Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2530-2532
Erythropoietin Receptor Mutations Associated With Familial
Erythrocytosis Cause Hypersensitivity to Erythropoietin in the
Heterozygous State
By
Stephanie S. Watowich,
Xiaoling Xie,
Ursula Klingmuller,
Juha Kere,
Mikael Lindlof,
Stig Berglund, and
Albert de la Chapelle
From the Department of Immunology, MD Anderson Cancer Center,
Houston, TX; Hans-Spemann-Laboratory, Max-Planck-Institute for
Immunobiology, Freiburg, Germany; the Department of Medical Genetics,
University of Helsinki, Helsinki, Finland; the Department of Medicine,
University of Lund, Malmo General Hospital, Malmo, Sweden; and Human
Cancer Genetics Program, James Cancer Hospital and Solove Research
Institute, and Comprehensive Cancer Center, Ohio State University,
Columbus, OH.
 |
ABSTRACT |
Inherited mutations in the erythropoietin receptor (EPOR) causing
premature termination of the receptor cytoplasmic region are associated
with dominant familial erythrocytosis (FE), a benign clinical condition
characterized by hypersensitivity of erythroid progenitor cells to EPO
and low serum EPO (S-EPO) levels. We describe a Swedish family with
dominant FE in which erythrocytosis segregates with a new truncation in
the negative control domain of the EPOR. We show that cells engineered
to concomitantly express the wild-type (WT) EPOR and mutant EPORs
associated with FE (FE EPORs) are hypersensitive to EPO-stimulated
proliferation and activation of Jak2 and Stat5. These results
demonstrate that FE is caused by hyperresponsiveness of
receptor-mediated signaling pathways and that this is dominant with
respect to WT EPOR signaling.
© 1999 by The American Society of Hematology.
| |
ARTICLE |
THE SWEDISH erythropoietin receptor
(EPOR) allele contains a direct tandem duplication of nucleotides 5968 to 5975. This results in a frameshift beyond residue 405, introducing
25 amino acids not related to the EPOR, and a premature stop codon, deleting 79 residues at the C-terminus of the receptor (data not shown). Affected family members (Fig 1A,
Table 1) were plethoric and often had
additional symptoms, including hypertension, headaches, dizziness,
nosebleeds, and exertional dyspnea, which were most pronounced in the
males. These symptoms were alleviated by phlebotomies, and phlebotomy
treatment has been recommended.
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| Fig 1.
Dominant erythrocytosis and EPO hypersensitivity. (A)
Pedigree of the Swedish family. (B) EPO-dependent proliferation in 32D
cell lines. Cells expressing WT ( ), mFin ( ), mSwed ( ),
mFin+WT ( ), or mSwed+WT ( ) were assayed for growth in
EPO-containing medium. (C and D) EPO-dependent Jak2 and Stat5
activation in 32D cell lines. Cells were stimulated with EPO and Jak2
and Stat5 activation determined by immunoprecipitation and
antiphosphotyrosine immunoblot assays. Methods: The inheritance pattern
of familial erythrocytosis among members of a 5-generation family from
southern Sweden was determined by clinical observation. In some cases
the presence of the mutant EPOR allele was assayed by direct genomic
sequencing or an allele-specific hybridization test. 32D cell lines
were established as described.12 Cells were grown in media
containing serial dilutions of EPO (from 10 4 to 10 U/mL), or in media containing interleukin-3 (IL-3). Viable cells were
counted after 3 days. The number of cells in EPO cultures is expressed
as a percentage of the cells present in IL-3 cultures (% IL-3
response). For Jak2 and Stat5 activity assays, 32D cells were
stimulated for 10 minutes at 37°C with 0.2, 1.0, 2.0, or 10.0 U/mL
EPO, or were left unstimulated, as indicated. Proteins were
immunoprecipitated from detergent cell extracts with an antibody
specific for Jak2 or Stat5, separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and transferred to
nitrocellulose. The filters were probed with antiphosphotyrosine
antibody 4G10 and developed with chemiluminescent
reagents.9,12 A computer-generated image of the region of
the gel containing Jak2 ( 130 kD) or Stat5 (90 kD) is shown.
Chemiluminescent signals were quantified on a lumi-imager and are
expressed as a percentage of the level of Jak2 or Stat5 phosphorylation
elicited by EPO stimulation of the WT receptor at 10 U/mL EPO
(arbitrarily set to 100%). The levels of protein phosphorylation were
normalized to the levels of immunoprecipitated Jak2 or Stat5 for each
sample.
|
|
To investigate the molecular mechanisms of erythrocytosis, the Swedish
mutation and a previously described mutation from a Finnish family
(6002G 
A)1,2 were engineered in the murine EPOR
(mSwed and mFin, respectively). Clonal 32D cell lines expressing these
EPOR isoforms were established. To approximate EPOR expression patterns
predicted for heterozygous individuals, clonal cell lines coexpressing
wild-type (WT) and mutant EPORs were also established (data not shown).
The mFin and mSwed EPORs rendered hypersensitive proliferative
responses to EPO and dominantly enhanced the proliferative response of
the WT EPOR (Fig 1B).
EPO hypersensitivity could be caused by differential receptor
metabolism or altered signal transduction. Cell-surface receptor expression did not vary more than 2-fold, as judged by flow cytometry analysis, and the intracellular processing of newly synthesized WT,
mFin, or mSwed was similar (data not shown). These results suggest that
differential receptor metabolism is unlikely to be the sole cause of
the observed EPO-hypersensitivity.
To examine EPOR signaling, we assayed EPO-dependent activation of Jak2
and Stat5 by immunoprecipitation and immunoblot
analysis.3-5 Cells expressing the mutant receptors alone or
coexpressing mutant and WT EPORs had elevated levels of activated Jak2
and Stat5 at all EPO concentrations tested, relative to cells
expressing the WT receptor alone (Fig 1C and D). Stat5 also
demonstrated hypersensitivity to EPO, with half-maximal activation at
0.2 U/mL EPO in cells expressing mutant EPORs compared to 1.0 U/mL
EPO in WT cells. Similar levels of Jak2 or Stat5 were present in all
samples (data not shown).
Heterozygous individuals with FE are predicted to express both WT and
mutant EPORs,1,6-8 although this has not yet been documented. Our study provides the first analysis of cells engineered to coexpress WT and FE EPOR isoforms. The results show that FE alleles
confer dominant and hypersensitive EPO-dependent proliferation and
activation of Jak2 and Stat5. The hypersensitive response may reflect
mechanistic differences in Jak2 activation or inactivation by FE EPORs,
compared with WT EPOR. For example, the mutant receptors may abrogate
one or more negative-regulatory signals, such as SHP-1 or
CIS,9-11 that normally suppress Jak2 activity, while still
stimulating positive regulatory signals. Collectively, our results
indicate that dominant FE is caused by enhanced receptor signaling
because expression of a mutant allele in the heterozygous state is
sufficient to unbalance WT signal transduction.
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ACKNOWLEDGMENT |
We thank Adriana Acurio, Hong Lu, and Melanie Wickert for excellent
technical assistance; Karen Ramirez for flow cytometry analysis; Dr
Joan Egrie (Amgen, Thousand Oaks, CA) for the generous gift of recombinant EPO; and Dr Harvey F. Lodish for initiating this
collaboration and for advice during the course of these studies.
| |
FOOTNOTES |
Submitted March 11, 1999; accepted May 24, 1999.
Supported by grants from the National Cancer Institute, National
Institutes of Health (CA-77447) and the Texas Higher Education Coordinating Board Advanced Research Program (15-120) (to S.S.W.), and
by grants from the National Cancer Institute, National Institutes of
Health (CA-67941 and CA-16058) (to A.d.l.C.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Stephanie S. Watowich, PhD, MD Anderson
Cancer Center, Box 178, 1515 Holcombe Blvd, Houston, TX 77030.
| |
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