The Role of Vasoactive Intestinal Peptide (VIP)
in Megakaryocyte Proliferation
Chaneun Nam & Adam J. Case & Bruce S. Hostager &
M. Sue O’Dorisio
Received: 26 February 2008 /Accepted: 2 June 2008 /Published online: 29 July 2008
# Humana Press 2008
Abstract Megakaryocytopoiesis is a multistage process
that involves differentiation of hematopoietic stem cells
through the myeloid lineage, ultimately producing mega-
karyocytes and platelets. Vasoactive intestinal peptide (VIP)
stimulates adenylate cyclase and induces differentiation in
multiple cell types; VIP is expressed in hematopoietic stem
cells and in megakaryocytes, but its function in these cells
has not yet been delineated. The present study was
designed to investigate whether the type 1 VIP receptor,
VPAC1, mediates VIP effects on megakaryocytopoiesis.
The human megakaryoblastic leukemia cell line (CMK)
was transfected with VPAC1 and the transgene expression
was confirmed by qualitative polymerase chain reaction and
immunohistochemistry. The rate of proliferation and the
patterns of differentiation were then compared for CMK
and CMK/VPAC1 through multiple growth cycles. Upre-
gulation of VPAC1 expression resulted in a decreased
proliferation rate (p=0.0003) and enhanced differentiation
with CMK/VPAC1 cells having twice the cell surface area
of control CMK cells (p=0.001), thus increasing potential
for proplatelet formation. These results suggest that VIP acts
in an autocrine fashion via VPAC1 to inhibit megakaryocyte
proliferation and induce proplatelet formation.
Vasoactive intestinal peptide (VIP) is a 28-amino acid
peptide that is widely distributed in mammalian tissues and
traditionally defined as a neuropeptide (Said 2007). VIP
and pituitary adenylate cyclase-acting polypeptide
(PACAP) transmit signals via three G-protein-coupled
receptors, VIP receptor 1 (VPAC1), VIP receptor 2
(VPAC2), and PACAP receptor (PAC1; Harmar et al.
1998). Widespread distribution of VIP and PACAP is
correlated with their involvement in multiple biological
processes (Goetzl 2006; Jonsson et al. 2007; Kimata et al.
1996; Petkov et al. 2003; Regard et al. 2007; Segerson et al.
1989; Vosko et al. 2007; Wang et al. 2007), including
neurotransmission in both central and peripheral nervous
systems, circadian rhythms, modulation of both innate and
adaptive immunity, regulation of water and electrolyte
balance, glucose homeostasis, and pituitary hormone
release. VIP binds with high affinity to both VPAC1 and
VPAC2 and activates adenylate cyclase via both receptors,
but cyclic adenosine monophosphate (cAMP) generation is
markedly more efficient via VPAC1 (Summers et al. 2003).
PACAP has highest affinity for PAC1, but can induce cAMP
formation via each of the three receptor subtypes (Laburthe
et al. 2002). When binding to VPAC1, VIP functions as a
neuronal growth factor, regulates intestinal secretion, sup-
ports neuronal differentiation, regulates platelet function
(Scurfield and Radley 1981), and inhibits proliferation of
colony-forming cells (van Geet 2004). Megakaryocytes are
highly specialized precursor cells that differentiate into
platelets via intermediate cytoplasmic extensions known as
proplatelets. Nitric oxide triggers platelet release in vivo
and also induces release of platelet-like bodies from the
megakaryocyte cell line Meg-01 in vitro (Battinelli et al.
2007; Topp et al. 1990). Megakaryocyte maturation is
J Mol Neurosci (2009) 37:160–167
C. Nam:A. J. Case:B. S. Hostager:M. S. O’Dorisio (*)
Department of Pediatrics, Division of Hematology/Oncology,
Carver College of Medicine, University of Iowa,
Iowa City, IA 52242, USA
regulated by thrombopoietin (TPO) produced in the liver
and marrow stroma (Choi et al. 1995; Italiano et al. 2007;
Kaushansky 2008; Leven 2004; Tablin et al. 1990; Vitrat
et al. 1998). Megakaryocytes cultured with TPO in vitro
appear morphologically very similar to bone marrow
megakaryocytes, are able to generate proplatelets, and
when injected into mice, are able to migrate from peripheral
circulation into bone marrow sinusoids (Lichtman et al.
1978; Tavassoli and Aoki 1989).
We and other investigators have previously documented
expression of both VIP and VPAC1 in megakaryocytes and
platelets (Ercal et al. 1988; Park et al. 1996); however, the
role of VIP and VPAC1 in megakaryocyte differentiation
and platelet production has not yet been elucidated. PACAP
is a known inhibitor of platelet activation, and recent
evidence suggests this effect is mediated via VPAC1
(Freson et al. 2004, 2008). We have transfected the human
megakaryocyte cell line, CMK, with human VPAC1 and
now report that the overexpression of VPAC1 inhibits
CMK proliferation and induces their differentiation.
Materials and Methods
The human megakaryocyte cell line, CMK, derived from a
megakaryocytic leukemia (ATTC, Bethesda, MD, USA)
was cultured in Roswell Park Memorial Institute (RPMI)
1640 medium, (GIBCO BRL, Grand Island, NY, USA)
supplemented with 10% heat-inactivated fetal bovine serum
(GIBCO BRL) and penicillin/streptomycin in fully humid-
ified atmosphere containing 5% CO2 at 37°C. Cultures
were stimulated by addition of 50 ng/ml TPO (R&D,
Minneapolis, MN, USA) and further cultured for 72 h.
Human peripheral blood (3 ml) was collected in ethyl-
enediamine tetraacetic acid tubes from healthy adult donors
after appropriate informed consent under a protocol
approved by the University of Iowa IRB. Anticoagulated
blood was centrifuged 20 min at 200×g to pellet eryth-
rocytes and leukocytes; platelet-rich plasma was aspirated,
and platelet count was determined by Coulter counting.
Flow Cytometry Analysis of Surface Marker Expression
Fluorescence-activated cell sorting (FACS) analysis was
performed using monoclonal antibodies against human
CD61 labeled with either fluorescein isothiocyanate (CD61-
FITC) or phycoerythrin (CD61-PE) and human CD62p
labeled with phycoerythrin (CD62p-PE) (PharMingen, San
Diego, CA, USA). Isotype controls (human IgG-FITC, IgG-
PE) were run in parallel. Cell debris was eliminated from the
analysis by forward and side scatter gating. The samples were
acquired on FACSCaliber flow cytometer (Becton Dickinson,
San Jose, CA, USA) and analyzed with CellQuest software
FACS analysis of ploidy was performed using propidium
iodide staining. CMK or CMK/VPAC1 cells (5×105cells)
suspended in RPMI 1640 were pelleted at 700×g for 5 min
and washed twice in 2 ml phosphate-buffered saline (PBS)
without Ca or Mg. Cells were then resuspended in the PBS
solution, 500 μl of 70% ethanol was added, and cells were
incubated 30 min at 37°C for fixation. The fixation step
was repeated. In the final step, cells were resuspended in
2 ml PBS plus 0.050 ml of 100 μg/ml RNase and 0.2 ml
propidium iodide (50 μg/ml). Cells were kept in dark until
FACS analysis on a BD LSR 610/20 BP (Becton-
Dickinson, San Jose, CA, USA).
Preparation of Vectors and Transfection
A pcDNA3 plasmid containing human VPAC1 comple-
mentary deoxyribonucleic acid (cDNA) was prepared in our
laboratory (Park et al. 1996); pcDNA3 alone was used as
control. Transfection efficiency was monitored using
pEGFP. CMK cells (approximately 1×105) were suspended
in 10 ml RPMI 1640, centrifuged at 700×g for 10 min at
room temperature (RT), and resuspended in 400 μl
transfection medium (RPMI 1640 supplemented with
5 mM glutathione) and 10 μg plasmid DNA. These
mixtures were subjected to electroporation at 225 V for
30 ms in 4-mm electroporation cuvettes (Genepulser Xcell,
Bio-Rad, Hercules, CA, USA) followed by cooling on ice
for 10 min. The cells were transferred to a T-25 flask
containing 10 ml RPMI 1640/15% fetal bovine serum
(FBS) and incubated for 16 h. Viable cell counts were
determined by trypan blue dye exclusion, and aliquots of
1,000, 2,000, 5,000, and 10,000 cells were seeded
separately in flat-bottom 48-well plates and incubated
overnight at 37°C in 500 μl RPMI 1640/10% FBS. The
next day, 500 μl complete medium containing 800 μg/ml
G418 was added to each well. After 3 days, half the
medium in each well was removed, being careful not to
resuspend cells. Fresh medium containing 400 μg/ml G418
was added to maintain 1 ml volume in each well. Half the
medium in each well was replaced with fresh G418-
supplemented medium every 7 days during the 23- and
27-day culture period. Transfection efficiency was moni-
tored by FACS analysis, using pEGFP as control. Green
fluorescent protein (GFP) expression in the parent CMK
cell line was insignificant and averaged 36% 18 h after
transfection with pEGFP in these control transfections.
J Mol Neurosci (2009) 37:160–167 161161
PCR Analysisof VIP Expression in CMK and CMK/VPAC1
Ribonucleic acid (RNA) was harvested from CMK and
CMK/VPAC1, reverse transcribed using random hexamers
as primers, and amplified by polymerase chain reaction
(PCR), 40 cycles (94°C for 30 s, primer annealing at 60°C
for 30 s, extension at 72°C for 30 s) using Temp-Tronic
thermal cycler (Thermolyne, Dubuque, IA, USA) with
primers designed on Primer Express software:
Real-time PCR Analyses of VPAC1 Expression
CMK, CMK/pEGFP, CMK/pcDNA3, and CMK/VPAC1
were cultured through five passages and harvested, and
total RNAwas extracted using TRIzol (Invitrogen, Carlsbad,
CA, USA). RNA was purified using Rneasy Mini Kit
(QIAGEN, Valencia, CA, USA), and 2 μg RNA was
converted to cDNA using the Superscript (Life Technologies,
Grand Island, NY, USA) preamplification system for first-
strand cDNA synthesis as previously described (Lara-
Marquez et al. 2001). Real-time PCR was performed using
the 5′–3′ nuclease activity of Taq DNA polymerase. VPAC1
primers and probe were designed in our laboratories (Lara-
Marquez et al. 2001) using published cDNA sequences and
synthesized by Integrated DNATechnology (IDX, Coralville,
IA, USA). Ribosomal RNA primers and probe were obtained
from previously published sequences (Taq Man Ribosomal
City, CA, USA). The probe for VPAC1 was labeled with 6-
carboxyfluorescein, and the ribosomal RNA (rRNA) probe
was labeled with VIC in order to analyze target gene and
Figure 1 Effect of thrombopoietin on CD61 expression in CMK.
CMK cells cultured with or without TPO for 72 h were harvested,
fixed, and incubated with anti-hCD61 as described in “Materials and
Methods.” a Resting CMK cells have no significant GPIIIa (CD61)
expression; b TPO-treated CMK have high GPIIIa expression (>93%
of cells CD61+); c Human platelets highly express GPIIIa, (>99%
Relative to CMK
Relative Expression of VPAC1
Relative Expression of VPAC1
Figure 2 VPAC1 expression in
CMK by real-time PCR. Cells
(2×106) were harvested, and
RNA was purified as described
in “Materials and Methods”;
2 μg RNA was reverse tran-
scribed using random hexamers;
2.5 μl cDNA was added to real-
time PCR reaction. a VPAC1
expression at day 23 after trans-
fection and b VPAC1 expression
at day 27 after transfection. Left
to right: SKNSH/VPAC1 con-
trol cell line, CMK parent cell
line, CMK/pcDNA3, CMK/
VPAC1, CMK/pEGFP. Values
shown are mean of two experi-
ments, each performed in tripli-
cate. Results of duplicate
experiments varied by less than
162J Mol Neurosci (2009) 37:160–167
rRNA in the same well. The rRNA was used to ensure the
quality of RNA preparation and to control any loading
variation of the initial cDNA amount. Reactions were
performed in a MicroAmp Optical 96-well reaction plate
(PE Applied Biosystems) using 2.5 μl cDNA, 12.5 μl 2×
Master Mix (8% glycerol, 1× TaqMan buffer A, 200 μM
deoxyadenosine triphosphate, 200 μM deoxycytidine triphos-
phate, 200 μM deoxyguanosine triphosphate, 400 μM
deoxyuridine triphosphate, 0.05 U/μl AmpErase uracil N-
glycosylase, 5 mM MgCl2) plus (0.01 U/μl) Gold
AmpliTaq DNA polymerase (PE Applied Biosystems),
forward/reverse primers (900 nM), and labeled probe
(200 nM) for the target gene. The reaction mixture also
contained forward/reverse primers (50 nM) and the labeled
probe (50 nM) for the internal control (rRNA) in a final
volume of 25 μl. The PCR reaction was performed using
the following amplification scheme: one cycle of 2 min at
50°C (AmpErase UNG activation), one cycle of 10 min at
95°C (activation of Gold AmpliTaq and inactivation of
AmpErase UNG), followed by 40 cycles of denaturation
15 s at 95°C and annealing/extension step of 1 min at
60°C. All reactions were carried out using 7700 Sequence
Detector thermocycler (PE Applied Biosystems) linked to
a Macintosh computer using Sequence Detector Software
(PE Applied Biosystems).
Effect of VIP Overexpression on CMK Proliferation
Megakaryocyte colonies developed around the edge of flat-
bottom 48-well plates on days 19–21 after transfection.
Proliferation of CMK, CMK/pcDNA3, and CMK/VPAC1
was monitored by colony counts under light microscopy
(Olympus, Melville, NY, USA) on days 23 and 27. CMK
and CMK/VPAC1 were harvested on day 27, and 5×104
cells were seeded into T-25 flasks at 37°C for 72 h in fully
humidified atmosphere containing 5% CO2in air. Cultured
Seeded Cell Number
# of Confluent Wells
Seeded Cell Number
# of Confluent Wells
Figure 3 Effect of VPAC1 overexpression on proliferation. CMK and
CMK/VPAC1 cultures were seeded at indicated density in 48-well
plates. Confluent wells monitored by light microscopy. a Day 23
cultures of CMK and CMK/VPAC1; b day 27 CMK and CMK/
VPAC1. Values shown are mean±SD of three experiments
Cell number (x100000)
Figure 4 Cell growth assay. CMK and CMK/VPAC1 were seeded at
5×104cells per well and cultured for 72 h as described in “Materials
and Methods” at which time cells were harvested and enumerated by
Coulter counter. Values shown are results from five independent
experiments, each performed in triplicate
J Mol Neurosci (2009) 37:160–167163163
cells were counted by Coulter counter (Beckman, Fullerton,
Cultured CMK and CMK/VPAC1 were deposited on glass
slides in a cytocentrifuge, fixed in 2% paraformaldehyde
in PBS, pH 7.2, washed in PBS for 5 min, and incubated
in block solution (10% goat serum, 3% bovine serum
albumin, 0.1% saponin) in PBS for 1 h at RT. Anti-VPAC1
antibody (Exalpha Biological, Watertown, MA, USA) was
added at a dilution of 1:100 in PBS for 2 h at 4°C; slides
were washed in PBS, incubated with anti-IgG (GAM
Alexa 488) at a dilution of 1:200 for 30 min at RT, washed
in PBS, incubated with propidium iodide solution for
10 min at RT, and washed with PBS for 5 min. Cell
morphology, VPAC1 expression, and cell size were
examined in a 1,024 confocal microscope (Bio-Rad,
Hercules, CA, USA).
All values are presented as mean+SD. Statistical signifi-
cance was determined using the Student’s t test and analysis
of variance (p<0.05).
Characterization of CMK
TPO regulates megakaryocyte proliferation by increas-
ing endomitosis and inhibiting apoptosis (Schmitz et al.
1994; Zauli et al. 1997). CD61 (GPIIIa) is widely used as
a specific biomarker for megakaryocytes (Stamler and
Loscalzo 1992). Flow cytometry analysis was performed
using human anti-CD61/FITC to monitor TPO stimula-
tion of megakaryocyte differentiation (Fig. 1). CD61
expression was detected in less than 5% of resting CMK
cells (Fig. 1a). After stimulation with TPO for 72 h, 93%
of the cells expressed CD61 (Fig. 1b). By comparison,
99% of freshly isolated human platelets expressed CD61
Upregulation of VPAC1 Expression in CMK
The expression level of VPAC1 was quantified in parent
and transfected CMK cells by real-time PCR using human
neuroblastoma cell line, SKNSH/VPAC1 (Balster et al.
2002) as positive control (Fig. 2). CMK/VPAC1 demon-
strated a 560-fold increase in VPAC1 expression at day 23
(Fig. 2a) and a 1,400-fold increase at day 27 (Fig. 2b)
compared to the parental cell line.
Effects of VPAC1 Upregulation on CMK Proliferation,
Differentiation, Ploidy, and Proplatelet Formation
The effects of VPAC1 upregulation on megakaryocyte
proliferation, morphology, and differentiation were ana-
lyzed. CMK, CMK/VPAC1, and CMK/pcDNA3 cells were
transfected by electroporation and cultured overnight in
T-25 flasks. At 18 h after transfection, viable cells were
Figure 5 Morphology and VPAC1 Expression in CMK and CMK/
VPAC1. Cells were harvested, fixed, and stained with anti-hVPAC11
antibody (green) with neutral fast red counterstain. Cell size and
VPAC1 expression were analyzed by confocal microscopy. a CMK
average cell diameter of 15.7 μm and (b) CMK/VPAC1 average cell
diameter of 19.7 μm
164J Mol Neurosci (2009) 37:160–167
harvested, enumerated by Coulter counting, and replated in
flat-bottom 48-well plates at densities of 1, 2, 5, and 10×
103viable cells per well. In each experiment, 48 wells were
seeded at each cell density. Megakaryocyte colonies were
first detected at around 20 days; the number of confluent
wells was determined at days 23 and 27. CMK/pEGFP
demonstrated a linear cell dose growth curve, with a mean
of 8, 11, 29, and 39 confluent wells at day 23; CMK/
VPAC1 grew more slowly with a mean of 0, 1, 8, and 13
confluent wells (Fig. 3a; p<0.001). Analysis at day 27
demonstrated similar inhibition of growth rate in CMK/
VPAC1 compared to CMK/pEGFP; the latter cultures
proliferated at a rate 12, 13, 37, and 44 confluent wells
when seeded with 1, 2, 5, or 10×103cells per well,
respectively. In contrast, CMK/VPAC1 cells seeded at the
same density produced 1, 2, 10, and 21 confluent wells
under the same growth conditions (Fig. 3b; p<0.0001).
These results indicate that increased expression of VPAC1
inhibits megakaryocyte proliferation. Growth rate of CMK/
pcDNA3 was not significantly different from CMK/pEGFP.
The growth rate of CMK/VPAC1 was also analyzed by
quantification of total cells using Coulter counter after
growth of stable transfectants through five cell passages
(Fig. 4). CMK proliferation rate was three- to fourfold
greater than CMK/VPAC1 (p=0.0003).
Morphology of CMK and CMK/VPAC1 as well as
VPAC1 expression was examined by confocal microscopy
as shown in Fig. 5. VPAC1 was expressed in the parent
CMK cell line as we had previously shown by reverse
transcriptase PCR and Western blot (Ercal et al. 1988; Park
et al. 1996). The average diameter of CMK cells was 15.2±
3.1 μm (Fig. 5a). Overexpression of VPAC1 resulted in a
significant increase in the size of megakaryocytes (19.7±
6.5 μm; p=0.010) and a concomitant increase in the level
of expression of VPAC1 (Fig. 5b). CMK/VPAC1 cells
demonstrated an average doubling in cell volume compared
Overexpression of VPAC1 also was associated with
increased ploidy of megakaryocytes as shown in Table 1.
Incubation with CMK with TPO for 72 h resulted in a
decrease in 2N cells and a concomitant increase in cells
with 4N and 8N. TPO induced a further increase in the
percent of CMK/VPAC1 cells with 8N ploidy (p<0.05)
compared to CMK cells with or without TPO.
Source of VIP
CMK/VPAC1 cells demonstrated significant changes in
proliferation and differentiation compared to the parent cell
line, without addition of exogenous VIP to cultures, raising
the question of whether or not megakaryocytes synthesize
VIP. Therefore, VIP gene expression was analyzed by PCR
as shown in Fig. 6. VIP messenger RNAwas not detectable
in either CMK or in CMK/pEGFP; however, VIP expres-
sion was highly upregulated in CMK/VPAC1 cells, sug-
gesting the presence of an autocrine loop for VIP and
VPAC1 in this cell line.
We have previously observed VPAC1 expression and
characterized VPAC1 receptor pharmacology on human
megakaryocytes and platelets (Ercal et al. 1988; Park et al.
Table 1 Effect of TPO and VPAC1 overexpression on megakaryocyte ploidy
Treatment Percent of cells with indicated ploidy
*p<0.05, **p<0.01CMKvsCMK+TPO;***p<0.05, ****p<0.001 CMK vs CMK/VPAC1+TPO;ap<0.05 CMK+TPO vs CMK/VPAC1+TPO
72 bp (VIP)
1 2 3 4 5 6
Figure 6 Effect of VPAC1 upregulation on VIP expression in
CMK cells. RT-PCR was performed as described in “Materials
and Methods,” and products were electrophoresed in 1.8% agarose
gel; lane 1, MW marker (100-bp ladder); lane 2, water control; lane
3, CMK; lane 4, CMK/pcDNA3; lane 5, CMK/VPAC1; lane 6,
J Mol Neurosci (2009) 37:160–167 165 165
1996). VPAC1 expression on human megakaryocytes and
platelets has recently been confirmed on both murine and
human platelets (Freson et al. 2008). We now demonstrate
that overexpression of VPAC1 in a human megakaryocytic
cell line results in upregulation of endogenous VIP
expression, similar to our previous observations in neuro-
blastoma cells (Balster et al. 2002). These data suggest that
VIP may play a critical role in CMK proliferation and
differentiation. Taken together, our data now strongly
suggest that VIP decreases megakaryocyte proliferation
and also induces differentiation and that effects of VIP are
mediated via VPAC1. Our previous data exclude VPAC2 as
the mediator of these effects (Park et al. 1996). While
Freson and coworkers attribute effects of PACAP to
VPAC1 rather than to PAC1, neither these investigators
nor our investigative team has directly quantified PAC1
expression on human megakaryocytes and platelets; thus,
PAC1-mediated effects have not been ruled out.
In conclusion, our study demonstrates expression of
VPAC1 in CMK cells, confirming our previous observations
in human bone marrow and cord blood megakaryocytes
(Park et al. 1996). More importantly, these results demon-
strate that increased expression of VIP and VPAC1 results
in inhibition of megakaryocyte proliferation while inducing
maturation, resulting in higher ploidy and increased volume
of individual megakaryocytes, thus increasing the potential
for proplatelet formation. Taken together, these results
support our conclusion that VIP plays an important role in
regulating megakaryocyte proliferation and proplatelet
formation and that these effects are mediated via VPAC1.
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