REGUIATION OF ERYTHROPOIESIS 
H. Franklin Bunn, M.D., Investigator 
Dr. Bunn and his colleagues have been investi- 
gating the biogenesis and the mechanism of action 
of erythropoietin, the hormone that regulates the 
production of red blood cells in humans and other 
mammals. Dr. James Cunningham's research fo- 
cuses on the murine ecotropic receptor for retro- 
viruses. 
I. Structure-Function Relationships of 
Erythropoietin. 
Erythropoietin (Epo) is a highly soluble protein 
containing 166 amino acids and 40% carbohydrate. 
Because it has not yet been crystallized, there is no 
information on its three-dimensional structure. Pre- 
liminary computer-based modeling, done in collab- 
oration with Dr. Fred Cohen (University of Califor- 
nia at San Francisco) suggests that Epo is 
homologous to human growth hormone (20 kDa) 
and assumes a right-handed four-helix bundle 
structure, with antiparallel adjacent a-helices. Dr. 
Jean-Paul Boissel and Dr. Bunn created a set of de- 
letion mutants that excised each of the potential 
helices. In all cases, A^- and O-glycosylation sites 
were preserved. Human Epo cDNA and the mu- 
tated cDNAs were inserted into a mammalian vector 
and transiently expressed in COS7 cells. When 
compared with the normal Epo construct, nearly 
the same mRNA levels were observed for all the 
mutants, but no protein was detected in either the 
cells or the media by both radioimmunoassay and 
in vitro bioassay. These results suggest that these 
deletions resulted in very unstable translation 
products. In contrast, partial deletion of a pre- 
dicted interconnecting nonhelical loop gave rise, 
after transient expression, to a protein produced 
and secreted nearly as effectively as the Epo wild 
type. This structural model predicts that this dele- 
tion should not destabilize the molecule. Moreover, 
this mutant exhibited full biological activity. An- 
other approach to studying structure-function rela- 
tionships is to generate and compare Epo se- 
quences from a variety of mammals. A large set of 
primers corresponding to sequences conserved be- 
tween mouse and human were synthesized in order 
to amplify by means of the polymerase chain reac- 
tion (PCR) genomic Epo fragments from cell lines 
of 10 mammalian species covering eight orders. Re- 
striction enzyme mapping verified that these ampli- 
fied fragments originated from the corresponding 
animals. Comparison of these sequences should 
provide insights into important functional domains 
in the Epo molecule. 
II. Regulation of the Erythropoietin Gene. 
The human hepatoma cell line Hep3B synthe- 
sizes large quantities of Epo in a regulated manner 
in response to hypoxia and cobaltous chloride 
(C0CI2). This regulation occurs at the Epo mRNA 
level. To delineate further the nature of the regula- 
tion of Epo gene expression, Dr. Mark Goldberg 
and Dr. Bunn have studied the effects of hypoxia 
(1% O^) and CoCl^ on the rate of Epo gene tran- 
scription. Although Northern blot analyses showed 
that steady-state Epo mRNA levels increase more 
than 100-fold in response to hypoxia or CoCl^, nu- 
clear runoff experiments demonstrated only a 5- to 
10-fold increase in Epo mRNA transcription in re- 
sponse to these stimuli. Experiments were there- 
fore undertaken to investigate post-transcriptional 
influences on steady-state Epo mRNA levels. When 
Hep3B cells were grown in 1% to increase Epo 
mRNA levels, and then switched to 21% O^, Epo 
mRNA had a rapid rate of decay, with steady-state 
levels falling by 50% within 1.5-2 h and with 
almost undetectable levels by 6 h. This finding is 
similar to reported changes in kidney Epo mRNA 
levels in mice switched from a hypoxic to a non- 
hypoxic environment. To distinguish between 
transcriptional and post-transcriptional contribu- 
tions to this observed fall in steady-state Epo mRNA 
levels, actinomycin D chase experiments were 
performed. Hep3B cells were made hypoxic to 
allow an increase in Epo mRNA levels. After the ad- 
dition of actinomycin D the cells were either kept 
hypoxic or allowed to equilibrate in 21% for var- 
ious periods of time. The stability of the Epo mRNA 
in the presence of actinomycin D at both ten- 
sions was much greater than that observed in the 
absence of actinomycin D and much greater than 
that which has been reported in vivo. If actino- 
mycin D was preventing the transcription of a 
rapidly turning over mRNA whose product destabi- 
lizes Epo mRNA, cycloheximide would be expected 
to have a similar effect on the stability of Epo 
mRNA. Subsequent experiments did demonstrate a 
reproducible increase in the stability of the Epo 
mRNA in the presence of cycloheximide. Thus it ap- 
pears that the induction of Epo mRNA in response 
Continued 
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