Adhesion of human myelomonocytic (HL-60) cells induced by 1,25-dihydroxyvitamin D3 and phorbol myristate acetate is dependent on osteopontin synthesis and the alpha v beta 3 integrin

Abstract

A key event in bone resorption is the attachment of osteoclasts on the bone surface. Accumulating data supports the notion that this interaction involves the matrix protein osteopontin on the bone surface interacting with a receptor of the integrin family (alpha v beta 3) at the osteoclast clear zone. Based on the recent observation that osteopontin phosphorylation appears to be required for this interaction, it is of considerable interest to delineate the structural requirements for osteopontin-mediated cell attachment. Although binding of isolated osteoclasts to osteopontin-coated surfaces involves the alpha v beta 3 integrin, this system suffers from considerable disadvantages to allow detailed studies in this respect. We have therefore turned to another cell system, HL-60 promyelocytic leukemia cells, to address these questions. In the presence of the phorbol ester TPA (10 nM) and 1,25-dihydroxyvitamin D3 (100 nM), 90% of the HL-60 cells became adherent within 24 hours and exhibited a macrophage-like appearance. Under these conditions, the osteopontin mRNA levels was elevated around 60-fold compared to the control, non-adherent cells. The absolute requirement of de novo osteopontin synthesis for the TPA and 1,25-vit D3-induced HL-60 cell adhesion was demonstrated by neutralisation of adhesion using an anti-osteopontin polyclonal antibody as well as following transfection of an antisense osteopontin phosphorothioate-modified oligonucleotide. Finally, inhibition of induced HL-60 cell adhesion by an RGD-containing peptide or by an antibody to the alpha v beta 3 integrin complex suggested that the cell-derived osteopontin interacts with this integrin. It is concluded that HL-60 cells induced to differentiate with the combination of TPA and 1,25-vit D3 can be utilised as a model system to delineate structural requirements involved in the interaction between osteopontin and the alpha v beta 3 integrin.