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)-dependent 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 study how PGHS-1 expression is regulated in platelets.


METHODS
Because platelets are anucleated, their protein content is a consequence of gene expression in precursor cells known as megakaryocytes. We used the immortalized human megakaryoblastic cell line MEG-01 as a model to study the expression of PGHS-1, because MEG-01 cells can be induced to differentiate into platelet-like structures by adding nanomolar concentrations of 12-0-tetradecanoylphorbol-13-acetate (TPA). We determined the expression profiles of PGHS-1 protein and mRNA in the cells comprising the three different populations of MEG-01 cultures: nucleated floating, nucleated attached, and platelet-like structures.


RESULTS
We determined that PGHS-1 protein levels were higher in the nucleated adherent population than in the nucleated floating population. PGHS-1 protein levels were greatest in the anucleated platelet-like population. In contrast, we found that PGHS-1 mRNA levels were highest in the cells that comprised the nucleated adherent population. Addition of TPA induced the expression of PGHS-1 protein and mRNA in all three populations but did not change 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 expression of PGHS-1 protein on a cell-by-cell basis in the nucleated MEG-01 populations. We found that the percentage of MEG-01 cells expressing PGHS-1 protein in the adherent population was greater than in the floating population. We measured a time-dependent increase in the percentage of cells that expressed PGHS-1 over a period of 8 days after singular addition of TPA (1.6x10(-8)M). Importantly, we observed that TPA treatment stimulated floating MEG-01 to adhere to the surface of the tissue culture vessel and that, after such treatment, only floating MEG-01 cells suffered a compromised viability. We found that a high percentage of control cells expressed glycoprotein IIb/IIIa and that TPA treatment did not significantly alter this percentage. We did not detect glycoprotein Ib in control cells but did measure a slight increase in the percentage of MEG-01 cells that expressed this antigen in the TPA-treated population.


CONCLUSIONS
We established a correlation between the level of PGHS-1 expression and the overall level of differentiation of MEG-01 cells. PGHS-1 protein expression, which increases consistently over the full course of differentiation, now may be used as an additional and perhaps better index by which to survey megakaryocytes.
Mroske
C
C
Department of Biochemistry, Microbiology and Immunology, Ottawa, Canada.
Plant
M H
MH
Franks
D J
DJ
Laneuville
O
O
D002448:Cell Adhesion; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D051545:Cyclooxygenase 1; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008565:Membrane Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000096:biosynthesis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000096:biosynthesis; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; D008565:Membrane Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D012333:RNA, Messenger; D051545:Cyclooxygenase 1; C496511:PTGS1 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D013755:Tetradecanoylphorbol Acetate