Expression pattern and crosstalk of G protein-coupled receptors in human erythroid progenitor cells

Abstract During erythropoiesis and human development different globin genes (α, β, γ, δ and ε) are expressed as a result of globin gene switching. We investigated globin gene expression in comparison to the expression of other genes in erythroid progenitor cells (EPC) during ontogenesis using in-house produced microarrays containing 16,659 oligonucleotides. Human primitive CD34+ cells were isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and mobilized peripheral blood (mPB), and developed into EPC in the presence of erythropoietin and other cytokines. The differentiation to EPC was confirmed by flow cytometry as 100% cells were CD71+. In microarray studies, a total of 2996 genes were highly expressed in FL, 2673 genes in CB, 2580 in mPB, 1465 in PB and 1259 in BM derived EPC. 661 of these genes were common for all type of cells. The high level of expression, beside globin genes, was observed for the following genes: transferrin receptor, proteoglycans, ALAS2, Charcot-Leyden crystal protein, nucleophosmin, eosinophil peroxidase, myeloperoxidase and ribonucleases. Most of the analyzed genes demonstrated down-regulation during ontogenesis (elastase 2, glutathione peroxidase 1, SERPINB1, nudix, mitochondrial proteins, ribosomal proteins, enthoprotin, serine proteinase inhibitor), but some showed up-regulation (hexokinase, superoxide dismutase 2, spectrin). Besides developmental changes of globin gene expression during ontogenesis, we also analyzed changes in their expression during erythropoiesis in these different tissues by quantitative PCR. Beta-globin gene expression reached the maximum levels in cells of adult blood origin: BM (176 fmol/μg) and PB (110 fmol/μg). Gamma-globin gene expression, of FL origin, had steady levels during erythroid differentiation (20 fmol/μg), whereas cord blood derived EPC demonstrated consistent up-regulation (60 fmol/μg) in contrast to cells originated from adult blood (3–15 fmol/μg at day 14th). G protein related genes and histone deacetylases were elevated in CB derived EPC, concomitant with increased gamma-globin gene expression. We also analyzed the gamma-globin induction by hydroxyurea, a well known inducer, and established which G protein-coupled receptors involved pathways are activated in PB derived EPC: dopamine receptors D1, D2 and D5, beta 2 adrenergic receptor, human DP prostanoid receptor and prostaglandin E receptor 1, as well as genes activated by cAMP/PKA, PI-3 kinase, MAP and NO/cGMP pathways. This study establishes concomitant changes in expression of globin genes and other known and/or previously unrecognized genes, which appear to be involved in erythropoiesis.

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Mortalin-2 (mthsp70/PBP74/Grp75) Mediates the Erythropoietin Signal To Stimulate Growth of Purified Human Erythroid Progenitor Cells.

Blood ◽

10.1182/blood.v108.11.4165.4165 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4165-4165

Author(s):

Rie Ohtsuka ◽

Yasunobu Abe ◽

Tomomi Fujii ◽

Junji Nishimura ◽

Koichiro Muta

Keyword(s):

Gene Expression ◽

Cell Growth ◽

Progenitor Cells ◽

Expression Pattern ◽

Gene Expression Pattern ◽

Janus Kinase ◽

Expression Vectors ◽

Brdu Incorporation ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

Abstract Erythropoietin (EPO) stimulates erythroid growth by enhancing the proliferation, maturation and survival of late stage erythroid progenitor cells. Intracellular signaling molecules such as Janus kinase 2 (JAK2) and phosphoinositide-3 kinase (PI-3K)/Akt are considered mediators of the EPO signal; however, the entire process of EPO stimulation remains undetermined. Previously, we used siRNA to show that mortalin-2, which is a member of the heat shock protein 70 family of chaperones, mediates EPO signaling to stimulate the growth of human erythroid colony forming cells (ECFCs). In the present study, we examined the relationship between cell growth and mortalin-2 overexpressed in ECFCs, and analyzed the gene expression pattern of ECFCs treated with mortalin-2 siRNA using a DNA microarray, to further clarify the intracellular mechanism by which EPO and mortalin-2 interact. In the presence of different concentrations of EPO (0–1.0 U/ml), the effect of mortalin-2 overexpression on ECFC growth was determined by MTT assay. There was tendency to better viability of ECFCs treated with mortalin-2 expression vectors than control cells, especially in the EPO 0 U/ml group (p=0.08). BrdU ELISA, used to investigate the effects of mortalin-2 on the DNA synthesis of ECFCs, revealed that when ECFCs were treated with mortalin-2 expression vectors, the cells showed an increase in the amount of BrdU incorporation into DNA without EPO. Next we analyzed the gene expression pattern using mRNA obtained from ECFCs cultured with or without EPO after treatment with mortalin-2 siRNA or control siRNA. When ECFCs were cultured with EPO after treatment with mortalin-2 siRNA, the expression of 19 genes was suppressed to less than 0.6 fold, and these genes included those involved in cell growth, apoptosis or transport, such as interleukin 6 receptor, ATP-binding cassette, Mdm2, BCL2 interacting protein and interleukin 10 receptor alpha. Furthermore, the expression of 8 genes was upregulated to over 1.5 fold, and these genes included transcription or signal transmission related genes, such as ubiquitin A-52 residue ribosomal protein fusion product 1 and serum/glucocorticoid regulated kinase 2. There were also some genes whose expressions overlapped with genes obtained from ECFCs cultured without EPO after treatment with control siRNA. Our data suggests that mortalin-2 expression depends on various pathways, and that one of these pathways mediates EPO signaling to stimulate mortalin-2 expression, which is related to the growth of erythroid progenitor cells.

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