Osteopontin regulates actin cytoskeleton and contributes to cell proliferation in primary erythroblasts

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 beta1 and alpha4, three known receptors for OPN, and that the integrin beta1 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.

FIGURE 1. Expression of OPN in highly purified human erythroblasts

A, flow cytometry analysis of human erythroblasts on day 8 of culture prior to sorting for GlyA and CD71 (transferrin receptor) (left panel) and after sorting (right panel). The purity of the sorted population was 98–99%. B, photomicrographs of differentiating human erythroblasts stained with hematoxylin and benzidine on day 6 (basophilic erythroblasts), day 9 (polychromatic), and days 12 and 16 (orthochromatic) of culture. C, expression of OPN in glycophorin A/CD71-sorted cells determined by real-time qPCR and normalized against 18 S rRNA. Levels are compared with the level of OPN expression in day 0 cells. The results are the mean of triplicate determinations. Bars indicate medians with S.D. *, p < 0.01 compared with day 0; **, p < 0.01 compared with day 9; and ***, p < 0.01 compared with day 12. D, qPCR analysis of OPN expression in bone marrow-derived and growth factor-mobilized peripheral blood (mPB)-derived erythroid progenitors cultured until day 9. Levels are compared with the level of OPN expression in mPB. Bars indicate medians with S.D. The results are the mean of triplicate determinations.

FIGURE 2. Expression, secretion, and localization of OPN in human erythroblasts

A, primary erythroblasts were collected and analyzed for the levels of OPN protein at various times during the differentiation program. The same blot was reprobed with an anti-tubulin antibody (middle panel) and an anti-band 3 antibody (bottom panel) to monitor the extent of differentiation and to establish protein loading controls, respectively. On a separate immunoblot, total lysates collected from erythroleukemia cell lines, K562 and HEL were probed with the same anti-OPN antibody as a negative control, followed by immunoblotting against anti-tubulin antibodies as the protein loading control. B, quantitation of OPN in the culture media during erythroid differentiation. ELISA was performed using an OPN ELISA kit to determine the amount of secreted OPN present in the culture media at various time intervals during erythroid differentiation. All data are presented as mean ± S.D. The results are the mean of triplicate determinations. *, p < 0.01 compared with media; **, p < 0.01 compared with day 8–11; and ***, p = 0.55 compared with day 11–14. C, OPN (green) was localized to cells expressing Gly A (red) by confocal immunofluorescence microscopy on day 10 of culture using specific antibodies to OPN and Gly A. The first panel shows DIC micrographs of the cells. Images of larger fields are shown for the OPN, Gly A, and secondary antibody controls. Images of two single cells expressing OPN and GlyA are shown under high power.

FIGURE 3. OPN regulates localization and distribution of F-actin in human and mouse erythroblasts

A and B, OPN expression was knocked down in human erythroblasts by transfection of OPN-specific siRNA into day 6 cells. Real time RT-PCR and immunoblot analysis were performed to confirm the efficient knock-down of the gene 24-h, post-transfection. A set of nonspecific siRNA was also transfected as a control. C, distribution of F-actin in human erythroblasts transfected with control siRNA (Ctrl) and cells transfected with OPN-specific siRNA 48-h post-transfection by immunofluorescence analysis using Texas Red-conjugated phalloidin. The top panel shows DIC micrographs of cells. D, localization of F-actin in erythroblasts of wild-type and OPN knock-out male mice. CD71-selected erythroblasts were cultured in vitro for 16 h with or without OPN as indicated in the case of OPN knock-out mice (OPN^−/−) and without added OPN in the case of WT mice. Cells were harvested and analyzed for F-actin filaments by Texas Red-conjugated phalloidin. DIC and fluorescence micrographs of two individual cells (under high power) and larger fields are depicted.

FIGURE 4. OPN-mediated signal transduction in human erythroblasts

A, OPN activates Rac-1 GTPase in erythroblasts. Erythroblasts on day 9 of culture were stimulated with OPN with or without pretreatment with the Rac inhibitor, NSC23766 (50 µm). Cells without OPN stimulation and Rac inhibitor pretreatment were used as a control (lane 1). Total cell lysates were used to determine the GTP-bound Rac-1 (active) by pull-down assays. A Rac-1 immunoblot was also performed using equal amounts of the original lysates. Levels of Rac-1 activity (association with Pax binding domain) were calculated after normalizing against the total Rac-1 band density. B and C, OPN phosphorylates adducin in human erythroblasts. Erythroblasts on day 9 of culture were serum-starved and then stimulated with indicated doses of OPN for 30 min or stimulated with 2 µg/ml of OPN for indicated times. Lysates were collected, analyzed by SDS-PAGE, and immunoblotted against an anti-phospho adducin antibody. The same blots were subsequently reprobed with anti-tubulin and/or actin antibodies as a control for protein loading. Levels of phosphorylation at each dose or time point were calculated after normalizing against the tubulin band density. D, OPN phosphorylates multiple intracellular proteins in human erythroblasts. Erythroblasts on day 10 of culture were serum-starved and then stimulated with OPN for indicated times. Lysates were subjected to immunoblot analysis using an anti-phosphothreonine-specific antibody. The same blot was then reprobed with anti-tubulin antibody as a protein loading control. Levels of phosphorylation at each time point were calculated after normalizing against the tubulin band density.

FIGURE 5. Efflux of intracellular calcium by OPN in human erythroblasts

Cultured cells (day 10) were loaded with membrane-permeable fluo-3/AM and stimulated with no stimulant (open circles), 1 µg/ml OPN (filled circles), 1 µm A23187 (open square), or 1 µg/ml OPN + 1 µm A23187 (filled square). Each trace represents the mean ± S.D. of triplicate determinations.

FIGURE 6. Expression of receptors for OPN in human erythroblasts

Flow cytometry analysis was performed on day 10 human erythroblasts to detect the expression of various OPN receptors. A, integrin β₁, CD44, and integrin α₄ expression in GlyA-positive erythroblasts. B, CD44v6, integrin β₅, and α_vβ₃ in GlyA-positive erythroblasts. C, PBMNC was used as a positive control for CD44v6 and integrin α_vβ₃ and CHO cells as a positive control for β₅ to confirm the presence of receptors that were not observed in erythroblast cells.

FIGURE 7. OPN promotes cell proliferation of erythroblasts

A, erythroblasts from bone marrows (CD71-selected) of WT and OPN^−/− mice were cultured with or without OPN as indicated. The cell proliferation was assessed by MTT assay after 48 h in culture. All data are presented as mean ± S.D. The results are the mean of triplicate determinations. *, p = 0.02 compared with WT cells; B, integrin β₁ receptor contributes to cell proliferation in human erythroblasts. Day 9 human erythroblasts were cultured for 24 h in the presence or absence of various neutralizing antibodies (20 µg/ml) as indicated prior to determining the level of proliferation by an MTT assay. All data are presented as mean ± S.D. The results are from the mean of determinations. *, p = 0.001 compared with control.