RICERCA ITALIANA     

Control mechanisms of erythropoiesis and congenital and familial polycythemias: role of oxygen-sensing pathways

Seconda Università degli Studi di Napoli

Abstract

Regulation of oxygen homeostasis is critical to survival. Hypoxia results in increased levels of hypoxia-inducible factor (HIF1), which is part of a widespread O2-sensing mechanism providing transcriptional regulation of erythropoietin (Epo), vascular endothelial growth factor (VEGF) and many other hypoxia-regulated genes. HIF1 is composed of two subunits, HIF1-alpha and HIF1-beta, which form a heterodimer; only HIF1-alpha is regulated by hypoxia. Normoxia-induced ubiquitin-mediated degradation of HIF1-alpha protein is the major regulator of HIF1alpha levels. Cellular HIF1-alpha protein levels are increased by hypoxia and HIF1-alpha protein decays rapidly with return to normoxia.
The ubiquitination of HF1-alpha requires a prolyl hydroxylation that commits the transcription factor for the interaction with an E3 ubiquitin-protein ligase. The E3 complex is formed by various proteins including: the von Hippel-Lindau protein (VHL), elongin B, elongin C, cullin 2, and RBX1. The 3 prolyl hydroxylases, identified in mammalian cells use O2 as a substrate to generate 4-hydroxyproline at residues 402 and 564 of HIF1-alpha.
Chuvash polycythemia is the only known congenital polycythemia due to an abnormality in the oxygen pathway. The autosomal recessive disorder,endemic in the mid-Volga River region (Chuvashia), is a non-benign hematological disease characterized by a high hemoglobin content, high plasma Epo, varicose veins, vertebral hemangiomas and low blood pressure. Patients affected by chuvash polycythemia die early, mainly for cerebral vascular events or peripheral thrombosis. Genome-wide screening and candidate gene characterization demonstrated that the Arg200Trp mutation (C598T) of the VHL gene causes the chuvash polycythemia. All of the patients with chuvash polycythemia are homozygotes for this mutation, while all obligate carriers are heterozygotes.
Recently, we demonstrated that in Ischia, an island of Naples bay, exists a cluster of chuvash polycythemia which shows a frequency of heterozygotes higher than in Chuvashia. Moreover, we observed that congenital polycythemia is a disease remarkably frequent in South Italy.
On these bases, the general aim of this proposal is to investigate the molecular bases of various form of congenital polycythemias due to genetic alterations of oxygen-sensing pathways.
Up to now, we collected more than 100 cases of hereditary polycythemias and identify several genetic alterations including: classical VHL mutation (C598T), novel VHL mutation and Epo receptor mutation. Moreover, we found congenital cases of polycythemia not due to VHL mutation but related to alteration of oxygen-depending pathways.
Aims of the projects include: i) the development of biochemical strategies for mechanistically identify the altered steps of the HIF1-alpha metabolism and/or the hypoxia-depending molecular response; ii) the development of an Italian bank of congenital polycythemia cases along with the establishment of lymphoblastoid cell lines from all the patients; iii) the identification of the mechanism(s) responsible for the increased growth response of polycythemic erythroid precursors to Epo, and iv) the characterization of the molecular bases of the polycythemia in subject with only one mutated VHL allele (heterozygotes). In addition, the availability of a polycythemic family with a rare case of truncated Epo receptor will allow us to investigate the processes by which Epo regulates the growth and differentiation of erythoid precursors. In summary, our study, by employing a number of altered genetic conditions and biochemical, cellular biology and genetic approaches will permit to shed new light on several aspects on the mechanisms which connect the blood oxygen pressure to the erythropoiesis control. Moreover, we will obtain novel information on congenital erythocytosis, a non begnign disease, whose pathogenetic mechanisms are largely unknown and clinical features still await for a rational classification. <<<

Principal Investigator

Fulvio Della Ragione Seconda Università degli Studi di NAPOLI

Research Objectives

The project has the general objective to investigate the O2-sensing molecular mechanisms and their alterations which result in an accelerated erythropoiesis and a concomitant polycythemia. The investigation will performed as an integration between clinical and basic expertizes.
It is well known that under hypoxic conditions, renal cells produce high amount of erythropoietin (Epo), a pivotal cytokine which stimulates the bone marrow to produce high amount of red cells. The response of erythroid precursors to Epo is correlated to the engagement of Epo receptor, that after the interaction with its ligand forms either homodimer and heterodimer with the c-Kit receptor. The Epo receptor activation causes a complex array of events, including the phosphorylation of several tyrosines localized in the cytosolic domain of the receptor. Subsequently, the activated receptor induces the up-regulation of a number of pathways with results into increased proliferation and differentiation of the erythroid precursors. Contemporaneously, the receptor is able to activate an intrinsic loop that causes the down-regulation of the receptor activity itself.
The mechanism by which hypoxia causes the up-regulation of Epo gene expression is extremely complex. In brief, low level of O2 induces the increase of a transcriptional factor, i.e. HIF1-alpha, and the consequent expression of the genes modulated by this protein (including Epo and VEGF). The control of oxygen on HIF1-alpha level works at post-translational level. Indeed, low level of 02 prevents the degradation of HIF1-alpha that normally occurs via an ubiquitinilation process employing as E3 activity the VHL (von Hippel-Lindau) protein. Normal level of oxygen, conversely, allows an almost complete removal of HIF1-alpha resulting in a very low level of the transcription factor.

Recently, the groups of research involved in this project have demonstrated that constitutive activation of the O2-responding pathways cause an increased Epo production and a significant erythrocytosis. The congenital genetic alteration involves VHL that, as described above, normally down-regulates the oxygen-responding mechanism and Epo production. The congenital erythrocytosis due to VHL alteration has been previously demonstrated only in Chuvashia, a region of Russia and has been accordingly defined as Chuvash polycythemia.
We have surprisingly demonstrated a very high frequency of Chuvash polycythemia in the South Italy and particularly in Ischia, an island of the Naples harbour (Perrotta et al. Blood. 107, 514-9: 2006).
The discovery that the hereditary disease was endemic in these areas prompted us to investigate in more details the molecular mechanisms at the basis of this form of polycythemia.
In addition, we have collected a significant number of polycythemic cases which show genetic alteration different from the Chuvash mutation. These aberrations involved either VHL gene (novel mutations) or non identified genes of the O2-sensing pathway.
Finally, we have also identified a dominant case of polycythemia due to the truncation of Epo receptor.
The availability for the first time of this large array of genetic conditions, all involving the pathways responding to the O2 blood pressure, might shed new light on the physiological molecular mechanisms regulated by the oxygen.


On the basis of the described findings, our project has the following specific goals.
1. Construction of a registry and a bank of the Italian cases of congenital polycythemia.
2. Development of biochemical methodologies for investigating functional alterations of the O2-depending pathways and the Epo receptor-related pathways
3. Preparation of established cell lines from the congenital polycythemia cases as well as of BFU-E and CFU-E cells from the polycythemic patients.
4. Molecular and clinical characterization of patients affected by polycythemia of Chuvash type (VHL C598T)
5. Molecular and clinical characterization of patients affected by polycythemia due to VHL mutation different from the Chuvash mutation
6. Molecular and clinical characterization of patients affected by polycythemia not due to VHL mutation
7. Characterization of the altered response to Epo observed in polycythemic erythroid precursors.
8. Molecular and clinical characterization of patients affected by polycythemia due to VHL mutation in heterozygosity
9. Molecular and clinical characterization of polycythemias due to truncation of Epo receptor

Several considerations suggest that the proposal goals might be reached..
First of all, a number of cases of the polycythemias above described are already available to the proponent investigators. Moreover, several new cases (not yet analyzed) have been collected in the last year.
Second, the collaboration between the units started several years ago and has resulted in a number of interesting data regarding erythropoiesis
Third, the units posses different expertise which cover basic, genetic and clinical aspects.
Final, all the units work in the same University, making the scientific collaboration very easy.


The results obtained will allow to get novel information on the several obscure aspects of the mechanisms controlled by the oxygen pressure. Moreover, we will shed new light on congenital polycythemias, an heterogeneous disease with a negative prognosis, which still needs both the understanding of the different molecular bases and a rationale classification. <<<

Timescale

24 months

National and international background

Polycythemias are a heterogeneous group of disorders defined by an absolute increase in red cell mass (1, 2). Congenital polycythemias can be:
1. Primary and result from a) inherited defects in hypoxia sensing mechanisms or b) from inherited intrinsic defects in red blood cell precursors that cause increased responsiveness to erythropoietin (Epo) (primary familial and congenital polycythemia).
2. Secondary and are due to inherited conditions that lead to increased serum Epo levels.

The control of oxygen homeostasis is critical to survival and tissue hypoxia might cause cellular dysfunction and ultimately can lead to cell death. Major causes of tissue hypoxia are (a) decreased blood oxygenation (such as occurs in certain pulmonary disorders); (b) altered oxygen release from hemoglobin (associated with some hemoglobinopathies), and (c) impaired blood delivery leading to localized anemia (i.e., ischemia) as a result of low cardiac output or vascular obstruction. In order to adapt to hypoxia, mammals use a number of physiological responses. These include, among others, (a) increased production of erythropoietin (Epo), which augments the production of red blood cells; (b) induction of tyrosine hydroxylase, which facilitates the control of ventilation through the carotid body, and (c) the stimulation of new blood vessels by upregulation of VEGF (1). At the cellular level, hypoxia induces a number of metabolic changes that allow for continued energy generation despite decreased oxygen availability.

One of the pivotal factors in the cellular response to hypoxia is the so-called hypoxia inducible factor (HIF), which transcriptionally activates genes that mediate adaptive responses to reduced oxygen availability. HIF is a heterodimer consisting of one of three alpha subunits (HIF1-alpha, HIF2-alpha, or HIF3-alpha) bound to a protein which is known as HIF1-beta. HIF1-alpha is a member of the basic helix-loop-helix (bHLH) superfamily, in which the HLH domain mediates the binding to DNA and a PAS domain that facilitates the heterodimerization of HIF1-alpha??with HIF1-beta (1–3).
HIF target genes play critical roles in metabolism, angiogenesis, cell proliferation, and cell survival. Examples of HIF target genes include angiogenic growth factors and survival factors (particularly, VEGF, Epo), cell surface receptors and transporters, extracellular matrix proteins and modifying enzymes, transcription factors, cytoskeletal proteins and glycolytic enzymes (aldolase, G6P isomerase; hexokinase, LDH A).
HIF binds to the hypoxia-responsive element, which contains the core recognition sequence 5’-TACGTG-3’ (5), in the cis-regulatory regions of hypoxia-inducible genes. Transcriptional activation by HIF is linked to its ability to recruit coactivator proteins such as CREB-binding (CBP), p300, steroid receptor coactivator-1, and translation initiation factor 2 (4–6). Whereas changes in oxygen levels do not affect HIF1-beta protein levels, hypoxia markedly increases the abundance of the HIF1-alpha subunits (7–11)

The unravelling of the mechanisms underlying the dramatic regulation of HIF1-alpha level has been one of the most intriguing results of the last years. The O2-dependent degradation of HIF1-alpha involves its initial ubiquitination and proteolysis by the 26S proteasome (12-14). The von Hippel-Lindau tumor suppressor protein (VHL) is required for this process, (15, 16). VHL forms a complex with elongin B and C, cullin 2, and RBX1 to form an E3 ubiquitin-protein ligase capable of functioning with E1 ubiquitin-activating and E2 ubiquitin-conjugating enzymes to mediate the ubiquitination of HIF1-alpha (17-20). Because the ubiquitination and degradation of other key regulatory proteins are generally dependent on phosphorylation, it has been supposed that residues of HIF1-alpha underwent this post-synthetic modifications, but no conclusions were reached.
Instead, Pro-564 is hydroxylated in an O2-dependent manner, and this modification is required for VHL binding (21, 22, 23). Pro-402 represents a second site of hydroxylation and VHL binding (24). Pro-402 and Pro-564 are each contained within a similar amino acid sequence (LXXLAP).

Three prolyl hydroxylases (PHD1, 2 and 3) were identified in mammalian cells and shown to use O2 as a substrate to generate 4-hydroxyproline at residue 402 and/or 564 of HIF1-alpha (27-29).
The hydroxylation reaction also requires 2-oxoglutarate as a substrate and generates succinate as a side product. Ascorbate is required as a cofactor. The PHD catalytic site contains an Fe(II) ion that is coordinated by two histidine and one aspartate residue. Unlike heme-containing proteins, the Fe(II) in 2-oxoglutarate- dependent oxygenases can be chelated or substituted by Co(II), rendering the enzyme inactive.

Most importantly, these PHDs have a relatively high Km for O2 that is slightly above its atmospheric concentration, such that O2 is rate limiting for enzymatic activity under physiological conditions (25, 28). As a result, changes in the cellular O2 concentration are directly transduced into changes in the rate at which HIF1-alpha is hydroxylated, ubiquitinated, and degraded. However, a thorough analysis of the relationship between O2 concentration and enzyme activity for each of the PHDs in living cells, and a comparison with the corresponding dose-response curve for HIF1-alpha expression (30), has not yet been reported. In particular, the plot of HIF1-alpha protein levels as a function of O2 concentration in HeLa cells yielded a sigmoidal curve suggestive of cooperativity (30), a finding that is not readily explained by the known biochemistry of the HIF1-alpha PHDs.

Remarkably, HIF1-alpha transactivation domain function is regulated by O2-dependent hydroxylation of Asn-803, which blocks its binding to the coactivators CBP and p300 (31). Factor inhibiting HIF-1 (FIH-1) is a protein that interacts with and inhibits the activity of the HIF1-alpha transactivation domain (32), functions as the asparaginyl hydroxylase. As in the case of the PHDs, FIH-1 appears to use O2 and 2-oxoglutarate and contain Fe(II) in its active site (33-35), although it has a Km for O2 that is three times lower than the prolyl hydroxylases (36-39).
Hydroxylation of Asn-803 is predicted to disrupt the these protein-protein interaction between HIF1-alpha and CBP or p300..Similarly, hydroxylation of Pro-564 has been shownto also function as a molecular switch to positively regulate the interaction of HIF-1? and VHL (40, 41).
Thus hydroxylation provides a mechanism for regulating protein-protein interactions, similar to the effect of phosphorylation and other posttranslational modifications. However, what sets hydroxylation apart is that the modification occurs in an O2-dependent manner, thus establishing a direct link between cellular oxygenation and HIF-1 activity.
One remarkable aspect of the O2-sensing system described above is its plasticity. Although O2 may be the limiting substrate for hydroxylation under physiological conditions, it appears that under altered conditions iron or ascorbate may also be limiting. Furthermore, the expression of the PHDs varies from one cell type to another as well as in response to various physiological stimuli, including hypoxia (28, 42-44). Moreover, the transcriptional response elicited by a hypoxic stimulus also demonstrates a remarkable degree of plasticity, because the battery of target genes that is regulated by HIF-1 is unique to each cell type (45). Thus the identification of the molecular components of the O2-sensing system represents a milestone, rather than a finish line, on the course to defining the physiology of oxygen homeostasis.

Patients affected by CP die early, mainly for cerebral vascular events or peripheral thrombosis. Erythroid progenitors of CP patients are hypersensitive to Epo but the molecular mechanism of the hypersensitivity remains totally obscure.
Genome-wide screening and candidate gene characterization demonstrated that the Arg200Trp (R200W) mutation (C598T) of the VHL gene causes the CP (20). All of the patients with CP are homozygotes for this mutation, while all obligate carriers are heterozygotes (22). Molecular studies in patients with CP indicate that VHL C598T leads to impairment of the interaction of VHL protein with HIF1-alpha and resulting in increased HIF1 levels and expression of downstream target genes (23).
Because CP is characterized by a germline mutation in the VHL gene, it has been hypothesized that homozygotes for this mutation might develop certain vascular tumors as occurs in the classic VHL syndrome. Conversely, VHL C598T homozygosity was associated with varicose veins, lower blood pressures and elevated serum VEGF and PAI-1 concentrations (p&lt;0.0005), as well as premature mortality related to cerebral vascular events and peripheral thrombosis. In no case, tumors typical of the classic VHL syndrome were found (22). VHL C598T homozygotes have been detected in patients with sporadic or familial congenital erythrocytosis from diverse ethnic groups (24-28). Particularly, 19 homozygotes have been identified among the over 150 known cases of non-Chuvash familial erythrocytosis. Furthermore, eight other VHL mutations (Arg79Cys, Gly104Val, Asp126Tyr, Val130Leu, Gly144Arg, Tyr175Cys, Leu188Val, His191Asp, Pro192Ala) have been detected in either homozygotes or compound heterozygotes (24, 25, 27-29) These mutations have been detected in a total of 10 cases, which indicates that the C598T transition is the major cause of VHL-related erythrocytosis. To address the question of whether the VHL C598T substitution occurred in a single founder or from recurrent mutational events, haplotype analysis of eight highly informative single nucleotide polymorphic markers covering 340 kb spanning the VHL gene has been performed on 101 subjects bearing the VHLC598T mutation and 447 normal unrelated individuals from Chuvash, South-East Asian, Caucasian, Hispanic and African-American ethnic groups. The differences in allele frequencies for each marker between 447 normal controls (598C) and 101 subjects bearing 598T were highly significant (p&lt;10-7), indicating strong linkage disequilibrium. In fact, its frequency in Chuvashia is about 0.057 (20), whereas the worldwide frequency of the Chuvash-associated haplotype is about 0.001377 (30). Thus, it has been estimated that the VHLC598T mutation arose in a single ancestor between 12,000 and 51,000 years ago. It is possible that this wide dissemination from the original founder may be associated with some survival advantages for heterozygotes carrying this mutation.
Such an advantage might be related to a subtle improvement of iron metabolism, erythropoiesis, embryonic development, energy metabolism or some other yet unknown effect. An intriguing possibility is raised by the recent demonstration of a protective role for HIF1-alpha in regulating VEGF in pre-eclampsia (31, 32), the leading cause of maternal and fetal mortality worldwide (33). Another positive role of a mildly augmented hypoxic response is an improvment in the bactericidal action of neutrophils, as recently observed in HIF1-alpha knock-in mice (34).
A few cases of CP that appear to have mutations of only one VHL allele confound an obvious pathophysiological explanation. In a Ukrainian family, two children with polycythemia were heterozygotes for VHL G376T (D126Y) but their heterozygotes fathers were not polycythemic (35). Peripheral blood erythroid progenitors from the children and father were hypersensitive to recombinant Epo in in vitro clonogenic assays in a way similar to what is seen in CP patients. The propositus peripheral granulocytes and platelets were polyclonal as determined by an X chromosome based transcriptional clonality assay (36), arguing against an additional somatic mutation of a hematopoietic progenitor leading to clonal hematopoiesis akin to polycythemia vera.
There are some reports describing separate VHL heterozygous patients in whom the inheritance of a null VHL allele in a trans position was excluded; the molecular mechanism of their polycythemic phenotype remains to be elucidated (26). Finally, more than half of patients affected by congenital polycythemias with elevated Epo levels do not have VHL mutations, and the molecular basis of disease remains to be elucidated. Lesions in genes linked to oxygen-dependent gene regulation and their interacting proteins are leading candidates for mutation screening in polycythemic patients with elevated Epo without VHL mutations. Some of these are inherited in a dominant fashion.

An additional form of congenital polycythemia, indirectly connected to an altered oxygen-sensing pathways, is due to a different aspect of the mechanisms controlling the erythropoiesis, namely the alteration of EpoR.

As a central hormonal regulator of red cell production, Epo is required for development beyond the colony forming unit–erythroid (CFUe) stage and functions primarily as an erythroblast survival factor (1). As accurately described before, via mechanisms regulated by HIF1, Epo is expressed in the adult kidney and is secreted as a complex sialoglycoprotein (3). Its actions on erythroid progenitor cells then depend on Epo binding to preformed EpoR dimers (4).

EpoR signals for erythroblast formation involve first the activation of Jak2, an essential upstream
kinase that preassembles at a conserved EpoR box-1 domain (3). Jak2 then mediates the phosphorylation of 8 conserved EpoR cytoplasmic phosphotyrosine (PY 343, 401, 429, 431, 443, 460, 464 and 479) motifs (3). These EpoR PY sites function as a scaffold for the binding of a complex, yet fairly well defined set of Src-homology 2 (SH2) and phosphotyrosine-binding protein domain signal transduction factors. One subset of EpoR PY site–recruited factors coordinates negative feedback. PY429 binds protein tyrosine phosphatase, nonreceptor type 6 (SHP-1), which can dephosphorylate Jak2 (12). PY401 together with PY429 and PY431 binds SOCS-3 and cytokine-inducible SH2-containing protein 1 (Cis-1) (13), which (as suppressors of cytokine signaling) can interfere with Jak2 and/or Stat5 activation and can also target interacting factors for ubiquitination (14). In addition, SH2-containing inositol phosphatase-1 (Ship-1; an inhibitory phosphatase for phosphatidylinositol 3,4,5-triphosphate) associates with activated EpoR complexes and downmodulates PI3K-stimulated events (15).
Epo’s predominating positive signals are linked to a distinct subset of EpoR PY sites, and coupled effectors (3). PI3K binding at EpoR PY479 leads to Akt, mammalian target of rapamycin (mTOR), and p70S6K activation (16, 17). Growth factor receptor– bound protein 2 (Grb2)/Shc binding at PY464 (together with Syp phosphatase binding at PY425) (18) has been linked to murine homologue of Drosophila son of sevenless (mSos)/Ras/Raf/MEK regulation, while phospholipase Cgamma1 activation and calcium flux mediated by transient receptor potential cation channel, subfamily C, member 2 (TRPC2) appear to couple to PY460 (19). EpoR PY site–dependent signals, in addition, have been implicated in Gab docking protein (20) and NF-kB modulation (21). Finally, Stat5 activation occurs predominantly via PY343 (22) and may promote Bcl-xL expression (23). <<<