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The Journal of Immunology, 2007, 178: 2075-2082.
Copyright © 2007 by The American Association of Immunologists, Inc.

Stromal Derived Growth Factor-1{alpha}: Another Mediator in Neural-Emerging Immune System through Tac1 Expression in Bone Marrow Stromal Cells1

Kelly E. Corcoran*, Nitixa Patel* and Pranela Rameshwar2,{dagger}

* Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, NJ 07107; and {dagger} Department of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Stromal cell-derived growth factor-1{alpha} (SDF-1{alpha}) is a member of the CXC chemokines and interacts with the G protein, seven-transmembrane CXCR4 receptor. SDF-1{alpha} acts as a chemoattractant for immune and hemopoietic cells. The Tac1 gene encodes peptides belonging to the tachykinin family with substance P being the predominant member. Both SDF-1{alpha} and Tac1 peptides are relevant hemopoietic regulators. This study investigated the effects of SDF-1{alpha} on Tac1 expression in the major hemopoietic supporting cells, the bone marrow stroma, and addresses the consequence to hemopoiesis. Reporter gene assays with the 5' flanking region of Tac1 showed a bell-shaped effect of SDF-1{alpha} on luciferase activity with 20 ng/ml SDF-1{alpha} acting as stimulator, whereas 50 and 100 ng/ml SDF-1{alpha} acted as inhibitors. Gel shift assays and transfection with wild-type and mutant I{kappa}B indicate NF-{kappa}B as a mediator in the repressive effects at 50 and 100 ng/ml SDF-1{alpha}. Northern analyses and ELISA showed correlations among reporter gene activities, mRNA (beta-preprotachykinin I), and protein levels for substance P. Of relevance is the novel finding by long-term culture-initiating cell assays that showed an indirect effect of SDF-1{alpha} on hemopoiesis through substance P production. The results also showed neurokinin 1 and not neurokinin 2 as the relevant receptor. Another crucial finding is that substance P does not regulate the production of SDF-1{alpha} in stroma. The studies indicate that SDF-1{alpha} levels above baseline production in bone marrow stroma induce the production of substance P to stimulate hemopoiesis. Substance P, however, does not act as autocrine stimulator to induce the production of SDF-1{alpha}. This study adds SDF-1{alpha} as a mediator within the neural-immune-hemopoietic axis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tac1 (also referred to as preprotachykinin I or PPT-I)3 is a single copy gene that is conserved by evolution (1). Tac1 is ubiquitously expressed, although its functions are organ specific (2). Tac1 peptides exhibit neurotransmission function in the nervous system, hemopoietic regulation in bone marrow (BM), and immunomodulatory properties in the immune system (3). Tac1 produces several peptides that belong to the tachykinin family with substance P (SP) and neurokinin-A as its major gene products (4). SP and neurokinin-A interact with different binding affinities to three related seven-transmembrane G protein-coupled receptors: neurokinin (NK) 1, NK2, and NK3 (5, 6).

NK1 and NK2 mediate stimulatory and inhibitory effects on hemopoiesis, respectively (5). NK2 is constitutively expressed in unstimulated BM stroma, whereas NK1 expression requires induction by cytokines with stimulatory effects on hemopoiesis and by broad-acting cytokines such as IL-1 (7). The expression of NK1 correlates with down-regulation of NK2 on normal hemopoietic cells and BM stroma (5). Although the mechanism involved in this yin-yang type of expression between NK1 and NK2 is unclear, the experimental evidence suggests mechanisms involving intracellular crosstalk (5). The regulated expressions of NK1 and NK2 in the BM stroma affect hemopoiesis, mainly because one receptor subtype appears to modulate the functions of the other (1, 4).

Similar to the tachykinins, the chemokine stromal-derived growth factor (SDF)-1{alpha} is also involved in hemopoiesis (8). The production of SDF-1{alpha} in BM stromal cells is membrane-bound and available for interaction with the CXCR4-expressing hemopoietic stem cells (HSCs) (9, 10). SDF-1{alpha} does not appear to act as a hormone because it is localized at the source of production. Specifically, SDF-1{alpha} is retained within the BM niche as membrane bound or is attached to the surrounding proteoglycans of extracellular matrix proteins (11).

The levels of SDF-1{alpha} follow a gradient pattern across the BM cavity (12). SDF-1{alpha} is important for the retention of HSCs within the stromal compartment under homeostatic conditions. However, changes in this gradient facilitate HSC mobilization of the BM; into the peripheral circulation (9). Stromal-derived SDF-1{alpha} is important for the quiescent state of HSC close to the BM niche, whereas changes in the levels of SDF-1{alpha} have been shown to stimulate hemopoiesis (8, 13).

In mice, SDF-1{alpha} has been shown to mobilize HSCs into the peripheral circulation (14). Similarly, antagonists to the SDF-1{alpha} receptor CXCR4 have also been shown to mobilize HSCs, suggesting that the concentration of SDF-1{alpha} determines the fate of HSC (15). Together, these findings suggest that at high levels of SDF-1{alpha} the interaction between stromal cells and HSCs could be broken, in part due to a localized imbalance in SDF-1{alpha} levels. Although a large increase in SDF-1{alpha} levels could mobilize the HSCs from the BM cavity to the periphery, it is unclear how a small increase around the stromal cell niche could affect hemopoiesis. This study addresses the functional relationship between SDF-1{alpha} and the Tac1 gene in BM stroma and the consequence to hemopoiesis. We show that a modest increase in SDF-1{alpha} levels stimulates stromal cells to induce the hemopoietic regulator gene Tac1, leading to the production of SP. The latter stimulates hemopoiesis, specifically through NK1 receptor. The relevance of these studies to neural-hemopoietic regulation is discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

{alpha}-MEM, IMDM, Ficoll-Hypaque, the bicyclam AMD3100 CXCR4 antagonist, and SP were purchased from Sigma-Aldrich. Antagonists to NK-1 (CP-99,994) and NK-2 (SR-48968) were provided by Pfizer and Sanofi Research, respectively. The methods for dissolving and storing SP and the antagonists were previously described (16).

Cytokines and antibodies

Recombinant human GM-CSF and SDF-1{alpha} were purchased from R&D Systems, prolyl-4-hydroxylase mAb from DakoCytomation, PE-anti-CD14 from BD Pharmingen, p50 from Promega, rabbit anti-SP from Biogenesis, alkaline phosphatase (Alk Phos)-goat anti-rabbit IgG from Kirkegaard & Perry, and rabbit anti-p65 and HRP-goat anti-rabbit IgG from Santa Cruz Biotechnology.

BM stromal cells

BM aspirates were obtained from healthy donors between 18 and 25 years. The use of BM aspirates followed the guidelines of a protocol approved by the Institutional Review Board of University of Medicine and Dentistry of New Jersey (Newark, NJ). Nucleated cells (107) were added to 25-cm2 tissue culture flasks (Falcon 3109) in stromal culture medium ({alpha}-MEM with 20% FCS). Flasks were incubated at 37°C. At day 3, the RBC and neutrophils were removed by Ficoll-Hypaque density gradient and the mononuclear cells were readded in fresh stromal medium. The flasks were incubated until confluence with a weekly replacement of 50% fresh stromal medium. At confluence, the trypsin-sensitive adherent cells were passaged. Cells were passaged at least five times before being used in experiments. Flow cytometry studies indicated >99% of the cells were negative for CD14 and positive for prolyl 4-hydroxylase.

Northern analysis

BM stromal cells were stimulated for 6 h with 20 ng/ml SDF-1{alpha} or were unstimulated. Northern blots were performed for Tac1 mRNA (beta-PPT-A) as described (16). Total RNA (15 µg) was analyzed with a specific cDNA probe for beta-PPT-I, as previously described (17). The probe was randomly labeled with [{alpha}-32P]dATP, 3,000 Ci/mM, (DuPont Pharmaceuticals), as described (18). Membranes were stripped and reprobed with cDNA for 18S rRNA. Hybrids were detected by exposures in PhosphorImager cassettes (Amersham Biosciences). Cassettes were scanned on a Typhoon 9410 Molecular Imager PhosphorImager (Amersham Biosciences).

ELISA

Competitive ELISA quantitated SP as described (19). Streptavidin-coated 96-well plates (Sigma-Aldrich) were incubated with biotinylated SP. Equal volumes (100 µl each) of cell-free medium and rabbit anti-SP at 1/3000 dilution were added to the wells. Each sample was tested in triplicate. After this, wells were incubated with Alk Phos-conjugated goat anti-rabbit IgG and Sigma 104 phosphatase substrate (Sigma-Aldrich). SP levels were calculated from a standard curve produced from 12 serial dilutions of known SP concentrations and the absorbance was read at an OD of 405 nm. SDF-1{alpha} levels were quantitated with a Quantikine ELISA colorimetric quantitation kit (R&D Systems) according to the manufacturer’s instruction.

Vectors

pGL3-Basic vectors containing inserts of the 5' flanking regions of Tac1 (PPT-I/1.2 and PPT-I/N0) were previously described (18). Briefly, the gene-specific insert of PPT-I/1.2 is equivalent to 1.2 kb and includes intron 1, exon 1, and upstream sequences. Exon 1 and intron 1 are omitted in PPT-I/N0. The pSEAP vector system, which consists of three vectors (pSEAP-Basic, pSEAP-Enhancer, and pSEAP-Promoter) was purchased from BD Clontech. The exon 1 construct of Tac1 was inserted into pSEAP-Basic, pSEAP-Enhancer, and pSEAP-Promoter in the sense orientation using primers linked to sequences for HindIII and EcoR1. Exon 1 was placed in the antisense orientation into pSEAP-Promoter by amplifying the insert with primers containing XhoI and KpnI linkers. Insert direction was confirmed by a restriction digest and sequencing. pCMV-I{kappa}B{alpha} (wild type) allows for activation of NF-{kappa}B, whereas pCMV- I{kappa}BM (mutant) sequesters NF–{kappa}B in the cytosol by preventing its phosphorylation. Both vectors are part of the Mercury I{kappa}B dominant-negative vector set (BD Clontech).

Transient transfection and reporter gene assay

BM stromal cells were cotransfected with plasmid beta-galactosidase (beta-gal) and PPT-I/1.2 or PPT-I/N0 using the Effectene reagent (Qiagen). At 16 h, the transfectants were stimulated with SDF-1{alpha} at 20, 50, or 100 ng/ml in serum-free medium. After 16 h, the cells were scraped in 30 µl of lysis buffer (Promega) and then lysed by repeated freeze-thaw cycles in a dry ice/ethanol bath. Cell-free lysates were obtained by centrifugation at 15,000 x g for 5 min at 4°C. Luciferase activities were quantitated with 10 µl of lysates using the Luciferase Assay System and beta-galactosidase using kits from Promega or the Luminescent beta-galactosidase detection kit II from Clontech, respectively. Luciferase activity was presented per microgram of total protein in levels normalized with cells transfected with vector alone. Total protein was determined with a kit from Bio-Rad.

Alk Phos reporter gene assays

pSEAP-Basic, pSEAP-Enhancer, or pSEAP-Promoter containing the exon 1 (sense and antisense) inserts of Tac1 was cotransfected with pbeta-gal-Control (0.5 µg each) in 80% confluent BM stroma using SuperFect (Qiagen). After 48 h, the culture medium was analyzed for Alk Phos using the Great EscAPe SEAP detection kit (BD Clontech). Cells were scraped in 1 ml PBS and then lysed by repeated cycles of freezing and thawing in a dry ice/ethanol bath. Cell-free lysates (24 µl) were obtained by centrifugation at 15,000 x g for 5 min at 4°C and then diluted with 5x cell culture lysis buffer (Promega). beta-gal activities were quantitated as described above.

Modified long-term culture-initiating cell (LTC-IC) assays

Modified LTC-IC assays were performed in 25-cm2 tissue flasks as described (13). The assay used supporting layers of stroma. At confluence, the supporting cells were {gamma}-irradiated with 150 gray. After 16 h, the medium was replaced with 5 ml of fresh medium containing BM mononuclear cells at 107/ml. The cultures were performed in the presence or absence of different concentrations of SDF-1{alpha} and/or NK1-specific antagonists. There were weekly replacements of 50% culture medium. At week 12, the adherent and nonadherent cells from each flask were combined and then studied in clonogenic cultures for granulocytic-monocytic progenitors (CFU-GM) as described (13). Briefly, 105 cells were resuspended in 1.2% methylcellulose in medium supplemented with 3 U/ml GM-CSF and each culture was assayed in duplicate. Colonies with >15 cells were counted on day 10.

Western blot

Nuclear proteins were extracted with the Nxtract kit (Sigma-Aldrich) and total protein concentrations were determined using the Bio-Rad DC protein assay. Extracts (15 µg) were analyzed by Western blotting using electrophoresis with 12% SDS-polyacrylamide gels and the proteins were transferred onto polyvinylidene difluoride membranes (PerkinElmer). The membranes were incubated overnight with primary Abs followed by 2-h incubation with HRP-conjugated IgG. The primary and secondary Abs were used at final dilutions of 1/1000 and 1/2000, respectively. HRP was developed with a chemiluminescence detection reagent (PerkinElmer).

EMSA

EMSA for NF-{kappa}B binding was performed as described (18). Double-stranded oligonucleotides were synthesized at the Molecular Core Facility of the New Jersey Medical School (Newark, NJ). Sequences were synthesized based on the 5' flanking region of Tac1 (GenBank accession no. AF252261). Wild-type (+791/+824) sequences was 5'-CCCGCGGGA CTGTCCGTCGCAGTAAGTGCCCGCG-3' (sense). Both the sense and antisense sequences have TG overhangs in the 5' ends that served as end fillings with reverse transcriptase (SuperScript; Invitrogen Life Technologies) and [32P]CTP and dATP (50 µCi of 3000 Ci/mM; PerkinElmer). The consensus sequence for NF-{kappa}B is underlined (18). Mutant sequences changed the third G to A (G->A and the eighth T to G (T->G). Double-stranded probes were prepared with 2.5 µg of the forward and reverse oligonucleotides. The reaction mix consisted of 2.5 µg of dsDNA, 3 µg of poly(deoxyinosinic-deoxycytidylic acid) (Sigma-Aldrich), and 25 µg of proteins in the presence or absence of anti-p65 at 1/2000 final dilution. The control reaction contained 1 µg of p50.

Data analyses

Statistical evaluations of the data were done with ANOVA and Tukey-Kramer multiple comparisons test. A p value of <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effects of SDF-1{alpha} on the activation of the 5' flanking region of Tac1

The effects of SDF-1{alpha} at levels above baseline in stromal cells on the expression of Tac1 were studied with reporter gene constructs. We first studied reporter gene activities of the 5' flanking region of Tac1, previously designated as pGL3-PPT-I/1.2 (18). The fragment includes 722 bp upstream of the transcription start site, exon 1, and part of intron 1 (Fig. 1A). We first performed dose-response studies by stimulating pGL-1-PPT-1.2-transfected BM stroma with SDF-1{alpha} ranging between 5 and 100 ng/ml. Luciferase activities indicated the optimum response at 20 ng/ml and decreased responses at 50 and 100 ng/ml (Fig. 1B). We next focused on 20, 50, or 100 ng/ml SDF-1{alpha} by repeating the studies in the presence or absence of the CXCR4 antagonist AMD3100 at 10 ng/ml. The concentration of AMD3100 was based on dose-response studies ranging between 0.1–100 ng/ml. The effects of SDF-1{alpha} were reversed by AMD3100, indicating specific effects via the CXCR4 receptor (Fig. 1C).


Figure 1
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  		FIGURE 1. Effects of SDF-1{alpha} on the activity of the 5' flanking regions of Tac1. A, Schematic showing the relative sizes within the 5' flanking region of Tac1. B, Stromal cells were cotransfected with pGL3-PPT-I-1.2 and pGL3-beta-gal. After 16 h, transfectants were stimulated with various concentrations of SDF-1{alpha}, and then 16 h later cell extracts were analyzed for luciferase activities and beta-gal. Luciferase activities were normalized based on beta-gal activities and presented as the mean ± SD, n = 4. C and D, Stromal cells were transfected as in B, except that pGL3-PPT-I/N0 were included and then subjected to similar analyses; the results are presented as the mean ± SD, n = 7.

 
Exon 1 has been shown to have a consensus sequence for NF-{kappa}B site and might be responsible for the suppression observed beyond 20 ng/ml SDF-1{alpha} (Fig. 1A) (18). To determine the significance of exon 1, we omitted this region in PPT-I-1.2, designated pGL3-PPT-I-N0 (Fig. 1A), and then transfected stroma. The results showed a dose-response effect (Fig. 1D), indicating that exon 1 is important in Tac1 regulation in BM stroma at concentrations of SDF-1{alpha} above the endogenous level.

Characterization of Tac1-exon 1 in BM stromal cells

The differences in luciferase activities between PPT-I-1.2 and PPT-I-N0 led us to examine exon 1 closer, because this region accounts for the variations in activities between the two fragments (Fig. 1, C and D). Luciferase levels (Fig. 1) suggest that exon 1 could have repressor activity at 50 and 100 ng/ml SDF-1{alpha}. If this region is indeed a repressor, then it should suppress the activity of a heterologous promoter. We therefore inserted exon 1 into pSEAP-Basic, which secretes Alk Phos, making its activity quantifiable at various times after transfection. At the end of the experiment, cellular beta-gal activities were quantitated for transfection efficiency. The results are shown for Alk Phos levels at 48 h after transfection. The fold change from transfectants with vector alone was 20-fold for pSEAP-Basic (Fig. 2A, open bar). Similar transfection with the upstream sequences resulted in >92-fold increase (not shown). Thus, in the absence of the upstream sequences, exon 1 appears to be a weak promoter.


Figure 2
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  		FIGURE 2. Function of Tac1 exon 1 in BM stromal cells. A, BM stromal cells were transfected with pSEAP-Basic, pSEAP-Enhancer, or pSEAP-Promoter containing exon 1. At different times up to 48 h, Alk Phos activities were quantitated with aliquots of culture medium. The results are shown for values at 48 h and presented as activity normalized with beta-gal, mean ± SD, n = 5. B, BM stromal cells were transfected with pSEAP-Promoter with exon 1 inserted in the sense (S) or antisense (AS) orientation. After 48 h, Alk Phos activities were determined and presented as normalized values, mean ± SD, n = 5. *, p < 0.05 vs unstimulated.

 
The next set of transfections confirms promoter activity for exon 1 by the insertion into pSEAP of a heterologous enhancer (pSEAP-Enhancer) or a promoter (pSEAP-Promoter). In both transfections, Alk Phos levels were significantly (p < 0.05) increased over p-SEAP-Basic (Fig. 2A, middle and right bars). The 3-fold increase for pSEAP-Promoter suggests two reasons: exon 1 could have an enhancer function, or perhaps the increase is due to the outcome of two promoters, the pSEAP-Promoter and exon 1 promoter activity, as suggested in the left open bar of Fig. 2A.

To ascertain that exon 1 is indeed a weak promoter and not an enhancer, we next inserted the sequence in the sense and anti-sense orientations in the pSEAP-Promoter vector and then transfected BM stroma. After 48 h, Alk Phos activities showed no significant (p > 0.05) change in reporter gene activity (Fig. 2B), indicating that exon 1 does not have enhancer functions in stroma and therefore supporting weak promoter function.

NF-{kappa}B as a mediator on the repressor effects at high SDF-1{alpha} levels

Because exon 1 shows weak promoter activity (Fig. 2), we next determined whether it is responsive to SDF-1{alpha} and, if so, whether the response is concentration dependent. We therefore transfected stromal cells with pSEAP-Basic-exon 1 and then stimulated the cells with SDF-1{alpha} at 20, 50, and 100 ng/ml. After 48 h, Alk Phos activities showed increased levels in the presence of 20 ng/ml SDF-1{alpha} and significant (p < 0.05) reduction at 50 and 100 ng/ml (Fig. 3A).


Figure 3
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  		FIGURE 3. Role of NF-{kappa}B in SDF-1{alpha}-mediated activation of reporter gene activities. A, Cells were transfected as in Fig. 2A except that the transfectants were stimulated with SDF-1{alpha} at 20, 50, and 100 ng/ml. The results are mean ± SD, n = 4. B, Representative of three gel shift assays done with nuclear extracts from IL-1{alpha}-stimulated stroma. Positive control was done with wild-type probe and 1 µg of purified p50. Specificity of binding was studied by supershift with anti-p65 at 1/2000 final dilution. Parallel studies were done with mutant probes. C, Representative of three Western blots for p65 done with nuclear extracts from stromal cells stimulated for 2 h with various concentrations of SDF-1{alpha}. The membrane was stripped and reprobed with anti-beta-actin. D, The studies described in A were repeated, except that the cells were cotransfected with wild-type or mutant I{kappa}B. The results are the mean ± SD, n = 4. E, Stromal cells were cotransfected with pGL3-PPT-I-1.2 and wild-type or mutant I{kappa}B. After this, the experimental steps followed those in Fig. 1B. The results are presented as the mean ± SD, n = 4. *, p < 0.05 vs 20 ng/ml SDF-1{alpha}; **, p < 0.05 vs mutant I{kappa}B.

 
Exon 1 has a consensus region for NF-{kappa}B (18). We now ask whether this region could be involved in the repression at high SDF-1{alpha} levels. To address this question, we needed to determine whether the consensus region is a functional NF-{kappa}B site. To this end, a gel shift assay was used to study the binding of p50 to wild-type and mutant 32P-labeled nucleotide probes spanning the consensus region. Controls with purified p50 showed a band with the wild-type probe but not with mutant probe (Fig. 3B, second and third lanes from the left). To test binding with NF-{kappa}B subunits present in nuclear extracts, we selected extracts from IL-1{alpha}-stimulated stroma, shown to be positive for p65 by Western blotting (not shown). The results showed a sharp band that supershifted with anti-p65 (Fig. 3B, second and third lanes from the right). There was a light to almost undetectable band when the nuclear extract was incubated with a mutant probe (Fig. 3B, right lane).

The role of NF-{kappa}B in the SDF-1{alpha}-mediated activity of exon 1 was studied by cotransfecting stroma with pSEAP-Basic/exon 1 and wild-type or mutant I{kappa}B. The levels of Alk Phos after 48 h showed increases at 50 and 100 ng/ml SDF-1{alpha} but no effect for unstimulated transfectants or for 20 ng/ml SDF-1{alpha} (Fig. 3D).

Because exon 1 acted as a repressor for PPT-I-1.2 (Fig. 1, C and D), we asked whether NF-{kappa}B can also mediates repressive functions for PPT-I-1.2 following stimulation with high SDF-1{alpha} levels. We repeated the studies described for Fig. 3D, except that the whole cell extracts were studied for luciferase activity. The results showed increased luciferase in the presence of mutant I{kappa}B, with 50 and 100 ng/ml SDF-1{alpha} (Fig. 3E, two right groups). Similar effects were not observed in the cases of 20 ng/ml SDF-1{alpha} and unstimulated transfectants (Fig. 3E, two left groups). In summary, NF-{kappa}B is shown to mediate the repressive activity of PPT-I in stromal cells at high SDF-1{alpha} concentrations.

Induction of endogenous Tac1 by SDF-1{alpha}

We next determined whether SDF-1{alpha} could induce the expression of endogenous Tac1. BM stromal cells were stimulated with 20 ng/ml SDF-1{alpha} and after 6 h total RNA was extracted and then analyzed by Northern analyses for Tac1 mRNA (beta-PPT-I). Compared with unstimulated stroma, there were significantly denser bands in stimulated stroma (representative blots are shown in Fig. 4A, n = 4). We next determined whether the increase in beta-PPT-I correlated with protein increase. SP levels served as indicators of beta-PPT-I expression because it could be produced by each of the four possible Tac1 transcripts (1).


Figure 4
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  		FIGURE 4. Effects of SDF-1{alpha} on the expression of Tac1. A, Representative of four Northern blots for Tac1 mRNA (beta-PPT-I) with total RNA from stroma, unstimulated or stimulated with 20 ng/ml SDF-1{alpha}. The lanes were normalized with 18S rRNA. B, ELISA quantitated SP in serum-free medium obtained from stroma, unstimulated or stimulated for various times with different levels of SDF-1{alpha}. The results are presented as the mean SP levels ± SD (pg/ml; n = 5).

 
Confluent stromal cells were stimulated with 20, 50, and 100 ng/ml SDF-1{alpha}. At 24, 36, and 48 h, culture medium was collected and then quantitated for SP levels by ELISA. At 20 ng/ml SDF-1{alpha}, there was significant (p < 0.05) increase in SP levels at 24 and 36 h, but a reduction at 48 h (Fig. 4B, left bars). At 50 ng/ml SDF-1{alpha}, there was similar trend but significantly (p < 0.05) reduced SP (Fig. 4B, middle bars). At 100 ng/ml SDF-1{alpha} the levels were minimal (Fig. 4B, right bars). Together, this section shows Tac1 induction by SDF-1{alpha} at 20 ng/ml over baseline production in BM stroma but reduced production at high levels.

Effects of exogenous SDF-1{alpha} on LTC-IC cultures

SP has been reported to induce the production of cytokines with hemopoietic stimulatory effects (20) and has also been shown to enhance hemopoiesis, both at the level of granulocytic-monocytic progenitors and at the level of immature progenitors/LTC-IC cells (21). We therefore determined whether SDF-1{alpha}, added exogenously at different levels (20, 50 and 100 ng/ml), could enhance LTC-IC proliferation. The studies were done for 6- and 12-wk cultures. These two time points were selected because the 6-wk assay evaluates hemopoietic progenitors that are relatively more mature than those in 12-wk cultures. Thus, the analyses would provide information on both the mature and immature hemopoietic progenitors. The results showed significant (p < 0.05) increases in CFU-GM (readout) for 6-wk cultures at all SDF-1{alpha} levels but only at 20 ng/ml for 12-wk cultures (Fig. 5).


Figure 5
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  		FIGURE 5. Effects of exogenous SDF-1{alpha} on hemopoiesis. BM mononuclear cells (107/ml) were added to confluent gamma-irradiated stroma in the presence or absence of 20, 50, and 100 ng/ml SDF-1{alpha}. At wks 6 and 12, aliquots of mononuclear cells were studied in clonogenic assays for CFU-GM. The number of CFU-GM colonies are presented as the mean ± SD, n = 5. Each experiment was performed with cells from a different donor.

 
SP is a mediator in the activation of LTC-IC cells by SDF-1{alpha}

SDF-1{alpha} has been shown to induce the production of SP in BM stroma (Fig. 4). We asked whether SP might mediate the enhanced hemopoietic effects by SDF-1{alpha} at 20 ng/ml (Fig. 5). The LTC-IC assays were repeated in the presence or absence of 100 nM antagonists specific for NK1 (CP-99,994) or NK2 (SR-48968). These concentrations were determined in dose-response studies. Furthermore, at 100 nM neither antagonist affected the viability of BM mononuclear cells and stromal cells in 12-wk cultures, as indicated by trypan blue exclusion. Positive controls were done in parallel with SP, which is known to stimulate LTC-IC cells (1). Baseline cultures (medium) showed no change in 6- and 12- wk cultures in the presence of NK1 antagonists (Fig. 6, A and B). However, the NK2 antagonist resulted in a significant (p < 0.05) increase in CFU-GM (Figs. 6, A and B).


Figure 6
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  		FIGURE 6. SP is a secondary mediator in the activation of LTC-IC cells by SDF-1{alpha}. BM mononuclear cells (107/ml) were studied in LTC-IC assays in the presence of 20 ng/ml SDF-1{alpha} and/or 100 nM an antagonist to NK1, CP-99,994 (A), or to NK2, SR-48968 (B). Positive controls were stimulated with 10 nM SP. CFU-GM were analyzed as for Fig. 5 and then presented as the mean CFU-GM ± SD, n = 5. *, p < 0.05 vs unstimulated or SDF-1{alpha} stimulated; **, p < 0.05 vs SDF-1{alpha} stimulated without NK1 antagonist.

 
LTC-IC cultures (6- and 12-wk) stimulated with SDF-1{alpha} in the presence of the NK1 antagonist showed significant reduction in CFU-GM (p < 0.05) as compared with cultures without the antagonist (Fig. 6, A and B). Similarly for LTC-IC with medium, in the presence of SDF-1{alpha} and the NK-2 antagonist there were significant (p < 0.05) increases in CFU-GM colonies (Fig. 6, A and B). The results showed that the increases in LTC-IC activity at 6- and 12-wk cultures by 20 ng/ml SDF-1{alpha} were mediated by the NK1 receptor. In the absence of NK2 activation, there was significant (p < 0.05) increase in CFU-GM over cultures with only SDF-1{alpha}.

Effect of SP on the induction of SDF-1{alpha} in BM stroma

It is possible that SP induced by SDF-1{alpha} in stroma could lead to autocrine regulation of hemopoiesis. In this case, SP production would be expected to increase SDF-1{alpha} production in stroma. To address this question, we stimulated stroma with 10 nM SP in the presence or absence of 100 nM NK1 antagonist. At different times the culture medium was collected and then quantitated for SDF-1{alpha} levels by ELISA. The lower end of the standard curve, 0.5 pg/ml, was included in the linear section of the graph. The results showed no significant (p > 0.05) change in SDF-1{alpha} levels over baseline (Fig. 7), indicating that, at least in a pure culture of BM stroma, SP does not induce the production of SDF-1{alpha}.


Figure 7
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  		FIGURE 7. Effects of SP on SDF-1{alpha} production in BM stroma. ELISA quantitated the levels of SDF-1{alpha} in BM stroma, unstimulated or stimulated, for 24 h with 10 nM SP in the presence or absence of 100 nM NK1 antagonist (CP-99,994) in sera-free medium. The results are presented as the mean SDF-1{alpha} levels ± SD (pg/ml, n = 5).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study reports on the link between SDF-1{alpha} and the Tac1 gene in BM stroma. We have found that the concentrations of SDF-1{alpha} affect the expression of Tac1, specifically via CXCR4 (Fig. 1). Although relatively low concentrations (20 ng/ml) of SDF-1{alpha} activated the 5' flanking region of Tac1, higher concentrations (50–100 ng/ml) were inhibitory (Fig. 1, B and C). Because the CXCR4 is a G protein-coupled receptor, one can argue that receptor desensitization could be operative at high SDF-1{alpha} levels (22). However, our studies have ruled out this mechanism because we showed a dose-dependent effect in reporter gene activities when the 3' region of reporter gene fragment was omitted (Figs. 1D). We concluded that mechanisms other than receptor desensitization could be possible. Analyses of exon 1 revealed weak promoter function in BM stroma (Figs. 2, A and B). Exon 1 could also act as a negative regulator of PPT-I-1.2 (Fig. 1).

The dual role of exon 1, weak promoter vs repressor activity, makes this fragment a region of interest with regard to Tac1 regulation. Analyses of the NF-{kappa}B region in exon 1 indicate that this transcription factor is relevant to the repressive function of high SDF-1{alpha} levels (Fig. 2). This does not, however, fulfill the broad role of NF-{kappa}B in the enhancing effects of low SDF-1{alpha} levels. Previous studies have reported enhancing functions of cAMP response element regions on Tac1 (18). NF-{kappa}B might be involved in an antagonistic effect on CREBs at high levels of SDF-1{alpha}. These mechanistic pathways are important and represent ongoing studies in the laboratory.

The verification of enhanced Tac1 promoter activity following SDF-1 stimulation confirmed enhanced expression of Tac1 at both the mRNA and protein levels (Fig. 4). These effects of exogenous SDF-1{alpha} on Tac1 were relevant to hemopoiesis as shown by increased hemopoietic activity in LTC-IC cultures (Fig. 5). Because the studies were done by the LTC-IC assays, we concluded that the level of hemopoietic regulation occurred at the most primitive level.

Although the NK1 antagonist blunted the hemopoietic effects, we showed a significant increase in hemopoietic activity in the presence of NK2 antagonists (Fig. 6). This suggested that NK2 activation is important to prevent exacerbated stimulation by NK1 on hemopoiesis. This observation is consistent with other studies showing functional crosstalk between NK1 and NK2 (5). Another possibility for this enhanced hemopoietic effect of the NK2 antagonist is that SP, in the absence of antagonist, could bind weakly to NK2 and might elicit negative effects through the production of cytokines with inhibitory effects on hemopoiesis (1).

These studies do not show evidence that SP regulates the production of SDF-1{alpha}, either negatively or positively. We have observed similar SDF-1{alpha} levels in stromal cells stimulated with exogenous SP and unstimulated stroma (Fig. 7). This ruled out the possibility that SP, in the presence of the NK1 antagonist, could act as negative feedback on SDF-1 production to reduce hemopoietic activity (Fig. 6). The observed lack of autocrine stimulation by SP not being able to induce the production of SDF-1{alpha} further in stroma might be explained by the normal biology of the BM. Specifically, hemopoietic stem cells and stroma interact close to the endosteum to retain the stem cells (9). SDF-1{alpha} increase could lead to mobilization of the stem cells into the periphery. Thus, physiologically it would be a disadvantage if SDF-1{alpha}-mediated production of SP causes autocrine production of SDF-1{alpha}. In fact, high levels of SDF-1{alpha} showed suppression of 12-wk LTC-IC (Fig. 5). Although there are several possible explanations for this observation, perhaps high SDF-1{alpha} might cause the movement of hemopoietic stem cells from the marrow region. If this occurs, then the 12-wk LTC-IC cells will undergo cell death due to the lack of cellular support of stroma. These are intriguing observations that should be explored further in future studies. Also, the lack of evidence for autocrine stimulation of stroma by SP to produce SDF-1{alpha} further supports receptor crosstalk between NK1 and NK2 (5). Despite the evidence of intracellular crosstalk between NK1 and NK2, the molecular mechanism of this occurrence is unknown.

These studies have implications for the neural-immune-hemopoietic axis. The BM is innervated by peptidergic fibers including those that are positive for SP. Because SP is a hemopoietic stimulator, it is logical to assume that this report showing a link between SP-SDF-1{alpha}-hemopoietic axis could be linked to the nervous system. Evidence has shown that often the signal to increase hemopoiesis comes from the central nervous system and is regulated secondarily by cytokines (23, 24). For example, the SDF-1{alpha} shown in this report could be a mediator in the neural-hemopoietic response through SP and perhaps other neurotransmitters. It would be interesting in future studies to determine whether SDF-1{alpha} and other mediators, through retrograde uptake, may serve as a negative feedback on the nervous system to turn off the signals coming from the central nervous system (25, 26, 27, 28).

This report also has direct relevance to functions of the BM microenvironment. In BM, HSCs are sequestered is a quiescent state close to the endosteum bound to stromal cells through SDF-1{alpha}/CXCR4 interaction (10). To enter hemopoiesis, the HSCs need to move from the stem cell niche into the proliferative niche (29). This occurs through up-regulation of various proteases and cytokines in the stem cell niche. Both SP and SDF-1{alpha} have been shown to function as hemopoietic enhancers (5, 8). Importantly, SP has been shown to up-regulate GM-CSF, which has been reported to mobilize HSCs into the periphery through down-regulation of SDF-1{alpha} (30, 31).

Therefore, the relationship that is demonstrated between SDF-1{alpha} and SP in hemopoiesis could involve secondary production of GM-CSF. This could result in hemopoietic stimulation or perhaps antagonize the increase in SDF-1{alpha} to move the HSC into the proliferative niche, consequently increasing hemopoiesis. In summary, this report shows a novel mechanism by which a neurotransmitter, SP, interacts with SDF-1{alpha} within the BM microenvironment to affect hemopoiesis. These studies have implications for the neural-hemopoietic axis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Department of Defense and the University Hospital Cancer Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School. This work was performed in partial fulfillment for a Ph.D. thesis by K.E.C. and was done at the Department of Medicine, Division of Hematology/Oncology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103. Back

2 Address correspondence and reprint requests to Dr. Pranela Rameshwar, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Medical Science Building, Room E-579, 185 South Orange Avenue, Newark, NJ 07103. E-mail address: rameshwa{at}umdnj.edu Back

3 Abbreviations used in this paper: PPT-I, preprotachykinin I; Alk Phos, alkaline phosphatase; BM, bone marrow; beta-gal, beta-galactosidase; HSC, hemopoietic stem cell; LTC-IC, long-term culture-initiating cell; NK, neurokinin; SDF, stromal-derived growth factor; SP, substance P. Back

Received for publication July 31, 2006. Accepted for publication November 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Greco, S. J., K. E. Corcoran, K. J. Cho, P. Rameshwar. 2004. Tachykinins in the emerging immune system: relevance to bone marrow homeostasis and maintenance of hematopoietic stem cells. Front. Biosci. 9: 1782-1793. [Medline]
  2. Rameshwar, P., A. Poddar, P. Gascon. 1997. Hematopoietic regulation mediated by interactions among the neurokinins and cytokines. Leuk. Lymphoma 28: 1-10. [Medline]
  3. Singh, D., D. D. Joshi, M. Hameed, J. Qian, P. Gascón, P. B. Maloof, A. Mosenthal, P. Rameshwar. 2001. Increased expression of preprotachykinin-I and neurokinin receptors in human breast cancer cells: implications for BM metastasis. Proc. Natl. Acad. Sci. USA 97: 388-393.
  4. Kang, H. S., K. A. Trzaska, K. Corcoran, V. T. Chang, P. Rameshwar. 2004. Neurokinin receptors: relevance to the emerging immune system. Arch. Immunol. Ther. Exp. 52: 338-347.
  5. Bandari, P., J. Qian, H. S. Oh, J. A. Potian, G. Yehia, J. S. Harrison, P. Rameshwar. 2003. Crosstalk between neurokinin receptors is relevant to hematopoietic regulation: cloning and characterization of neurokinin-2 promoter. J. Neuroimmunol. 138: 65-75. [Medline]
  6. Bellucci, F., F. Carini, C. Catalani, P. Cucchi, A. Lecci, S. Meini, R. Patacchini, L. Quartara, R. Ricci, M. Tramontana, et al 2002. Pharmacological profile of the novel mammalian tachykinin hemokinin 1. Br. J. Pharmacol. 135: 266-274. [Medline]
  7. Rameshwar, P., P. Gascon. 1995. Substance P (SP) mediates production of stem cell factor and interleukin-1 in bone marrow stroma: potential autoregulatory role for these cytokines in SP receptor expression and induction. Blood 86: 482-490. [Abstract/Free Full Text]
  8. Arai, A., A. Jin, W. Yan, D. Mizuchi, K. Yamamoto, T. Nanki, O. Miura. 2005. SDF-1 synergistically enhances IL-3-induced activation of the Raf-1/MEK/Erk signaling pathway through activation of Rac and its effector Pak kinases to promote hematopoiesis and chemotaxis. Cell. Signal. 17: 497-506. [Medline]
  9. Lapidot, T., A. Dar, O. Kollet. 2005. How do stem cells find their way home?. Blood 106: 1901-1910. [Abstract/Free Full Text]
  10. Kucia, M., R. Reca, K. Miekus, J. Wanzeck, W. Wojakowski, A. Janowska-Wieczorek, J. Ratajczak, M. Z. Ratajczak. 2005. Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis. Stem Cells 7: 879-894.
  11. Neiva, K., Y. X. Sun, R. S. Taichman. 2005. The role of osteoblasts in regulating hematopoietic stem cell activity and tumor metastasis. Braz. J. Med. Biol. Res. 38: 1449-1454. [Medline]
  12. Laurence, A. D.. 2006. Location, movement and survival: the role of chemokines in haematopoiesis and malignancy. Br. J. Haematol. 132: 255-267. [Medline]
  13. Moharita, A. L., M. Taborga, K. E. Corcoran, M. Bryan, P. S. Patel, P. Rameshwar. 2006. SDF-1{alpha} regulation in breast cancer cells contacting bone marrow stroma is critical for normal hematopoiesis. Blood 108: 3245-3252. [Abstract/Free Full Text]
  14. Perez, L. E., O. Alpdogan, J. H. Shieh, D. Wong, A. Merzouk, H. Salari, R. J. O’Reilly, M. R. van den Brink, M. A. Moore. 2004. Increased plasma levels of stromal-derived factor-1 (SDF-1/CXCL12) enhance human thrombopoiesis and mobilize human colony-forming cells (CFC) in NOD/SCID mice. Exp. Hematol. 3: 300-307.
  15. Flomenberg, N., J. DiPersio, G. Calandra. 2005. Role of CXCR4 chemokine receptor blockade using AMD3100 for mobilization of autologous hematopoietic progenitor cells. Acta Haematol. 114: 198-205. [Medline]
  16. Rameshwar, P., A. Poddar, G. Zhu, P. Gascon. 1997. Receptor induction regulates the synergistic effects of substance P with IL-1 and PDGF on the proliferation of bone marrow fibroblasts. J. Immunol. 158: 3417-3424. [Abstract]
  17. Oh, H. S., A. Moharita, J. G. Potian, I. P. Whitehead, J. C. Livingston, T. A. Castro, P. S. Patel, P. Rameshwar. 2004. Bone marrow stroma influences transforming growth factor-beta production in breast cancer cells to regulate c-myc activation of the preprotachykinin-I gene in breast cancer cells. Cancer Res. 64: 6327-6336. [Abstract/Free Full Text]
  18. Qian, J., G. Yehia, C. Molina, A. Fernandes, R. J. Donnelly, D. J. Anjaria, P. Gascón, P. Rameshwar. 2001. Cloning of human preprotachykinin-I promoter and the role of cAMP response elements in its expression by IL-1 and stem cell factor. J. Immunol. 166: 2553-2561. [Abstract/Free Full Text]
  19. Rameshwar, P., P. Gascon. 1996. Induction of negative hematopoietic regulators by neurokinin-A in bone marrow stroma. Blood 88: 98-106. [Abstract/Free Full Text]
  20. Rameshwar, P., G. Zhu, R. J. Donnelly, J. Qian, H. Ge, K. R. Goldstein, T. N. Denny, P. Gascón. 2001. The dynamics of bone marrow stromal cells in the proliferation of multipotent hematopoietic progenitors by substance P: an understanding of the effects of a neurotransmitter on the differentiating hematopoietic stem cell. J. Neuroimmunol. 121: 22-31. [Medline]
  21. Gascon, P., J. Qian, D. D. Joshi, T. Teli, A. Haider, P. Rameshwar. 2000. Effects of preprotachykinin-I peptides on hematopoietic homeostasis. A role for bone marrow endopeptidases. Ann. N Y Acad. Sci. 917: 416-423. [Abstract/Free Full Text]
  22. Dar, A., P. Goichberg, V. Shinder, A. Kalinkovich, O. Kollet, N. Netzer, R. Margalit, M. Zsak, A. Nagler, I. Hardan, et al 2005. Chemokine receptor CXCR4-dependent internalization and resecretion of functional chemokine SDF-1 by bone marrow endothelial and stromal cells. Nat. Immunol. 6: 1038-1046. [Medline]
  23. Miyan, J. A., C. S. Broome, A. M. Afan. 1998. Coordinated host defense through an integration of the neural, immune and haemopoietic. Domest. Anim. Endocrinol. 15: 297-304. [Medline]
  24. Wrona, D.. 2006. Neural-immune interactions: an integrative view of the bidirectional relationship between the brain and immune systems. J. Neuroimmunol. 172: 38-58. [Medline]
  25. Felten, D. L., S. Y. Felten, D. L. Bellinger, D. Lorton. 1992. Noradrenergic and peptidergic innervation of secondary lymphoid organs: role in experimental rheumatoid arthritis. Eur. J. Clin. Invest. 1: 37-41.
  26. Weihe, E., D. Nohr, S. Michel, S. Muller, H. J. Zentel, T. Fink, J. Krekel. 1991. Molecular anatomy of the neuro-immune connection. Int. J. Neurosci. 59: 1-23. [Medline]
  27. Campenot, R. B.. 1994. NGF and the local control of nerve terminal growth. J. Neurobiol. 25: 599-611. [Medline]
  28. Köbbert, C., R. Apps, I. Bechmann, J. L. Lanciego, J. Mey, S. Thanos. 2000. Current concepts in neuroanatomical tracing. Prog. Neurobiol. 62: 327-351. [Medline]
  29. Heissig, B., K. Hattori, S. Dias, M. Friedrich, B. Ferris, N. R. Hackett, R. G. Crystal, P. Besmer, D. Lyden, M. A. Moore, et al 2002. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 109: 625-637. [Medline]
  30. Hattori, K., B. Heissig, S. Rafii. 2003. The regulation of hematopoietic stem cell and progenitor mobilization by chemokine SDF-1. Leuk. Lymphoma 44: 575-582. [Medline]
  31. Gazitt, Y.. 2002. Comparison between granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the mobilization of peripheral blood stem cells. Curr. Opin. Hematol. 9: 190-198. [Medline]



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