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In vivo and in vitro regulation of erythropoietin mRNA: measurement by
competitive polymerase chain reaction
J Fandrey and HF Bunn
Department of Medicine, Brigham and Women's Hospital, Harvard Medical
School, Boston, MA.
The regulation of erythropoietin (Epo) production was investigated by
competitive polymerase chain reaction, a highly sensitive and accurate
means of measuring Epo mRNA levels. Co-amplification of the test sample
with added mutant Epo cDNA template corrects for variability in the
efficiency of amplification. Epo mRNA levels were determined in tissues of
normal rats and in animals with varying degrees of anemia. Reduction of the
hematocrit level from 0.40 to 0.15-0.20 resulted in a 300-fold increase in
kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from
the liver. In contrast, very low levels detected in lung and spleen were
not significantly increased by anemia. The human hepatoma cell line, Hep3B,
secretes high levels of Epo in response to hypoxia. This regulation is, to
a large extent, transcriptional. When Hep3B cells were incubated in the
presence of decreasing O2 tension from 160 to 7 mm Hg, there was a
monotonic increase in Epo mRNA to 50 to 100 times the normoxic level.
Hyperoxia did not suppress basal expression. When cells were incubated at a
PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was
maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic
incubation, cells were then exposed to normoxia, which shut off further
transcription of the Epo gene. The decay of Epo mRNA levels closely
followed first order kinetics with a half-life of 2 hours, an effective
measurement of message stability.
Volume 81,
Issue 3,
pp. 617-623,
02/01/1993
Copyright © 1993 by The American Society of Hematology
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