The Gene Encoding the Hematopoietic Stem Cell Regulator CCN3/NOV Is under Direct Cytokine Control through the Transcription Factors STAT5A/B

Abstract

Cytokines control the biology of hematopoietic stem cells (HSCs) and progenitor cells in part through the transcription factors STAT5A/B. To investigate the target genes of STAT5A/B activated by cytokines in HSCs and progenitors, we performed microarray analyses using Lineage⁻ Sca-1⁺ c-Kit⁺ (KSL) cells in the presence and absence of STAT5A/B. Stimulation with a mixture containing IL-3, IL-6, stem cell factor, thrombopoietin, and Flt3 ligand induced Ccn3/Nov mRNA over 100-fold in WT (control) but not Stat5a/b-null KSL cells. CCN3/NOV is a positive regulator of human HSC self-renewal and development of committed blood cells. Without stimulation, the Ccn3/Nov signal level was low in control KSL cells similar to Stat5a/b-null KSL cells. To determine which cytokine activates the Ccn3/Nov gene, we analyzed Lineage⁻ c-Kit⁺ (KL) and 32D cells using quantitative PCR and ChIP assays. Although stimulation with a mixture lacking IL-3 prevented the induction of Ccn3/Nov in control KL cells, IL-3 alone could induce Ccn3/Nov mRNA in control KL and 32D cells. ChIP assays using 32D cells revealed IL-3-induced binding of STAT5A/B to a γ-interferon-activated sequences site in the Ccn3/Nov gene promoter. This is the first report that Ccn3/Nov is directly induced by cytokines through STAT5A/B.

Introduction

STAT5A and STAT5B (referred to as STAT5A/B) are two closely related members of the family of signal transducers and activators of transcription (STAT) proteins. STATs are activated upon binding of a ligand to the receptor by phosphorylation of a critical tyrosine residue through Janus kinases (JAK). Activated STATs bind to specific DNA sequences (TTCnnnGAA), named γ-interferon-activated sequences (GAS² sites), and initiate transcription of target genes (1). Genetic evidence links STAT5A/B to the normal physiology and pathophysiology of a wide range of cell types (1), including murine and human HSCs (2–5). Notably, cell-specific ablation of the Stat5a/b locus in mice resulted in a complete loss of T and B cells (6, 7), defective granulopoiesis (8), and erythropoiesis (9). In HSCs, STAT5A is activated by thrombopoietin, IL-3, and granulocyte-macrophage colony-stimulating factor (10). Activation of STAT5A/B is also required for self-renewal and quiescence of HSCs (4, 11), but little is known about the underlying molecular mechanisms and the target genes downstream of STAT5A/B. To investigate STAT5A/B target genes upon stimulation with cytokines in HSCs and progenitor cells, we sorted Lineage⁻ Sca-1⁺ c-Kit⁺ (KSL) cells from wild type (control) and Stat5a/b-null mice and performed microarray analyses.

CCN3/Nov, originally characterized as a site of proviral integration in an avian model of nephroblastoma, belongs to the CCN family of multimodular matricellular signaling proteins (12, 13). The Ccn3/Nov gene encodes a secreted protein that associates with the extracellular matrix and is thought to be involved in the regulation of proliferation, differentiation, survival, adhesion, and migration through integrin-mediated signaling pathways (14). Ccn3/Nov has been reported to act as a tumor suppressor gene in solid tumors including hepatocellular carcinomas, Wilm tumors, Ewing sarcomas, gliomas, and adrenocortical carcinomas (15–17). On the other hand, CCN3/Nov has been suggested as a candidate marker for HSCs, and it has been shown to be essential for HSC and hematopoietic progenitor cell functional integrity (18, 19). It has been reported that CCN3/Nov is essential for self-renewal of HSCs and progenitors and development of committed blood cells after bone marrow transplantation using human cord blood stem cells (19). Therefore, CCN3/Nov and STAT5A/B can be considered positive regulators in normal hematopoietic cells. Here we show that Ccn3/Nov is a cytokine-inducible gene and a direct target of STAT5A/B.

EXPERIMENTAL PROCEDURES

Animals and Fetal Liver Cells

Stat5a/b^−/− (Stat5a/b-null) mice have been described (34). 60 female and 15 male Stat5a/b^+/− mice on the C57BL/6 background were mated to prepare Stat5a/b-null and WT (control) fetal liver cells for each microarray analysis. 10–15 fetal livers were harvested from control or Stat5a/b-null fetuses on embryonic day 14.5, and KSL cells or Lineage⁻ c-Kit⁺ (KL) cells were sorted. Fetal liver mononuclear cells were purified by ACK lysing buffer (Lonza Walkersville, Inc.) and stained with biotinylated antibodies specific for the following lineage markers: CD4 (H129.19), CD8 (53-6.7), B220 (RA3-6B2), Gr-1 (RB6-8C5), TER119 (Ter-119), and IL-7Rα chain (B12-1 or A7R34). Lineage⁺ cells were partially removed with goat anti-rat IgG-conjugated magnetic beads (BioMag; Qiagen), and the remaining cells were stained with streptavidin-PE-Cy5.5 (eBioscience) and anti-c-Kit-allophycocyanin (APC) with or without anti-Sca-1-PE antibodies. To exclude dead cells, propidium iodide was used. Cells were sorted using a MoFlo^TM high performance cell sorter (Dako). All antibodies were purchased from BD Biosciences except streptavidin-PE-Cy5.5 antibody.

Sorting for LT-HSCs and ST-HSCs

Fetal liver cells were harvested from 19 wild type C57BL/6 fetuses at embryonic day 14.5, and adult bone marrow cells were harvested from 20 wild type C57BL/6 mice (11–18 weeks old). Mononuclear cells were purified by ACK lysing buffer (Lonza Walkersville, Inc.) and stained with biotinylated antibodies specific for the following lineage markers: CD4 (H129.19), CD8 (53-6.7), B220 (RA3-6B2), Gr-1 (RB6-8C5), TER119 (Ter-119), and IL-7Rα chain (B12-1 or A7R34). Anti-CD11b antibody (M1/70) was also used only for bone marrow cell staining. Lineage⁺ cells were partially removed with goat anti-rat IgG-conjugated magnetic beads (BioMag; Qiagen), and the remaining cells were stained with streptavidin-Pacific Blue (Invitrogen), anti-c-Kit-PE-Cy7, anti-Sca-1-PE-Cy5.5, anti-CD34-Alexa Fluor 647, anti-CD150-FITC, anti-CD48-PE, and anti-CD135-PE antibodies. To exclude dead cells, propidium iodide was used. KSL CD34⁻CD48⁻CD150⁺CD135⁻ cells were sorted for long term (LT)-HSCs, and KSL CD34⁺ cells were sorted for short term (ST)-HSCs. Cells were sorted using a FACSAria II cell sorter (BD Biosciences). All antibodies were purchased from BD Biosciences except streptavidin-Pacific Blue antibody.

32D Cells

The IL-3-dependent mouse 32D cell line (CRL-11346, ATCC) was used in this study. Cells were grown according to the manufacturer's recommendations in the following complete culture medium: RPMI 1640 with 2 mm l-glutamine adjusted to contain 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, 10 mm HEPES, and 1 mm sodium pyruvate supplemented with 10% heat-inactivated fetal bovine serum and 10% mouse IL-3 culture supplement (354040, BD Biosciences). Cells were starved in complete culture medium without IL-3 for 6 h and stimulated with IL-3 (100 ng/ml) for the indicated times.

Affymetrix Microarray

KSL cells derived from control and Stat5a/b-null fetal livers were stimulated with a cytokine mixture consisting of 100 ng/ml IL-3, IL-6, stem cell factor, thrombopoietin, and Flt3 ligand for 16 h or left untreated. Total RNA from four different samples (Stat5a/b-null-unstimulated, Stat5a/b-null-stimulated, control-unstimulated, and control-stimulated) was prepared with RNeasy Plus mini kits (Qiagen) and processed/analyzed by the NIDDK Core Facility at the National Institutes of Health. Gene Chip expression 3′ amplification reagents in a two-cycle cDNA synthesis kit (Affymetrix) were used. Affymetrix gene expression analysis arrays for the Mouse430_2 were used. The microarray signals were referred to by the Affymetrix robust multichip average algorithm. Up- and down-regulated genes were selected based on p values (<0.05), and -fold changes (>2.0 or <−2.0) were assessed by analysis of variance with the Partek Pro software (Partek). Potential STAT5A/B-regulated genes were identified by comparison of the different experimental groups (see Fig. 1, A–C, and Table 1). The microarray analysis was performed with three independent biologic sample sets. The microarray data have been uploaded to the Gene Expression Omnibus (GEO) under accession number (GSE20684).

FIGURE 1.

Venn diagrams of microarray analyses. Genes significantly up-regulated or down-regulated were selected as -fold change >2.0, p value <0.05. +, stimulated sample; −, unstimulated sample. WT+ versus WT− means the comparison of stimulated control samples with unstimulated control samples. A, genes significantly induced by stimulation in control KSL cells, but unchanged in Stat5a/b-null KSL cells, were selected (560-gene group). B, genes significantly induced through STAT5A/B with or without stimulation were selected (369- and 58-gene group). C, genes significantly induced through STAT5A/B upon stimulation in KSL cells were selected (100-gene group).

TABLE 1.

mRNA levels of STAT5A/B target genes induced in KSL cells upon stimulation with cytokine mixture determined by microarray analysis

-Fold changes and p values were analyzed using three independent experimental datasets. +, stimulated sample; −, unstimulated sample; WT+ vs. WT− means the comparison of stimulated control samples with unstimulated control samples. The sample on the right-hand side of “vs.” was used as the standard in each comparison. Genes were selected according to Fig. 1 as STAT5A/B target genes induced by cytokine-stimulation. Unnamed genes are not shown.

Probe set ID	Gene symbol	WT+ vs. WT−		KO+ vs. WT+
		-Fold change	p value	-Fold change	p value
1426852_x_at	Nov	431.0	0.000	−405.8	0.000
1426851_a_at	Nov	118.4	0.000	−135.3	0.000
1434360_s_at	Ptprg	70.1	0.001	−14.4	0.011
1424800_at	Enah	47.7	0.000	−80.3	0.000
1418186_at	Gstt1	39.8	0.000	−16.0	0.001
1424801_at	Enah	35.3	0.000	−48.0	0.000
1454849_x_at	Clu	21.0	0.000	−7.7	0.004
1418626_a_at	Clu	18.9	0.000	−10.5	0.000
1437689_x_at	Clu	18.1	0.000	−8.7	0.001
1419524_at	Tph1	16.0	0.005	−9.5	0.013
1429691_at	Ptprg	15.3	0.003	−5.7	0.024
1456070_at	Ptprg	14.1	0.006	−5.5	0.038
1417216_at	Pim2	13.5	0.001	−8.2	0.002
1417103_at	Ddt	12.8	0.001	−7.6	0.002
1444177_at	Gm6648	12.2	0.001	−11.4	0.001
1460469_at	Tnfrsf9	12.1	0.000	−9.1	0.000
1420692_at	Il2ra	11.8	0.008	−15.9	0.005
1449033_at	Tnfrsf11b	10.5	0.002	−9.7	0.002
1437458_x_at	Clu	9.5	0.000	−6.8	0.001
1420353_at	Lta	7.6	0.000	−5.1	0.001
1420463_at	Clnk	7.5	0.000	−3.7	0.002
1418666_at	Ptx3	7.3	0.001	−4.7	0.003
1455885_at	Amz1	7.0	0.000	−2.6	0.007
1435396_at	Stxbp6	6.9	0.000	−10.6	0.000
1434180_at	Fermt2	6.7	0.000	−6.7	0.000
1454899_at	Lpp	6.4	0.006	−4.1	0.018
1421473_at	Il1a	6.1	0.007	−5.5	0.009
1449199_at	Muc1	5.9	0.004	−3.4	0.022
1451461_a_at	Aldoc	5.6	0.003	−2.7	0.034
1438796_at	Nr4a3	5.6	0.001	−3.4	0.006
1419226_at	Cd96	5.4	0.003	−8.4	0.001
1440047_at	Socs1	5.2	0.002	−2.7	0.019
1436714_at	Lpp	5.2	0.008	−3.3	0.030
1424097_at	Elovl7	5.2	0.000	−3.4	0.002
1442586_at	Socs2	5.2	0.006	−5.6	0.005
1421624_a_at	Enah	4.9	0.000	−5.3	0.000
1431004_at	Loxl2	4.8	0.050	−5.1	0.043
1448737_at	Tspan7	4.7	0.021	−6.5	0.009
1442223_at	Enah	4.7	0.041	−4.9	0.037
1455314_at	Lpp	4.6	0.013	−3.1	0.043
1427878_at	0610010O12Rik	4.6	0.000	−2.6	0.001
1425253_a_at	Madcam1	4.5	0.001	−3.2	0.004
1427918_a_at	Rhoq	4.4	0.000	−2.1	0.012
1438470_at	Socs2	4.2	0.000	−4.3	0.000
1459903_at	Sema7a	4.0	0.002	−5.4	0.001
1422000_at	Akr1c12	4.0	0.001	−7.3	0.000
1452565_x_at	LOC641050	3.9	0.000	−5.0	0.000
1444062_at	2900056L01Rik	3.8	0.000	−2.6	0.003
1448110_at	Sema4a	3.8	0.007	−2.8	0.021
1449335_at	Timp3	3.8	0.008	−3.5	0.011
1419136_at	Akr1c18	3.8	0.014	−3.6	0.017
1444176_at	Atp6v0d2	3.8	0.013	−2.7	0.039
1459914_at	Mcart6	3.7	0.028	−5.9	0.008
1435901_at	Usp40	3.6	0.001	−3.2	0.002
1457342_at	Ikzf4	3.5	0.021	−5.3	0.006
1429625_at	2900054C01Rik	3.4	0.004	−2.1	0.038
1452436_at	Loxl2	3.4	0.019	−2.7	0.045
1426816_at	Ccdc64	3.4	0.001	−3.1	0.001
1425673_at	Lpp	3.3	0.016	−2.7	0.034
1454711_at	Trio	3.3	0.005	−4.2	0.002
1441891_x_at	Elovl7	3.3	0.001	−2.7	0.002
1440312_at	Elovl7	3.3	0.000	−4.0	0.000
1424098_at	Elovl7	3.2	0.001	−4.1	0.000
1436178_at	Leprel1	3.2	0.027	−3.4	0.022
1438306_at	Rnf180	3.2	0.028	−3.0	0.033
1456429_at	Malt1	3.1	0.014	−2.3	0.045
1444235_at	1700025G04Rik	3.1	0.006	−2.6	0.012
1417883_at	Gstt2	3.1	0.011	−5.2	0.002
1431110_at	Plxdc2	3.1	0.000	−7.9	0.000
1418421_at	Bcl6b	3.0	0.001	−2.4	0.003
1449187_at	Pdgfa	2.8	0.007	−2.3	0.019
1424032_at	Hvcn1	2.8	0.004	−2.6	0.006
1452009_at	Ttc39b	2.7	0.001	−2.0	0.006
1435332_at	Htr7	2.7	0.001	−2.0	0.005
1437235_x_at	Lpp	2.7	0.007	−2.7	0.008
1438242_at	Usp40	2.7	0.001	−2.7	0.001
1427020_at	Scara3	2.7	0.005	−2.8	0.004
1425029_a_at	Mboat2	2.6	0.032	−7.8	0.001
1426712_at	Slc6a15	2.6	0.038	−2.7	0.030
1430069_at	4921517L17Rik	2.6	0.021	−2.7	0.018
1440167_s_at	Lpp	2.6	0.015	−2.3	0.023
1456629_at	Kank3	2.5	0.002	−2.5	0.003
1417749_a_at	Tjp1	2.4	0.003	−3.1	0.001
1433745_at	Trio	2.4	0.010	−4.7	0.001
1432011_at	2900052L18Rik	2.4	0.016	−2.2	0.023
1424030_at	Grhl1	2.4	0.002	−2.4	0.002
1425749_at	Stxbp6	2.3	0.001	−2.6	0.000
1419184_a_at	Fhl2	2.3	0.015	−2.0	0.027
1446770_at	Pik3cd	2.2	0.021	−2.1	0.028
1440910_at	C77370	2.1	0.001	−2.1	0.002
1418350_at	Hbegf	2.0	0.041	−2.1	0.031

qPCR

Sorted KL cells derived from control and Stat5a/b-null mice were stimulated with a complete cytokine mixture or an IL-3-depleted cytokine mixture for 16 h (see Fig. 2B). Control KL cells were stimulated with IL-3 for 3 or 16 h (see Fig. 2C). LT-HSCs and ST-HSCs derived from fetal liver cells or bone marrow cells were stimulated with IL-3 for 16 h (see Fig. 2D). Each cytokine was used at a 100 ng/ml concentration. RNA was isolated and subjected to qPCR. Reverse transcription of total RNA was performed using the SuperScript III protocol (Invitrogen), and TaqMan PCR reactions were done consecutively by using a 7900 HT fast real-time pcr system (Applied Biosystems). TaqMan probes for Ccn3/Nov (Mm00456855-m1) and β-actin (4352341E) were used (Applied Biosystems) for qPCR.

FIGURE 2.

Ccn3/Nov is induced through STAT5A/B upon IL-3 stimulation in KL cells. A, IL-3 activated STAT5A in KL cells. Control KL cells were stimulated with IL-3 (50 ng/ml) followed by fixation and detection of phosphorylated STAT5A by flow cytometry. B, a cytokine mixture induced Ccn3/Nov mRNA in control KL cells but not Stat5a/b-null KL cells. Depletion of IL-3 from the mixture reduced the induction of Ccn3/Nov in control KL cells. Control or Stat5a/b-null KL cells were stimulated with a cytokine mixture or a mixture without IL-3 for 16 h. Ccn3/Nov mRNA was detected by qPCR. -Fold changes were determined as compared with unstimulated samples. Data are shown as mean ± S.D. of triplicates as log scale. C, Ccn3/Nov was induced in control KL cells after stimulation with IL-3 (100 ng/ml) for the indicated time periods. Data are shown as mean ± S.D. of triplicates as log scale. D, Ccn3/Nov was induced in LT-HSCs and ST-HSCs derived from fetal liver and adult bone marrow of wild type mice after stimulation with IL-3 (100 ng/ml) for 16 h. Data are shown as mean ± S.D. of triplicates as log scale.

Detection of Phosphorylated STAT5a by Flow Cytometry

Control KL cells were stimulated with IL-3 (50 ng/ml) for 15 min followed by fixation (2% paraformaldehyde) and permeabilization (cold methanol/acetone, 1:1 v/v). Phosphorylated STAT5 was detected with anti-phosphorylated STAT5 antibody (BD Biosciences) by flow cytometry.

Chromatin Immunoprecipitation

After IL-3 stimulation, a chromatin immunoprecipitation (ChIP) assay was performed using a ChIP assay kit (Upstate Biotech Millipore) according to the manufacturer's recommendations. For immunoprecipitation, normal rabbit serum (negative control), anti-STAT5A, or anti-STAT5B (R&D Systems) antibodies were used. The following primers were used for qPCR: GAS1 forward, 5′-CCCCTTCCATAGAAACATGC-3′; GAS1 reverse, 5′-TTGGACACAATGCGAAAATC-3′; GAS2 forward, 5′-TGGGACATTTTTGGTTCCAT-3′; GAS2 reverse, 5′-ATTTCATGTTCGTGGACTGG-3′. Primers for a region outside of a suspected STAT5A/B binding region were: NS forward, 5′-GAACTGAGTTTCCCATCAACC-3′ and NS reverse, 5′-TTGGCATAGTCAAGCCAGTCC-3′.

Statistical Analyses

Statistical significance was determined by Student's t test (two-tailed, unpaired/unequal variances).

RESULTS

Populations of KSL Cells Derived from Control and Stat5a/b-null Fetal Livers

To explore the function of STAT5A/B in the HSC-enriched population, Stat5a/b-null mice were used. Because Stat5a/b-null mice die perinatally, we used fetal livers at embryonic day 14.5. We sorted KSL cells, which are composed of LT-HSC, ST-HSC, and multipotent progenitor populations. KSL populations of Stat5a/b-null and control samples in Lineage-negative cells were 5.1 ± 1.7 and 2.7 ± 0.5% each (p value <0.01, eight independent experiments).

STAT5A/B Regulate Ccn3/Nov upon Cytokine Stimulation of KSL Cells

Total RNA was isolated after stimulation with a cytokine mixture for 16 h to investigate STAT5A/B target genes by microarray analyses. First we compared stimulated control samples with unstimulated control samples (WT+ versus WT−) to determine the level of stimulation in KSL cells (Fig. 1A). 2171 genes were significantly induced in control KSL cells. Then we identified the genes that were significantly induced by stimulation in control but not in Stat5a/b-null cells (3163 down-regulated genes and 2423 up-regulated genes) (Fig. 1A). The expression of well known STAT5A/B target genes, such as Socs2, pim2, and IL-2Rα, was highly induced in control cells but not Stat5a/b-null cells by stimulation (in the 560-gene group shown in Fig. 1A). Genes significantly induced through STAT5A/B were identified as down-regulated in the comparison of (KO− versus WT−) and (KO+ versus WT+) (Fig. 1B). 369 genes were induced through STAT5A/B upon stimulation with a cytokine mixture, only 58 genes were induced through STAT5A/B without stimulation, and 25 genes were induced through STAT5A/B unrelated to the stimulation. To select STAT5A/B target genes induced by cytokine stimulation, common genes of 560 genes and 344 genes were selected (100 genes) (Fig. 1C, Table 1). These 100 genes met the criteria of “significantly induced by stimulation in control as compared with unstimulated control KSL cells, but unchanged in Stat5a/b-null KSL cells” and “significantly induced in control as compared with Stat5a/b-null KSL cells upon stimulation.” The most strongly induced gene among those 100 genes was Ccn3/Nov, which encodes a positive regulator of normal HSCs and progenitor cells (19). Although the Ccn3/Nov signal level in control KSL cells was similar to Stat5a/b-null KSL cells and low in the absence of cytokine mixture, treatment with cytokines resulted in a more than 100-fold elevation of Ccn3/Nov mRNA only in control but not Stat5a/b-null cells.

IL-3 Is the Key Cytokine for Induction of Ccn3/Nov

To investigate which cytokine is responsible for the induction of Ccn3/Nov through STAT5A/B, KL cells (HSC- and multipotent progenitor-enriched population) were sorted. Phosphoflow cytometry and qPCR were performed using control KL cells. IL-3 activated STAT5a in control KL cells (Fig. 2A). The cytokine mixture induced Ccn3/Nov mRNA in control but not in Stat5a/b-null KL cells (Fig. 2B). IL-3 depletion from the cytokine mixture abolished the induction of Ccn3/Nov mRNA in control KL cells (Fig. 2B). Notably, IL-3 alone induced Ccn3/Nov mRNA in control KL cells (Fig. 2, B and C). Although each individual cytokine in the mixture could induce Ccn3/Nov in KL cells, the strongest activator was IL-3 (supplemental Fig. 1). In addition, single cytokine depletion from the mixture demonstrated IL-3 as the stimulating cytokine of Ccn3/Nov expression (supplemental Fig. 1). The induction of Ccn3/Nov mRNA by IL-3 was also observed in LT-HSCs and ST-HSCs derived from fetal liver cells and bone marrow cells (Fig. 2D). These data demonstrate that IL-3 induced the expression of the Ccn3/Nov mRNA via STAT5A/B in HSCs and progenitors and that IL-3 is the key inducer of Ccn3/Nov among the cytokines in a mixture.

Ccn3/Nov Is under Direct Control through STAT5A/B upon IL-3 Stimulation

To examine the mechanism underlying the induction of Ccn3/Nov mRNA by IL-3 stimulation, STAT5A/B binding to the Ccn3/Nov gene promoter was evaluated using the IL-3-dependent lymphoblast cell line 32D. Elevated Ccn3/Nov mRNA levels were observed after 1 h, and mRNA levels were increased more than 1000-fold by 16 h (Fig. 3A). STAT5A/B consensus binding sites are located at −9560 bp (GAS1: TTCTAAGAA) and −8466 bp (GAS2: TTCTGAGAA) upstream of the transcriptional start site of the murine Ccn3/Nov gene. To test binding of STAT5A/B to these GAS sites, ChIP was combined with qPCR (Fig. 3B). 32D cells were treated with IL-3 for 0, 1, or 6 h followed by ChIP assays. Primers for two potential STAT5A/B binding sites in the Ccn3/Nov gene promoter, GAS1 and GAS2, were used to determine STAT5A/B binding. After 1 h and 6 h, STAT5A/B binding to the GAS1 region was increased by 7- and 15-fold, respectively, as compared with unstimulated cells. However, no increased binding was observed in the GAS2 region.

FIGURE 3.

STAT5A/B bind to GAS sites located upstream of the Ccn3/Nov gene upon IL-3 stimulation. A, IL-3 induced Ccn3/Nov in 32D cells. 32D cells were starved for 6 h and stimulated with IL-3 (100 ng/ml) for 1, 6, or 16 h. Ccn3/Nov mRNA levels were assessed by qPCR as compared with unstimulated samples. Data are shown as mean ± S.D. of triplicates as log scale. B, ChIP assays demonstrated STAT5A/B binding to GAS sites located upstream of the Ccn3/Nov gene. STAT5A/B bound to GAS1 after stimulation with IL-3 (100 ng/ml) for 1 and 6 h in 32D cells. Nonspecific primers (NS) were used for the locus devoid of a GAS site upstream of Ccn3/Nov gene as a negative control. Data are shown as mean ± S.D. of triplicates.

DISCUSSION

STAT5A/B are evidently important for blood cells including HSCs. A critical role of cytokines and STAT5A/B in the self-renewal and expansion of human and mouse HSCs has been reported (2–5). Decreased absolute numbers of KSL cells, lymphocytes, granulocytes, and erythrocytes were observed in the mouse model lacking STAT5A/B in blood cells. Additionally, strong activation of STAT5A/B by Flt3 mutations (internal tandem duplications) has been observed in 30% of patients with acute myelogenous leukemia (20). Constitutive STAT5 activity was demonstrated in BCR-ABL-positive chronic myelogenous leukemia (CML) cell lines, lymphoblastic cell, and hematopoietic cell lines transfected with Bcr-Abl, leading to malignant transformation (21–23). The essential role of STAT5 in BCR-ABL-induced cell growth and transformation was confirmed by several studies that suggest a critical role of STAT5A/B in the initiation and/or progression of CML (24–26). There is no doubt that STAT5A/B play an important role in normal and malignant hematopoiesis. Notably, we and others (4, 7) showed that the KSL population, but not the absolute number, of Lineage-negative liver cells in Stat5a/b-null fetuses was significantly greater than in control littermates. This suggests that in normal hematopoiesis, STAT5A/B might be more important for the maintenance of the precursor pool than the HSC and progenitor pool.

Our ChIP assays demonstrated IL-3-dependent STAT5A/B binding to the Ccn3/Nov gene promoter specifically at the GAS1 site. While the GAS1 sequence is conserved between mouse and human, located −5491 bp upstream of the transcriptional start site of the human Ccn3/Nov gene, but GAS2 is not. This suggests that CCN3/NOV might be induced via STAT5A/B in humans as well. It has been reported that CCN3/NOV plays a role as a positive regulator of HSC self-renewal in human cord blood cells (19). In that study, CCN3/NOV was required for the development of committed lineages. In addition to the role of CCN3/NOV in normal HSCs, CCN3/NOV has been reported as a tumor suppressor in malignant disease. Decreased expression of the CCN3/NOV has been reported in BCR-ABL- positive human CML samples at diagnosis (27). It has been shown that CD34⁺ CML cells treated with imatinib in vitro had increased intracellular and secreted CCN3/NOV protein upon entering remission and that CCN3/NOV induction reduced the colony formation ability of BCR-ABL-positive cells (27). These data suggest that CCN3/NOV is a suppressor of cell growth in CML stem cells similar to its function in solid tumors. However, the role of CCN3/NOV in CML stem cells is opposite to that in normal stem cells. Further studies should focus on elucidating the difference in CCN3/NOV signaling in malignant and normal cells.

Gene expression profiles of human umbilical cord blood-derived hematopoietic stem progenitor cells, murine embryonic stem-derived HSCs, murine HSCs, LT-HSCs, ST-HSCs, and multipotent progenitors have been published, but neither CCN3/NOV nor Ccn3/Nov have been identified in these studies (28–33). We report here that the expression of the Ccn3/Nov gene in various murine cell types, fetal liver-derived LT-HSC and ST-HSC, adult bone marrow-derived LT-HSC and ST-HSC, KSL, KL, and 32D cells, requires the inductive stimulus of cytokines that activate STAT5A/B. It is therefore likely that the absence of the Ccn3/Nov expression in the published HSC gene expression profiles is the result of a lack of cytokine stimulation in these systems. Therefore, we also report that attention should be paid to the design of gene expression profiles.

In summary, this study reveals a novel STAT5A/B target gene in HSCs and progenitor cells that is involved in the regulation of normal hematopoiesis. This is the first report that the expression of the Ccn3/Nov gene, encoding a positive regulator of hematopoiesis in HSCs, is induced after cytokine stimulation through STAT5A/B. However, the mechanism regulating CCN3/NOV expression in CML cells probably involves additional layers as CCN3/NOV levels are suppressed in CML cells despite a constitutive activation of STAT5A/B. In the future, it will be necessary to study CCN3/NOV signaling upon stimulation in adult normal HSCs and in CML cells to understand its diverse roles in normal hematopoiesis and leukemogenesis.