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

Jeong-Ah Kang,Ying Zhou,Tahlia L Weis,Hui Liu,Jodie Ulaszek,Nilesh Satgurunathan,Li Zhou,Koen Van Besien,John Crispino,Amit Verma,Philip S Low,Amittha Wickrema

doi:10.1074/jbc.m706712200

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.

Highlights

Provides signals for cell proliferation (4 – 6)
Our studies show that OPN receptors CD44 and several integrins are expressed in these cells and suggest that integrin ␤1 is responsible for transmitting the proliferative signal
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 [17]

Summary

EXPERIMENTAL PROCEDURES

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). 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 ␣-thioglycerol, IMDM, and 1 unit /ml EPO for 16 h in the presence or absence of 1 ␮g/ml OPN prior to immunofluorescence analysis for F-actin. 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 washed in the same buffer before the incubation of Alexa Fluor-conjugated anti-mouse IgG antibodies at room temperature for 30 min. Cell Proliferation Assays—In the experiments where proliferation effects of OPN were determined, mouse CD71-positive erythroblasts from WT and OPNϪ/Ϫ mice were cultured for 48 h prior to determining the level of proliferation. 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)

RESULTS

13 K562 HEL

DISCUSSION