Demonstration of osteonectin mRNA in megakaryocytes: the use of the
polymerase chain reaction
XC Villarreal, BW Grant and GL Long
Department of Biochemistry, University of Vermont, Burlington 05405.
Platelets have been shown to release osteonectin on thrombin stimulation.
The origin of platelet osteonectin was unclear as it may have been
synthesized by megakaryocytes or it may have been endocytosed from plasma
as other platelet alpha-granule constituents are. Platelet osteonectin has
a larger apparent molecular size than the bone species, although the
molecular basis for this difference has not been elucidated. These two
issues have been addressed here by (1) examining the potential for
osteonectin biosynthesis in human megakaryocytes by demonstrating the
presence of osteonectin mRNA in purified megakaryocytes, and (2) comparing
the coding portion of osteonectin transcript in megakaryocytes to the size
of its bone counterpart. Because of the limitations of cell population
purity and in obtaining sufficient numbers of megakaryocyte cells for
Northern analysis, we have used the polymerase chain reaction (PCR) to
detect the presence of human osteonectin mRNA in megakaryocyte and
megakaryocyte-depleted bone marrow cells. Isolation of RNA, cDNA synthesis,
and PCR were performed on human osteosarcoma SaOS-2 cells, enriched
megakaryocytes, and megakaryocyte-depleted cells. Restriction enzyme
analysis of PCR DNA products confirmed the identity of the products as
those encoding osteonectin for all three cell populations studied. In
addition, the sizes of DNA indicate that osteonectin genomic DNA, nuclear
RNA, or altered transcript were not amplified, and that the transcript from
megakaryocytes is the same size as that from bone cells. These data suggest
that the difference in protein size between platelet and bone osteonectin
is due to posttranslational modification. To overcome the possibility that
megakaryocyte signal originated from contaminating cells (less than 5% by
cell count), all three cell populations were diluted to less than one cell
per tube and PCR amplification was performed. Limiting dilution analyses
demonstrated the presence of osteonectin mRNA in single megakaryocytes as
well as in single cells from the cell population depleted of
megakaryocytes, suggesting the capacity for osteonectin biosynthesis in all
cells studied. The procedure we describe in this report can be used to
examine specific characteristics of mRNA molecules in heterogeneous cell
populations and in situations where only small quantities of cells can be
obtained.
Volume 78,
Issue 5,
pp. 1216-1222,
09/01/1991
Copyright © 1991 by The American Society of Hematology
