RICERCA ITALIANA     

Research program

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

University Co-ordinator

Seconda Università degli Studi di NAPOLI - MEDICINA SPERIMENTALE - ()

Research Unit Leader

Francesca Rossi

Description

On the basis of the Background and the interaction with the other units the task of this unit will be:

TASK 1 Development of cultures of BFU-E and CFU-E from erythroid precursors from peripheral blood
TASK 2 Identification and characterization of polycythemic patients with truncated erythropoietin receptor
TASK 3 Analysis of the transduction pathways altered in the polycythemic subject already identified with truncated erythropoietin receptors.

EXPERIMENTAL PLANE

TASK 1 Development of cultures of BFU-E and CFU-E from erythroid precursors from peripheral blood
The development of BFU-E and CFU-E samples will be an essential goal of the proposed project. Following it is reported briefly the preparation scheme which we will follow. Preliminar results confirmed that this methodology is able to prepare samples arriched of a specific cell populations.

1) Culture of erythroid cells
Heparinized peripheral blood mononuclear cells (PBMCs) from normal volunteers and polycythemic patients will be isolated on Ficoll-Histopaque gradient.
Early hematopoietic progenitors CD34+ cells will be separated by using a CD34+ cell magnetic sortine kit. The CD34+ cell fraction will be cultured in Iscove’s modified Dulbecco’s medium containg 20% of fetal bovine serum (FBS) and supplemented with 20 ng/ml SCF, 3 IU/mL Epo, 1 ng/ml IL-3 , dexamethasone (10-6 M) and estradiol (10-6 M). CD34+ cells will be seeded at a concentration of 1 x 100000 cells /ml. The cultures will be diluted over time with a much fresh medium to mantain the cell concentration in the range of 1-2 x 1000000 cell/ml. To induce differentiation, the cells will be harvested from the culture, washed with IMDM and cultured in fresh IMDM in presence of 20% FCS, 1 u/ml EPO and 10 ng/ml human ricombinant insulin.
After some days (5-7 for the BFU-E and 10-14 for the CFU-E), the cells will be collected and phenotypically characterized.

2) Phenotypic Analysis of Cells
Cell morphology will be analyzed accordino to standard criteria on cytocentrifuged stained with May-Grunwald-Giemsa. The antigenic profile will be analyzed by flow cytometry with a Coulter Elite ESP Cell Sorter according to standard protocols. The cells will be resuspended in Ca2+ and Mg2+ free phosphate buffered saline supplemented with 1% bovine serum albumin, 2 mM EDTA, and 0,01% NaN3 and labeled on ice with phycoerythrin(PE)-conjugate CD34 and fluorescein isothiocynate (FITC)-conjugate CD71 and anti-glycophorin A. Cells incubated with the corresponding isotope-matched antibodies will be used for gating nonspecific fluorescence and dead cells will be excluded by propidium iodide staining.


TASK 2 Identification and characterization of polycythemic patients with truncated erythropoietin receptor

As reported in the preliminary results we have recently identified a subject affected by congenital and familial erythocytosis showing an alteration at the Epo receptor. The mutation results in the loss of the last 80 amino acids. Up to now, not more than 8 mutations have been identified, namely G>A transition at 6002 (corresponding to 70 aa truncation), G insertion at 5975 (64 aa truncation), 7 bases deletion between 5975-5991 (64 aa truncationa), T insertion at 5967 (69 aa truncation), C>T transition at 5986 (74 aa truncation), CT transition 5881 (110 aa truncation).

In our investigation, however, we have screened up to now for EpoR mutations not more than 40 % of the cases we have already collected except and thus it is completely possible that we will identify other patients with the alterations. Moreover, we plan to obtain additional cases on the basis of our collaboration with AIEOP and Italian Society of Hematology.
To select the cases to investigate, we will analyze only those cases lacking functional and genetics modifications in the oxygen-sensing pathways. This initial screening will be performed by the unit of Dr. Fulvio Della Ragione (functional investigation on the oxygen-sensing processes, level and activity of HIF1-alpha, ubiquitination of HIF1-alpha etc.) and by the unit of Dr. Silverio Perrotta (genetic studied on genes of the oxygen-responding pathways, i.e. VHL, HIF1-alpha, PHDs etc).
We also carried out EpoR genetic analysis on the cases of congenital

In all the instances, we have already discovered one family (two subjects) with a mutation at Epo receptor. In these patients we will investigate the main growing and differentiation characteristics of erythoid progenitors, since it is extremely possible that these cells will growth in the presence of extremely low (or in the absence) of erythropoietin. We will also investigating the dependence of erythroid precursors to other molecule (SCF and glucorticoids) probably involved in erythroid differentiation.
A second major point of investigation, will be the analysis of the expression of the normally and mutated receptor. This is a goal particularly interesting since no data are available in literature regarding the occurrence of the truncated receptor in the erythroid precursors or in any other cell phenotypes.

Initially, by means of real time PCR, we will analyze the relative expression of the two allele. This approach will give to us information the transcriptional efficiency of the two alleles. We will also perform experiments of transcription in vitro employing nuclear preparations of cell from control and the two patients. Finally, by means of treatment with actinomycin D, we will evaluate the half-life of the normal and mutated allele mRNA. This experiment will furnish information on the possible occurrence of different metabolism between the normal and altered transcripts.

In a second part of the task, by means of immunoblotting, we will study the occurrence of the truncated form of the receptor as well as its localization on the membrane. Indeed, so far

it is still completely unknown wheter the mutated protein is stable, is normally synthesized. maturated (EpoR is a glycoprotein), and localized on the membrane. Indeed, it has to be remembered that the single chain of EpoR forms both homodimers and heterodimers with c-Kit.
Thus, it will important to evidentiate whether the truncated form will associate with a) the normal form, b) only with the truncated form, and c) c-Kit. It is also conceivable that the alterd chain was unable to interact with any other chain.

All these features have never been investigated in the other few cases of Epo receptor altered described in literature, thus leaving the biological results of the alteration completely undefined.


TASK 3 Analysis of the transduction pathways altered in the polycythemic subject already identified with truncated erythropoietin receptors.

In this task, we will face questions related to the molecular mechanisms by which alterations of the EpoR influences the growth and differentiation of erythroids cells.
Indeed, as detailed explained in the background section, the intracellular domain of Epo receptor (the C terminus) contains 8 tyrosine whose phosphorylation strongly influences the interaction of the receptor with various of its effectors.

The initial part of the task will be to investigate the effect of mutation on the postive pathways. As in almost all part of the project, we will use erythroid precursors (BFU-E and CFU-E) as model system. It is to underline, that the two patients with the mutation at the Epo receptor as subject to blood withdrawal every two weeks and thus, we will have sufficient materials to perform the planned experiments. The possible constitutive activation of the transduction pathways which drive the antiapoptic activity of the receptor will be evaluated by estimating some of the putative terminal effector including Bcl-x, Pim-1, oncostatin M and SOCS-3.
The role of Bcl-x as antiapoptotic molecule is well established and it clear correspond to the activity of stimulated EpoR. In addition, Pim kinases can confer resistance to rapamycin (an mTOR inhibitor) and can act in parallel with mTOR to modulate eIF4E, an important factor of erythoid differentiation. For oncostatin-M, present analyses reveal substantial expression by erythroblasts, as well as novel survival effects on late-stage erythroblasts. In oncostatin-M–deficient mice, decreases in circulating erythrocytes, interestingly, have been observed.
Then, the complex between the receptor and the positive protein (particularly complexes including Jak2 and Stat5) will be determined by immunoprecipitation and analysis of the immunoprecipitate by selective antibodies.

In addition of evaluating the constitutive activation of the pathways inducing the antiapoptotic response, we will study the effect on the pathways that down-regulate the activation of the receptor.
In this case the activity of SHP-1) (which can dephosphorylate Jak2) will be evaluated as well as the level of Cis-1. The last factor can interfere with Jak2 and/or Stat5 activation and can also target interacting factors for ubiquitination.

Moreover the absence of interaction between the EpoR and SHIP-1 will be studied, by means of immunoprecipitation followed by the analysis of the immunoprecipitate materials by immunoblotting.
Finally, we will investigate the status of the proteins which regulate the cell cycle by comparing sample derived by erythroid progenitors from normal subject and those from the subjects affected by the mutation to the Epo receptor. Indeed, recent data suggest that the modulation of the receptor induces the variation of some of the pivotal protein of the cell cycle including p27Kip1. This part of the project will be performed in collaboration with the unit of Dr Fulvio Della Ragione.