Epo and Non-hematopoietic Cells: What Do We Know? - Tissue-Protective Cytokines: Methods and Protocols - page 13

1 Epo Expression Is Regulated by Hypoxia-Inducible Factors

Epo expression is hypoxia inducible and regulation occurs via the
hypoxia responsive element (HRE) present in the 3¢ region of the
gene which is bound by heterodimeric transcription factors namely
hypoxia-inducible factors (HIFs). Three members of the HIF tran-
scription factor family HIF-1, -2, and -3 have now been identified.
HIF-1 was discovered in 1991 by its ability to bind and stimulate
transcription of the Epo gene during hypoxia ( 16, 17 ) and for sev-
eral years, was assumed to be the primary stimulus for Epo produc-
tion in response to acute hypoxia. Later a second hypoxia-inducible
transcription factor termed HIF-2 was discovered ( 18-20 ).
Subsequent data from in vivo ( 21 ) and in vitro ( 22 ) experiments
suggested that despite the fact that HIF-1 clearly binds the HRE
of the Epo gene in response to hypoxia and both have the potential
to bind many of the same genes, in vivo HIF-2 is the primary
mediator of Epo expression in kidneys in response to hypoxia. In
agreement downregulation of HIF-2 in the brain, but not HIF-1,
drastically reduced hypoxia-induced Epo expression ( 23 ) and more
recently Haase and colleagues ( 24 ) clearly demonstrated the pri-
mary role of HIF-2 in promoting the hypoxic renal Epo response.
The HIFs are heterodimers composed of a constitutively
expressed b subunit (also known as aryl hydrocarbon receptor
nuclear translocation, ARNT) and an oxygen-regulated a subunit
(reviewed by ref. 25-27 ). Regulation of HIF activity occurs at dif-
ferent levels including protein stability, phosphorylation, nuclear
translocation, and activity, all being influenced by alterations in
oxygen levels. Under normoxic conditions the a subunit is
degraded. In contrast, under hypoxic conditions the a subunit is
stabilized and translocated to the nucleus where it dimerizes with
ARNT and subsequently binds to hypoxic binding sites (HBS) of
target genes. The HBS is a conserved consensus sequence (A/G)
CGTG within the HRE present in oxygen-regulated target genes
involved in cell survival, glycolysis, angiogenesis, erythropoiesis,
and iron metabolism ( 25 ). Degradation of HIF-a is triggered by
oxygen-dependent hydroxylation of prolyl residues located in the
oxygen-dependent degradation domain by a family of prolyl
hydroxylases, namely PHD1, PHD2, and PHD3. These enzymes
are specific HIF prolyl hydroxylases that require Fe(II) as a cofac-
tor as well as oxygen and 2-oxoglutarate as co-substrates ( 28, 29 ).
Prolyl hydroxylation promotes the recruitment of the tumor sup-
pressor protein von Hippel Lindau, which is part of the E3 ligase
ubiquitination complex, priming HIFs for degradation in the pro-
teosomes (reviewed by ref. 30, 31 ).
Other regulatory elements in the 5¢ promoter of the Epo
gene include a highly conserved GATA sequence as well as NFkB
binding motifs ( 32, 33 ). Both these sites seem to have inhibitory