Osteopontin Regulates Actin Cytoskeleton and Contributes to Cell Proliferation in Primary Erythroblasts

Erythropoietin and stem cell factor are the key cytokines that regulate early stages of erythroid differentiation. However, it remains undetermined whether additional cytokines also play a role in the differentiation program. Here, we report that osteopontin (OPN) is highly expressed and secreted by erythroblasts during differentiation. We also demonstrate that OPN-deficient human and mouse erythroblasts exhibit defects in F-actin filaments, and addition of exogenous OPN to OPN-deficient erythroblasts restored the F-actin filaments in these cells. Furthermore, our studies demonstrate that OPN contributes to erythroblast proliferation. OPN knock-out male mice exhibit lower hematocrit and hemoglobin levels compared with their wild-type counterparts. We also show that OPN mediates phosphorylation or activation of multiple proteins including Rac-1 GTPase and the actin-binding protein, adducin, in human erythroblasts. In addition, we show that the OPN effects include regulation of intracellular calcium in human erythroblasts. Finally, we demonstrate that human erythroblasts express CD44 and integrins β1 and α4, three known receptors for OPN, and that the integrin β1 receptor is involved in transmitting the proliferative signal. Together these results provide evidence for signal transduction by OPN and contribution to multiple functions during the erythroid differentiation program in human and mouse. Erythropoietin and stem cell factor are the key cytokines that regulate early stages of erythroid differentiation. However, it remains undetermined whether additional cytokines also play a role in the differentiation program. Here, we report that osteopontin (OPN) is highly expressed and secreted by erythroblasts during differentiation. We also demonstrate that OPN-deficient human and mouse erythroblasts exhibit defects in F-actin filaments, and addition of exogenous OPN to OPN-deficient erythroblasts restored the F-actin filaments in these cells. Furthermore, our studies demonstrate that OPN contributes to erythroblast proliferation. OPN knock-out male mice exhibit lower hematocrit and hemoglobin levels compared with their wild-type counterparts. We also show that OPN mediates phosphorylation or activation of multiple proteins including Rac-1 GTPase and the actin-binding protein, adducin, in human erythroblasts. In addition, we show that the OPN effects include regulation of intracellular calcium in human erythroblasts. Finally, we demonstrate that human erythroblasts express CD44 and integrins β1 and α4, three known receptors for OPN, and that the integrin β1 receptor is involved in transmitting the proliferative signal. Together these results provide evidence for signal transduction by OPN and contribution to multiple functions during the erythroid differentiation program in human and mouse. Early stages of erythroid cell differentiation are regulated by multiple growth factors including interleukin-3, erythropoietin (EPO), 4The abbreviations used are:EPOerythropoietinMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideWTwild typePBSphosphate-buffered salineOPNosteopontinSCFstem cell factorqPCRquantitative PCRHcthematocritGlyAglycophorin AHbhemoglobinELISAenzyme-linked immunosorbent assaysiRNAsmall interfering RNA.4The abbreviations used are:EPOerythropoietinMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideWTwild typePBSphosphate-buffered salineOPNosteopontinSCFstem cell factorqPCRquantitative PCRHcthematocritGlyAglycophorin AHbhemoglobinELISAenzyme-linked immunosorbent assaysiRNAsmall interfering RNA. and stem cell factor (SCF) (1Koury M.J. Sawyer S.T. Brandt S.J. Curr. Opin. Hematol. 2002; 9: 93-100Crossref PubMed Scopus (155) Google Scholar, 2Kelley L.L. Koury M.J. Bondurant M.C. Koury S.T. Sawyer S.T. Wickrema A. Blood. 1993; 82: 2340-2352Crossref PubMed Google Scholar). EPO and SCF have distinct functions. The predominant role of EPO is to deliver survival signals and maintain cell viability (3Koury M.J. Bondurant M.C. Science. 1990; 248: 378-381Crossref PubMed Scopus (719) Google Scholar, 4Muta K. Krantz S.B. Bondurant M.C. Wickrema A. J. Clin. Investig. 1994; 94: 34-43Crossref PubMed Scopus (153) Google Scholar), whereas SCF provides signals for cell proliferation (4Muta K. Krantz S.B. Bondurant M.C. Wickrema A. J. Clin. Investig. 1994; 94: 34-43Crossref PubMed Scopus (153) Google Scholar, 5Muta K. Krantz S.B. Bondurant M.C. Dai C.H. Blood. 1995; 86: 572-580Crossref PubMed Google Scholar, 6Jacobs-Helber S.M. Penta K. Sun Z. Lawson A. Sawyer S.T. J. Biol. Chem. 1997; 272: 6850-6853Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Together these two growth factors guide the erythroid differentiation program from the early basophilic stage through the late polychromatic stage of maturation. However, the effects of these cytokines explain only the early stages of erythropoiesis. We were interested in identifying additional cytokines and/or factors involved in the erythroid differentiation program, especially factors that regulate the remodeling of the cytoskeleton. To achieve these objectives we developed methods to obtain extremely pure primary erythroblasts that synchronously differentiate into reticulocytes. Utilizing these cells, we screened a cDNA microarray and identified OPN as one of the cytokines that is highly expressed by erythroblasts during differentiation. erythropoietin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide wild type phosphate-buffered saline osteopontin stem cell factor quantitative PCR hematocrit glycophorin A hemoglobin enzyme-linked immunosorbent assay small interfering RNA. erythropoietin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide wild type phosphate-buffered saline osteopontin stem cell factor quantitative PCR hematocrit glycophorin A hemoglobin enzyme-linked immunosorbent assay small interfering RNA. OPN is a multifunctional cytokine that is highly expressed during bone remodeling and has pro-inflammatory effects (7Koury S.T. Koury M.J. Bondurant M.C. J. Cell Biol. 1989; 109: 3005-3013Crossref PubMed Scopus (114) Google Scholar, 8Ashkar S. Weber G.F. Panoutsakopoulou V. Sanchirico M.E. Jansson M. Zawaideh S. Rittling S.R. Denhardt D.T. Glimcher M.J. Cantor H. Science. 2000; 287: 860-864Crossref PubMed Scopus (964) Google Scholar, 9Denhardt D.T. Guo X. Faseb. J. 1993; 7: 1475-1482Crossref PubMed Scopus (1010) Google Scholar, 10Denhardt D.T. Noda M. O'Regan A.W. Pavlin D. Berman J.S. J. Clin. Investig. 2001; 107: 1055-1061Crossref PubMed Scopus (915) Google Scholar, 11Rittling S.R. Denhardt D.T. Exp. Nephrol. 1999; 7: 103-113Crossref PubMed Google Scholar, 12Razzouk S. Brunn J.C. Qin C. Tye C.E. Goldberg H.A. Butler W.T. Bone. 2002; 30: 40-47Crossref PubMed Scopus (72) Google Scholar). OPN has anti-apoptotic, chemotactic, and proliferative properties, depending on the cell type and context. It also plays a vital role in the delayed-type immune response and is known to be secreted by activated T cells and macrophages (13Denhardt D.T. Giachelli C.M. Rittling S.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 723-749Crossref PubMed Scopus (307) Google Scholar). OPN knock-out mice are viable and live a normal life span but suffer from bone defects and problems with wound and fracture healing (14Duvall C.L. Taylor W.R. Weiss D. Wojtowicz A.M. Guldberg R.E. J. Bone Miner Res. 2007; 22: 286-297Crossref PubMed Scopus (167) Google Scholar). To date, OPN has not been shown to be expressed by erythroblasts, nor has it been implicated in functions associated with erythroid cell maturation. Here, we demonstrate that OPN is expressed by erythroblasts and contributes to the regulation of actin cytoskeleton and proliferation. We also demonstrate that stimulation of erythroblast cells by OPN induces activation and/or phosphorylation of Rac-1 GTPase and other intracellular proteins, including efflux of intracellular calcium. Finally, our studies show that OPN receptors CD44 and several integrins are expressed in these cells and suggest that integrin β1 is responsible for transmitting the proliferative signal. Collectively, our data define a cytokine important in the regulation of multiple functions during the erythroid differentiation program. Antibodies and Reagents—Initial microarray studies that identified OPN expression by erythroblasts were carried out by Memorec Biotec Inc. in Cologne, Germany (a Miltenyi Biotec company). Both fluorochrome-conjugated and non-conjugated glycophorin A (GlyA) antibodies were purchased from BD Biosciences, Inc. The transferrin receptor (CD71) antibody was purchased from Beckman Coulter, Inc. Recombinant OPN and recombinant SCF were from R & D, Inc. The anti-phospho adducin Ser-724 antibody (cat. no. 05-587), which also recognizes phospho adducin Ser-726 in human cells, was purchased from Upstate Biotechnology, Inc. The anti-phosphothreonine (cat. no. 71-8200)-specific antibody was purchased from Zymed Laboratories Inc. The anti-OPN antibody used for immunoblot analysis and immunofluorescence was from R & D, Inc. Fluorescently labeled phalloidin was purchased from Molecular Probes. Fluo-3/AM was purchased from VWR International. The A23187 calcium ionophore was purchased from Calbiochem, Inc. The Rac inhibitor, NSC23766 (cat. no. 553502) was purchased from Calbiochem Inc. Primary Human Erythroid Cultures and Flow Cytometry—Human primary erythroblasts were generated by culturing CD34+ early hematopoietic progenitors initially isolated from growth factor-mobilized peripheral blood (purchased from ALL Cells, Inc.) using an Isolex 300i cell selection device. The culture contained 15% fetal bovine serum, 15% human serum, Isocove's modified Dulbecco's medium (IMDM), 10 ng/ml interleukin-3, 2 units/ml EPO, and 50 ng/ml SCF. During the initial 8 days of culture, cells were fed on days 3 and 6 by adding equal volumes of fresh culture media supplemented with growth factors. However, no new interleukin-3 was added after the initial addition on day 0, and the amount of SCF added to the fresh media was gradually decreased at each feeding day 10 day 2 The amount of EPO added was 2 units/ml during each day 8 of culture, cells were by for or cells using a The of the isolated by was cells were in the media as with EPO and the of SCF was to 2 were fed one on day 10 of culture by adding equal volumes of fresh media with only EPO during were at during the culture and with and as to the differentiation program A. Krantz S.B. J.C. Bondurant M.C. Blood. PubMed Google Scholar). Bone erythroblasts were isolated by selection of cells from cells from bone of cells were used for PCR PCR was using PCR Inc.) on the using OPN and PCR were as for by of and The analysis was by the from to at and the at the levels of the in each were by the and the of in each OPN and erythroblast cells on day or 10 of culture were and with media to secreted OPN from the culture were in media bovine for to stimulation with analysis was as A. S. A. S. Sawyer S.T. K. M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). for OPN was using medium from cells been at a of days of culture, the medium was and used in the an OPN was using a to the and of from no. were purchased from The The of mice was by PCR of using as by the mice were purchased from The as was by the of and To obtain bone cells, mouse and were with phosphate-buffered saline and through a to obtain a cell To erythroblasts, the cells were with antibodies and using a cell or for using the selection and/or cells were and with and for and on for immunofluorescence analysis to F-actin In the the of OPN on F-actin cytoskeleton was erythroblasts were in bovine serum, and EPO for in the or of OPN to immunofluorescence analysis for a mouse erythroblasts were also but In for mouse blood was of mouse blood were from the mouse was used to for blood 3 was on human and mouse cells after and cells on were in in for and to with fetal bovine in for at In OPN was in human erythroblasts, cells were with anti-OPN antibody in fetal bovine and at for and in were with antibody at for in the as with antibody was at for were in the the of antibodies at for were in the were to and were with medium In the of F-actin was human and mouse erythroblasts were using phalloidin to the were using a a 6 erythroblasts or day cells were at a of cells in of growth medium in a In a of medium and 3 of the were and at for of or OPN Inc.) were added and at for The was added to the cells. The was and for The of expression was by PCR or The of OPN expression was for to Rac-1 in culture were in fresh medium for 2 with or the Rac to stimulation with of were with in the of at for in a The were for Rac-1 by immunoblot analysis using an 10 human erythroblasts were in medium at and the medium was were three in 2 10 and at and of cells were added to of in a The was with to of 2 were added to the The was at for with additional of 2 were added to each The was at for an additional with The was at for at The was in 10 bovine and at for at The was to in and at for at The was The cells were in 2 of and at for to of cells were added to of in a were at with no OPN, or A was with OPN for 3 and at with cells were at and the was at using a 2 were for each of for OPN 10 erythroblasts were used to the cell expression of integrins α4, and with glycophorin A by Antibodies integrin β1 integrin and integrin were from Inc. The antibody was purchased from R & Inc. The antibody integrin was from Inc. blood cells and cells were used as for receptors that not show expression in erythroblasts. Cell the proliferation effects of OPN were mouse erythroblasts from and mice were for to the of proliferation. In the the of antibodies on cell proliferation was day human erythroblasts were for in the or of for each of the antibodies in cell culture contained which is used as an in Cell proliferation was by bromide assay to the OPN by Human obtain an extremely pure erythroid cell that in a we used a primary human erythroid cell culture which was modified from our S. J. J. D. Wickrema A. S. A. PubMed Scopus Google Scholar). We initially early hematopoietic cells that and differentiation to the erythroid During the of in culture, we a highly erythroblast by for and cells cells were for the We also the of other and cell after by analysis not cells were for days for differentiation to were at several during culture and for hemoglobin to differentiation The OPN expression was using for OPN at the stages of the differentiation cells polychromatic erythroblasts erythroblasts and late stage erythroblasts for OPN were in early hematopoietic cells and at stages of but the levels were at on day of culture To the that OPN is expressed by erythroblasts as a of growth factor of our we also erythroblasts isolated from bone of studies that OPN is expressed by bone erythroblasts, to a in to peripheral cells We OPN expression by immunoblot analysis using cell at during The OPN was at stages of erythroid differentiation The two cell and which were used as not express We the immunoblot antibody to equal and with antibodies 3 to differentiation. 3 is an erythroid protein, which late during the differentiation program, and is an of erythroid differentiation. and of OPN is a secreted we whether erythroblasts OPN during their differentiation OPN was by in culture medium of erythroblasts The of OPN was especially days 8 and of culture, during which these cells hemoglobin as as several other cytoskeleton proteins A. Koury S.T. Dai C.H. Krantz S.B. J. 1994; PubMed Scopus Google Scholar). OPN during the late stages of erythroid differentiation and/or functions We the intracellular of OPN in erythroblasts on day 10 of labeled antibodies OPN and we the of OPN in cells that OPN is expressed by erythroid progenitors OPN a in cells and was the whereas was to the cell antibody was used as a which not with erythroblast cells OPN F-actin and the of OPN we OPN expression in human erythroblasts by cells with a of OPN, we were to the expression of OPN at the and levels A and OPN F-actin in the we the of OPN on F-actin during erythroid differentiation. In erythroblasts, F-actin is in the of the cells. The of OPN in a of the and of F-actin in erythroblasts as by using The of OPN in human erythroblasts in the of actin filaments in the of the cells and the of the which was the We erythroblasts from the bone of and which that in were no F-actin filaments the compared with In these cells, F-actin the of cells Furthermore, we the erythroblasts with recombinant mouse OPN, we were to the F-actin filaments in the of the cells The in actin filaments were only at of to the expression in the cell by the responsible for the regulation of actin by OPN in erythroblasts, we whether Rac-1 a known of actin is activated by Rac-1 to the of and to the Rac-1 activation by V. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We Rac-1 activation in response to OPN by a Rac-1 assay on cell from human erythroblasts. by exogenous OPN in a in Rac-1 to compared with the of cells with a Rac GTPase to OPN stimulation the of that Rac-1 GTPase is a of OPN We on adducin, an actin-binding that plays an important role in actin regulation in the erythroid cytoskeleton. of adducin are in the erythroblast cytoskeleton and have phosphorylation that are for multiple A and are known to adducin V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), phosphorylation of adducin is not To OPN stimulation to adducin cell were after OPN stimulation or for with of and with an that OPN stimulation to phosphorylation of and/or in a and that phosphorylation as by the of the and To whether other proteins are also in response to OPN, we used anti-phosphothreonine antibody to phosphorylation in erythroblasts. that at three other proteins were at the of OPN as a signal in erythroid cells The of these proteins were and The of phosphorylation of proteins a of phosphorylation and after that the OPN on erythroblasts in of signal transduction is by OPN has been to regulation of in we used a assay to the role of OPN in the regulation of intracellular OPN stimulation of erythroblasts efflux of from the cells, as by decreased compared with that were not by OPN a cells were with a which a in to The of OPN in cells in a of compared with the calcium as a OPN stimulation of erythroblasts induces in or calcium in OPN-deficient whether are in OPN knock-out mice the blood of mice and wild-type mice we that and levels were lower in male mice compared with the wild-type were in knock-out The of and wild-type mouse were not nor were in the blood cell and were normal and blood normal not We also the levels and in male two factors that are associated with and that and levels were to the mice not in wild-type and OPN-deficient at at at at in a new of OPN in Human of the effects of OPN on erythroblasts, we the expression of known OPN receptors in human erythroblasts. The receptors we were integrins α4, and 10 human erythroblasts were with antibodies each of these receptor proteins with and expression levels were by α4, and CD44 but not integrins and were expressed on day 10 erythroblasts A and and cells were used as in these OPN to but to and the role of OPN in cell or of To whether OPN erythroblast proliferation we bone erythroblasts from and mice for in and an cell proliferation We also a from mice in the of added The OPN cells at a the early erythroblast cells from mice during the culture The addition of exogenous OPN to cells restored the proliferation to the of cells that OPN-deficient cells are to to exogenous OPN to the as cells OPN We also whether OPN contributes to erythroblast differentiation and/or of To whether OPN contributes to erythroblast we the erythroblasts from and mice for and the levels of by expression is highly during erythroid differentiation. We not in the of differentiation and cells that OPN not play a role in erythroblast differentiation not Finally, we whether OPN contributes to erythroblast survival using human erythroblast cells. We day 8 erythroblasts for with or EPO but added OPN into these to whether OPN provide by the of of these cells by and for that OPN not provide not β1 the Cell in Human the receptors responsible for the in cell we carried out cell proliferation assay on human erythroblast cells after culturing for with antibodies integrin α4, and the three receptors that were expressed on these cells. We antibody as a is not expressed in erythroblasts. data that only to the integrin β1 antibody the proliferation of erythroblasts on these of the integrin β1 by OPN, to to the proliferative signal in erythroblasts, studies whether OPN receptor in erythroblasts. We the regulation of using a culture of pure erythroblasts with maturation. was in that erythroblasts not cells as and which are known to and cytokines including osteopontin (13Denhardt D.T. Giachelli C.M. Rittling S.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 723-749Crossref PubMed Scopus (307) Google Scholar). these cells, we that OPN is expressed erythroid differentiation and were to role in erythroid cell OPN was expressed in cells, at lower levels compared with cells that were at late stages of the differentiation program. Furthermore, we that OPN expression was not to growth factor as OPN are also in bone erythroid OPN expression days 6 and of culture, to the of but cells to OPN at late stages of differentiation. We that OPN is in polychromatic cells by OPN in cells. studies have shown that OPN is secreted by several hematopoietic cell as and cells in the of and immune (13Denhardt D.T. Giachelli C.M. Rittling S.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 723-749Crossref PubMed Scopus (307) Google Scholar, M. C. Blood. PubMed Scopus Google Scholar). To our is the report expression and of OPN by a hematopoietic cell type not associated with an of the role of F-actin in the cytoskeleton and role in bone we whether OPN plays a role in the regulation of F-actin in erythroblast cells. We and of actin in human OPN and mouse OPN knock-out erythroblasts compared with and wild-type cells. In erythroblasts, F-actin from the and the addition of OPN to mouse erythroblasts and of F-actin the OPN role in the regulation of the actin cytoskeleton and the of cells to to we that OPN plays an important role in the regulation of actin in erythroid and proteins are of the involved in the regulation of the actin cytoskeleton in multiple cell M. Curr. Opin. Cell Biol. 2002; PubMed Scopus Google Scholar, S. C.H. Blood. PubMed Scopus Google Scholar). studies have shown that or are of phosphorylation of adducin in but the role of cytokines in phosphorylation has not been V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that activation of Rac-1 GTPase and phosphorylation of the actin-binding protein, adducin, are by It has been shown that EPO Rac-1 in the EPO cell and in the cell but these effects have not been in primary erythroid progenitors A. 2002; PubMed Scopus Google Scholar). In addition to phosphorylation of adducin and activation of we also that at three other proteins and are at in response to OPN in human erythroid The at is it has been shown that adducin is also at in other cell V. K. J. Cell Biol. 1999; PubMed Scopus Google Scholar). The other two proteins to be on the these proteins not to be or which have been shown to be of OPN in cells D.T. S.M. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In additional we that erythroid progenitors efflux in response to exogenous results are in with a by A. J. A. M. S. A. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar), a of intracellular in in response to OPN the in to the activation of the responsible for efflux is regulated by and A in other cell it is that a is involved in erythroid it was that mice Rac-1 and have defects in the F-actin cytoskeleton to is not with the of only Rac-1 S. C.H. Blood. PubMed Scopus Google Scholar). We compared the blood of wild-type mice with OPN knock-out mice and that have lower hematocrit and hemoglobin levels compared with their wild-type counterparts. the OPN knock-out mice not exhibit a in or compared with the wild-type was no in the OPN knock-out and the wild-type male no defects as or other were in OPN knock-out mice in to in knock-out mice S. C.H. Blood. PubMed Scopus Google Scholar). However, our data that OPN contributes to erythroblast proliferation in explain the for lower and in mice compared with the SCF is for erythroblast the data in our suggest that additional cytokines as OPN also to cell in the erythroid In additional we the expression of several receptors that have been shown to with OPN and deliver including cell and (13Denhardt D.T. Giachelli C.M. Rittling S.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 723-749Crossref PubMed Scopus (307) Google Scholar). in were identified as OPN receptors of their through the in receptors as CD44 and also have been shown to OPN through G.F. S. Glimcher M.J. Cantor H. Science. PubMed Scopus Google Scholar, J. H. H. K. S. H. K. M. Res. 1999; Google Scholar). studies that integrins α4, and CD44 receptors are highly expressed in erythroblasts, and integrin β1 is important for cell proliferation. studies have that OPN cell proliferation in addition to cell survival in several hematopoietic cell as cell and mouse bone cells through the of CD44 S.T. Biol. 2000; PubMed Scopus Google Scholar). on our OPN not to have a role in cell EPO, which is for of cell viability in erythroblasts. studies whether CD44 and integrin receptors OPN in erythroblasts and their role in signal transduction and expression in In our studies erythroblasts as a of OPN We also that OPN multiple in erythroblasts, and plays an important role in F-actin A of OPN is lower and in male mice in addition to decreased cell proliferation in mouse erythroblasts. Finally, we that the of OPN on cell proliferation is through the integrin actin plays a role in cell and in with integrin receptors it is that OPN as a to erythroblasts and the bone by the of into the peripheral studies are to the functions of OPN in the of erythroid and blood the bone We for the We and for the mouse We are also to and for and the of the of and for