Abstract
Osteopontin (OPN) is a secreted protein shown to have multiple functions depending on cell type and context. The cell types known to express OPN are typically assocoated with immune and /or bone remodeling functions. Here we demonstrate that OPN is also expressed and secreted by primary human erythroid progenitors and that it is involved in actin remodeling in erythroid cells. We initially identified OPN as an important cytokine in erythroid differentiation by screening a cytokine/receptor cDNA microarray. Subsequently using a culture system of highly purified primary erythroid progenitors (sorted for glycophorin A) we examined OPN mRNA and protein expression during terminal erythroid differentiation. We demonstrated that OPN mRNA is expressed throughout differentiation and that OPN protein is also detectable at basophilic, polychromatic and orthochromatic stages during the maturation program. Furthermore by ELISA we were able to detect secretion of OPN at each of the major stages of differentiation. In addition we show that CD44 and integrin alpha 4, two of the receptors for OPN are highly expressed in these cells. Stimulation of these cells with rOPN resulted in phosphorylation of several proteins one of which was identified as the heat shock protein-27 (Hsp27). In subsequent studies we were able to demonstrate that OPN regulates phosphorylation of Hsp27 at serine-78 site through the p38 MAP kinase pathway. We then effectively knocked down the expression of OPN by siRNA methodology, which resulted in formation of distinct F-actin bundles in the perimembrane region of the cell. By contrast in cells transfected with control siRNA maintained F-actin in a punctate appearance. Altogether our studies suggest a critical signaling function for OPN in regulation of atleast one of the erythrocyte cytoskeletal elements during erythroid differentiation.
Author notes
Corresponding author