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486 Part VI: The Erythrocyte Chapter 32: Erythropoiesis 487

This degradation of HIFs α subunits is initiated by a posttransla- substitute for EPO in BFU-E cultures. Furthermore, anephric, nonane-
tional hydroxylation event at residue proline 564 (P564) that is medi- mic patients with no detectable EPO have elevated levels of IGF-1. 135
ated by one of several iron-containing proline hydroxylases (PHDs).
The hydroxylation of HIFs α subunits facilitates binding to the VHL REMOVAL OF ERYTHROCYTE
protein and subsequent ubiquitination and proteasomal degradation.
Osteosarcoma protein 9 (OS-9) binds to both HIF-1α and PHD2 and ORGANELLES
126
is required for efficient prolyl hydroxylation. Under hypoxic condi-
tions, HIF-1 and HIF-2 α proteins are not degraded and are translocated NIX-DEPENDENT CLEARANCE OF
to the cell nucleus where they dimerize with HIF-β to form the HIF MITOCHONDRIA BY AUTOPHAGY
heterodimer that activates transcription through binding to specific
hypoxia-responsive elements on target genes. Another regulatory step During terminal erythroid differentiation, erythroid cells discard all
involves O -dependent asparaginyl-hydroxylation of asparagine (N) 803 their internal organelles including mitochondria. Autophagy has been
2
in HIF-1α that requires the enzyme HIF-3, also known as FIH-1 (factor suggested to play an important role in this process based on early
inhibiting HIF-1). Hydroxylation of N803 during normoxia blocks the morphologic studies. 136,137 This is confirmed by studies using chemical
binding of transcription factors p300 and CBP to HIF-1, resulting in inhibitors of autophagy and small interfering ribonucleic acid (siRNA)
inhibition of HIF-1–mediated gene transcription. Under hypoxic con- knockdown of essential autophagy associated genes. 138,139 At the initial
ditions, HIF α subunits are not hydroxylated. The unmodified protein stage of autophagy, a double-membrane structure is formed to seques-
escapes VHL-binding, ubiquitination, and degradation (see Fig. 32–7). ter cytoplasmic components in autophagosomes. Autophagosomes
When N803 of HIF-1α is not asparaginyl-hydroxylated, p300 and CBP then fuse with lysosomes and become autophagolysosomes to degrade
can bind to the HIF-1 heterodimer, allowing transcriptional activation the sequestered components. Mice deficient in a bcl-2 family member,
of HIF-1 target genes. Nix, a protein that is expressed during erythropoiesis and regulates
HIF-2 Transcription Factor HIF-1α and HIF-2α exhibit a high mitochondrial apoptosis (autophagy), display defects in the clearance
degree sequence homology but have differing mRNA expression pat- of mitochondria. Interestingly, the formation of autophagosomes in
terns: HIF-1α is expressed ubiquitously, whereas HIF-2α expression reticulocytes is normal in the absence of Nix, suggesting that Nix is not
is restricted to certain tissues. 118,127 The kidney is the main site of EPO required for the initiation of autophagy or the formation of autopha-
production (i.e., renal interstitial cells), and HIF-2 and, to a lesser gosomes. Instead, mitochondria remain clustered outside of autopha-
138
degree, HIF-1 are the principal regulator of EPO transcription in the gosomes in Nix−/− reticulocytes. This indicates that Nix is required
87
kidney. 118,127 In other tissues, such as brain and liver (which generates for the sequestration of mitochondria by autophagosomes. Another
128
approximately 15 percent of circulating EPO), EPO gene transcription study using virally transformed Nix−/− erythroid cells also reported
140
127
is HIF-2–dependent. The discovery of an iron-responsive element defective inclusion of mitochondria by autophagosomes. Therefore,
in the 5′ untranslated region of HIF-2α reveals a novel regulatory link Nix deficiency leads to a specific defect in mitochondrial removal
between iron availability and HIF-2α expression that may also influ- without causing a general block in autophagy or erythroid maturation.
129
ence control of erythropoiesis. The importance of HIF-2α in regulation Nix−/− reticulocytes are defective in the loss of mitochondrial mem-
of EPO gene was demonstrated by a gain-of-function HIF-2α mutation brane potential during in vitro maturation. 138
causing erythrocytosis. 130
Hypoxia-Independent Regulation of Hypoxia-Inducible Factor DOWNREGULATION OF CELL SURFACE
While O -dependent regulation of the HIF-1α subunit is mediated PROTEINS AND OTHER ORGANELLES
2
by prolyl hydroxylases, VHL protein, and the proteasomal complex,
hypoxia-independent regulation of HIF-1α has been uncovered. This During erythroid maturation, reticulocytes release small vesicles con-
novel mechanism involves the receptor of activated protein kinase C taining cellular proteins. These vehicles are exosomes that are involved
(RACK1) as a HIF-1α–interacting protein that promotes prolyl hydroxylase/ in the clearance of cell-surface proteins, such as acetyl cholinesterase,
VHL-independent proteasomal degradation of HIF-1α. RACK1 com- CD71 transferrin receptor and integrin α β . 141,142 The enrichment of
4 1
petes with heat shock protein 90 (HSP90) for binding to the PAS-A these surface molecules in exosomes suggests that the exosomal path-
domain of HIF-1α. HIF-1α degradation is abolished by loss-of-function way plays a major role in the clearance of these surface molecules. Other
RACK1. RACK1 binds to the proteasomal subunit, elongin-C, and pro- cellular components, such as lysosomes, endoplasmic reticulum, Golgi
motes ubiquitination of HIF-1α (see Fig. 32–7). Therefore, RACK1 and apparatus, ribosomes, and RNA, are also cleared during erythroid mat-
HSP90 are the essential components of an O /PHD/VHL-independent uration. The precise mechanisms for the removal of these components
2
mechanism for regulating HIF-1α. 131 are unclear. In Nix-deficient mice, the loss of CD71 and ribosomes are
The rapid degradation of HIF is complex and tightly regulated, and normal during erythroid maturation, suggesting that exosomal path-
mutations affecting the genes that encode the regulatory factors may ways play a major role in the clearance of CD71 or ribosomes. 138
underlie some of the unexplained congenital polycythemias.
This complex (Chaps. 34 and 57) constitutes the oxygen sensor MICRORNAS IN ERYTHROPOIESIS
(see Fig. 32–7). 124,125,132
MicroRNAs (miRNAs) are small 18- to 22-nucleotide noncoding RNAs
INSULIN-LIKE GROWTH FACTOR-1, RENIN– that regulate gene expression by inhibiting protein translation or by
ANGIOTENSIN SYSTEM, AND HEMATOPOIESIS destabilizing target mRNAs; they are important regulators of hemato-
poiesis. The role of miRNAs in regulation of erythropoiesis is being
Although in vitro studies of erythropoiesis have provided crucial infor- actively defined. Some miRNAs are mainly expressed in early stages of
mation about the regulation of erythropoiesis, many experiments were erythropoiesis, others in late stages, and some have biphasic expression
performed in the presence of serum and serum-component proteins during erythroid differentiation. Some appear to have erythroid spe-
capable of stimulating and inhibiting erythropoiesis. 133,134 Using serum- cific expression. The critical role of miRNAs and their relationship to
143
free conditions, insulin-like growth factor-1 (IGF-1) can partially the essential transcription factors regulating erythropoiesis is outlined

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