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HEMATOPOIESIS
From the Information System and
Technology-Academic Computer Center and the Department of Pathology
and Laboratory Science, University of Medicine and Dentistry of
New Jersey-New Jersey Medical School; Department of Biological
Sciences, Rutger's University, Newark, NJ; and Ciphergen Biosystems,
Fremont, CA.
Hematopoietic regulation is a complex but dynamic process regulated
by intercellular and intracellular interactions within the bone marrow
(BM) microenvironment. Through neurokinin-1 (NK-1) and NK-2 receptors,
peptides (eg, substance P [SP]) encoded by the preprotachykinin-I
gene mediate distinct hematopoietic effects. Cytokines, associated with
hematopoietic stimulation, and SP regulate the expression of each other
in BM mesenchymal and immune cells. Neutral endopeptidase (NEP) uses SP
as a substrate to produce SP(1-4), which inhibits the proliferation of
matured myeloid progenitor. This study determines whether the
degradation of SP to SP(1-4) by endogenous NEP in BM stroma could be a
feedback on hematopoietic stimulation by stem cell factor (SCF).
SP(1-4) induced the production of transforming growth factor (TGF)- In the adult, the bone marrow (BM) is the major
site of hematopoiesis. Biological activities in the BM are complexed,
albeit controlled, through cell-cell interactions among the
hematopoietic stem cell, progenitors, mesenchymal cells, and accessory
cells.1 Cellular interactions in the BM could lead to the
induction of soluble hematopoietic regulators, such as cytokines,
neuropeptides, and neurotrophic factors.2 Other
hematopoietic regulators could be derived as neurotransmitters from the
innervated nerve fibers in the BM and as hormones from the peripheral
circulation.2 Despite the distant source of hormones, they
modulate biological responses in the BM through specific receptors on
resident BM cells.3
The general consensus is that a finite pool of hematopoietic stem cells
produces mature immune and blood cells through a complexed but
controlled network that includes cells and soluble factors. Regardless
of the pathways that lead to hematopoietic stimulation, the process of
feedback is of utmost importance to maintain homeostasis and to protect
the pool of hematopoietic stem cells. Understanding this dynamic
process in the BM would have an impact on several clinical areas such
as gene therapy, chemotherapy, and BM
transplantation.4 In this study, we describe a model that
might explain the mechanism for negative feedback by the amino terminal
of substance P (SP), SP(1-4), on the effects of 2 hematopoietic
stimulators: stem cell factor (SCF) and SP. These 2 stimulators
regulate the induction of each other in BM cells.2,5
SP(1-4) could be derived from degradation of SP by endogenous
endopeptidases in the BM.6,7 This study also reports on
the functional plasticity of neurokinin-1 (NK-1) receptor to regulate
hematopoiesis.8
SP, an undecapeptide, is evolutionarily conserved and is the
major peptide derived from the preprotachykinin-I (PPT-I)
gene.9,10 PPT-I peptides exert both stimulatory and
inhibitory hematopoietic effects,5 which are mediated
through G-protein-coupled receptors: NK-1, NK-2, and
NK-3.5,11,12 NK-1 is induced in BM cells by cytokines and
other stimulatory hematopoietic regulators.2 NK-2 is
constitutively expressed in BM cells that are unstimulated or
stimulated with suppressive hematopoietic regulators.5
NK-1 and NK-2 are not coexpressed in BM cells since NK-1 induction by
cytokines is correlated with the down-regulation of
NK-2.3,5
Endopeptidases are ubiquitously expressed in hematopoietic
cells.7 Dipeptidyl-peptidase IV (CD26),
aminopeptidase, angiotensin-converting enzyme, and neutral
endopeptidase (NEP)/CD10 use SP as their
substrate.6,7,13,14 The carboxyl portion of the truncated
peptide can be further digested by aminopeptidases
N/CD13,15,16 resulting in Arg-Pro and Arg-Pro-Lys-Pro: SP(1-4). Cytokines, SP, and endopeptidases regulate the expression of
each other in BM cells.13,14,17 The proinflammatory
properties of SP and cytokines have been linked to the activities of
various endopeptidases.14,17
SP fragments could exert immune and hematopoietic
regulation.18,19 This study shows that SP(1-4) inhibits
the proliferation of late and early hematopoietic progenitors. The
parent peptide, SP, and cytokines associated with hematopoietic
stimulation regulate the expression of each other, leading to positive
hematopoiesis.2 To this end, we investigated whether
digestion of SP to SP(1-4) could be a mechanism of hematopoietic
feedback on the effects of SCF. This particular cytokine represents a
model hematopoietic stimulator, interacting with SP to regulate the
expression of each other.20,21 Induction of the following
was studied: negative hematopoietic regulators, transforming growth
factor (TGF)- Reagents, cytokines, and antibodies
Clonogenic assays for granulocyte-macrophage colony-forming
units
Modified long-term culture-initiating cells Confluent stromal cells were cultured in 25-cm2 flasks and then subjected to 150 Gy, delivered by a cesium source. We added 107 bone marrow mononuclear cells to the flask containing the -irradiated stroma. Beginning at week 5 of
culture, aliquots of cells were assayed for CFU-GMs every week up to
12 weeks.
Preparation and stimulation of BM stroma Stromal cultures were prepared with BM aspirates from healthy donors as described.24 Briefly, stromal cells were cultured at 33°C, and at day 3, the granulocytes and red blood cells were removed by Ficoll-Hypaque density gradient. Cultures were reincubated with weekly replacement of 50% culture media until confluence.Cytokine, NK receptor, and c-kit expressions were studied in confluent
stroma, stimulated with 10 nM SP(1-4) and/or 10 ng/mL SCF. Stroma was
stimulated in 3 mL sera-free The relative expression of c-kit in BM stroma was determined by immunofluorescence. BM stroma was cultured on coverslips (Fisher Scientific, Springfield, NJ) and then stimulated with 10 ng/mL SCF. At different times after cell stimulation, cells were labeled with 50 ng/mL biotinylated goat anti-c-kit and FITC-avidin. Nonspecific binding was determined with biotinylated nonimmune goat anti-IgG. Cells were immediately examined on an Olympus Probis (New York/New Jersey Scientific, Middlebush, NJ) microscope. The fluorescence intensity at an excitation of 595 nm was dim in unstimulated stroma and increased to bright in cells stimulated for 6 hours to 24 hours. Quantitation of SP immunoreactivity Competitive enzyme-linked immunosorbent assay (ELISA) quantitated SP immunoreactivity (SP-IR) as described.3 Briefly, Immulon 96-well plates (Dynatech Laboratories, Chantilly, VA) were precoated with streptavidin and then incubated with biotinylated SP (Chiron Mimotopes, Emeryville, CA). Equal volumes (50 µL) of unknown samples and optimum rabbit anti-SP were added to quadruplicate wells. Each sample was assayed as undiluted and in 3 serial dilutions. Complexed anti-SP was detected with alkaline phosphatase-conjugated goat antirabbit IgG and Sigma 104 phosphatase substrate. SP-IR levels were calculated from a standard curve developed with optic density (OD) at 405 nm versus 12 serial dilutions of known SP concentrations, ranging from 100 to 0.08 pg/mL.Quantitative reverse transcriptase-polymerase chain reaction Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) for NK-1, NK-2, and -PPT-I mRNA was performed as
described.3,24 Briefly, 2 µg total RNA from BM stroma
was reverse transcribed, and 200 ng complementary DNA (cDNA) was used
in PCR reactions with standard DNA at log10-fold dilutions
ranging between 10 2 and 106 attomole per
liter. PCR products (10 µL) were separated on agarose containing
ethidium bromide. The DNA was scanned with a Fluorimager (Molecular
Dynamics, Sunnyvale, CA), and the densities were analyzed with
ImageQuant software version 5.2 (Molecular Dynamics). A
standard curve was established for each unknown sample: band densities of unknown/standard DNA versus log10 standard DNA
concentration. The concentration of the unknown sample was read at the
point where the unknown and standard were equivalent.
Construction of DNA standards was previously
described.24
Immunofluorescence for NK receptors BM stroma was stimulated with 10 ng/mL SCF. At 24 hours, cells were washed and then immediately incubated at 4°C for 2 hours with 200 ng/mL biotin-SP or biotin-NK-A. After this, cells were labeled with FITC-avidin for 30 minutes and then examined for fluorescence intensity with an Olympus Provis AX-70 at 480 ± 40 nm and 535 ± 50 nm per emission. Minimal fluorescence was observed in cells colabeled with excess unconjugated SP or NK-A or in cells labeled with FITC-avidin alone. The optimal experimental conditions were determined in dose-response and time-course studies. Specificity of binding by SP or NK-A was studied in competition labeling with the pan-tachykinin antagonist spantide.TNF-  beginning at 1.5 nM. Plates were incubated
overnight, and the next day, cell viability was determined by means of
the MTT assay. Briefly 20 µL MTT at 5 mg/mL was added to each well. The plates were incubated for 2 hours at 37°C and then centrifuged at
800g for 5 minutes. Supernatants were removed and cells
washed once with phosphate-buffered saline (PBS) by centrifugation at 2000g for 5 minutes. Thiazolyl blue crystals were dissolved
with 100 µL isopropanol, and the OD was determined at 570 to 650 nm with a Kinectic microplate reader (Molecular Devices, Menlo Park, CA).
TGF- .25 Each
sample was tested in triplicate with 25 µL, 50 µL, or 100 µL
supernatants. TGF- levels were determined from a standard curve
established with TGF- concentrations ranging from 0.001 to 10 ng/mL
versus cell concentration. The presence of TGF-1 was verified by
reanalyses of samples containing more than 20 ng/mL TGF- with
neutralizing anti-hTGF-1.25 Neutralization was
determined if the anti-TGF-1 reversed the growth-inhibitory effect
of the supernatants.
Biopanning of phage display library and selection of clones Peptide-binding sites were determined by screening a random dodecapeptide library, FliTrx, as per manufacturer's instruction (Invitrogen, Carlsbad, CA). Briefly, bacteria were induced with tryptophan and then passaged for 6 hours in 60-mm Petri dishes (Nunclon Delta, Nalge Nunc, Rochester, NY) that were precoated with 20 µg SP(1-4). After the seventh passage, the adherent bacteria were expanded in liquid culture and then subcultured on agar. Forty colonies were expanded, and the pellet from each bacterial culture was boiled in sample buffer and then spotted on methanol-soaked polyvinylidene fluoride (PVDF) transfer membranes (NEN, Boston, MA). The membranes were consecutively incubated with 10 µg/mL SP(1-4) overnight at room temperature and rabbit anti-SP for 2 hours. Anti-SP was detected with alkaline phosphatase-conjugated goat antirabbit IgG followed by incubation for 15 minutes with 5-bromo-4-chloro-3-indolyl-phosphatase/nitroblue tetrazolium phosphate substrate system (Kirkegaard and Perry Laboratories). A single blinded observer scored the relative intensities of the spots, with zero for no detection and 4 for the highest intensity. Twenty clones with assignments between 1 and 4 were selected and further analyzed in Western blots by means of gradient gels of 4% to 20% (Invitrogen).25 Seven clones were finally selected on the basis of the development of a single, dense band at the predicted molecular mass (Mr).DNA was extracted from each clone (described above) and then sequenced in both orientations at the Molecular Core Facility, UMDNJ-New Jersey Medical School. DNA sequencing was performed with the FliTrx forward and Rsr reverse primers, provided with the peptide library. Each clone was analyzed with the Wisconsin Genetics Computer Group (Madison, WI) package of DNA/protein sequence analysis programs (version 10). Six clones were selected on the basis of greater than 40% identity to the protein sequence of NK-1.26 Computer modeling of SP and NK-1 The transmembrane (TM) portion of NK-1 was modeled by means of WHAT-IF (Inspire Pharmaceuticals, Durham, NC) in conjunction with Swiss-Model (GlaxoSmithKline, Geneva, Switzerland) at the Swiss Institute of Bioinformatics (Geneva) and European Molecular Biology Laboratory (EMBL) (Heidelberg, Germany). The structures of both SP and NK-1 were generated by means of SYBYL 6.6 (Tripos Associates, St Louis, MO). A homolog of the TM region of NK-1 was modeled at the EMBL and Swiss-Model repository by means of WHAT-IF, an algorithm that maps the sequence onto a template structure. WHAT-IF optimizes the structure while adjusting for side-chain collisions and interactions. The template used was the alpha-carbon chain from the crystallized structure of the G-protein- coupled receptor, bacteriorhodopsin.27 The arrangement of the TM helices in bacteriorhodopsin is the conventional model for all members of the G-protein-coupled receptor. The intracellular and extracellular loops were constructed by means of a loop search. SP was constructed ab initio on the basis of its sequence. A molecular dynamics run was performed with the use of a time step of 5, carried out through 500 iterations.Energy minimization was performed. Docking of SP into NK-1 was performed with SYBYL's Docking engine and Flexidock. All energetics calculations were performed by means of Kollman-United charges with the Tripos force-field engine. Profiling for SP and SP(1-4) by protein-chip analyses SP and SP(1-4) were profiled in stromal cell extracts by means of Ciphergen's ProteinChip Technology (Ciphergen Biosystems, Fremont, CA). Weak Cation Exchanger (WCX2) ProteinChip array (for profiling) and the preactivated chip surface, PS1 (for affinity studies) were used for the identification of SP(1-4). The basic method, which was provided by Ciphergen, was adjusted to optimize the analyses that follow. Stromal cells were washed with PBS and then subjected to repeated freeze-thaw. Cell-free lysates were prepared by centrifuging at 4°C for 30 minutes/15 000g. WCX2 was pretreated with 10 mM HCl, and 500 µL each sample was spotted on the array by means of a bioprocessor. The chips were incubated at room temperature for 30 minutes with vigorous shaking to ensure binding of sample to array. Subsequently, chips were washed with 5% Triton/PBS (2×) and with PBS (1×). A saturated solution of-cyano-4-hydroxy cinnamic acid (CHCA)
(Ciphergen Biosystems) was diluted at 1:50 in 50% acetonitrile and
0.5% trifluoroacetic acid. Diluted CHCA (0.5 µL) was added to the
spots of the arrays and the chips were dried at room temperature. After
this, chips were read on Ciphergen's surface-enhanced laser desorption
ionization-time of flight mass spectrometer (SELDI TOF-MS). Accurate
mass was determined by collecting 150 averaged laser shots. Stromal
extracts from 48-hour-stimulated cultures were selected for further
identification of immunoreactive SP(1-4). PS1 chips were pretreated
with 50% acetonitrile and then incubated for 45 minutes with 10 µL
anti-SP at 1:500 dilution in PBS. Control spots contained nonimmune
rabbit serum. The arrays were blocked for 25 minutes with 1M
ethanolamine and washed with PBS + 0.5% Triton X (2×) and a final PBS
wash step. Five hundred µL of cell extracts was incubated with the use of the bioprocessor. The chips were washed with PBS + Triton X,
PBS, rinsed with 5 mM HEPES, and dried. CHCA was applied and SP(1-4)
bound to the PS1 chip was analyzed as described above for the
profiling studies.
Northern analysis Northern analyses for steady-state NEP and NK-1 mRNA were performed as described.28 For NEP mRNA, stromal cells were stimulated with SCF for 16 hours in sera-free-MEM supplemented with
insulin-transferrin-selenium-A. At different times afterwards, total
RNA was extracted and 10 µg was used for Northern analyses. Membranes
were consecutively hybridized with [-32P]-d-adenosine
5'-triphosphate-labeled cDNA probes for NEP and 18S
ribosomal RNA (rRNA) as described.3 NK-1 mRNA was
determined with total RNA from stromal cultures that were sequentially
stimulated with SCF and SP(1-4), described above. The cDNA for NK-1 was
previously described.29 NK-2 cDNA was prepared by RT-PCR
with total RNA from BM stroma as template and the same primers as were
used for quantitative RT-PCR (described above). The NK-2 fragment,
which was equivalent to 274 base pairs, was subcloned into pNoTA/T7 (5 Prime  3 Prime, Boulder, CO). The cDNA for human NEP and
18S rRNA were purchased from American Type Culture Collection
(Manassas, VA).
Statistical analysis Data were analyzed by means of the Student t test to determine the significance (P value) between experimental values.
Interactions between SCF and SP(1-4) on the induction of TGF- .5 We therefore determined whether SP(1-4) could
induce 2 hematopoietic suppressors in BM stroma: TGF- and
TNF-.28,30,31 Table 1 shows that SP(1-4) significantly (P < .05) increases the
induction of both cytokines in BM stroma: unstimulated, less than 5 pg/mL TGF- and less than 104 pM TNF-;
stimulated, 90 ± 15 pg/mL TGF- and 40 ± 4 pM TNF-.
The next set of experiments determined whether SP(1-4) and SCF could
affect the role of each other on the induction of TGF- To determine if SCF could affect the induction of TNF- Effects of SP(1-4) on early and late hematopoietic progenitors SP(1-4) inhibited the proliferation of hematopoietic progenitors (CFU-GMs) in sera-containing cultures.8 To verify that the sera did not contribute to this inhibitory effect of SP(1-4), clonogenic assays were repeated for CFU-GMs (n = 7) in sera-free cultures. The percentage of inhibition by SP(1-4) on CFU-GMs in the sera-free cultures was significant (P < .05): 55% ± 4% for 1 nM SP(1-4) and 65% ± 6% for 10 nM SP(1-4). This indicates that the inhibitory effect of SP(1-4) on CFU-GMs was independent of the sera in the experimental assay. Since SP(1-4) induces the production of TGF- and TNF- (Table 1), which are
inhibitors of hematopoiesis,30 studies were performed to
determine if these cytokines could mediate the suppression of SP(1-4)
on CFU-GMs. Clonogenic assays were performed with 10 nM SP(1-4) and/or
various concentrations of anti-TGF-1 or anti-TNF-. The data,
shown in Figure 1A, represent the point
of maximal effect by the anti-TGF- (0.5 µg/mL). Anti-TGF-
showed significant (P < .05) change in reversing the
suppression of SP(1-4) on CFU-GMs, whereas anti-TNF- showed no
change (Figure 1A). Equivalent concentrations of nonimmune rabbit and
goat IgG showed no change in CFU-GMs cultured with 10 nM SP(1-4) (data
not shown). These results indicate that part of the suppressive effect
of SP(1-4) on CFU-GMs could be indirectly mediated by the production of
TGF-1 in BM stroma.
TGF- Effects of SP(1-4) on NK-1 and NK-2 expression Since NK-1 and NK-2 receptors mediated the hematopoietic effects of the tachykinins,5 this section describes the results of studies designed to further determine the interacting receptor for SP(1-4). Owing to the lack of specific antibodies to the human NK-1 and NK-2 receptors, their detection by immunofluorescence was performed with the preferred ligand that was conjugated to biotin: SP for NK-1, and NK-A for NK-2.12 Spantide, a pan-tachykinin antagonist that competes with the ligand for NK receptor binding, was used to determine the specificity of binding. Representative results of 5 different labeling experiments are shown in Figure 2. Consistent with published reports,5 the results showed that SP binds to stromal cells in which NK-1 receptor was induced (SCF stimulation, Figure 2B). NK-A did not bind to the SCF-stimulated stroma (Figure 2C). These results are internal controls that show that the induction of NK-1 by cytokines correlated with reduced expression of NK-2.2,5 Interestingly, stromal cells stimulated with SCF for 16 hours and then a second time with SP(1-4) showed a reduction in NK-1 expression (SP binding, Figure 2E) and relatively brighter staining for NK-2 (NK-A binding, Figure 2F). These results show that SP(1-4) reduced the expression of NK-1 in SCF-stimulated stroma with concomitant increase in the expression of NK-2.
Quantitative RT-PCR was used to determine if the phenotypic expression
of NK receptors (Figure 2) correlated to the respective mRNA levels.
The levels of NK-2 mRNA in unstimulated and SP(1-4)-stimulated stroma
were similar (Table 2). The level of NK-2
mRNA was, however, reduced following a first stimulation with SCF
(Table 2). This reduction in NK-2 mRNA was reversed following a second
stimulation by SP(1-4) (Table 2). NK-1 mRNA levels were quantitated in
BM stroma stimulated with SCF (first stimulation). After 16 hours, the
point at which NK-1 mRNA levels were increased (Table 2), stromal cells
were restimulated with SP(1-4) (second stimulation). Compared with
unstimulated stroma, the second stimulation showed a significant
(P < .05) increase in NK-1 mRNA levels (Table 2): 275 ± 14 molecule per microgram total RNA in stimulation with SCF
plus SP(1-4) versus less than 1 molecule per microgram total RNA in
unstimulated cells. The increase in NK-1 mRNA level by SCF plus SP(1-4)
was significantly reduced (P < .05) compared with stroma
stimulated with SCF alone: 275 ± 14 versus 1480 ± 34 molecules
per microgram total RNA (Table 2). Northern blots with total RNA from
selected experimental points supported the difference in NK-1 and NK-2
expression in unstimulated cultures (Figure
3, lane 1); SCF stimulation (Figure 3,
lane 2); and cultures stimulated consecutively with SCF and SP(1-4)
(Figure 3, lane 3). The results showed that SP(1-4) reduced the
expression of NK-1 induced by SCF, with a concomitant increase in NK-2
expression.
Induction of NEP and correlation with detectable SP and SP(1-4) Total RNA from unstimulated and stimulated (10 ng/mL SCF) BM stroma was analyzed for steady-state NEP mRNA levels by Northern analyses. The results (Figure 4A) indicate single bands at the predicted size of 1.8 kilobases. Strong bands were observed at 16 and 24 hours, with no detectable band at 48 hours. Figure 4B shows the fold change of normalized (18S rRNA) mRNA in stimulated over unstimulated stroma. The data indicate that the steady-state mRNA for NEP is optimally increased by SCF after 16 hours and remained detectable up to 24 hours.
To determine if the induction of NEP correlated with the degradation of
SP, timeline profiling studies were used to determine the change of SP
to SP(1-4) in stromal cell extracts stimulated with 10 ng/mL SCF.
Analyses for SP and SP(1-4) were performed at different times by means
of Ciphergen's ProteinChip arrays. ELISA determined the levels of
SP-IR in the supernatant and cell extracts of the stimulated stroma
(5 × 106 stroma). An affinity protein chip with
mobilized anti-SP determined the presence of SP(1-4) in extracts from
48-hour-stimulated stroma. The results showed fewer than 2 ng SP-IR in
unstimulated stroma (n = 5). At 24 hours, the total levels of SP-IR
(supernatants and cell extracts) in stimulated stroma were 185 ± 8
ng. SP levels were reduced to 12 ± 5 ng at 48 hours and to baseline
levels (less than 2 ng) at 72 hours (Table
3).
Since the small size of SP(1-4) limits its detection by methods similar
to SP, ProteinChip arrays were determined to be sensitive for the
detection of SP(1-4). Stromal extracts were analyzed at 24, 48, and 72 hours of SCF stimulation for peaks that are equivalent to SP and
SP(1-4). A peak corresponding to the size of SP (Figure 5A) was detected in 5 different samples,
which disappeared at 48 and 72 hours. During these same periods (48 and
72 hours), peaks corresponding to SP(1-4) were detected (Figure 4B). To
ascertain that the peak corresponding to 495 d in the 48-hour
extract was indeed SP(1-4), the analyses was further studied with an
affinity chip in which anti-SP was mobilized. Previous studies
indicated that this antibody binds to SP(1-4), which explained a single peak at 495 d (Figure 5C) with no detection at approximately
1395 d. Binding of nonimmune rabbit sera to similar chips showed
no detectable protein (data not shown). Figure 5D summarizes the dynamics of SP and detectable SP(1-4) in SCF-stimulated stroma. The
time spans of reduced SP correlated with up-regulation of NEP mRNA and
the detection of molecules corresponding to 495 d, the size of
SP(1-4).
Three-dimensional molecular model of SP/SP(1-4)-NK-1 interactions Functional studies with specific NK-1 and NK-2 receptor antagonists indicated that the NK-1 subtype mediated the suppressive effects on hematopoiesis by SP(1-4).8 Studies described in this report, however, showed low levels of NK-1 mRNA in BM stroma stimulated with SP(1-4) compared with SCF (Table 2). Relatively reduced fluorescence was also observed for NK-1 labeling in stroma costimulated with SCF and SP(1-4) (Figure 2). Owing to the small size of SP(1-4), the use of standard binding assays poses technical problems for the study of its binding kinetics. We therefore used other approaches to understand SP(1-4)-NK-1 interactions. The first set of studies screened a random dodecapeptide library for SP(1-4) interacting sequences, which resulted in 6 clones that shared significant homology to NK-1 protein residues: clones 7, 23, and 28 aligned with exon 1, and clones 12, 19, and 26 with exon 5 (Figure 6). Alignment of the residues from the clones retrieved from the peptide library with NK-1 is shown at the bottom of Figure 6.
To determine how the residues shown in Figure 6 interact with SP(1-4),
we used computer-assisted molecular modeling to generate a
3-dimensional structure of SP and NK-1. This model was also needed to
understand the paradigm of NK-1 mediating both positive and negative
hematopoietic effects.2,8 We are unaware of a crystal or
nuclear magnetic resonance structure for the extracellular and
intracellular loops of NK-1, which is a 7-TM, 407-residue, G-protein-coupled receptor.10 The spatial arrangement of
its secondary structure is shown in Figure
7,33 and the sequences shown
for the TM regions were used to model the intracellular and
extracellular loops, by means of a loop search (SYBYL). Given a certain
root mean square distance between 2 end points, the loop search
algorithm searched a database of loops and the highest scoring homolog
was selected between 24.3% and 46%.
A crystallized structure of SP was not available in any public
database. There are interesting aspects of the sequence of SP, which
has an amide group at the carboxyl terminal (Figure 8A). The position of the 2 phenyl rings
of the adjacent Phe7,8 is optimal when they are
trans to each other, and their spatial arrangement lowers
the probability for folding of the amino and carboxyl tails toward each
other. Pro2 and Pro4 create a kink in the head
structure and the electrostatic interaction between positive and
negative residues helps to stabilize the kink.
After the 3-dimensional structures of SP and NK-1 were generated, they
were docked by liphophilic, electrostatic, and H-bond interactions. The
3 types of interactions were performed to ensure that the docking was
proper. Docking was done with and without the solvent-accessible
surfaces, which were generated in SYBYL. MOLCAD (Tripos Associates) was
used to map the lipophilic, electrostatic, and H-bond potentials on the
surfaces. DOCK and Flexidock were used to prevent collision while
docking the solvent accessible surfaces of SP and NK-1. The results
(Figure 8B-C) are consistent with a previous report,33
which indicated that the contact sites between SP and NK-1 are mainly
on the extracellular loops and a few residues in the extracellular
matrix-TM interface. The results are also consistent with the
interacting sites retrieved from the peptide library, which indicated
that exons 1 and 5 of NK-1 are important for contact with SP(1-4)
(Figures 6, 8C). Exon 1 encodes TM 1-3, shown as
A diagram representing the findings of this study is shown in
Figure 9. We showed the potential for an
additional mechanism in which the PPT-I gene could be
involved in modulating hematopoiesis. In the BM, SP(1-4) could be
derived through enzymatic digestion by endogenous
endopeptidases.6,7 Since SP(4-11) exerts
stimulatory effects similar to the parent peptide,8 the
effects of the amino terminal fragment raises the question of whether
SP(1-4) is effective as a hematopoietic feedback while the carboxyl
fragment might be in the vicinity of the receptor. Although this
question has to be addressed in more detailed study, the experimental
evidence suggests that SP(5-11) might be quickly degraded to a
nonfunctional form by other endopeptidases that can use the longer
carboxyl fragment as a substrate.15,16 Unpublished and
ongoing studies using Ciphergen's ProteinChip technique showed no
evidence of SP(5-11). This might be caused by rapid
degradation of the carboxyl fragment by other endopeptidases, which
would result in SP(1-4) alone.
We reported that the negative hematopoietic effects of SP(1-4) were mediated through NK-1.8 This was a paradox since NK-1 receptor mediates positive hematopoietic responses by interacting with SP.2 Although it was expected that a negative hematopoietic response would be mediated through NK-2 receptor,5 screening of the peptide library did not show a potential interacting site for SP(1-4) within NK-2 (Figure 6). The lack of evidence for NK-2 as a mediator in the effect of SP(1-4) was puzzling since SP(1-4) did not induce the other NK subtype (NK-1) in stromal cells (Table 2). An understanding of the role of NK-1 in the SP(1-4) effect was provided by the appearance of SP(1-4) in SCF-stimulated cells (Figure 5) while NK-1 expression was still up-regulated (Figure 2, Table 2). Analyses of these results suggest that the induction of NK-1 by SCF was blunted after the change of SP to SP(1-4) and that, with time, membrane expression of NK-1 was reduced (Figure 2). The significance of this reduction is the provision of a feedback mechanism on the effects of SCF. Further studies that are beyond the scope of this report are needed to determine if this feedback mechanism is common to other stimulatory hematopoietic growth factors. This question would be pertinent for further understanding of the data shown in this report since several cytokines, associated with inflammatory responses and hematopoietic stimulation, can induce the production of SP.2 Figure 5C summarizes the dynamics between SP and SP(1-4). SP is first
detected in stromal cells stimulated with SCF. This is followed by the
appearance of NEP and then the degradation of SP to SP(1-4). Further
elucidation of these findings and quantitation of SP, SP fragments, and
various endopeptidases are the subject of ongoing studies in the
laboratory. This model does not explain the signaling pathway mediated
by SP(1-4) to induce TGF- The 3-dimensional structure of NK-1 and SP allows assumptions regarding
interactions between NK-1 and SP and provides a possible explanation
for the function of SP(1-4) in hematopoietic feedback. Both SP and its
fragment, SP(1-4), can occupy the same pocket within NK-1
(Figure 8B-C). A common pocket for both SP and SP(1-4) indicated that
in the presence of SP(1-4), the fit for SP within NK-1 would be
hindered. In the absence of SP-NK-1 interactions, the production of
stimulatory hematopoietic regulators such as cytokines could be
down-regulated.2 Since these cytokines induce the
expression of NK-1, the loss of SP-NK-1 could result in
down-regulation of NK-1 and a switch to alternative functions by
SP(1-4)-NK-1 (Figure 8C). A possibility for this is shown by the
production of TGF- Previous studies5 as well as this report show that a
feedback effect on hematopoiesis by the PPT-I gene is not a
one-way process, but reversible. The induction of TGF- When the conventions of head (residues 1-4) and tail (residues 5-11)
are used for SP, certain assumptions can be made about the structure of
SP. The model, shown in Figure 8A, indicates no helical structure of SP
but a curve in the head region. However, Cowsik et al40
reported a structure of SP that is partly helical in the tail region
when bound to lipids. Although we used a different 3-dimensional
structure of SP, the structure of Cowsik et al40 could
also dock to the NK-1 model, shown in Figures 8B and 8C. Using
molecular dynamics, the model shown in this study is the lowest in
energy ( SP binds to NK-1 within an interface between the cell membrane and the extracellular matrix.33,41 The confirmation of SP-NK-1, shown in the 3-dimensional model (Figure 8B-C) allows easy access for endopeptidases to cleave SP. The model accounts for a structural difference between SP(1-4) and SP(5-11) and also for electrostatic (Figure 8B) and lipophilic (not shown) complementarities between SP and NK-1. We hypothesize that the message sequence of SP, located within the carboxyl terminal,42 cannot activate NK-1 if the address sequence, which includes SP(1-4), occupies the pocket (Figure 8C). Without proper configuration of the message, the peptide would not be able to activate the G-protein through a classical mechanism. However the SP(1-4) address alone mediates signaling through the NK-1 receptor. This is demonstrated by the biological functions mediated by SP(1-4) (Table 1, Figure 1). The negative effect of SP(1-4) on hematopoiesis is not unusual for a small peptide since similar effects were reported for other small peptides.43,44 The biology of PPT-I peptides and their fragments is applicable not only to the BM but also to other lymphoid organs2 and would unravel the finely tuned interplay between NK-A, endopeptidases, SP, and other hematopoietic/inflammatory factors. The inhibitory hematopoietic effects of SP(1-4) suggest that this peptide can influence the self-renewal capabilities of the hematopoietic stem cells. Further research in this area is necessary since such information would be important for application in malignancies that are phenotypically late-stage progenitors with the adapted self-renewal capability, such as leukemia and lymphoma.4
Submitted April 16, 2001; accepted June 25, 2001.
Supported by National Institutes of Health grants HL-54973, HL-57675, and CA89868.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Pranela Rameshwar, PhD, UMDNJ-New Jersey Medical School, 185 South Orange Ave, MSB, Rm E-579, Newark, NJ 07103; e-mail: rameshwa{at}umdnj.edu.
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© 2001 by The American Society of Hematology.
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  S. J. Greco and P. Rameshwar Enhancing Effect of IL-1{alpha} on Neurogenesis from Adult Human Mesenchymal Stem Cells: Implication for Inflammatory Mediators in Regenerative Medicine J. Immunol., September 1, 2007; 179(5): 3342 - 3350. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
). The fate of SP(4-11) is yet to be
determined (?).
B as a central mediator in the induction of TGF-