Characterization of prostaglandin endoperoxide H synthase-1 enzyme expression during differentiation of the megakaryocytic cell line MEG-01

Objective. Because the prostaglandin endoperoxide H synthase-1 (PGHS-1)-dep endent formation of thromboxane A(2) is an important modulator of platelet function, this pathway represents a pharmacologic target for the inhibition of platelet function by aspirin. The objective of our research was to stud y how PGHS-1 expression is regulated in platelets. Materials and Methods. Because platelets are anucleated, their protein cont ent is a consequence of gene expression in precursor cells known as megakar yocytes. We used the immortalized human megakaryoblastic cell line MEG-01 a s a model to study the expression of PGHS-1, because MEG-01 cells can be in duced to differentiate into platelet-like structures by adding nanomolar co ncentrations of 12-0-tetradecanoylphorbol-13-acetate (TPA). We determined t he expression profiles of PGHS-1 protein and mRNA in the cells comprising t he three different populations of MEG-01 cultures: nucleated floating, nucl eated attached, and platelet-like structures. Results. We determined that PGHS-1 protein levels were higher in the nuclea ted adherent population than in the nucleated floating population, PGHS-1 p rotein levels were greatest in the anucleated platelet-like population. In contrast, we found that PGHS-1 mRNA levels were highest in the cells that c omprised the nucleated adherent population. Addition of TPA induced the exp ression of PGHS-1 protein and mRNA in all three populations but did not cha nge the relationship of the amount of PGHS-1 protein or mRNA expressed in a given population relative to the other two fractions. We measured the expr ession of PGHS-1 protein on a cell-by-cell basis in the nucleated MEG-01 po pulations. We found that the percentage of MEG-01 cells expressing PGHS-1 p rotein in the adherent population was greater than in the floating populati on. We measured a time-dependent increase in the percentage of cells that e xpressed PGHS-1 over a period of 8 days after singular addition of TPA (1.6 x 10(-8)M). Importantly, we observed that TPA treatment stimulated floatin g MEG-01 to adhere to the surface of the tissue culture vessel and that, af ter such treatment, only floating MEG-01 cells suffered a compromised viabi lity. We found that a high percentage of control cells expressed glycoprote in IIb/IIIa and that TPA treatment did not significantly alter this percent age. We did not detect glycoprotein Ib in control cells but did measure a s light increase in the percentage of MEG-01 cells that expressed this antige n in the TPA-treated population. Conclusion. We established a correlation between the level of PGHS-1 expres sion and the overall level of differentiation of MEG-01 cells. PGHS-1 prote in expression, which increases consistently over the full course of differe ntiation, now may be used as an additional and perhaps better index by whic h to survey megakaryocytes. (C) 2000 International Society for Experimental Hematology, Published by Elsevier Science Inc.