD34+ cells from UCB and ABM was 2- to 3-fold higher (respectively) than in these same cells after 7 days' culture (Figure 1B). Investigation of ORP-3 in the more primitive subset of cells defined by the CD34+38 immunophenotype6,7 indicates higher expression than in the more mature CD34+38+ subset (Figure 1C). Taken together, ORP-3 expression data indicate higher expression in a less differentiated subset of hematopoietic cells. The physiological significance of this differential expression and the function of ORP-3 in these cell populations remain to be elucidated. Translation of the ORP-3 open-reading frame yields an 887-amino acid protein that has a high degree of homology to OSBP. OSBP is a well-characterized and highly conserved protein that has been demonstrated to bind oxysterols in a dose-dependent fashion. ORP-3 and OSBP harbor the same modular domainsa C-terminal OSBP domain that is required for OS binding8 and an N-terminal pleckstrin homology domain9 that is required for translocation of the ligand protein to the Golgi apparatus.10,11 As do some OSBPs, ORP-3 contains a putative leucine zipper region.12 It must be noted however, that though leucine zipper regions may be associated with DNA binding,13 this has not been demonstrated in any OSBPs.12,14,15 To date, the complete coding sequence of only one human OSBP (OSBP-Hm) has been published; however, at least 8 other human ORP partial and complete gene sequences (designated ORP 1-8) exist on the GenBank database.1 The partial sequence for the human OSBP homologue, ORP-4, was identified by Fournier et al16 using dd-PCR as a screen to identify genes associated with metastatic potential. They designated the sequence HeLa metastatic gene (HLM). Northern blot analysis of ORP-4/HLM expression demonstrated that ORP-4/HLM expression is significantly associated with metastatic potential. OSBPs have also been identified in lower species, and their expression has been strongly associated with cell cycle progression.17,18 Given our observations of differential expression of ORP-3 in cells with different cell cycle profiles (such as CD34+CD38+ and CD34+CD38 cells),19 we are investigating the possibility that ORP-3 may be involved in cell cycle regulation. Interestingly, it has been hypothesized that levels of OSBP-Hm vary as a function of cell cycle.20 This finding has not been confirmed, nor has its significance been reported. Given the homology between ORP-3 and OSBP, it is likely that ORP-3 also plays a role in mediating oxysterol effects on cells. Oxysterols are hydroxylated derivatives of cholesterol that have been demonstrated to inhibit the transcription of many genes involved in cholesterol biosynthesis and cell replication and to be apoptotic to a variety of cell types.21-23 Additionally, investigations in our laboratory have demonstrated that oxysterols are potent inhibitors of HL60 and granulocyte macrophage-colony-forming unit cell growth and that they induce apoptosis in CD34+ cells (manuscript in preparation). Given the importance of these processes in hematopoiesis and the differential expression of ORP-3, we believe that characterization and investigation of the function of ORP-3 may provide useful insights into a possible regulatory role for oxysterols and their binding proteins in hematopoietic stem cell proliferation, differentiation, and self-renewal.     Acknowledgments We thank the medical and nursing staff of The Geelong Hospital and St John of God (Geelong, Victoria, Australia) for the collection of UCB and ABM samples.     Footnotes Submitted March 13, 2001; accepted June 7, 2001. Supported in part by the Australian Red Cross Blood Service. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Claudia Gregorio-King, Stem Cell Laboratory, Douglas Hocking Research Institute, Barwon Health, The Geelong Hospital, Deakin University, Geelong 3220, Victoria, Australia; e-mail: ccgk{at}deakin.edu.au.     References Top Abstract Introduction Study design Results and discussion References 1. Laitinen S, Olkkonen VM, Ehnholm C, Ikonen E. Family of human oxysterol binding protein (OSBP) homologues: a novel member implicated in brain sterol metabolism. J Lipid Res. 1999;40:2204-2211[Abstract/Free Full Text]. 2. Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34+ cell determination by flow cytometry: International Society of Hematotherapy and Graft Engineering. J Hematother. 1996;5:213-226[Medline] [Order article via Infotrieve]. 3. Morrison SJ, Uchida N, Weissman IL. The biology of hematopoietic stem cells. Annu Rev Cell Dev Biol. 1995;11:35-71[CrossRef][Medline] [Order article via Infotrieve]. 4. Graham GJ, Wright EG. Haemopoietic stem cells: their heterogeneity and regulation. Int J Exp Pathol. 1997;78:197-218[CrossRef][Medline] [Order article via Infotrieve]. 5. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood. 1993;81:2844-2853[Abstract/Free Full Text]. 6. Cairo MS, Wagner JE. Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation. Blood. 1997;90:4665-4678[Free Full Text]. 7. Xiao M, Dooley DC. Cellular and molecular aspects of human CD34+ CD38 precursors: analysis of a primitive hematopoietic population. Leuk Lymphoma. 2000;38:489-497[Medline] [Order article via Infotrieve]. 8. Ridgway ND, Dawson PA, Ho YK, Brown MS, Goldstein JL. Translocation of oxysterol binding protein to Golgi apparatus triggered by ligand binding. J Cell Biol. 1992;116:307-319[Abstract/Free Full Text]. 9. Saraste M, Hyvonen M. Pleckstrin homology domains: a fact file. Curr Opin Struct Biol. 1995;5:403-408[CrossRef][Medline] [Order article via Infotrieve]. 10. Lagace TA, Byers DM, Cook HW, Ridgway ND. Altered regulation of cholesterol and cholesteryl ester synthesis in Chinese-hamster ovary cells overexpressing the oxysterol-binding protein is dependent on the pleckstrin homology domain. Biochem J. 1997;326:205-213. 11. Levine TP, Munro S. The pleckstrin homology domain of oxysterol-binding protein recognises a determinant specific to Golgi membranes. Curr Biol. 1998;8:729-739[CrossRef][Medline] [Order article via Infotrieve]. 12. Dawson PA, Ridgway ND, Slaughter CA, Brown MS, Goldstein JL. cDNA cloning and expression of oxysterol-binding protein, an oligomer with a potential leucine zipper. J Biol Chem. 1989;264:16798-16803[Abstract/Free Full Text]. 13. Landschulz WH, Johnson PF, McKnight SL. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988;240:1759-1764[Abstract/Free Full Text]. 14. Taylor FR, Shown EP, Thompson EB, Kandutsch AA. Purification, subunit structure, and DNA binding properties of the mouse oxysterol receptor. J Biol Chem. 1989;264:18433-18439[Abstract/Free Full Text]. 15. Taylor FR. Correlation among oxysterol potencies in the regulation of the degradation of 3-hydroxy-3-methylglutaryl CoA reductase, the repression of 3-hydroxy-3-methylglutaryl CoA synthase and affinities for the oxysterol receptor. Biochem Biophys Res Commun. 1992;186:182-189[CrossRef][Medline] [Order article via Infotrieve]. 16. Fournier MV, Guimaraes FC, Paschoal ME, Ronco LV, Carvalho MG, Pardee AB. Identification of a gene encoding a human oxysterol-binding protein-homologue: a potential general molecular marker for blood dissemination of solid tumors. Cancer Res. 1999;59:3748-3753[Abstract/Free Full Text]. 17. Alphey L, Jimenez J, Glover D. A Drosophila homologue of oxysterol binding protein (OSBP)implications for the role of OSBP. Biochim Biophys Acta. 1998;1395:159-164[Medline] [Order article via Infotrieve]. 18. Hull CM, Johnson AD. Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans. Science. 1999;285:1271-1275[Abstract/Free Full Text]. 19. Reems JA, Torok-Storb B. Cell cycle and functional differences between CD34+/CD38hi and CD34+/38lo human marrow cells after in vitro cytokine exposure. Blood. 1995;85:1480-1487[Abstract/Free Full Text]. 20. Beseme F, Astruc ME, Defay R, Descomps B, Crastes de Paulet A. Characterization of oxysterol-binding protein in rat embryo fibroblasts and variations as a function of the cell cycle. Biochim Biophys Acta. 1986;886:96-108[Medline] [Order article via Infotrieve]. 21. Smith LL, Johnson BH. Biological activities of oxysterols. Free Radic Biol Med. 1989;7:285-332[CrossRef][Medline] [Order article via Infotrieve]. 22. Thompson EB, Ayala-Torres S. Oxysterols and apoptosis: evidence for gene regulation outside the cholesterol pathway. Crit Rev Biochem Mol Biol. 1999;34:25-32[CrossRef][Medline] [Order article via Infotrieve]. 23. Schroepfer GJ Jr. Oxysterols: modulators of cholesterol metabolism and other processes. Physiol Rev. 2000;80:361-554[Abstract/Free Full Text]. 24. Applied Biosystems. Relative quantitation of gene expression. ABI PRISM 7700 Sequence Detection System: User Bulletin 2. Foster City, CA: Author; 1997:11-16. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gregorio-King, C. C. Articles by Kirkland, M. 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	Copyright © 2001 by American Society of Hematology         Online ISSN: 1528-0020