Globin gene expression in correlation with G protein-related genes during erythroid differentiation
© Čokić et al.; licensee BioMed Central Ltd. 2013
Received: 1 June 2012
Accepted: 11 February 2013
Published: 20 February 2013
The guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs) regulate cell growth, proliferation and differentiation. G proteins are also implicated in erythroid differentiation, and some of them are expressed principally in hematopoietic cells. GPCRs-linked NO/cGMP and p38 MAPK signaling pathways already demonstrated potency for globin gene stimulation. By analyzing erythroid progenitors, derived from hematopoietic cells through in vitro ontogeny, our study intends to determine early markers and signaling pathways of globin gene regulation and their relation to GPCR expression.
Human hematopoietic CD34+ progenitors are isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and G-CSF stimulated mobilized PB (mPB), and then differentiated in vitro into erythroid progenitors. We find that growth capacity is most abundant in FL- and CB-derived erythroid cells. The erythroid progenitor cells are sorted as 100% CD71+, but we did not find statistical significance in the variations of CD34, CD36 and GlyA antigens and that confirms similarity in maturation of studied ontogenic periods. During ontogeny, beta-globin gene expression reaches maximum levels in cells of adult blood origin (176 fmol/μg), while gamma-globin gene expression is consistently up-regulated in CB-derived cells (60 fmol/μg). During gamma-globin induction by hydroxycarbamide, we identify stimulated GPCRs (PTGDR, PTGER1) and GPCRs-coupled genes known to be activated via the cAMP/PKA (ADIPOQ), MAPK pathway (JUN) and NO/cGMP (PRPF18) signaling pathways. During ontogeny, GPR45 and ARRDC1 genes have the most prominent expression in FL-derived erythroid progenitor cells, GNL3 and GRP65 genes in CB-derived cells (high gamma-globin gene expression), GPR110 and GNG10 in BM-derived cells, GPR89C and GPR172A in PB-derived cells, and GPR44 and GNAQ genes in mPB-derived cells (high beta-globin gene expression).
These results demonstrate the concomitant activity of GPCR-coupled genes and related signaling pathways during erythropoietic stimulation of globin genes. In accordance with previous reports, the stimulation of GPCRs supports the postulated connection between cAMP/PKA and NO/cGMP pathways in activation of γ-globin expression, via JUN and p38 MAPK signaling.
KeywordsG protein G protein-coupled receptors Erythroid progenitors Ontogeny Globins
The guanine nucleotide binding protein (G protein)-coupled receptor (GPCRs) family represents the largest group of cell surface receptors that regulate cell growth, proliferation, and differentiation . The silencing of Gpr48, as GPCR is highly expressed in the fetal liver (FL) and premature erythroblast, has no effects on primitive erythropoiesis but significantly reduces definitive erythropoiesis through the cAMP/PKA/CREB pathway . Inactivation of Gpr48 induces remarkable decreases in the proliferation of definitive erythroid progenitors and erythroblast islands in FL . GPCRs are linked via G proteins to adenylyl cyclase, phospholipases, and ionic conductance channels . Thus, the Gαs protein is known to couple GPCRs to adenylyl cyclase to stimulate formation of the second messenger cAMP. It has been found that, upon activation of the cAMP pathway, expression of the gamma (γ)-globin gene is induced in adult erythroblasts . Once formed, cAMP consecutively stimulates cAMP-dependent protein kinase (PKA). According to our previous results, cytostatic hydroxycarbamide (hydroxyurea) also induces phosphorylation of endothelial nitric oxide synthase (eNOS) in a PKA-dependent manner . Hydroxycarbamide, as a γ-globin inducer, increases intracellular cAMP levels as well as cGMP levels in human erythroid progenitor cells . Fetal hemoglobin induction by hydroxycarbamide is mediated by the nitric oxide (NO)-dependent activation of soluble guanylyl cyclase (sGC) .
G proteins also couple the receptors to other cellular effectors systems. Thus, Gαo has been shown to link GPCRs to Ca2+ conductance channels to regulate the influx of Ca2+ to cells . Hydroxycarbamide-induced rise in intracellular Ca2+ demonstrates dependence on the calcium leak from endoplasmic reticulum . In addition to G proteins, GPCRs also couple with β-arrestins involved in termination of receptor activation after prolonged agonist binding . Furthermore, β-arrestins facilitate the internalization of GPCRs, followed by ubiquitination and proteasome degradation with consequential GPCR down-regulation . We showed that hydroxycarbamide inhibited the proteasome activity, which also supports the correlation between GPCRs and globin genes control .
Several groups have examined the gene expression profile of human CD34+ hematopoietic progenitor cells from bone marrow (BM), peripheral blood (PB) and cord blood (CB) using microarray technology [12, 13]. The modulation of gene expression during ontogeny in FL- and CB-derived hematopoietic progenitor cells appears to overlap largely with early response genes of growth factor stimulated adult BM hematopoietic progenitor cells . Recent studies have begun to define general gene expression profiling of human erythroid cells from different origins - adult BM and PB [15, 16]. In general, it has been hypothesized that globin gene switching may be mediated by proteins expressed during different stages of ontogeny.
A previous report demonstrated that stromal feeder layers of human FL, CB, and adult BM did not change hemoglobin types during erythroid differentiation of CD34+ hematopoietic progenitor cells derived from the equivalent tissues . Instead of this approach, we perform erythroid differentiation of only CD34+ hematopoietic progenitor cells originated from fetal to adult hematopoietic cells. The erythroid cells growth and differentiation markers have been determined in in vitro liquid cultures. The γ-globin gene expression is the most increased in CB-derived erythroid cells, while beta (β)-globin gene expression is the highest in adult blood cells (BM, PB) during erythroid differentiation. We compare the G proteins and GPCRs gene expression at several stages in ontogeny by array analyses. During γ-globin induction, we identify GPCRs related genes that were activated via the cAMP/PKA, p38 MAP kinase and NO/cGMP signaling pathways.
Characterization of erythroid cell cultures
Globin genes expression during erythroid differentiation
Effect of hydroxycarbamide on G protein-coupled receptor signaling pathways in human erythroid progenitor cells
Human G protein-coupled receptor signaling pathway profile in erythroid progenitor cells, of peripheral blood origin, after treatment with hydroxycarbamide
G protein-coupled receptors:
Beta 2 adrenergic receptor
D1 dopamine receptor
D2 dopamine receptor
D5 dopamine receptor
Sphingosine-1-phosphate receptor 2
Human DP prostanoid receptor
Prostaglandin E receptor 1, EP1 subtype
PI-3 kinase pathway:
v-akt murine thymoma viral oncogene homolog 1
Adiponectin, C1Q and collagen domain containing
PRP18 pre-mRNA processing factor 18 homolog
PKC pathway (Ca 2+ ,MEK, etc.):
Luteinizing hormone beta polypeptide
Jun B proto-oncogene,
ETS-domain protein (SRF accessory protein 1)
Heat shock 70 kDa protein 4
Heat shock 90 kDa protein 1, alpha
Suppressor of cytokine signaling 1
MAP kinase pathway (p42/p44MAP, p38MAP):
Plasminogen activator inhibitor, type I
v-jun avian sarcoma virus 17 oncogene homolog
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein
Human helix-loop-helix zipper protein
G-protein related genes in erythroid progenitor cells during ontogenesis
To reveal the essential mechanisms in erythropoiesis several studies have performed gene expression profiling in erythroid cells from hematopoietic tissues through ontogeny. The findings in these reports are heterogeneous, reflecting the variation in the experimental systems used. By choosing early erythroid progenitors differentiated from purified CD34+ cells, we extend those studies to evaluate globin gene expression in correlation to GPCR-coupled genes from fetal to adult erythropoiesis. The γ-globin gene expression is most prominent in CB-derived erythroid progenitors, whereas β-globin gene expression is major in adult blood-derived (BM, PB, mPB), low in CB-derived and almost completely absent in FL-derived erythroid progenitors. The GPCR-coupled genes have been studied in erythroid progenitor cells during stimulation of γ-globin gene production. Hydroxycarbamide stimulates the expression of several genes (ADIPOQ, SOCS1, HSP90AA1, PRPF18) activated by GPCRs via cAMP/PKA, p38 MAPK, and NO/cGMP signaling pathways. A certain number of G-proteins (α, β, γ isoforms) and GPCRs demonstrate variation or stability of gene expression in erythroid progenitor cells during ontogeny. Genes GPR45 and ARRDC1 have the most prominent expression in FL-derived, genes GNL3 and GRP65 in CB-derived (with high γ-globin gene expression), GNL2, GPR110, ARRDC2 and GNG10 in BM-derived (with β-globin gene expression), GNAQ, GNA13 and GPR44 in mPB-derived, GPR89C and GPR172A in PB-derived erythroid progenitors.
The G-protein α15 (GNA15) is expressed particularly in hematopoietic cells . Beta 2-adrenergic receptor (ADRB2), induced by hydroxycarbamide, can specifically couple to GNA15 upregulated in erythroid progenitors of CB and adult cells origin in our microarray study . It has been reported that calcium-sensing receptor-mediated MAP kinase (ERK1/2) activation requires GNAI2 coupling . Inhibition of the ERK pathway lead to increased hemoglobin levels . Furthermore, according to our results GNAI2 gene expression is decreased in every examined erythroid progenitors. The activation of p38 MAPK signaling pathway, induced by GNA12/13, is also involved in butyrate-mediated erythroid differentiation, another γ-globin inducer as well as inhibitor of histone deacetylases . Additionally, the inhibition of histone deacetylases induced a high increase of γ-globin mRNA and activated p38 signaling during fetal hemoglobin stimulation .
While mechanisms involved in globin gene expression have been recognized at different levels within the regulatory hierarchy, relations among these molecular pathways are only emerging. We associate our new results with the NO/cGMP pathway described in our previous publications [6, 7], and demonstrate the induction of G-proteins and GPCR-coupled genes during γ-globin stimulation and erythropoiesis through ontogeny. These genes and related signaling pathways, involved in the mechanism of globin activation, might be targets for the therapeutic agents to upregulate γ-globin gene expression and fetal hemoglobin levels in hemoglobinopathies. Therefore, further direct studies are required to confirm that modifications in the level of expression of GPCRs lead to significant changes in NO/cGMP and other signaling pathways important for γ-globin gene expression in erythroid cells.
Liquid erythroid cell cultures
For erythroid progenitor cell cultures, blood was obtained from consenting normal volunteers from the National Institutes of Health, Department of Transfusion Medicine according to the regulations and guidelines of the Office of Human Subjects Research. Adult PB mononuclear cells are isolated from buffy coats of healthy donors (3 individuals per experiment) using Lymphocyte Separation Medium (BioWhittaker, Walkersville, MD). We wash mononuclear cells twice with Dulbecco’s phosphate-buffered saline (PBS, Invitrogen Corporation, Carlsbad, CA), and CD34+ hematopoietic progenitors are purified by positive immunomagnetic selection using the MACS cell isolation system (Miltenyi Biotec, Auburn, CA). Commercial FL- (Cambrex Bio Science, Inc., Walkersville, MD), CB-, BM- and granulocyte-colony stimulating factor (G-CSF) stimulated mobilized PB- (mPB, AllCells LLC, Berkeley, CA) derived CD34+ cells are also collected by positive immunomagnetic selection (Miltenyi Biotec). To stimulate erythroid differentiation, the labeled CD34+ cells of all samples are cultured in the medium that contains 30% fetal bovine serum (FBS), 2 mmol/L glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% deionized bovine serum albumin, 10 mmol/L mercaptoethanol, 1 mmol/L dexamethasone, 33 μg/ml holo-transferrin, 10 ng/ml SCF, 1 ng/ml IL-3 and 1 ng/ml GM-CSF (Sigma, St. Louis, MO), and 1 U/ml human recombinant EPO (Amgen Inc, Thousand Oaks, CA) . For microarray analysis, erythroid progenitors are isolated at day 6 of erythroid cell culture at 37°C and 5% CO2 with balanced 95% room air. At different time points during in vitro erythroid differentiation, the viable cell counts are performed with the use of a trypan-blue exclusion technique (BioWhittaker).
After 6 days of erythroid culture, 5×105 cells are washed in PBS containing 0.5% FCS and 0.02% sodium azide and incubated for 20 minutes at the ambient temperature in the presence of the appropriate monoclonal antibodies at a twofold saturating concentration. Anti-glycophorin-A (GlyA) FITC, anti-CD34 PE, anti-CD71 Tricolor, anti-CD36-APC markers are used for cell staining at day 6 of erythroid culture (Beckman-Coulter, Miami, FL). Erythroid cells are then washed, fixed in PBS containing 4% formaldehyde, and acquired on an LSRII flow cytometer (BD Biosciences, San Jose, CA) equipped with lasers emitting at wavelengths 355, 488, 532, 407 and 638 nm using DIVA4.1.2 software. The saturation of unspecific binding sites is achieved by normal mouse serum control present in the staining buffer. Data are analyzed with Flowjo software (Tree Star, San Carlos, CA).
Isolation of total RNA
After 6 days of erythropoietin treatment and incubation at 37°C (5% CO2, 95% humidity), we use the RNeasy protocol for isolation of total RNA from erythroid progenitor cells (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Concentration and integrity of total RNA is assessed using an 8453 UV/Visible Spectrophotometer (Hewlett-Packard GmbH, Waldbronn, Germany) and Agilent 2100 Bioanalyzer Software (Agilent Technologies, Waldbronn, Germany) comparing the ratio of 28S and 18S RNA peaks to ensure that there is minimal degradation of the RNA sample. One microgram of total RNA is reverse-transcribed with SuperScript II RNase H- Reverse Transcriptase (Invitrogen Corporation).
Quantitative real-time PCR assay of γ- and β-globin mRNA transcripts is carried out with the use of gene-specific double fluorescently labeled probes in a 7700 Sequence Detector (Applied Biosystems, Foster City, CA). The specific primers and TAQMAN probes (synthesized by the NIDDK core oligonucleotide facility) are designed using Primer Express software (Applied Biosystems) and prepared on an ABI 394 synthesizer (Applied Biosystems) as previously described . Platinum Quantitative PCR SuperMix-UDG (Invitrogen Corporation) is used for each of the primer pairs containing a final concentration of 200 μM dNTPs, 0.5 μM Rox reference dye (Invitrogen Corporation), 0.2 μM each of TAQMAN probe, forward and reverse primers. Expression levels are determined using the associated SDS software (ABI Prism, Applied Biosystems) and Microsoft Excel (Redmond, WA). Standard curves are constructed using dilutions of an accurately determined plasmid containing the cDNA of interest as template.
Total human universal RNA (HuURNA) isolated from a collection of adult human tissues to represent a broad range of expressed genes from both male and female donors (BD Biosciences, Palo Alto, CA) serve as a universal reference control in the competitive hybridization. All 5 blood tissues are hybridized against HuURNA. The correlation coefficients among those biological repeats themselves are consistently ≥ 0.8, which documented the quality of hybridization and consistency of expression among the replicates of all 5 tissues. Labeled cDNA probes are produced as described .
For hybridization, 36 μl hybridization mixture (cDNA mixture, 10 μg COT-1 DNA, 8–10 μg poly(dA), 4 μg l yeast total RNA, 20X SSC and 10% SDS) is pre-heated at 100°C for 2 minutes and cooled for 1 minute. Total volume of probe is added on the array and covered with cover slip. Slides are placed in hybridization chamber and 20 μl water is added to the slide, and incubated overnight at 65°C. Slides are then washed for 2 minutes each in 2X SSC, 1X SSC and 0.1X SSC and spin-dried.
iii) Data Filtration, normalization, and analysis
Microarray slides are scanned in both Cy3 (532 nm) and Cy5 (635 nm) channels using Axon GenePix 4000B scanner (Axon Instruments, Inc., Foster City, CA) with a 10-micron resolution. Scanned microarray images are exported as TIFF files to GenePix Pro 3.0 software for image analysis. For advanced data analysis, gpr and jpeg files are imported into microarray database, and normalized by software tools provided by NIH Center for Information Technology (http://nciarray.nci.nih.gov). We gathered a set of 8,719 erythroid cells gene expression data derived from 11 datasets that have been posted on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database.
Human G Protein-coupled receptors signaling pathway finder gene array
Erythroid progenitor cells, of PB origin, are treated with hydroxycarbamide at day 4 after stimulation with EPO and incubated 48 hours at 37°C. At day 6, total RNA is isolated. GPCRs array is completed using the GEArray Q Series Chemiluminescence Detection User system (SABiosciences, Frederick, MD). Briefly, the preheated annealing mix with 2 μg of total RNA is added to labeling mix with biotin-16-dUTP (Roche Applied Science, Indianapolis, IN) and reverse transcriptase (Promega Corporation, Madison, WI). The biotin labeled cDNA probe is denatured before addition of the probe to the hybridization solution with the GEArray Q Series membrane. After hybridization with continuous agitation, and washing, the chemiluminescent detection is performed. We use a digital imaging system (FluorChem Imaging system, Alpha Innotech Corporation, San Leandro, CA) to record the chemiluminescent image of the array. The relative abundance of a particular transcript is estimated by directly comparing its signal intensity to the signal derived from a housekeeping gene cyclophilin A. The list of 110 genes for human GPCRs signaling pathway finder gene array is available from the manufacturer (SABiosciences, Frederick, MD).
The one way ANOVA Tukey’s Multiple Comparison tests and paired t test are applied using Prism 4 software (GraphPad Software Inc., San Diego, CA) for measurement of statistical significance in cell growth and antigen levels among blood tissues, as well as for γ- and β-globin expression. For microarray data management and analysis, we use NCI/CIT microArray database (mAdb) system.
This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases and by grant from the Serbian Ministry of Education and Science .
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