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(Journal of Leukocyte Biology. 2002;71:641-651.)
© 2002 by Society for Leukocyte Biology

Physiological levels of 1{alpha}, 25 dihydroxyvitamin D3 induce the monocytic commitment of CD34+ hematopoietic progenitors

Alexis Grande*, Monica Montanari*, Enrico Tagliafico*, Rossella Manfredini*, Tommaso Zanocco Marani*, Michela Siena*, Elena Tenedini*, Andrea Gallinelli{dagger} and Sergio Ferrari*

* Dipartimento di Scienze Biomediche, Sezione di Chimica Biologica, and
{dagger} Dipartimento di Scienze Ginecologiche, Ostetriche e Pediatriche, Università di Modena e Reggio Emilia, Modena, Italy

Correspondence: Sergio Ferrari, Dipartimento di Scienze Biomediche, Sezione di Chimica Biologica, Università di Modena e Reggio Emilia, Via Campi 287, 41100, Modena, Italy. E-mail: sergio{at}unimo.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although supraphysiological levels of 1{alpha}, 25 dihydroxyvitamin D3 (VD) have been demonstrated extensively to induce the monomacrophagic differentiation of leukemic myelo- and monoblasts, little is known about the role that physiological levels of this vitamin could play in the regulation of normal hematopoiesis. To clarify this issue, we adopted a liquid-culture model in which cord blood CD34+ hematopoietic progenitors, induced to differentiate in the presence of different combinations of cytokines, were exposed to VD at various concentrations and stimulation modalities. The data obtained show that physiological levels of VD promote a differentiation of CD34+ hematopoietic progenitors characterized by the induction of all the monomacrophagic immunophenotypic and morphological markers. This effect is not only exerted at the terminal maturation but also at the commitment level, as demonstrated by the decrease of highly undifferentiated CD34+CD38- hematopoietic stem cells, the down-regulation of CD34 antigen, and the increase of monocyte-committed progenitors. Molecular analysis suggests that the VD genomic signaling pathway underlies the described differentiation effects.

Key Words: vitamin D3 • hematopoietic stem cells • monocytic differentiation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone marrow (BM) is the principal source of mature blood-cell formation in normal adults. To assure this function, BM contains cells at all stages of hematopoietic cell development, including the most primitive stem cells. A key property of hematopoietic stem cells is represented by their capacity to self-renew or to differentiate to committed hematopoietic progenitors. In turn, these last cells are able to generate the more mature hematopoietic precursors and blood cells [1 ]. The complex process of hematopoiesis appears to be locally regulated by interaction of hematopoietic cells with secreted cytokines produced by adjacent BM stromal cells, a heterogeneous cell population composed of macrophages, endothelial cells, fibroblasts, and adipocytes [1 ]. Some of these cytokines, such as stem-cell factor (SCF), FLT3-ligand (FLT3-l), thrombopoietin (TPO), interleukin-11 (IL-11), IL-6, and IL-3, are mainly responsible for pluripotent stem-cell proliferation and commitment [2 , 3 ]. Other cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, erythropoietin (Epo), and again TPO, are involved in the differentiation of specific hematopoietic lineages instead [1 , 2 , 4 , 5 ].

Several lines of evidence suggest that some vitamins, i.e., compounds that are biochemically distinct from cytokines, can also regulate aspects of normal hematopoiesis, perhaps by cooperating with hematopoietic growth factors. These compounds are able to exert their biological effects through the interaction with specific nuclear receptors belonging to the steroid receptor superfamily [6 ] and characterized by the ability to promote a ligand-dependent activation of gene transcription [7 ]. Based on these properties, they have also been defined as nuclear hormones. An example of a vitamin able to control normal hematopoiesis is represented by all-trans retinoc acid (ATRA). In fact, when added to a liquid culture of murine hematopoietic stem cells, pharmacological concentrations (10-6 M) of ATRA delay the differentiation of the most primitive stem cells and at the same time, enhance the terminal granulocytic maturation of granulo-monocyte-committed progenitors [8 ]. A distinct nuclear hormone, 1{alpha}, 25 dihydroxyvitamin D3 (VD), can also regulate the differentiation of normal hematopoietic cells. Initially, VD was demonstrated to be a powerful differentiation inducer for a large variety of neoplastic cells, including carcinoma cells of different origin, and acute myeloid leukemia (AML) blasts having a M2-M3 or M5 phenotype [9 ]. In fact, the in vitro treatment of AML cells with a sole administration of VD at supraphysiological concentrations (10-7/10-8 M) results in a monocyte-macrophage differentiation that is achieved within 5 days from stimulation [9 10 11 ]. By using comparable experimental conditions, it was subsequently shown that high concentrations of this vitamin also enhance the capacity of normal BM cells to generate macrophagic colonies in semisolid media [12 13 14 ]. In general, two distinct intracellular pathways, genomic and nongenomic, respectively, are responsible for VD-induced biological effects. The interaction of this vitamin with the corresponding nuclear VD receptor (VDR) mediates the genomic actions [15 ]. As a heterodimer with the retinoid-X receptor (RXR), the ligand-occupied form of VDR binds to transcription-control sequences known as VD response elements (VDRE), leading to the induction of downstream VD target genes. Recently, the interaction between the VDR/RXR heterodimers and several transcriptional coactivators has been demonstrated to be necessary to activate a VDR-dependent gene expression [15 ].

In addition to the genomic effects described above, VD has also been shown to modulate nongenomic actions that lead to a rapid intracellular influx of Ca2+ and involve the activation of protein kinase C (PKC) [9 , 15 ] and PI-3 kinase (PI-3K), based on a recent observation [16 ]. To date, it is still controversial which of the two signaling pathways described is responsible for the differentiation effects exerted by VD on hematopoietic cells, and several lines of evidence support the possibility of a cross-talk between the genomic and nongenomic actions of this vitamin [16 , 17 ].

In spite of the evidence that supraphysiological concentrations of VD can induce the mono-macrophagic differentiation of late hematopoietic progenitors and precursors, it remains to be clarified whether physiological levels of this vitamin are able to influence the late as well as early phases of hematopoiesis. For this purpose, we performed liquid cultures of cord blood (CB) CD34+ hematopoietic stem cells. In fact, when cultured in the presence of early acting hematopoietic cytokines, these cells differentiate to mature myeloid cells within 14 days. This differentiation is characterized by a decline of the CD34+ and the highly undifferentiated CD34+CD38- cell populations, which are almost complete at day 7, and by the gradual appearance of granulocytic and mono-macrophagic differentiation markers, culminating at day 14 of culture. Then, the capacity of VD to modulate the proliferation and differentiation of these primary hematopoietic cells has been analyzed by cytofluorimetry, morphology, and a colony-forming cell (CFC) assay. This last assay was used simply to evaluate the proportion of the different hematopoietic progenitors upon stimulation with the investigated compound and not as an experimental model, as reported previously [12 13 14 ]. In addition, a molecular analysis has been performed aimed at characterizing the intracellular pathway activated as a result of the stimulation with the vitamin used. The data obtained provide novel evidence that physiological levels of VD induce the mono-macrophagic differentiation of CD34+ progenitors through an effect that is exerted not only at the terminal maturation level but also at the commitment level, highlighting the role played by this nuclear hormone in the regulation of normal hematopoiesis. Furthermore, our results suggest that VD genomic pathway is involved in this differentiation effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
CD34+ stem cell purification, culture, and differentiation
Human CD34+ cells were purified from umbilical CB samples collected after normal deliveries, according to institutional guidelines for discarded material. Initially, mononuclear cells were obtained by a Ficoll-Hypaque (Lymphoprep; Nycomed Pharma, Oslo, Norway) gradient separation. CD34+ hematopoietic stem cells were then separated using the Direct CD34 progenitor cell isolation kit (MiniMacs; Miltenyi, Auburn, CA), based on a magnetic cell-sorting procedure, normally yielding a purity higher than 95%. To achieve an optimal expansion and differentiation of these primary hematopoietic cells, a 14-day liquid culture was performed by seeding CD34+ cells in 24-well plates at a density of 5–10 x 104/ml (1.5–2 ml/well) in Iscove’s modified Dulbecco’s medium (Gibco-BRL, Grand Island, NY), added with 20% fetal calf serum (FCS; Gibco-BRL) in the presence of human (hu) hematopoietic cytokines as: SCF (50 ng/ml), FLT3-l (50 ng/ml), IL-11 (50 ng/ml), IL-6 (10 ng/ml), and IL-3 (10 ng/ml; all from R&D Systems, Minneapolis, MN). After the first week of culture, hematopoietic cells were expanded further by seeding each sample in 4–5 ml/well fresh medium and cytokines in six-well plates always at a density of 5–10 x 104/ml. In a distinct set of experiments, 50 ng/ml human TPO (Euroclone, UK) was added to the suspension culture to obtain a long-term self-renewal of CD34 hematopoietic progenitors as described [18 , 19 ]. In this last case, the culture was prolonged up to 1 month, subculturing the cells weekly. VD (Hoffman-Laroche, Basel, Switzerland) was added to the different CD34 cell cultures at concentrations ranging from 10-10–10-8 M, weekly or daily, according to the experimental planning.

CFC assays
CFC assays were carried out by plating 500—1000 CD34+ cells in 35-mm Petri dishes 72 h after the beginning of liquid culture in 1 ml methylcellulose medium containing 30% FCS, 5% giant cell tumor-conditioned medium (used as a source of hematopoietic growth factors), and 3 U/ml huEpo (Gibco-BRL). Colonies were scored 14 days after plating in methylcellulose as erythroid [burst forming unit-erythroid (BFU-E)/colony-forming units (CFU)-E], CFU-GM, CFU-G, or CFU-M. At least 200 colonies were scored for each sample analyzed.

Morphological and immunophenotypic analysis
Differentiation of CD34+ cells has been monitored by morphological analysis of May-Grumwald-Giemsa (MGG)-stained cytospins and by flow cytometric analysis of CD34, CD38, CD14, and CD11b surface-antigen expression, performed at days 7 and 14 of liquid culture as described [20 ]. The following monoclonal antibodies (mAb) were used for labeling cell samples: fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34 mAb, phycoerythrin (PE)-conjugated mouse anti-human CD38 mAb, PE-conjugated mouse anti-human CD14 mAb, and PE-conjugated mouse anti-human CD11b mAb (all from Becton Dickinson Systems, Mountain View, CA). We performed negative controls by staining cells with isotype-matched, nonspecific antibodies (Becton Dickinson). Briefly, each antibody was incubated at the proper dilution with cell samples in phosphate-buffered saline (PBS) containing 5% FCS and 1% FcR-blocking reagent (Miltenyi) for 30 min at 4°C. Cells were then washed twice, resuspended with PBS, and analyzed by a Coulter Epics XL flow cytometer (Coulter Electronics, Hialeah, FL). At least 10,000 events were counted for each sample to ensure statistical relevance. Analysis was performed in terms of positivity percentage or mean fluorescence intensity (MFI).

Nuclear extract (NE) preparation
NE of the analyzed cell populations, untreated or VD-treated, were obtained from 2–3 x 106 cells as described previously [21 ] with minor modifications [10 ]. Protein concentration was evaluated by the Lowry method.

Western blotting
VDR protein expression in NE of the examined cell samples was assessed by Western blot analysis as described [22 ] with some modifications [10 ]. Briefly, 10–20 µg NE of each sample was dissolved in 1x reducing-loading buffer [50 mM Tris, pH 8, 5% ß-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 0.1% bromophenol blue, 10% glycerol], loaded onto 7.5–10% SDS-polyacrylamide gel electrophoresis, and electrophoresed in TGS buffer (25 mM Tris, pH 8.3, 250 mM glycine, 0.1% SDS). The separated proteins were transferred at 4°C onto a nitrocellulose sheet by an electroblotting procedure in TGM buffer (25 mM Tris, pH 8.3, 250 mM glycine, 20% methanol) for 2 h at 1 ampere. To monitor the electroblotting efficiency, the membrane was stained in 0.2% Ponceau S/0.3% trichloroacetic acid (TCA) and destained in 0.3% TCA. Membranes were preblocked in blocking solution (10 mM Tris, pH 8, 150 mM NaCl, 0.05% Tween 20, Tris-buffered saline/Tween 20, supplemented with 4 mg/ml normal serum) for 1 h at room temperature and then incubated with a 1:500 dilution of the primary 9A7{gamma} rat anti-VDR mAb (Affinity Bioreagents, Golden CO), followed by a 1 h incubation at room temperature with a secondary goat anti-rat immunoglobulin antibody, horseradish peroxidase-conjugated (1:5000 diluted). The detection was carried out by the enhanced chemiluminescence method (Amersham Life Science, Little Chalfont, England).

RNA analysis
Total cellular RNA was extracted from 0.5–1 x 106 cells of each analyzed sample by means of the guanidinium-cesium chloride centrifugation technique [23 ]. Expression of 24-OHase VD target-gene expression at the mRNA level was then assessed using reverse transcriptase-polymerase chain reaction (RT-PCR), performed as described previously [24 ]. Briefly, each RNA sample was used to generate a corresponding cDNA by means of the Moloney murine leukemia virus RT enzyme. Subsequently, the obtained cDNAs were PCR-amplified using oligonucleotide primers that had been compared previously with the N.I.H. GenBank database through the DNAsis software (Hitachi, Brisbaine, CA) to avoid homologies with other gene sequences. All the oligonucleotide primers used in the RT-PCR experiments were designed on distinct exons to exclude a possible genomic DNA contamination of the RNA samples. The following oligonucleotide primers were used to analyze 24-OHase gene [25 ] expression: 24-OHase direct primer (DP), 5'-TCTACGGCGTACACGTCCCCTCAGC-3'; 24-OHase reverse primer (RP), 5'-AAATGGTGTCCCAGGCCAGAGTGTG-3'. The cDNAs used in the RT-PCR reaction were normalized assessing the expression levels of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) "housekeeping" gene [26 ]. For this purpose, we used the following oligonucleotide primers: GAPDH DP, 5'-GAAGGTGAAGGTCGGAGTC-3'; GAPDH RP, 5'-GAAGGCCATGCCAGTGAGCT-3'. All of the amplified fragments were analyzed by agarose-gel electrophoresis, followed by ethidium-bromide staining.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of CB CD34+ hematopoietic progenitors with scalar concentrations of VD
To assess the effects exerted by VD on normal CD34+ hematopoietic cells, we initially adopted treatment conditions used previously for AML cells and normal BM cells. Therefore, a preliminary experiment was performed in which CD34+ cells were exposed to weekly administrations of supraphysiological (10-8 M) to physiological (10-10 M) concentrations of VD [27 ]. Thereafter, the effects of VD stimulation were monitored by CFC assay, performed at day 3, and by immunophenotypic and morphological analysis, performed at days 7 and 14 of liquid culture.

Estimation of highly undifferentiated CD34+CD38- hematopoietic progenitors, performed at day 7 of liquid culture by multiparameter flow cytometry (Fig. 1) , shows that these cells represent, as expected, a small fraction (1.6%) of untreated control cells. It is interesting that treatment with 10-8 M VD results in a remarkable reduction of CD34+CD38- cells (0.1%), at the same time promoting a concomitant down-regulation of CD34 antigen expression (3% vs. 14% of untreated, control cells), and a clear induction of the CD14 monocyte-specific antigen (49%/MFI 234 vs. 24%/MFI 101 of control cells). Apparently, the expression of this last antigen is no further modified during the second week of culture, except for a MFI increase that is observed at day 14 in all of the analyzed samples. Lower VD concentrations (10-9 M and 10-10 M) affect CD34 and CD14 antigen expression only slightly, and they seem to promote a certain depletion of the highly immature CD34+CD38- cell population (0.3 and 1.1%, respectively, vs. 1.6% of control cells).



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  		Figure 1. Flow cytometry analysis of CD34+ hematopoietic progenitors stimulated weekly with scalar concentrations of VD. This analysis was performed at days 7 and 14 of liquid culture to assess percentages of CD34+CD38-, CD34+, and CD14+ cells upon treatment of CD34+ hematopoietic progenitors with weekly additions of VD at scalar concentrations (10-8–10-10 M). Cells were stained with FITC-conjugated anti-CD34, PE-conjugated anti-CD38, and PE-conjugated anti-CD14 mAb. The used amounts of VD are shown on the left; CONTROL is represented by cells cultured in the presence of early hematopoietic cytokines without VD. Percentages of CD34+CD38- cells are shown inside square boxes, whereas percentages of CD34+ and CD14+ cells are shown above line markers in each histogram. MFI values of CD14 expression are shown below line markers.

 
Few cells having a mature monocyte/macrophage morphology have been detected in day 7 hematopoietic cell samples, regardless of the treatment condition considered, whereas morphological analysis performed at day 14 discloses the presence of a macrophage population whose proportion is consistent with the different percentages of CD14 antigen expression observed by flow cytometry (unpublished results).

To assess the effects of VD at different levels of hematopoietic cell differentiation, we also treated day 7 myeloid precursors, which, in our experimental conditions, appear to be composed predominantly of granulocytic precursors (late myeloblast-promyelocytic cells). Surprisingly, stimulation of these cells with 10-8 M VD results in a mono-macrophagic maturation, which as assessed by flow cytometry and morphology, is comparable to the differentiation obtained by analogous treatment of day 0 CD34+ cells (Fig. 2 ). This VD-induced "differentiaton shift", implying the maturation of normal, late myeloblasts/promyelocytes to monocyte-macrophages, has been observed already by others using more homogeneous preparations of granulocytic precursors [28 ].



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  		Figure 2. VD induced differentiation of normal myeloid precursors. Myeloid precursors, obtained by a 7-day liquid culture of CD34+ hematopoietic progenitors in the presence of early acting hematopoietic cytokines (DAY7, left panels), were treated for 1 week with a sole administration of VD at supraphysiological concentrations (10-8 M; DAY14, right panels). Differentiation was monitored by flow cytometry analysis of the monocyte-specific CD14 antigen expression (upper panel) and by morphology (MGG staining, lower panel). Representative results from three independent experiments are described: average ± SD values were 23 ± 2% and 51 ± 4% for control and VD-treated cells, respectively.

 
A CFC assay, performed by seeding cells at day 3 of liquid culture in methylcellulose, demonstrates that treatment of CD34+ cells with 10-8 M VD promotes an increase of CFU-M and a parallel decrease of BFU-E, consistently with the flow cytometry and morphology data, and the other types of hematopoietic colonies (CFU-GM and CFU-G) appear to be unaffected (Fig. 3 ). Lower concentrations of VD (10-9 and 10-10 M) produce no appreciable effects on the clonogenic capacity of CD34+ cells (Fig. 3) .



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  		Figure 3. Clonogenic activity of CD34+ hematopoietic progenitors stimulated weekly with scalar concentrations of VD. CFC assay was performed by plating in methylcellulose CD34+ cells cultured for 3 days under the different tested conditions. Cells used for this assay were untreated (CONTR.) or treated with the indicated concentrations of VD, administered once at the beginning of liquid culture. The different types of scored colonies (BFU-E, open bars; CFU-GM, shaded bars; CFU-G, dotted bars; CFU-M, gridded bars) are shown as percentage of total colonies. Average results from a triplicate experiment are shown together with the corresponding SD values.

 
Differentiation capacity of CB CD34+ hematopoietic progenitors exposed to supraphysiological or physiological levels of VD
The results obtained so far raise the question of whether a weekly administration of 10-8 M VD induces the mono-macrophagic differentiation of CD34+ cells, because supraphysiological levels of VD are required for a short period, coinciding with hematopoietic commitment at least for the first week of treatment, or because this modality of stimulation is able to assure physiological levels of this vitamin for a prolonged period of time. This last hypothesis appears to be especially convincing if we consider that the physiological concentration of VD in human serum is 0.5–2 x 10-10 M (ranging up to 5x10-10 M in some paraphysiological conditions) [27 ], and the half-life of this nuclear hormone in biological fluids has been estimated to be 3–6 h in vivo and in vitro [27 , 29 ]. Based on these considerations, we performed a set of experiments in which CB CD34+ hematopoietic stem cells were stimulated with 2.5 x 10-10 M VD added daily to the liquid culture or for comparison, with a weekly administration of 10-8 M VD. The extent of mono-macrophagic differentiation attained with the different examined culture conditions was monitored again by flow cytometry (analysis of CD14 and CD11b surface-markers expression), morphology, and CFC assay.

Flow cytometry results reveal that the supraphysiological or the physiological modality of stimulation with VD at day 7 of culture induces a marked depletion of very primitive CD34+CD38- hematopoietic stem cells (average 0.2% and 0.3%, respectively, vs. 1.9% of control cells), whereas they diverge to some degree in the extent of CD34 expression down-regulation (average 1% and 3.5%, respectively, vs. 6% of control cells; Fig. 4A ). Comparable effects are detected when the expression of surface-differentiation markers is analyzed. In fact, although physiological levels of VD promote a CD14 antigen expression that is slightly lower at day 7 of culture compared with supraphysiological concentrations of this vitamin (average 37% and 47%, respectively; Fig. 4B ), progression of culture up to day 14 gives rise to a CD14 positivity averaging 48%, regardless of the VD stimulation modality used (Fig. 4B) . Conversely, the mean percentages of CD14 antigen expression in untreated control cells are 18% and 22% at days 7 and 14, respectively (Fig. 4B) , as expected on the basis of the experiment described previously. Superimposable results are obtained by flow cytometry analysis of the myeloid-related CD11b antigen expression (Fig. 4B) .



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  		Figure 4. Flow cytometry analysis of CD34+ hematopoietic progenitors exposed to supraphysiological or physiological VD levels. Histograms showing the results of flow cytometry analysis were performed on CD34+ hematopoietic progenitors untreated (CONTR.) and treated with VD administered weekly at supraphysiological (VD 10-8 M) or daily at physiological concentrations (VD 10-10 M daily). (A) Percentage of CD34+CD38- (open bars) and CD34+ (shaded bars) cells at day 7 of treatment. (B) Expression of CD14 (dotted bars) and CD11b (gridded bars) myeloid differentiation markers at days 7 and 14 is shown as percentage of positive cells. Percentages of positive cells were calculated on the basis of isotype controls performed as described in Materials and Methods. Mean values with the corresponding SD obtained from three distinct experiments are shown for each surface antigen analyzed.

 
Although with a slower differentiation rate, taken together, our data indicate that CD34+ hematopoietic progenitors stimulated with physiological levels of VD undergo a mono-macrophagic maturation, whose extent is comparable to that induced by supraphysiological concentrations of this vitamin.

Morphological changes induced by treatment of CD34+ cells with daily administration of 2.5 x 10-10 M VD (Fig. 5 ) are substantially comparable to those obtained using a weekly stimulation with 10-8 M VD, described in the previous section. In fact, physiological concentrations of VD generate only a small percentage of monocytes/macrophages at day 7 of culture, whereas 50–60% of analyzed cells are monocytes/macrophages at day 14, implying that a high proportion of CD14+ cells are still at the blast stage of differentiation at day 7, achieving a mature phenotype only at completion of culture (day 14).



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  		Figure 5. Morphological analysis of VD physiologically stimulated CD34+ cells. This analysis (MGG staining) was performed on freshly separated (DAY 0) and VD-treated (DAY 7 and DAY 14) CD34+ hematopoietic progenitors. Stimulation was accomplished in liquid culture by adding physiological doses of VD daily. At day 7, the majority of cells is represented by myeloid precursors, and only few monocytoid elements are detected. At day 14, a high proportion of cells exhibit a mono-macrophagic morphology. A lower and superimposable proportion of monocyte/macrophages was observed in untreated or 10-8 M weekly treated CD34+ cells, respectively (not shown).

 
In other terms, these results demonstrate that treatment of CD34+ hematopoietic progenitors with VD leads to the acquisition of a complete mono-macrophage phenotype in experimental conditions representative of normal hematopoiesis.

CFC assays, performed by plating CD34+ cells exposed for 3 days to daily addition of 2.5 x 10-10 M VD, exhibit a percentage of macrophage colonies (49%) that is about twofold higher as compared with control cells (25%) and is comparable with those produced by a sole treatment with 10-8 M VD (55%; Fig. 6 ). Conversely, this last modality of VD stimulation promotes a more pronounced inhibition of erythroid colonies (13% vs. 26% of VD physiologically stimulated cells and 42% of control cells). Apparently, granulo-macrophagic and granulocytic colonies are not influenced significantly by any of the stimulations used (Fig. 6) .



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  		Figure 6. Clonogenic capacity of CD34+ hematopoietic progenitors exposed to supraphysiological or physiological VD levels. CFC assay was performed using day 3 CD34+ cells untreated (CONTR.) and VD-treated with a sole administration at supraphysiological concentrations (VD 10-8 M) or by daily addition of this vitamin at physiological levels (VD 10-10 M daily). The different types of scored colonies (BFU-E, open bars; CFU-GM, shaded bars; CFU-G, dotted bars; CFU-M, gridded bars) are shown as percentage of total colonies. Average results and SD values from a triplicate experiment are shown.

 
Stimulation of CD34+ hematopoietic progenitors with M-CSF, VD, and M-CSF/VD association
In a distinct set of experiments, the capacity of VD to deplete CD34+CD38- cells and to promote the differentiation of CD34+ hematopoietic progenitors was analyzed in comparison with M-CSF, a growth factor that plays a key role in the physiological regulation of mono-macrophagic differentiation. As assessed by flow cytometry, M-CSF treatment induces a very weak increase of CD34+CD38- cell percentage in VD-untreated or VD physiologically stimulated cells (Fig. 7A ). In addition, these last cells undergo an extent of CD14 antigen up-regulation (44% at day 7; 47% at day 14), which is clearly higher as compared with M-CSF (28% at day 7; 33% at day 14; Fig. 7B ). Combined stimulation with M-CSF and VD results in an additive effect, more evident at the end of culture, raising CD14 expression up to 60% of cells at day 14 (Fig. 7 B) . These data indicate that VD promotes differentiation effects that are distinct to those of M-CSF, at least in our experimental conditions in terms of level and efficiency of action.



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  		Figure 7. Flow cytometry analysis performed to compare VD and M-CSF treatment of CD34+ cells. Histograms showing flow cytometry analysis results were obtained upon treatment of CD34+ hematopoietic progenitors with M-CSF, VD, or M-CSF + VD. Untreated cells were used as control (CONTR.). VD treatment was performed by daily addition of this vitamin at physiological concentrations. (A) Percentage of CD34+CD38- cells at day 7 is shown. (B) Percentage of CD14+ cells at days 7 and 14. Mean values with the corresponding SD obtained from three independent experiments are shown.

 
Effects of physiological doses of VD on a TPO-supported liquid culture of CD34+ cells
To confirm VD-induced CD34+CD38- differentiation in a more appropriate experimental model, we performed a stroma-free liquid culture of CD34+ stem cells, obtained by including TPO in the mix of the cytokines used so far because of its ability to support the self-renewal of very primitive hematopoietic stem cells [18 , 19 ]. This assay was performed by using serum-free conditions of culture for two main reasons: preliminary experiments indicated that these culture conditions delay the differentiation of CD34+ cells, promoting an enrichment of the CD34+CD38- cell fraction as already described by others [30 ], and are permissive for erythroid and megakaryocytic differentiation [20 ], thus allowing to assess the effects exerted by VD on a normal, multilineage hematopoiesis.

Flow cytometry results, shown in Figure 8 , evidence that 60%, 27%, and 6% of control untreated cells are CD34+CD38- at days 3, 5, and 7 of culture, respectively. A daily exposure to 2.5 x 10-10 M VD promotes a remarkable decrease of these cells (5%, <1%, 0%, respectively), which is apparently a result of their differentiation to CD34+CD38+ cells initially and to CD34-CD38+ cells subsequently, as shown by the shift of the corresponding analyzed dot plots. The former effect is evident especially at day 3 of culture, where VD stimulation leads to a clear inversion of the CD34+CD38-/CD34+CD38+ ratio (<0.1 vs. 8.5 of untreated control cells). After 3 weeks of culture in the same experimental conditions, 1–2% of control cells are still CD34+/CD38-, whereas VD-treated cells are completely CD34- (unpublished results).



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  		Figure 8. Flow cytometry analysis of CD34+ hematopoietic progenitors exposed to physiological VD levels in a TPO-supported, liquid-culture model. Flow cytometry analysis was performed at days 3, 5, and 7 of liquid culture in serum-free medium (X-VIVO 20) to better characterize the effect induced on CD34+CD38- cells by stimulation with daily addition of VD at physiological levels (VD 10-10 M/d). This effect was compared with control cells (CONTROL) cultured in the presence of early hematopoietic cytokines but without VD. Cells were stained with FITC-conjugated anti-CD34 (y axis) and PE-conjugated anti-CD38 (x axis) mAb. Isotype control, shown at the top of the figure, was performed as described in Materials and Methods. Percentages of the different analyzed cell subsets are described and discussed in Results. Representative results from three independent experiments are shown. Mean ± SD values for CD34+38- cells in control and VD 10-10 M/d samples were 60 ± 7 0% and 2 ± 0.5% at day 3; 30 ± 3% and 0.3 ± 0.1% at day 5; and 5 ± 1% and 0.1 ± 0.0% at day 7, respectively.

 
Morphological analysis performed at day 21 of serum-free liquid culture shows that hematopoietic cells at different stages of differentiation belonging to myeloid, erythroid, and megakaryocytic lineages are detected in control sample, whereas over 70% of VD-treated cells exhibit a macrophagic phenotype, the remaining being represented by granulocytic cells (unpublished results).

Involvement of VD genomic pathway in mono-macrophagic differentiation of CD34+ hematopoietic progenitors induced by VD
As mentioned above, several intracellular transducers, nuclear (VDR) or cytoplasmic (PKC and PI-3K), account for the so-called genomic or nongenomic biological actions of VD, respectively. To date, the molecular events, underlying the effects exerted by VD on cell differentiation, are still controversial, although most studies corroborate the involvement of the nuclear pathway to this regard. Once activated, the VD genomic pathway leads, by definition, to the induction of VD target-gene transcription, preceded by a massive accumulation of VDR protein in the nuclear compartment of stimulated cells. Based on these considerations, we investigated whether the genomic pathway was responsible for VD-induced mono-macrophagic differentiation of CD34+ cells, analyzing VDR nuclear protein expression by Western blot and the induction of a VD primary-response gene by RT-PCR.

Western blot analysis, performed on nuclear extracts of hematopoietic cells under different conditions of VD treatment at day 14 of liquid culture, demonstrates that VDR is highly expressed following a weekly 10-8 M stimulation with VD (Fig. 9A , lane 2) but not expressed in untreated control cells (Fig. 9A , lane 1). A lower expression is detected upon a weekly stimulation with 10-9 M VD (Fig. 9A , lane 3), whereas treatment with 10-10 M VD in the same experimental conditions induces levels of VDR protein that are barely detectable (Fig. 9A , lane 4). It is interesting that a weekly 10-8 M VD stimulation applied to day 7 myeloid precursors in liquid culture promotes levels of VDR protein induction (Fig. 9A , lane 5) that are superimposable to those obtained by a comparable treatment of day 0 hematopoietic progenitors (Fig. 9A , lane 2). Furthermore, when CD34+ hematopoietic stem cells are treated daily with physiological levels of VD, they undergo an extent of nuclear VDR induction (Fig. 9B , lane 3) that although slightly lower, is comparable with that observed by stimulating these cells weekly with 10-8 M VD (Fig. 9B , lane 2).



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  		Figure 9. VDR protein expression in the nuclear compartment of CD34+ hematopoietic progenitors exposed to VD treatment at various stimulation conditions. Western blot analysis was performed on nuclear extracts of primary hematopoietic cells at day 14 of liquid culture to assess VDR protein expression. The different tested culture conditions are shown on the top of each panel. Molecular weight of VDR protein is shown on the right side of each panel. (A) The effects exerted by weekly additions of VD at scalar concentrations (lanes 2–4, VD 10-8–10-10 M) are analyzed and compared with control, untreated cells (lane 1, CONTR.). Lane 5 refers to a 10-8 M VD treatment administered at day 7 of culture as described in Figure 2 . (B) The levels of VDR protein, promoted by treatment with the supraphysiological (lane 2, VD 10-8 M) and the physiological (lane 3, VD 10-10 M/d) modalities of stimulation, are shown and compared again with control, untreated cells (lane 1, CONTR.).

 
To investigate the activation of VDR-dependent gene transcription, we assessed the expression of the 24-OHase VD primary-response gene by RT-PCR [25 ], because our unpublished data indicate that it is associated strictly with VD-induced mono-macrophagic differentiation in AML cell lines. The data obtained also indicate that mRNA expression of 24-OHase is not detectable in untreated control cells at day 7 or at day 14 of culture (Fig. 10 , upper panel, lanes 1 and 5, respectively). Conversely, when CD34+ cells are stimulated with 10-8 M VD added weekly to the culture, the expression of this gene is remarkably induced at day 7 (Fig. 10 , upper panel, lane 2), persisting unchanged up to day 14 of culture (Fig. 10 , upper panel, lane 6), and a comparable up-regulation is obtained upon a daily administration of 10-10 M VD, although at lower levels (Fig. 10 , upper panel, lanes 3 and 7).



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  		Figure 10. Messenger RNA expression of the 24-OHase VD target gene upon treatment of CD34+ cells with supraphysiological or physiological VD concentrations. Messenger RNA expression of 24-OHase VD target gene (upper panel) and GAPDH housekeeping gene (lower panel) were analyzed by RT-PCR in primary hematopoietic cells under the different conditions tested at days 7 and 14 of liquid culture. Agarose gels stained with ethidium bromide are shown. The size of amplified gene fragments is indicated on the right side of each panel. Treatments are indicated at the top of the figure. The following samples have been analyzed: untreated cells (lanes 1 and 5, CONTR.); cells treated weekly with supraphysiological concentration of VD (lanes 2 and 6, VD 10-8 M); cells treated daily with physiological concentration of VD (lanes 3 and 7, VD 10-10 M/d); negative control (lanes 4 and 8, NEG.), obtained performing RT-PCR amplification without cDNA template.

 
These results clearly suggest that different conditions of VD treatment, including the physiological mode of stimulation with this vitamin, are able to massively recruit VDR protein to the nucleus of primary hematopoietic cells, consequently leading to a complete activation of the genomic pathway, as demonstrated by the transactivation of an endogenous VD primary-response gene is related to mono-macrophagic differentiation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the capacity of VD to induce the mono-macrophagic differentiation of AML cells has been characterized widely in recent years [9 10 11 ], only few studies have tried to address the possible role played by this vitamin and its nuclear receptor VDR in normal, hematopoietic-progenitor differentiation [9 ]. These studies substantially evidenced the effects exerted by supraphysiological VD levels on the clonogenic capacity of normal BM mononuclear cells, i.e., a heterogeneous population of primary hematopoietic cells. We adopted an alternative, experimental model consisting of a liquid culture in which a homogeneous cell population (CB CD34+ hematopoietic progenitors) was grown in the presence of early acting hematopoietic cytokines. These culture conditions allow dissection of the differentiation effect of a given compound in terms of level of action (hematopoietic commitment or terminal-maturation) and time, provide physiological levels of VD persistently, by replacing and/or readding this vitamin during the course of the experiment. In addition, the combined use of morphological and immunophenotypic analysis allows a more accurate evaluation of differentiated cells. In the context of our experimental design, CFC assays were used simply to monitor the effects promoted by VD treatment, in liquid culture, on the proportion of the different hematopoietic progenitors. Based on VD half-life in vivo and in vitro, CD34+ cells were treated with daily administrations of 2.5 x 10-10 M VD or for comparison, with weekly addition of 10-8 M VD aimed at exposing them to physiological or supraphysiological levels of this vitamin, respectively. Untreated cells not receiving any VD treatment were used as control to estimate the basal, cytokine-induced, monocytic differentiation.

The data obtained show that the physiological modality of VD treatment clearly promotes a mono-macrophagic differentiation of CD34+ hematopoietic stem cells, characterized by a remarkable induction of the expected immunophenotypic (CD14, CD11b) and morphological markers. This differentiation effect is exerted not only at the terminal maturation but also at the commitment level, as demonstrated by the CD34 antigen down-regulation and the CFU-M increase observed after only 3 days of incubation with VD.

Based on the observation that the appearance of the CD38 antigen on the CD34+ cell surface is associated strictly with the commitment of very primitive, uncommitted, hematopoietic stem cells to the more differentiated committed progenitors [31 ], we analyzed by multiparameter flow cytometry whether VD stimulation exerted any effect on CD34+CD38- stem cells. For this purpose, we used the current culture conditions or a distinct serum-free, TPO-supported liquid culture, which determines an enrichment of the highly undifferentiated CD34+CD38- cell fraction based on our preliminary results and findings described previously [30 ]. Regardless of the experimental settings used, treatment with VD elicits a strong depletion of the CD34+CD38- cells, which is clearly the consequence of their differentiation to CD34+CD38+ cells. In addition, by using the serum-free/TPO culture model, a conversion of a trilineage to a virtually unilineage (macrophage-committed) hematopoiesis is observed when cells are incubated in the presence of VD. These data indicate that the investigated vitamin plays a physiological role in the regulation of early hematopoietic commitment.

To better contextualize this role in the hierarchy of hematopoietic cell differentiation, we compared the effects of VD stimulation with those of M-CSF, a growth factor specifically promoting mono-macrophagic differentiation in physiological conditions. Consistently with the expression of its receptor shown to begin at the CFU-GM/CFU-M stage of myeloid commitment [32 , 33 ], M-CSF affects CD34+CD38- cells only weakly in our experimental conditions, at the same time enhancing VD-induced, terminal macrophagic maturation of CD34+ cells. These data suggest that the effects evoked by physiological doses of VD are distinct from those of M-CSF, at least in terms of level of action, because they seem to regulate the early and late events of hematopoietic commitment, respectively. Conversely, M-CSF enhances VD-induced mono-macrophagic differentiation of hematopoietic precursors, showing that this vitamin is able to cooperate with a growth factor involved physiologically in the regulation of monocytopoiesis.

Taken together, the data shown demonstrate that VD promotes mono-macrophage maturation by acting on different levels of hematopoietic-cell differentiation, spanning from uncommitted progenitors to mature cells. Because this study was carried out by using CB CD34+ stem cells and because of the growing body of evidence indicating a biological diversity between CB and adult hematopoietic cells [34 ], the relevance of our data may be limited to CB cells.

The differentiation effects of VD are likely to be mediated by the genomic signaling pathway, because a remarkable and sustained increase of VDR nuclear levels is detected, coupled with a clear transactivation of the 24-OHase VD primary-response gene. The participation of the nongenomic actions of VD mediated by PKC or PI-3K, cannot be excluded in this regard and might be explored by using specific enzyme inhibitors.

Numerous studies indicate that spontaneous or exogenously induced up-regulation of master regulator transcription factors is responsible for hematopoietic commitment by activating genetic programs that lead to the induction of specific cytokine receptors [35 , 36 ]. Standing on this "stochastic" or "permissive" model of hematopoiesis, the role of cytokines (as M-CSF) would simply be to deliver survival and expansion signals to lineage-specific hematopoietic progenitors/precursors [4 ]. Our data show that ligand-induced up-regulation of VDR protein in the nuclear compartment of normal CD34+ hematopoietic progenitors is associated with an increase of terminally differentiated monocytes/macrophages. In our opinion, these findings allow us to hypothesize that VDR, being a transcription factor, might be a master regulator of mono-macrophagic differentiation, as already shown for MafB [37 ]. VDR overexpression or inactivation experiments, performed by virally transducing CD34+ hematopoietic progenitors, could help to verify this possibility.


   ACKNOWLEDGEMENTS
 
This work has been supported by a grant from A.I.R.C. and from M.U.R.S.T.-Cofin 2000.

Received August 23, 2001; revised December 11, 2001; accepted December 17, 2001.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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