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• W2038658082 abstract "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 3, 25 ng/ml; day 6, 10 ng/ml; day 8, 2 ng/ml). The amount of EPO added was 2 units/ml during each feeding. On day 8 of culture, cells were further purified by flow cytometry sorting for Gly A/CD71 or CD71 cells using a MoFlo high speed flow cytometer. The purity of the population isolated by this method was 98-99%. Sorted cells were cultured in the same media as before with EPO and SCF, except the concentration of SCF was reduced to 2 ng/ml. Cells were fed one more time on day 10 of culture by adding equal volumes of fresh media with only EPO (2 units/ml) during this final feeding. Cells were collected at various time points during the culture and stained with benzidine and hematoxylin, as described previously, to monitor the differentiation program (15Wickrema A. Krantz S.B. Winkelmann J.C. Bondurant M.C. Blood. 1992; 80: 1940-1949Crossref PubMed Google Scholar). Bone marrow-derived erythroblasts were isolated by selection of CD71-positive cells from mononuclear cells obtained from bone marrow aspirates of volunteer donors. CD71-positive cells were cultured until used for qPCR analysis. PCR Amplification—Real-time PCR was performed using QuantiTect SYBR Green PCR kit (Qiagen, Inc.) on the MyiQ instrument (Bio-Rad) using OPN and 18 S ribosomal RNA gene primers (OPN: forward, 5′-TTGCAGTGATTTGCTTTTGC-3′; reverse, 5′-GTCATGGCTTTCGTTGGACT-3′; 18s: forward, 5′-ATGCCCGTTCTTAGTTGGTG-3′; reverse, 5′-CGCTGAGCCAGTCAGTGTAG-3′). PCR conditions were as follows: 95 °C for 15 min followed by 40 cycles of 94 °C, 15 s, 55 °C, 30 s, and 72 °C, 30 s. The melting curve analysis was performed by increasing the temperature from 55 to 95 °C at 0.5 °C/10 s and measuring the fluorescence intensity at the interval. Relative RNA levels of the target gene in each sample were determined by the ΔΔCT method and normalized against the RNA level of 18 S in each sample. OPN Stimulation, Immunoblot Analysis, and ELISA—Primary erythroblast cells on day 9 or 10 of culture were collected and washed twice with IMDM media to eliminate any secreted OPN from the culture media. Cells were then incubated in serum-free media (IMDM/1% bovine serum albumin) for 4.5 h prior to stimulation with OPN. Immunoblot analysis was performed as described previously (16Wickrema A. Uddin S. Sharma A. Chen F. Alsayed Y. Ahmad S. Sawyer S.T. Krystal G. Yi T. Nishada K. Hibi M. Hirano T. Platanias L.C. J. Biol. Chem. 1999; 274: 24469-24474Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). An ELISA for OPN was performed using medium collected from cultures where cells had been seeded at a concentration of 1 × 106/ml. After 3-4 days of culture, the medium was collected and subsequently used in the ELISA assay. Also, an OPN ELISA was performed using a commercially available kit according to the manufacturer's instructions (Assay Design, Inc.). Isolation and Culture of Mouse Erythroblasts from OPN-/- Mice—Mice (B6.Cg-Spp1tm1Blh/J, cat. no. 004936) were purchased from The Jackson Laboratory. The genotype of mice was confirmed by PCR of tail DNA using primers, as suggested by the Jackson Laboratory. Pure C57BL/6 mice (WT) were purchased from The Jackson Laboratory as experimental controls. All animal research was approved by the University of Chicago Institutional Animal Care and Use Committees. To obtain bone marrow cells, mouse femurs and tibiae were flushed with phosphate-buffered saline (PBS) containing citrate and passaged through a 20-gauge syringe to obtain a single cell suspension. To isolate erythroblasts, the cells were stained with PE-conjugated Ter119 antibodies and sorted using a MoFlo-HTS cell sorter (Dako Cytomation) or selected for CD71 using the EasySep magnetic selection method. Selected and/or sorted cells were cytospun onto slides and stained with benzidine and hematoxylin for morphological evaluation and immobilized on Alcian Blue (Sigma)-treated coverslips for immunofluorescence analysis to evaluate F-actin distribution. In the experiment where the effect of OPN on F-actin cytoskeleton was determined, CD71-positive erythroblasts were cultured in 30% bovine serum, 0.1 mmol/liter α-thioglycerol, IMDM, and 1 unit /ml EPO for 16 h in the presence or absence of 1 μg/ml OPN prior to immunofluorescence analysis for F-actin. As a control, WT mouse erythroblasts were also cultured under similar conditions but without OPN. In experiments where Complete Blood Count (CBC) for mouse blood was obtained, about 20 μl of mouse blood were collected from the mouse tail vein. HEMVET 850 was used to determine CBC for blood samples within 3 h. Immunofluorescence Microscopy—Immunofluorescence microscopy was performed on human and mouse cells after immobilizing and fixing cells on Alcian Blue-treated coverslips. Cells were permeabilized in 0.1% Triton X-100 in PBS for 5 min and washed 5 min prior to blocking with 10% fetal bovine serum in PBS for 45 min at room temperature. In experiments where OPN was localized in human erythroblasts, cells were incubated with anti-OPN antibody diluted in PBS containing 1% fetal bovine serum and 0.01% Triton X-100 at 37 °C for 1 h, and washed in PBS containing 0.01% Triton X-100. Cells were then incubated with Alexa Fluor-conjugated anti-goat IgG antibody at room temperature for 45 min before washing in the same buffer as before. Incubation with anti-GlyA antibody was performed at room temperature for 1 h. Cells were washed in the same buffer before the incubation of Alexa Fluor-conjugated anti-mouse IgG antibodies at room temperature for 30 min. Cells were washed in PBS, the coverglasses were allowed to air-dry, and were mounted with Prolong Gold mounting medium (Invitrogen). In experiments where the localization of F-actin was performed, both human and mouse erythroblasts were stained using Texas Red-conjugated phalloidin according to the manufacturer's suggested procedure (Molecular Probes). All photographs were taken using a Leica SP2 AOBS spectral confocal microscope under a ×63 oil immersion lens. siRNA Transfection—Day 6 erythroblasts (basophilic) or day 9 GlyA/CD71 cells (polychromatic) were plated at a density of 1 × 106 cells in 0.5 ml of growth medium per well in a 12-well plate. In a 1.5-ml tube, 100 μl of serum-free medium and 3 μl of the TransIT-siQUEST transfection reagent (Mirus, Bio Corp.) were mixed and incubated at room temperature for 20 min. 50-100 nm of either control siRNA or OPN siRNA (Dharmacon, Inc.) were added and incubated at room temperature for 20 min. The TransIT-siQUEST reagent/siRNA complex mixture was added dropwise to the cells. The plate was gently rocked and incubated for 24-72 h. The knock-down of target gene expression was tested by real-time PCR or Western blot analysis. The suppression of OPN expression was effective for up to 72 h. Rac-1 Assay—Erythroblasts (day 9) in culture were placed in fresh serum-free medium for 2 h with or without the Rac inhibitor prior to stimulation with OPN. Equal amounts of lysates (500 μg) were incubated with GST-Pak1-PBD (to pull-down active GTP-bound Rac-1) in the presence of SwellGel™-immobilized glutathione at 4 °C for 1 h in a spin column (Pierce EZ-Detect Rac1 Activation kit). The samples were subsequently analyzed for bound Rac-1 by immunoblot analysis using an anti-Rac-1 antibody. Calcium Efflux Assay—Day 10 human erythroblasts were centrifuged in medium at 400 × g, and the medium was aspirated. Cells were washed three times in HEPES-G (123 mm NaCl, 5 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 25 mm HEPES, 10 mm glucose, pH 7.4) and consecutively centrifuged at 800, 600, and 500 × g.40 μl of cells were added to 20 ml of HEPES-G in a 50-ml sterile conical tube (0.2% Hct). The tube was covered with aluminum foil to exclude light. 20 μl of 2 mm fluo-3/AM stock were added to the tube. The sample was incubated at 37 °C for 15 min with shaking. An additional 20 μl of 2 mm fluo-3/AM stock were added to each tube. The sample was incubated at 37 °C for an additional 45 min with shaking. The sample was centrifuged at 400 × g for 5 min at 4 °C. The sample was washed twice in PBS-G (PBS, 10 mm glucose, 0.5% bovine serum albumin, pH 7.4) and centrifuged at 400 × g for 5 min at 4 °C. The sample was transferred to 15-ml conical tubes, washed once in HEPES-G, and centrifuged at 400 × g for 5 min at room temperature. The supernatant was aspirated. The cells were resuspended in 2 ml of HEPES-G (1% Hct) and incubated at room temperature for 15 min. Prior to flux studies, 100 μl of loaded cells were added to 1400 μl of HEPES-G in a glass cuvette. Cells were stimulated at 60 s with no stimulant, 1 μg/ml OPN, or 1 μm A23187. A fourth sample was preincubated with 1 μg/ml OPN for 3 min and stimulated at 60 s with 1 μm A23187. Loaded cells were excited at 506 nm, and the fluorescence emission was recorded at 530 nm using a fluorimeter (Aminco Bowman Series 2 Luminescence Spectrometer). Triplicate trials were run for each sample. Detection of Receptors for OPN Engagement—Day 10 erythroblasts were used to detect the cell surface expression of CD44, CD44v6, integrins β1, α4, β5, and αvβ3 along with glycophorin A by flow cytometry. Antibodies against CD44, integrin β1 (CD29), integrin α4 (CD49d), and integrin β5 were from eBioscience Inc. The antibody against CD44v6 was purchased from R & D Inc. The antibody against integrin αvβ3 was from Santa Cruz Biotechnology Inc. Peripheral blood mononuclear cells (PBMNC) and Chinese hamster ovary (CHO) cells were used as positive controls for receptors that did not show expression in erythroblasts. Cell Proliferation Assays—In the experiments where proliferation effects of OPN were determined, mouse CD71-positive erythroblasts from WT and OPN-/- mice were cultured for 48 h prior to determining the level of proliferation. In the experiment where the effect of neutralizing antibodies on cell proliferation was determined, day 9 human erythroblasts were cultured for 24 h in the presence or absence of 20 μg/ml for each antibody. None of the antibodies utilized in cell culture contained sodium azide, which is used as an antibacterial agent in most commercial antibodies. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay according to the manufacturer's suggested protocol (Sigma). OPN Expression by Highly Purified Human Erythroblasts—To obtain an extremely pure erythroid cell population that differentiates in a synchronous manner, we used a primary human erythroid cell culture system, which was modified from our previous work (17Uddin S. Ah-Kang J. Ulaszek J. Mahmud D. Wickrema A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 147-152Crossref PubMed Scopus (132) Google Scholar). We initially cultured CD34-positive early hematopoietic cells under conditions that promote commitment and differentiation to the erythroid lineage. During the mid-phase of in vitro culture, we selected a highly synchronous erythroblast population by flow cytometry sorting for GlyA- and CD71-positive cells (Fig. 1A). Sorted cells were 98-99% positive for the GlyA/CD71 population. We also confirmed the absence of other myeloid and lymphoid cell types after purification by flow cytometry analysis (data not shown). These cells were then recultured for another 6-8 days under conditions optimal for terminal differentiation to reticulocytes (Fig. 1B). Cells were collected at several time points during culture and stained for hemoglobin (benzidine) to monitor terminal differentiation (Fig. 1B). The OPN expression was evaluated using qPCR for OPN mRNA at the following stages of the differentiation program: CD34-positive cells (day 0), polychromatic erythroblasts (day 9), orthochromatic erythroblasts (day 12), and enucleating late stage erythroblasts (day 13) (Fig. 1C). Transcripts for OPN were present in CD34-positive early hematopoietic cells and at all stages of differentiation, but the highest levels were observed at later time points peaking on day 12 of culture (Fig. 1C). To exclude the possibility that OPN is expressed by erythroblasts as a result of growth factor mobilization (source of our CD34 cells), we also cultured erythroblasts (CD71-selected) isolated directly from bone marrow aspirates of volunteer donors. These studies confirmed that OPN is expressed by bone marrow-isolated erythroblasts, although to a lesser extent in comparison to peripheral blood-derived cells (Fig. 1D). We then determined OPN protein expression by immunoblot analysis using cell lysates collected at various time points during culture. The OPN protein was detectable at all stages of erythroid differentiation (Fig. 2A). The two erythroleukemia cell lines, K562 and HEL, which were used as negative controls, do not express OPN. We then reprobed the same immunoblot against anti-tubulin antibody to verify equal protein loading and with antibodies against protein band 3 (anion exchanger) to verify terminal differentiation. Band 3 is an erythroid transmembrane protein, which appears relatively late during the differentiation program, and is therefore an excellent marker of erythroid differentiation. Cellular Secretion and Localization of OPN—Because OPN is a secreted cytokine, we examined whether purified erythroblasts secrete OPN during their differentiation process. OPN was readily detectable by ELISA in culture medium of GlyA/CD71-positive erythroblasts (Fig. 2B). The level of OPN was especially high between days 8 and 11 of culture, during which time these cells rapidly synthesize hemoglobin as well as several other cytoskeleton proteins (18Wickrema A. Koury S.T. Dai C.H. Krantz S.B. J. Cell. Physiol. 1994; 160: 417-426Crossref PubMed Scopus (48) Google Scholar). Interestingly, OPN secretion persisted even during the very late stages of erythroid differentiation (days 14-19), suggesting continued autocrine and/or paracrine functions (Fig. 2B). We then examined the intracellular localization of OPN in differentiating erythroblasts on day 10 of culture. Using fluorescently labeled antibodies against OPN and GlyA (both indirect fluorescent conjugates), we observed the localization of OPN in GlyA-positive cells (day 10), further confirming that OPN is expressed by erythroid progenitors (Fig. 2C). OPN had a punctate appearance in cells and was distributed throughout the cytoplasm, whereas GlyA was localized to the cell membrane. Fluorochrome-labeled secondary antibody alone was used as a control, which did not react with erythroblast cells (Fig. 2C). OPN Regulates F-actin Filament Formation and Distribution—To investigate the functional relevance of OPN expression, we depleted OPN expression in human erythroblasts by siRNA. By transfecting cells with a pool (Smart Pool™) of siRNA directed against OPN, we were able to effectively inhibit the expression of OPN at the RNA and protein levels (Fig. 3, A and B). Because OPN regulates F-actin polymerization in the bone, we examined the impact of OPN depletion on F-actin reorganization during erythroid differentiation. In erythroblasts, F-actin is normally present in the peri-membrane region of the cells. The depletion of OPN resulted in a dramatic rearrangement of the distribution and appearance of F-actin in differentiating erythroblasts as determined by fluorescence microscopy using Texas-Red-conjugated phalloidin. The knock-down of OPN in human erythroblasts resulted in the reduction of actin filaments in the peri-membrane region of the cells and the appearance of aggregated bundles throughout the cytoplasmic region, which was evident under the oil immersion lens (Fig. 3C). We then examined cultured erythroblasts from the bone marrow of WT and OPN-/- mice, which showed that in OPN-/- mice, there were no definitive F-actin filaments along the peri-membrane region compared with WT mice. In these OPN-/- cells, F-actin had diffusely spread throughout the cytoplasm of cells (Fig. 3D). Furthermore, when we cultured the OPN-/- erythroblasts with recombinant mouse OPN, we were able to restore the F-actin filaments in the peri-membrane region of the cells (Fig. 3D). The changes in actin filaments were visible only at high magnification, although photomicrographs of larger fields allowed us to observe the overall expression in the total cell population. Signal Transduction by OPN—To identify the upstream signaling elements responsible for the regulation of actin polymerization by OPN in erythroblasts, we investigated whether Rac-1 GTPase, a known signaling intermediate of actin remodeling, is activated by OPN. Rac-1 belongs to the Rho family of GTPases and binds its downstream effector PAK-1 to initiate the signaling cascade upon Rac-1 activation by extracellular ligands (19Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). We tested Rac-1 activation in response to OPN by performing a Rac-1 binding assay on total cell lysates from human erythroblasts. Stimulation by exogenous OPN resulted in a 50% increase in Rac-1 binding to PAK-1, compared with the unstimulated sample (Fig. 4A). Moreover, pretreatment of cells with a Rac GTPase inhibitor (NSC23766) prior to OPN stimulation suppressed the extent of binding, demonstrating that Rac-1 GTPase is a target of OPN (Fig. 4A). We then focused on adducin, an actin-binding protein that plays an important role in actin filament regulation in the erythroid cytoskeleton. Three isoforms (α, β, γ) of adducin are present in the erythroblast cytoskeleton and have phosphorylation sites that are substrates for multiple kinases. Although protein kinases A and C are known to phosphorylate adducin (20Matsuoka Y. Hughes C.A. Bennett V. J. Biol. Chem. 1996; 271: 25157-25166Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), cytokine-induced phosphorylation of adducin is not known. To determine if OPN stimulation leads to adducin phosphorylation, total cell lysates were collected after OPN stimulation (for various times or for 30 min with increasing concentrations of OPN) and immunoblotted with an anti-phosphoserine 726 antibody. These experiments showed that OPN stimulation leads to phosphorylation of α and/or β isoforms (120/110 kDa) in a dose- and time-dependent manner that exceeds any basal phosphorylation levels, as indicated by the relative ratios of the band intensities (Fig. 4, B and C). To further explore whether other proteins are also phosphorylated in response to OPN, we used pan anti-phosphothreonine antibody to investigate general phosphorylation patterns in erythroblasts. This experiment revealed that at least three other proteins were phosphorylated at threonine sites, suggesting the involvement of OPN as a signal transducer in erythroid cells (Fig. 4D). The molecular sizes of these proteins were ∼120, 30, and 27 kDa. The kinetics of phosphorylation of all proteins showed a maximum level of phosphorylation between 5 and 15 min after exposure, indicating that the OPN effect on erythroblasts in terms of signal transduction is quite rapid (Fig. 4D). Intracellular Calcium Is Regulated by OPN—Because OPN has been linked to regulation of Ca2+ absorption in bone, we used a fluorescence assay to investigate the role of OPN in the regulation of intracellular Ca2+. Exogenous OPN stimulation of erythroblasts induced efflux of Ca2+ from the cells, as demonstrated by decreased fluorescence intensity compared with control samples that were not stimulated by OPN (Fig. 5). As a positive control, cells were stimulated with A23187, a Ca2+ ionophore, which induced a dramatic increase in fluorescence intensity corresponding to rapid Ca2+ influx. The incubation of OPN in A23187-treated cells resulted in a relative reduction of Ca2+ influx compared with the positive control. Because calcium often acts as a signaling mediator, OPN stimulation of erythroblast" @default.
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• W2038658082 date "2008-03-01" @default.
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• W2038658082 title "Osteopontin Regulates Actin Cytoskeleton and Contributes to Cell Proliferation in Primary Erythroblasts" @default.
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