Báo cáo khoa học: Cytokine properties of prokineticins Justin Monnier and Michel Samson pptx

REVIEW ARTICLE
Cytokine properties of prokineticins
Justin Monnier and Michel Samson
INSERM U620, Universite
´
de Rennes 1, IFR 140, Rennes Cedex, France
Introduction
Prokineticin 1 (PROK1) and prokineticin 2 (PROK2),
otherwise known as endocrine gland vascular endo-
thelial factor (EG-VEGF) and Bombina variegata 8
(Bv8), respectively, are a novel family of peptides that
are highly conserved across species. Indeed, prokineti-
cin-like peptides are present in invertebrates (crayfish,
shrimp), vertebrates (black mamba snake, frogs, trout,
fugu) and mammals (rodents, bull, humans) [1,2]. One
of the first biological activities reported was the ability
of the amphibian orthologue of PROK2 (Bv8) to elicit
hyperalgesia in rats and to induce gastrointestinal
motility of guinea-pig ileum [3]. Subsequently, the
human recombinant form of prokineticins was shown
to have similar gastrointestinal motility activities in
guinea-pigs [3,4]. Both studies confirm that prokinetic-
ins are structurally conserved across species and show
highly conserved bioactivities. Not long after, it was
demonstrated that PROK1 and PROK2 also had
potent angiogenic properties independent of vascular
endothelial growth factor [5]. Since then, numerous
other biological activities have been associated with
prokineticins, such as angiogenesis, neurogenesis,
ingestive behaviours and hormone release, gastrointes-
tinal motility, circadian rhythms, pain sensation, and

blood cell function and development [2,6–11]. Further-
more, the most elegant demonstration of the implica-
tion of prokineticins in a human disease has been
brought by the identification that different point muta-
tions in the genes encoding PROK2 or the Gprotein-
coupled prokineticin receptor-2 (PROKR2) can lead to
Kallmann syndrome [6,12,13].
This review aimed to retrace all the cytokine proper-
ties of prokineticins. This novel family of peptides
share many common features with the chemokine
superfamily, such as their small size (8 kDa), receptors
[G-protein coupled receptors (GPCRs)], signaling
Keywords
chemokine; chemokine receptor; cytokine;
dendritic cells; GPCR; innate immunity;
macrophages; myeloid cell; prokineticin;
prokineticin receptor
Correspondence
M. Samson, INSERM U620, Universite
´
de
Rennes 1, 2 avenue du Prof. Le
´
on Bernard,
35043 Rennes cedex, France
Fax: (+33) 02 23 23 47 94
Tel: (+33) 02 23 23 48 06
E-mail:
(Received 26 March 2008, revised 20 May
2008, accepted 17 June 2008)

doi:10.1111/j.1742-4658.2008.06559.x
Prokineticins are a novel family of secreted peptides with diverse regulatory
roles, one of which is their capacity to modulate immunity in humans and
in other species. Prokineticins are small peptides of 8 kDa that mediate
their biological activities by signaling through two homologous G-protein-
coupled receptors (prokineticin receptor 1 and prokineticin receptor 2).
This family of peptides is characterized by a completely conserved N-termi-
nal hexapeptide crucial for their bioactivities and a unique structural motif
comprising five disulfide bonds. Prokineticins and their receptors are highly
expressed in bone marrow, in peripheral circulating leukocytes, in inflamed
tissues and in resident organ immune cells. Their structure, size, signaling
and biological activities are reminiscent of the chemokine superfamily.
In this review, emphasis is placed on the properties of prokineticins as
cytokines and their role in the immune system.
Abbreviations
Bv8, Bombina variegata 8; GM-CSF, granulocyte–macrophage colony-stimulating growth factor; GPCR, G-protein coupled receptor; IL,
interleukin; LPS, lipopolysaccharide; MIP, macrophage inflammatory protein; PROK1, prokineticin 1; PROK2, prokineticin 2; PROKR1,
prokineticin receptor 1; PROKR2, prokineticin receptor 2; TNF-a, tumor necrosis factor-a.
4014 FEBS Journal 275 (2008) 4014–4021 Journal compilation ª 2008 FEBS. No claim to original French government works
mechanisms, and chemotactic and immunomodulatory
activities. Therefore, in the present study we first
described the gene, protein structure, signaling and
biochemical properties of prokineticins, which showed
similarity to some cytokines, notably chemokines and
defensins. Second, we overviewed the cellular source of
both prokineticins and their receptors throughout
immune cells. Finally, their numerous activities as
cytokines were overviewed.
Comparison of gene and protein struc-
ture of prokineticins and prokineticin

receptors with chemokines
Ligands PROK1 and PROK2
The gene that encodes PROK1 (86 amino acids) is
located on chromosome 1p21. It is composed of three
exons, with no known alternative splicing product [14].
The PROK2 gene maps to chromosome 3p21.1 and it
is composed of four exons, which gives rise to two
mature proteins: PROK2 (81 amino acids) (exons 1, 2
and 4) and a splice variant with a 21-amino acid insert
called PROK2L (102 amino acids) (exons 1, 2, 3 and
4). PROK1 and PROK2 share approximately 44%
amino acid identity [9]. In contrast to PROK1 and
PROK2, which are located on different chromosomes,
the genes encoding chemokines are often on the same
chromosome and their loci are physically very close
(i.e. CXC chemokines are almost all located on chro-
mosome 4q21) [15]. Furthermore, the genetic loci of
both prokineticins are not physically near any cytokine
family genes and it thus appears unlikely that both
prokineticin and cytokine genes could be involved in
translocation phenomenon pathologies.
After signal peptide processing, the secreted peptide
contains distinctive structural motifs that are highly
conserved across species. One such motif is the N-termi-
nal AVIT sequence and another comprises 10 conserved
cysteine residues [1,9]. This N-terminal AVIT sequence
is essential for the correct binding of receptors [16].
However, it has not yet been identified if prokineticins
can undergo in vivo proteolytic cleavage by extracellular
proteases. As observed with chemokines, the N-terminal

domain is also essential for receptor binding because
cleavage of peptides from the N terminus by extra-
cellular proteases regulates chemokine bioavailability by
either increasing or decreasing the biological activity of
the chemokine [17]. Furthermore, prokineticins are
highly basic. Indeed, PROK1 binds with high affinity to
heparin–sepharose, and PROK2 and PROK2L are
predicted to be highly basic with a pI of 8.85 and a pI
of 10.68, respectively. Thus, prokineticin activity may
also be regulated through the binding of extracellular
components, such as sulphate proteoglycans [14]. Inter-
estingly, the members of the chemokine family are also
highly basic, and their bioactivity is tightly regulated
through interactions with heparin sulfates of the extra-
cellular matrix [18]. Chemokines contain four to six cys-
teine residues, and this is a marked structural difference
from prokineticins, which contain 10 cysteine residues.
The 10 cysteine residues form five disulphide bonds that
confer a compact structure on the molecule, with N-and
C-termini present on the surface. One side of the
roughly ellipsoid protein has a positive net charge,
whereas the opposite side is hydrophobic [2].
We performed a phylogenic study in order to com-
pare the degree of similarity among prokineticins,
chemokines and defensins, (which, similarly to proki-
neticins, also contain a high number of cysteine resi-
dues) [19–22]. Interestingly, the results of the pylogenic
study revealed a higher similarity of amino acid
sequence between defensins and prokineticins than
with chemokines (Fig. 1A).

Receptors PROKR1 and PROKR2
There are two prokineticin receptors, named prokineti-
cin receptor 1 (PROKR1) and prokineticin receptor 2
(PROKR2); they are two closely related seven-trans-
membrane GPCRs that belong to the family of the
neuropeptide Y receptor. PROKR1 is located on
chromosome 2p13.1 and PROKR2 is located on chro-
mosome 20p12.3. No genes encoding cytokine receptors
are located near prokineticin receptors. Interestingly,
similarly to their ligands, both receptors are on different
chromosomes, and this contrasts with chemokine recep-
tors that are often located on the same chromosome,
such as CCR1, CCR2, CCR3, CCR4, CCR5 that are
located very closely on chromosome 3p21.3-24 [23].
Furthermore, all chemokine receptors are also
G protein seven-transmembrane-coupled receptors,
which is another common element shared between
chemokines and prokineticins. However, they diverge
in sequence with PROKRs, as can be seen in the
phylogenetic tree (Fig. 1B), where the closest related
chemokine receptor to PROKRs is XCR1.
Expression and regulation of
prokineticins and prokineticin
receptors in immune cells
Ligand expression
Several studies have reported a differential expression
of PROK1 and PROK2 within immune cells. Indeed,
J. Monnier and M. Samson Cytokine properties of prokineticins
FEBS Journal 275 (2008) 4014–4021 Journal compilation ª 2008 FEBS. No claim to original French government works 4015
LeCoulter et al. showed, in 2004, that within human

peripheral leukocytes only PROK2 was detected, and
the highest expression was observed in bone marrow,
neutrophils, dendritic cells and monocytes [24]. In con-
trast, another study reported a significant expression
of PROK1 in CD14
+
cells, T cells and B cells; how-
ever, PROK2 expression was not evaluated [24]. We
measured the expression of PROK1 and PROK2 in
fresh human monocytes obtained from 10 different
healthy donors and our results consistently showed
very high expression of PROK2 but undetectable
expression of PROK1 (data not shown). Furthermore,
we recently showed that the resident macrophage
population of human liver (called Kupffer cells) was
the specific source of PROK2 in liver, whereas PROK1
was weakly expressed [25].
Prokineticins have been associated differentially with
inflammatory and immune tissues. Transcript analysis
showed higher expression of PROK1 in rheumatoid
arthritis synoviocytes and in Crohn’s disease compared
with normal tissue [24]. In situ localization in appendi-
citis and tonsillitis samples revealed high expression of
PROK2 in infiltrating neutrophils [26]. Interestingly,
PROK1 transcripts were detected in tumor-infiltrating
T lymphocytes in ovarian carcinoma [27].
In other species, immunohistochemical staining
revealed that PROK1 was expressed in macrophages
of bovine corpus luteum regression and follicular
atresia [27,28]. In BALB ⁄ c mice, peritoneal macro-

phages were shown to express only PROK2 [29]. How-
ever, in C57BL6 mice, PROK1 was shown to bind
monocyte ⁄macrophage cells in the spleen [24].
Recently, Shojaei et al. [30] demonstrated in BALB ⁄c
nude mice implanted with several tumor cell lines that
the bone marrow mononuclear cells subset enriched in
PROK2 were CD11b
+
GR1
+
cells, consisting mainly
of macrophage and neutrophil lineage cells. These
results might reflect intraspecies variations.
Taken together, from the literature and from our
own results it seems that PROK2 is preferentially
expressed by cells from the monocyte–granulocyte line-
age and PROK1 seems to be less specifically expressed
by immune cells.
Receptor expression
PROKR2 has been shown to be expressed more highly
than PROKR1, in particular in CD8 cells, monocytes
and neutrophils [26]. In contrast, Dorsch et al.
reported a low expression of PROKR2 in monocytes,
but a substantial expression of both receptors in B
cells [24]. Recently, we studied the expression of PRO-
KR1 and PROKR2 in fresh human monocytes from
10 different donors at the protein level using flow
cytometry and we observed that both receptors were
expressed on the monocyte surface (J. Monnier,
V. Quillien, C. Piquet-Pellorce, C. Leberre, L. Preisser,

H. Gascan & M. Samson, unpublished data). In addi-
tion, we showed that in liver hepatic cells both PRO-
KR1 and PROKR2 mRNA were only expressed at
high levels by Kupffer cells [25]. In mice peritoneal
A
B
CCR6
0.7
0.8
CXCL8
CXCL9
CXCL11
CXCL10
CXCL3
CXCL1
CXCL2
CCL4
CCL5
CCL18
CCL3
CCL13
CCL8
CCL7
CCL2
XCL2
XCL1
CX3CL1
PROK1
PROK2
PROK2L

DEFb1
DEFa4
DEFa3
DEFa1
DEFb4
DEFb2
CXCR6
CXCR4
CXCR5
CXCR3
CCR10
CXCR2
CXCR1
CCR9
CCR7
CCR4
CCR5
CCR2
CCR3
CCR1
CCR8
CX3CR1
CXCR7
XCR1
PKR2
PKR1
Fig. 1. Phylogenic study comparing prokineticins with chemokines
and defensins. Sequence alignment was performed using
CLUSTALW
[22]; phylogeny was performed using the approximate likelihood-

ratio test for branches,
PHYML [20,21], visualization of phylogenic
trees was performed using
TREEDYN [19]. (A) Dendrogram represent-
ing amino acid sequence similarity among prokineticins, chemo-
kines and defensins. (B) Dendrogram representing amino acid
sequence similarity among prokineticins receptors and chemokine
receptors.
Cytokine properties of prokineticins J. Monnier and M. Samson
4016 FEBS Journal 275 (2008) 4014–4021 Journal compilation ª 2008 FEBS. No claim to original French government works
macrophages a predominance of PROKR1 transcripts
was found, and macrophage cells extracted from PRO-
KR1 knockout mice were unable to respond to
PROK2 [29].
In humans, it has not yet been determined which
receptor is essential for the immunoactivities linked to
PROK1 or PROK2. Future directions for research
could come from the PROKR knockout mice that
have been recently generated. Indeed, while PRO-
KR1
) ⁄ )
mice have been obtained at the expected
Mendelian rate without critical abnormalities, > 50%
of PROKR2
) ⁄ )
mice died at an early neonatal stage
and the surviving mice presented abnormalities similar
to Kallmann syndrome [31]. Challenging the PRO-
KR1
) ⁄ )

and PROKR2
) ⁄ )
mice with various pathogens
could be a very informative model for studying the
role of prokineticin receptors in the immune system.
Regulation
Very little is known about the regulation of prokineticin
and prokineticin receptors within immune cells.
However, very recently PROK2 was shown to be posi-
tively regulated in CD11b
+
Gr1
+
myeloid cells (con-
sisting mainly of neutrophils and cells of the
macrophage lineage), specifically by granulocyte
colony-stimulating factor and not by any other cytokine
tested [interleukin (IL)-4, IL-10, IL-13, monocyte
chemotactic protein-1, macrophage inflammatory
protein (MIP)-1a, MIP-1b, MIP-2, interferon-c, kerati-
nocyte-derived chemokine, fibroblast growth factor,
vascular endothelial factor, PROK2, granulocyte–mac-
rophage colony-stimulating growth factor (GM-CSF),
granulocyte colony-stimulating factor, tumor necrosis
factor-a (TNF-a), stromal cell-derived factor 1a and
transforming growth factor-b] [30]. Furthermore, we
evaluated if the expression levels of PROK2 and
PROK1 could be regulated according to the differentia-
tion state of monocytic cells. To achieve this we com-
pared the expression of PROK1 and PROK2 in

monocytes and monocyte-derived macrophages, in
undifferentiated and differentiated THP1 cells, and in
undifferentiated and differentiated U937 cells. Our
results showed that PROK1 was undetectable in all
samples measured; however, PROK2 was highly
expressed in all cells in the undifferentiated state and
showed a large decrease in cells in the differentiated
state (data not shown).
For the regulation of PROKR1 and PROKR2 in
myeloid cells there is still much to be learned. However,
we recently showed, by flow cytometry, that receptors
PROKR1 and PROKR2 were present on the surface of
monocytes, but that their expression was almost absent
on the same monocytes derived into macrophages by
GM-CSF or into dendritic cells by GM-CSF + IL-4
(J. Monnier, V. Quillien, C. Piquet-Pellorce, C. Leberre,
L. Preisser, H. Gascan & M. Samson, unpublished
data). Altogether, these data suggest that the differenti-
ation status of monocytic cells may have an impact
on the regulation of expression of prokineticin and
prokineticin receptors.
Functions of prokineticins and
prokineticin receptors in the immune
system
Prokineticin receptor signaling in immune cells
The affinity of prokineticins for their receptors are in a
similar range, with PROK2 showing a moderately
higher affinity for both receptors: the K
d
(nm) values for

PROK1 and PROK2 binding to PROKR1 are
12.3 ± 4.2 and 1.4 ± 0.5, respectively, and the K
d
(nm)
values for PROK1 and PROK2 binding to PROKR2
are 1.8 ± 0.1 and 2.0 ± 0.7, respectively [9,32–34].
Based on the literature it seems that intracellular
calcium mobilization and Gq protein activation is one
of the major signaling mechanisms of prokineticin
receptor activation [33–37]. However, other studies
have shown that prokineticin receptors can also couple
to Gi and Gs [26,32,33]. Only a few reports have
studied the prokineticin signaling mechanisms in
immune cells. It has been shown that human mono-
cytes exposed to PROK2 induced extracellular signal-
regulated kinase phosphorylation that was abolished
by pertussis toxin, suggesting involvement of the Gi
protein signaling pathway [26]. Interestingly, in mouse
macrophages, it seems that pertussis toxin was unable
to block the actions of PROK2, but rather inhibition
of the Gq protein pathway blocked the secretion of
cytokines mediated by PROK2 [29]. Recently, we
tested the effect of inhibitors of Gi protein (pertussis
toxin) and calcium [using the intracellular calcium
chelator 1, 2-bis (2-aminophenoxy) ethane-N, N, N’,
N’-tetraacetic acid (BAPTA)] on human monocytes
for their ability to block PROK1-mediated CXCL8
secretion. Our results show that PROK1-mediated
CXCL8 monocyte production was sensitive to pertussis
toxin and BAPTA (J. Monnier, V. Quillien, C. Piquet-

Pellorce, C. Leberre, L. Preisser, H. Gascan &
M. Samson, unpublished data). Taken altogether, the
results suggest that multiple pathways are involved in
prokineticin signaling in monocytes, and that there
might be some species-to-species variation.
Furthermore, another molecular mechanism that
could influence signaling is GPCR dimerization.
J. Monnier and M. Samson Cytokine properties of prokineticins
FEBS Journal 275 (2008) 4014–4021 Journal compilation ª 2008 FEBS. No claim to original French government works 4017
Indeed, it is now well described for chemokine
receptors such as CCR2 to CCR5 or CXCR4 to
CCR2 that dimerization can modulate signaling by
negative or positive binding cooperativity [38]. Thus, it
would be very informative to determine if prokineticin
receptors can homodimerize or heterodimerize, and
how this would influence signaling.
Hematopoietic activity of prokineticins
Identification that a prokineticin-like peptide (Astaki-
ne) induced a strong hematopoietic response in vivo,as
well as in vitro growth and differentiation of hemato-
poietic cells in invertebrates (shrimp and in crayfish),
suggests a primitive role for prokineticins as hemato-
poietic cytokines (Table 1) [1]. In vertebrates, the sys-
temic expression of PROK1 or PROK2 by injection of
adenovirus into nude mice resulted in a potent
hematopoietic response. Indeed, the total leukocyte,
neutrophil and monocyte count was increased, and
the mouse spleen was enlarged as a result of the large
number of immune cells produced (Table 1) [26].
Migration

In addition to their hematopoietic properties, PROK1
and PROK2 were also shown to be potent chemoattrac-
tants for human monocytes (Table 1). Indeed, migra-
tion of monocytes to prokineticins was observed at
concentrations as low as 10
)8
m [26], and mouse macro-
phages migrated in response to even lower concentra-
tions of PROK2 (10
)12
m) [29]. This is another property
that prokineticins share with chemokines, which are
first and foremost characterized by their ability to
induce the migration of immune cells at very low con-
centrations.
Differentiation
PROK1 has been shown to induce the differentiation
of both human and mouse bone marrow cells into the
monocyte ⁄ macrophage lineage (Table 1). In vitro,
human and mouse hematopoietic stem cells treated
with either PROK1 or PROK2 showed an increase in
the number of granulocytic and monocytic colony-
forming units [26]. This was further observed in
another study where human and mouse CD34
+
cells
showed a decrease in expression of CD34 and increase
in expression of CD14 after treatment with PROK1.
As well as inducing monocyte survival for 7 days,
PROK1 differentiated monocytes into macrophage-like

cells, as observed by the morphological changes
induced in monocytes and the down-regulation of sur-
face expression of B7-1, CD14, CXCR4 and CCR5
[24].
Cytokine and chemokine induction
By observing the cytokine signature induced in mono-
cyte ⁄ macrophages by prokineticins, several studies
Table 1. Summary of the effects induced by prokineticins on blood cells.
PK1 PK2 Astakine
1. Action of PKs on blood cells
Hematopoietic response › Monocyte + neutrophil count
› CFU-G and CFU-M
(Lecouter et al. [26])
› Monocyte + neutrophil count
› CFU-G and CFU-M
(Lecouter et al. [26])
› Hematopoiesis in vivo and
in vitro (Soderhall et al. [1])
Chemotaxis Monocytes
(Lecouter et al. [26])
Monocytes (Lecouter et al. [26])
Macrophages (Martucci et al. [29])
Survival ⁄ differentiation › Monocyte survival
› Macrophage differentiation
(Dorsch et al. [24]; Lecouter et al. [26])
› Monocyte survival
(Lecouter et al. [26])
Morphological changes › B7-2, fl B7-1, CCR5, CXCR4, CD14
(Dorsch et al. [24])
2. Monocyte/macrophage cytokine production

Alone › CXCL8, CCL4, CXCL1, TNF-a, IL-1b
(Monnier et al. [25]; Kisliouk et al. [27])
With LPS › CCL18, CCL20
(Monnier et al. [25])
› IL-1, fl IL-10
(Martucci et al. [29])
With LPS and IFN-c › IL-12
(Martucci et al. [29)]
7 days with PK1, then
24 h with LPS
› TNF-a, IL-12, fl
IL-10
(Dorsch et al. [24])
Cytokine properties of prokineticins J. Monnier and M. Samson
4018 FEBS Journal 275 (2008) 4014–4021 Journal compilation ª 2008 FEBS. No claim to original French government works
have demonstrated that prokineticins function as pro-
inflammatory mediators (Table 1) [24,27,29]. Indeed,
monocytes treated for 7 days with PROK1 are primed
to release TNF-a and IL-12, and to decrease IL-10
after treatment with lipopolysaccharide (LPS) [24].
In mouse macrophages, PROK2, in conjunction with
LPS, induced IL-1 and decreased IL-10 production,
and co-stimulation of PROK2 with LPS plus inter-
feron-c induced IL-12 [29]. Interestingly, those two
studies only demonstrated an indirect response to
PROK1 or PROK2 in differentiated monocytes.
However, other reports using fresh monocytes were
able to show a direct response for cytokine produc-
tion by prokineticins. In bovine monocytes, incuba-
tion with either PROK1 or PROK2 for 48 h

increased the levels of integrin b2, elevated the num-
ber of adherent cells, and stimulated TNF-a mRNA
expression, implying that prokineticins participate in
the activation of bovine monocytes [27]. We observed,
in human monocytes treated with 1 lgÆmL
)1
of
PROK1 for 8 h just after plating, that IL-1b and
TNF-a transcripts were strongly induced (data not
shown). Furthermore, we observed that monocytes
treated with PROK1 for 24 h secreted the chemokines
CXCL1, CXCL8 and CCL4. In addition, costimula-
tion with LPS and PROK1 showed synergy for
CCL18 and CCL20 production. Finally, we observed
that CXCL1 and CXCL8 secretion after PROK1
induction is only observed in monocytes and not in
monocyte-derived macrophages or dendritic cells,
probably because of the decrease of receptors in
macrophages and dendritic cells described previously
(J. Monnier, V. Quillien, C. Piquet-Pellorce, C. Leb-
erre, L. Preisser, H. Gascan & M. Samson, unpublished
data). Taken together it seems that, in vitro, the
differentiation state of monocytes has an impact on
the ability of prokineticins alone to induce chemo-
kines and cytokines.
Conclusion/perspectives
In conclusion, this review attempted to present the
many similar traits between prokineticins and cyto-
kines. Indeed, prokineticins show greater similarity to
the chemokine family than to members of the inter-

leukin family. They share with chemokines many
similar aspects: they are small secreted peptides, they
are highly basic and bind heparane sulfates, they both
contain cysteine residues, their N terminus is essential
for proper signaling, they signal through GPCRs, they
show multiple receptors with ligand cross-reactivity
and they are potent chemoattractants. Furthermore,
prokineticins also induce survival, differentiation and
activation of the granulocytic and monocytic lineages,
and they can be considered as pleiotropic chemokine-
like cytokines. However, there is still much to be
understood on how prokineticins modulate the innate
and adaptive immune systems.
Acknowledgements
Justin Monnier was supported by a PhD fellowship
from the Region Bretagne. Michel Samson was
supported by the Institut National de la Sante
´
et de
la Recherche Me
´
dicale (INSERM).
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