Proteinase-activated receptor-2: two potential inflammatory mediators of the gastrointestinal tract in Atlantic salmon
Journal of Inflammation
Proteinase-activated receptor-2: two potential inflammatory mediators of the gastrointestinal tract in Atlantic salmon
Jim Thorsen 0
Einar Lilleeng 0
Elin Christine Valen 0
shild Krogdahl 0
0 Address: Aquaculture Protein Centre, Basic Science and Aquatic Medicine, Norwegian School of Veterinary Science , Oslo , Norway
Proteinase-activated receptor 2 (PAR-2), activated by trypsin and other serine proteinases, is a key initiator of inflammatory responses in the intestine of mammals. Atlantic salmon fed diets with standard qualities of soybean meal (SBM) show enteritis of the distal intestine as well as increased activity of trypsin in both luminal contents and wall tissue. Luminal trypsin activity may possibly be involved in immune related disorders of the intestine also in Atlantic salmon via activation of PAR 2. In the present study our aim was to investigate if PAR-2 play a role in SBM induced enteritis. We performed multiple alignments based on nucleic acid sequences of PAR-2 from various animals available from public databases, and designed primers for use in cloning of the Atlantic salmon PAR2 transcript. We further cloned and characterized the full length sequence of Atlantic salmon PAR2 and investigated the expression in both early and chronic stages of SBM induced enteropathy. Two full length versions of PAR-2 cDNA were identified and termed PAR-2a and PAR-2b. Expression of the two PAR-2 transcripts was detected in all 18 tissues examined, but most extensively in the intestine and gills. A significant up-regulation in the distal intestine was observed for the PAR-2a transcript after 1 day feeding diets containing SBM. After 3 weeks of feeding, PAR2a was down-regulated compared to the fish fed control diets. These findings may indicate that PAR-2a participates in inflammatory responses in both the early and later stages of the SBM enteropathy. In the chronic stages of the enteropathy, down-regulation of PAR-2a may indicate a possible desensitization of the PAR-2a receptor. Expression of PAR-2b was not altered in the first 7 days of SBM feeding, but a significant up regulation was observed after 3 weeks, suggesting a putative role in chronic stages of SBM induced enteritis. The expression differences of the two PAR-2 transcripts in the feed trials may indicate that they have different roles in the SBM induced enteritis.
By ingesting feed, the gastrointestinal tract (GI tract) is
presented to food components and microorganisms
carried with the feed exposing the organism to allergens and
pathogens that can cause disease and hence affect animal
welfare. The GI tract is one of a few major entry points for
microorganisms and pathogens, and hence, animals have
well developed physical and chemical barriers in
combination with an effective mucosal immune system . The
mucosal and chemical barriers can be breached by
microorganisms and pathogens, and once breached, circulating
innate immune cells will form a second important basis
for defence. The gut immune apparatus represents
therefore a major element in the defence of an animal, and is
considered the largest immunological organ in man . A
well functioning immune apparatus in the
gastrointestinal tract is therefore of utmost importance for the
function and wellbeing of all animals.
SBM induced enteritis
Standard qualities of soybean meal (SBM), the most
important and cheapest protein rich feedstuff on the
world market, can only be used at limited levels in
salmonid diets because it challenges the gut immune systems
and fish health. Salmon, when fed diets with standard
qualities of SBM develop an inflammation like condition
(enteritis) in the distal intestine characterized by
inflammatory infiltrate in the intestinal mucosa, atrophy of
primary and secondary mucosal folds and decrease of
epithelial vacuolization [3-5]. A previous study
investigating the development of the enteritis using a 33% SBM feed
showed minor changes in intestinal histology in some of
the samples after two days of feeding . After 7 days of
feeding SBM the fish displayed all the signs present in the
fully developed condition including increased width and
marked reduction in height of simple mucosal folds as
well as increased cell infiltration of the lamina propria .
Further, upon feeding SBM for 1421 days the enteritis
was fully developed in all the fish examined . The SBM
induced enteritis may also be a key factor in the decrease
in growth as well as nutrient digestibility and absorption
observed at higher inclusion levels [6-9] and has been
suggested to have a negative effect on disease resistance .
Salmonids fed diets with standard qualities of SBM show
elevated activity of trypsin-like enzymes [11,12]
suggesting that trypsin might be involved in the development of
SBM induced enteritis. Further, studies with partly
fractionated soybean extracts have shown that the feed
substances participating in the enteropathy in salmon are
soluble in alcohol [13,14]. Later studies has suggested that
saponins, present in the soy alcohol extract, is a
compound that may cause some, but not all, of the intestinal
alterations seen in Atlantic salmon fed soybean meal .
Saponins are known to increase permeability of intestinal
tissue and thereby increase exposure to immune
stimulants . But still, more than 18 years after the SBM
induced enteritis was reported, the causative molecular
agents present in SBM responsible for the pathogenesis
have not been identified.
PAR-2 receptors and inflammation
Several studies in mammalian species have shown that
activation of cell surface receptors termed
proteinase-activated receptors (PARs) are key activators of inflammatory
responses in a wide range of tissues including the
gastrointestinal tract (GI) and airways [17-23]. These cell surface
receptors are G-protein coupled and belongs to a family of
seven transmembrane receptors that can be activated by
serine proteinases, such as trypsin. So far, four
proteinaseactivated receptors have been cloned and studied in man
[24-27], but to our knowledge no proteinase-activated
receptors have been studied in teleost fish. Activation of
PAR-2 (proteinase-activated receptor 2) in mammals is
achieved by proteolytic cleavage of an extracellular
peptide sequence hereby exposing an N-terminal tethered
ligand domain that binds to and activates the receptor
[28,29]. Upon activation of the PAR-2 receptor in
mammals, the receptor is internalized and targeted to
lysosomes for degradation [30,31]. To resensitize the cells
from the irreversible receptor cleavage new receptors are
mobilized by large Golgi stores as well as synthesis of new
receptors . The PAR-2 receptor has been detected in
various diverse tissues such as brain, eye, airway, heart, GI
tract, pancreas, kidney, liver, prostate, skin and in cells
such as epithelial cells, endothelial cells, as well as in
immune cells such as T-cells, neutrophils, mast cells and
eosinophils [32,33]. Some of the PAR-2 mediated effects
on leukocytes involve leukocyte rolling and adhesion 
as well as leukocyte migration in vivo . Activation of
PAR-2 by serine proteinases have been shown, in vitro, to
stimulate bone marrow progenitor cells to develop into
dendritic cells . Hence, serine proteinases might
participate in adaptive immune responses in vivo. Activation
of PAR-2 by trypsin in luminal colonocytes in mice affect
the permeability and hence could play an important role
in pathogenesis of different mammalian gastrointestinal
disorders . In humans, elevated colonic luminal
serine proteinase activity of irritable bowels syndrome (IBS)
patients has been suggested to involve PAR-2 activation
and mediate epithelial barrier dysfunction and
pathogenesis of IBS .
Mechanisms of immune responses in fish, whether
stimulated by dietary components or pathogens are not well
described. A better understanding of these responses is
expected to lead the way to develop healthier and more
productive diets and found the basis for improvements in
disease prevention and treatments.
The reported importance of PAR-2 activation in
mammalian inflammatory diseases motivated the cloning,
sequencing and expression analysis of the PAR-2
transcript in Atlantic salmon fed SBM diets. The data
presented indicates a possible role of PAR-2 as a mediator of
inflammatory responses in the distal intestine of Atlantic
Materials and methods
In order to study the impact of diets containing SBM on
the mRNA expression of PAR-2, samples from both a
Short term trial
Farmed Atlantic salmon (Salmobreed strain), weighing
214 g (average) on the termination of the experiment,
were kept in fibreglass tanks (1 1 0.6 m, water depth
4050 cm) containing running seawater (salinity 3234 g
L-1) under 24 h light conditions. The water temperature
was between 810C during the experimental period.
During the experiment the fish were fed either a fishmeal
(FM) diet or a diet containing 46% SBM (Table 1) for 1, 3
or 7 days. Prior to the feeding trial the fish were allocated
in the fibreglass tanks and fed the fish meal diet for 27
days. Further details on formulation, chemical
composition and production of the diets are given in . The
experiment was done at AKVAFORSK, The Institute of
Aquaculture Research in Sunndalsra (Norway).
Long term trial
Farmed Atlantic salmon with an initial weight of
approximately 176 g were distributed into fibreglass tanks (1 1
0.6 m, water depth 4050 cm) containing running sea
water (5.6C). The fish were fed either a FM diet or a diet
containing 30% SBM for 3 weeks (Table 1). Before the
start of the trial the salmon were fed a commercial diet
(Skretting AS, Stavanger, Norway). Further details on
formulation and chemical composition of the diets as well as
fish and rearing conditions are given in . The
experiment was done at AKVAFORSK, The Institute of
Aquaculture Research in Sunndalsra (Norway).
aNorsECO (Egersund Sildeoljefabrikk AS, Egersund, Norway).
bDeno-Soy F, soybean meal with hull that is hexane extracted and
toasted (Denofa, Fredrikstad, Norway).
cSkretting Australia (Cambridge, TAS, Australia).
dExtruded soybean meal, Skretting Australia (Cambridge, TAS,
Collection of tissue samples
Fish were randomly selected, anesthetized in tricaine
methansulphate (MS222), weighed, measured and killed
with a sharp blow to the head followed by abdominal
evisceration. The intestines were cleaned of all fatty tissue
and intestinal content prior to collection of samples. The
intestinal regions were defined as follows: the pyloric
intestine (PI) included the intestine with the caeca; the
mid intestine (MI) included the intestine between the
most distal pyloric caecum and the appearance of
transverse folds of the luminal surface and the increase in
intestinal diameter; the distal intestine (DI) included the
intestine between the distal end of the MI and anus.
For characterization of PAR-2a and PAR-2b mRNA
expression in various tissues 300 mg of the following tissues
were sampled from one fish fed a FM based diet:
oesophagus, stomach, pancreas, PI, MI, DI, liver, head kidney,
kidney, heart, spleen, thymus, brain, eye, gill, gonads, muscle
and skin. All samples except for pancreas were stored in
RNAlater (Ambion Inc.) at -20C until RNA isolation.
Pancreas was collected as follows: approximately 300 mg
of pancreatic tissue, i.e. diffuse pancreas embedded in the
fatty tissue surrounding the pyloric caeca, was gently
scraped off with a spatula and immediately snap frozen in
liquid nitrogen then transferred to ten times the volume
of RNAlater-ICE (Ambion, Inc.) and stored at -80C until
For quantification of PAR-2 mRNA expression
approximately 300 mg of the DI from the short-term and the
long-term trial was collected and stored on RNAlater
(Ambion Inc.) at -20C until RNA isolation. The
following diets and points of time were collected from the
shortterm trial: from the FM group (control fish) 2 fish were
collected on day 1, 3 and 7 respectively (6 in total); from
the SBM group 6 fish were collected at day 1, 3 and 7
respectively (6 fish per day). Nine DI samples were
collected from the FM and SBM group respectively in the
Total RNA extraction
Total RNA was isolated from oesophagus, stomach,
pancreas, PI, MI, DI, liver, head kidney, kidney, heart, spleen,
thymus, brain, eye, gill, gonads, and muscle using Trizol
(Invitrogen Ltd, Paisley, UK) according to the
manufacturer's protocol. A modified protocol was used for
pancreas with three times the volume of reagents.
First strand cDNA synthesis
cDNA was generated from five microgram of total RNA
using PowerScript Reverse Transcriptase (BD
Biosciences, Franklin Lakes, NJ, USA) according to the
manufacturer's protocol, primed with a mixture of oligo dT (25
ng/l) and random hexamer primers (2.5 ng/l).
Cloning and sequencing of PAR-2 mRNA sequences
Multiple DNA sequence alignments was performed from
Homo sapiens [GenBank:NM_005242], Danio rerio
[GenBank:XM_678622], Hippoglossus hippoglossus
[GenBank:EB034068], Oncorhynchus mykiss
[GenBank:BX861951] and Xenopus laevis
[GenBank:BX850546] PAR-2 sequences using the publicly
available web browser based bl2seq (Blast 2 Sequences,
). From these alignments we manually designed one
degenerated (PAR-2R_Deg) and one regular PCR primer
(PAR-2F) based on the identification of potential nucleic
acid conservation of PAR-2 sequences. All PCR products
amplified with Advantage 2 PCR enzyme mix (Clontech,
Takara Bio Inc, Shiga, Japan) were used in a
post-amplification procedure with addition of 2 U of Taq polymerase
(Biotools, B&M Labs, Madrid, Spain) in 1 PCR buffer
(Biotools) for 15 min at 72C before use in TOPO TA
cloning (TOPO TA Cloning Kit; Invitrogen, Carlsbad, CA,
USA). The PAR-2 primers and cDNA generated from the
distal intestine were used in PCR amplification using
Advantage 2 PCR enzyme mix (Clontech) in a total
reaction volume of 25 l with the following cycling
parameters: 35 cycles of 94C for 30 s, 60C for 30 s, and 72C
for 30 s. A positive PCR product of 149 bp was cloned
using TOPO TA Cloning kit (Invitrogen) according to the
manufacturers' instructions. From the cloning, five clones
were selected and grown for 16 h at 37C in Luria-Bertani
media containing 50 g/ml ampicilin. Plasmid DNA was
isolated (E.Z.N.A plasmid miniprep kit I, OMEGA
BioTek, Inc, GA, USA) and sent for sequencing (GATC
Biotech, Konstanz, Germany). From the PAR-2 sequence
obtained we manually designed specific PCR primers
unique for each PAR-2 versions for use in 5' and 3' RACE
(rapid amplification of cDNA ends). mRNA was isolated
from total RNA following the manufacturers instructions
(MicroPoly(A)Purist Kit, Ambion, Austin, TX, USA), and
approximately 1 g of was used for reverse transcription
using SMART RACE cDNA amplification Kit (Clontech).
The PCR reactions were performed using the Advantage 2
PCR enzyme mix (Clontech) with the following
touchdown PCR setup; 3 min at 94C followed by: (30 s at
94C, 3 min at 72C) five cycles, (30 s at 94C, 30 s at
70C, 3 min at 72C) five cycles, (30 s at 94C, 30 s at
68C, 3 min at 72C) 32 cycles. From each
transformation, 8 clones were selected and grown for 16 h at 37C in
Luria-Bertani media containing 50 g/ml ampicilin,
plasmids were isolated (E.Z.N.A plasmid miniprep Kit I) and
sequenced (GATC Biotech). The sequence
chromatograms were imported to the free software ContigExpress
(Vector NTI Advance 10, Invitrogen), trimmed for vector
and RACE primer sequences and assembled into contigs.
Quantitative real-time RT-PCR
Total RNA was extracted from DI as previously described.
Prior to reverse transcription, total RNA from all samples
were subjected to DNase treatment using a TURBO
DNAfree kit (Ambion) in accordance with the manufacturer's
First strand cDNA synthesis was performed with 0.8 g
total RNA from each sample using Superscript III
(Invitrogen) and Oligo(dT)20 primers (Invitrogen) in accordance
with the manufacturer's instructions.
Real-time RT-PCR primers for the two PAR-2 transcripts
were designed based on the full-length sequence using the
free available software FastPCR . Real-time RT-PCR
primers for the housekeeping genes were designed using
Primer3 software . PCR reactions were performed in a
total volume of 10 l using the LightCycler FastStart DNA
MasterPLUS SYBR GREEN I kit (Roche Diagnostics) using
4.5 l PCR-grade water, 0.5 l of each PCR primer (10
M), 2.5 l (6.25 ng) cDNA template and 2 l master mix.
The following program was used: Denaturation (10 min
at 95C), amplification and quantification program
repeated 40 times (10 sec. at 95C, 15 sec. at the
appropriate annealing temperature for the gene specific primers
(Table 2) and 10 sec. at 72C with a single fluorescence
measurement), melting curve program (60C to 99C
with a heating rate of 0.1C/sec.) and cooling program
down to 40C.
For determination of the crossing point (CP) the "second
derivative maximum method" measuring maximum
increase rate of newly synthesized DNA per cycle was used
on the basis of the LightCycler software 4.0 (Roche
Diagnostics). To confirm amplification specificity the PCR
products from each primer pair were subjected to melting
curve analysis and manual inspection of PCR products
after each run by agarose gel electrophoresis.
Relative quantification analyzes
The relative expression ratio of target mRNAs was
calculated using the LightCycler software 4.0 (Roche
Diagnostics) with calibrator-normalized relative quantification
and PCR efficiency correction based on a linear regression
fit. RNA from tissues of a fish from the FM group was used
as calibrator. Four reference genes; Elongation factor 1
alpha, Glyceraldehyde-3-phosphate dehydrogenase, 18S
RNA and -actin (Table 2) were analyzed for stability of
expression in the samples intended for relative
quantification analyses using the geNorm software . Relative
standard curves were generated on the basis of cDNA
pooled from 2 samples from each diet and sample time,
diluted in 5-fold or 10-fold dilution steps to cover the
expected detection range of the target and housekeeping
Gene name Accession number Primer name
SS-EF1-alpha F1 GTGCTGTGCTTATCGTTGCT
SS-EF1-alpha R1 GGCTCTGTGGAGTCCATCTT
SS beta-aktin F1 CAAAGCCAACAGGGAGAAGATGA
SS beta-aktin R1 ACCGGAGTCCATGACGATAC
GAPDH F1 AAGTGAAGCAGGAGGGTGGAA
GAPDH R1 CAGCCTCACCCCATTTGATG
SS-18SrRNA F1 TACAGTGAAACTGCGAATGG
SS-18SrRNA R1 GCATGGGTTTTGGGTCTG
PAR-2: Proteinase-activated receptor-2, ELF-1 : Elongation factor 1 alpha, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase. * PCR product
size after RACE amplification
To examine the evolutionary relationship of the cloned
Atlantic salmon sequences we aligned them with
published PAR-2 sequences from a set of animal species using
MEGA 4 , and used Jalview  to visualize the
aligned sequences. The following sequences were used;
human [GenBank:NM_001992, GenBank:NM_005242,
GenBank:NM_004101, GenBank:NM_003950], dog
GenBank:XM_844773, GenBank:XM_541962] mouse
GenBank:NM_010170, GenBank:NM_007975], rat
GenBank:NM_053313, GenBank:NM_053808], zebrafish
[GenBank:XM_694943], frog [GenBank:NM_001085783,
GenBank:NM_001086070]. In MEGA 4 we produced a
cladogram using Neighbor-joining with the following
settings; bootstrap = 10000 seed = 38877, complete deletion
for gaps/missing data, Poisson correction for amino acid
substitution and uniform rates among sites.
The Shapiro-Wilk W test was used to test conformity with
the normal distribution. Student's t test was used to
compare the relative expression of the respective genes
between diets in the three week feed trial. To correct for
multi comparisons of means, analysis of variance was
followed by Tukey's Honestly Significant Difference (HSD)
test. All results are presented as mean values with bars
representing SEM. All tests were carried out two-tailed, with a
significance level of 5%. The statistical analyses were
performed using JMP 5.0.1 software package (SAS Institute
Inc. Cary, NC, USA).
Results and discussion
Full-length cloning of PAR-2 transcripts
Using cloning and sequencing we identified two
fulllength PAR-2 mRNA transcripts termed PAR-2a
[GenBank:FJ184031] and PAR-2b [GenBank:FJ184032]
respectively, with 78% nucleic acid identity to each other in the
deduced open reading frame (ORF). The large difference
between the two transcripts indicate that they are likely to
be derived from two genes, probably caused by divergence
of the duplicated genome of Atlantic salmon . Several
expressed genes have previously been shown to be
duplicated in Atlantic salmon [46-49], but little is known about
what fraction of the reported duplicated genes are
functional. Deduced amino acid similarities of the two PAR-2
receptors were compared to other species by performed
multiple alignments with known PAR-2 sequences from
Homo sapiens and Danio rerio (Figure 1). To visualize the
phylogenetic sequence relations we produced a
cladogram using Neighbour joining with BLOSUM62 matrix
(Figure 2). From the alignments and the cladogram, both
deduced Atlantic salmon PAR-2 sequences show
similarity to other known PAR-2 sequences. However, the
PAR2a sequence show more similarity to Danio rerio PAR-2
sequence than to the alternative Atlantic salmon PAR-2b
sequence, which may indicate PAR-2a as the putative
ancestral gene. We also observed considerable differences
in the first 145 and 200229 aa (amino acids) of the two
deduced Atlantic salmon PAR-2 proteins. These regions of
the protein represent the putative N-terminal domain and
extra cellular loop 2 region, both being essential in the
activation of this receptor in mammals. Both deduced
Atlantic salmon PAR-2 protein sequences have a serine
proteinase cleavage site in the N-terminal part of the
protein (Figure 1) in a comparable position of the serine
proteinase site in the human PAR-2 protein.
Expression studies of PAR-2 transcripts
Thorough testing of the PCR primer pairs with diluted
plasmid templates for both PAR-2 genes in real-time
RTPCR experiments showed high specificity for the two
PAR2 transcripts (data not shown). Expression of both PAR-2
transcripts was seen in all the tissues examined, with
about 10100 fold higher expression in gills, pyloric-,
mid-, and distal intestine (Figure 3). Our findings are
similar to reports of high expression of PAR-2 in the colon
and small intestine of man [25,50]. In man, PAR-2 has
been demonstrated to mediate infiltration of leukocytes
as well as hyperreactivity in allergic inflammation of the
airway . Hence, the high expression of the PAR-2
receptor transcripts observed in the gills of Atlantic
salmon could point to analogous functions in the
respiratory organ of fishes.
Transcription level for PAR-2a showed a rapid and
significant up-regulation at day 1 in fish fed diet with SBM
whereas no expression differences was seen at day 3 or 7
(Figure 4). For PAR-2b there was no significant expression
change during the first 7 days in fish fed diets containing
SBM (Figure 4). Histopathological changes in the distal
intestine of the fish fed the SBM diets showed similar
features as previously described in another study . No
histopathological changes were observed after one day of
feeding SBM, and only minor changes in some fish were
seen at day three (results not shown). However, after 7
days most fish displayed the features of SBM induced
enteritis (results not shown). Most fish fed SBM diet for 21
aFMniugdluthirpuelme1anlig(nHmse)n[tGseonfBdaendku:NceMd_P0A0R52-242a]mviinsouaaliczieddseuqsiunegnJcaelvsiefrwo m[44A]tlantic salmon (Ss), zebrafish (Dr) [GenBank:XM_694943]
Multiple alignments of deduced PAR-2 amino acid sequences from Atlantic salmon (Ss), zebrafish (Dr)
[GenBank:XM_694943] and human (Hs) [GenBank:NM_005242] visualized using Jalview 44. The following percentage
amino acid identity between the compared sequences is indicated with blue color, <40% identity has no color, >40% is light
blue, >60% is medium dark blue, and >80% is dark blue.
itFAaoirgrcislu)1a,rdmeo4go2(ruPasAmeR(s-M1h,ouPwsAminRgu-s2tch,uePluArse)R,l-ar3taitoan(RdshaPtiptAuosRfn-4Ao)rtvleagnitciucss),alzmebornaf(iSshal m(Doasnaiolare,rinio)readndleftrtoegrs()X,ehnuompauns l(aHeovims)opsraoptieinnsa)s,ed-oagct(ivCaatneids
fraemceil-pA cladogram showing the relationship of Atlantic salmon (Salmo salar, in red letters), human (Homo sapiens),
dog (Canis familiaris), mouse (Mus musculus), rat (Rattus norvegicus), zebrafish (Danio rerio) and frog (Xenopus
laevis) proteinase-activated receptor 14 (PAR-1, PAR-2, PAR-3 and PAR-4). The cladogram was created in MEGA
4  using Neighbor-joining (BLOSUM62 matrix).
days displayed fully developed enteritis after histological
investigations as been reported previously .
Interestingly, at three weeks feeding a diet containing SBM, a
significant down-regulation was seen for PAR-2a, and a
significant up-regulation was detected for PAR-2b
compared to fish fed the control diet. Even though many
duplicated genes in Atlantic salmon might be classified as
pseudogenes, the observed response for the two PAR-2
genes may suggest involvement of both genes in the SBM
induced enteritis. Overexpression of PAR-2 has been
RFeiglautrivee3expression of PAR-2a (A) and PAR-2b (B) respectively
Relative expression of PAR-2a (A) and PAR-2b (B) respectively. Expression levels are relative to muscle tissue
samples. The following tissues were examined; ST: stomach, GI: gills, LI: liver, ES: esophagus, PI: pyloric caeca, MI: mid intestine, DI:
distal intestine, HK: head-kidney, KI: kidney, SK: skin, MU: muscle, GO: gonads, PA: pancreas, EY: eye, BR: brain, TH: thymus,
SP: spleen, HE: heart.
RFeiglautrivee4mRNA expression of PAR-2 in the distal intestine of Atlantic salmon
Relative mRNA expression of PAR-2 in the distal intestine of Atlantic salmon. The gene expression was normalized
to both elongation factor 1 and -actin, and an average normalized ratio for each individual was calculated. The x-axis
represents days after introduction to SBM and the y-axis represents the normalized ratio. Relative expression of PAR-2a (A) and
PAR-2b (B) in fish fed fishmeal (FM) (n = 6) day 0 or a diet with inclusion of soybean meal (SBM) at day 1 (n = 6), at day 3 (n =
6) and at day 7 (n = 6) days. Relative expression of PAR-2a (C) and PAR-2b (D) of fish fed FM diet and diet with inclusion of
SBM after 3 weeks of feeding. Error bars indicate S.E.M (standard error of the mean). Different lower case letters denote
significant differences (P < 0.05) between the means.
observed in biopsies from IBD (inflammatory bowels
disease) patients and PAR-2 could play an important role in
the development of colonic inflammation in man . A
pro-inflammatory role for PAR-2 was first shown in the
colon of mice where acute PAR-2 activation led to an
increase in epithelial permeability and bacterial
translocation . Patients with ulcerative colitis treated with a
tryptase inhibitor shown relieved symptoms or remission
of the disease , suggesting involvement of PAR-2 and
tryptase in the gastrointestinal disease in these patients.
Luminal proteinases has been demonstrated to regulate
colonic paracellular permeability in mice, and that
bacterial flora influences the degranulation of mucosal mast
cells . Further, it was suggested that the increased
expression of PAR-2 observed in colonocytes is influenced
by increased luminal proteinase activity rather than the
release of proteinases such as tryptase . Increased
luminal trypsin-like activity in the distal intestine of
Atlantic salmon fed diets with SBM [11,12] may suggest a
similar mode of PAR-2 activation by serine proteases in
In mouse colon and small intestine a higher expression of
PAR-2 has been reported in the surface epithelial cells
lining the upper two thirds of the villi compared to cells
located in the crypt region . An increased proliferative
compartment length as well as lower mucosal fold height
have been reported in fish fed SBM feed for three weeks or
more . As a consequence, the observed reduction of
PAR-2a expression after 3 weeks on a SBM diet may be
caused by a reduced number of proliferated cells
expressing PAR-2 towards the tip of the villi compared to the
control fish. If the two PAR-2 transcripts in Atlantic salmon
are not expressed equally in the same cell populations, the
increased expression of PAR-2b in fish fed SBM feed for 3
weeks might be caused by the number of proliferated cells
or the reported leukocyte infiltrate in fish fed diets
containing SBM. The expression profile of the two transcripts
in different cell populations of the intestine needs to be
Recent studies have shown that inflammation of the gut
disrupts the normal microbiota and dramatically boost
colonization of pathogenic bacteria in man [53,54]. An
inflamed gut is therefore an open invitation to several
species of pathogenic bacteria further promoting the
inflammation and a factor in causing disease. The microbiota in
Atlantic salmon changes upon exposure to SBM and a
more diverse population of adherent bacteria has been
reported after 3 weeks feeding a diet containing SBM .
The change of PAR-2a expression detected after one day of
exposure to feed containing SBM suggest PAR-2 receptor
activation and could therefore be responsible for an
initiation of inflammation. Such an inflammation in concert
with the new feed components could allow colonization
of new bacteria and ultimately changing the normal
microbiota. It is not known however if the microbiota of
Atlantic salmon fed feed containing SBM is altered as early
as one day after feeding, and a putative involvement of
bacteria in the early phases of the development of enteritis
merits further investigation.
The putative role PAR-2 seems to have in intestinal
inflammation in fishes makes it a potential important
marker for enteritis. Soybean meal appears as a promising
tool for studies of PAR-2, intestinal inflammation
responses as well as intestinal cell populations in Atlantic
In our study we have demonstrated that Atlantic salmon
have putative duplicated gene versions of the PAR-2
receptor. Both transcripts are highly expressed in the
gastrointestinal tract and the gills. In Atlantic salmon fed inclusion
levels of SBM the expression of the two PAR-2 transcripts
is altered. We have shown that PAR-2a has a significant
change of expression after one day of feeding diets
containing SBM, but that the expression is significantly
decreased in a three week feeding trial. The expression of
PAR-2b did not show altered expression in the first seven
days of feeding but a significant increase in expression is
observed after three weeks. The altered expression of the
two PAR-2 transcripts in the gut of fish fed diets
containing SBM suggests that PAR-2 may have an important role
in inflammation in fishes and other lower vertebrates. The
identification of the PAR-2 genes in Atlantic salmon, a
known initiator of inflammation in the gut of mammals,
opens up for future studies to further shed light on
molecular causes of the SBM induced enteritis observed in
The authors declare that they have no competing interests.
JT planned the experiments, conducted the primer design,
cloning of PAR-2 sequences, performed multiple
alignment and phylogenetic analysis, participated in the
realtime RT-PCR and drafted the manuscript. ECV isolated
RNA, performed real-time RT-PCR and participated in the
sampling. EL contributed in the real-time RT-PCR, in the
sampling and in the drafting of the manuscript. K
participated in the planning of the experiments, drafting of the
manuscript and contributed to the intellectual content.
1. Dommett R , Zilbauer M , George JT , Bajaj-Elliott M : Innate immune defence in the human gastrointestinal tract . Mol Immunol 2005 , 42 : 903 - 912 .
2. Brandtzaeg P , Halstensen TS , Kett K , Krajci P , Kvale D , Rognum TO , Scott H , Sollid LM : Immunobiology and Immunopathology of Human Gut Mucosa - Humoral Immunity and Intraepithelial Lymphocytes . Gastroenterology 1989 , 97 : 1562 - 1584 .
3. Baeverfjord G , Krogdahl A : Development and regression of soybean meal induced enteritis in Atlantic salmon, Salmo salar L, distal intestine: A comparison with the intestines of fasted fish . J Fish Dis 1996 , 19 : 375 - 387 .
4. Ingh TSGA van den, Krogdahl A : Negative Effects of Antinutritional Factors from Soya Beans in Salmonids . Tijdschr Diergeneeskd 1990 , 115 : 935 - 938 .
5. Ingh TSGA van den, Krogdahl A , Olli JJ , Hendriks HGCJ , Koninkx JGJF : Effects of Soybean-Containing Diets on the Proximal and Distal Intestine in Atlantic Salmon (Salmo-Salar) - A Morphological-Study . Aquaculture 1991 , 94 : 297 - 305 .
6. Krogdahl A , Bakke-McKellep AM , Baeverfjord G : Effects of graded levels of standard soybean meal on intestinal structure, mucosal enzyme activities, and pancreatic response in Atlantic salmon ( Salmo salar L.). Aquacult Nutr 2003 , 9 : 361 - 371 .
7. Nordrum S , Bakke-McKellep AM , Krogdahl A , Buddington RK : Effects of soybean meal and salinity on intestinal transport of nutrients in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss) . Comp Biochem Phys B 2000 , 125 : 317 - 335 .
8. Olli JJ , Krogdahl A , Vandeningh TSGA , Brattas LE : Nutritive-Value of 4 Soybean Products in Diets for Atlantic Salmon (SalmoSalar , L). Acta Agr Scand A-An 1994 , 44 : 50 - 60 .
9. Olli JJ , Krogdahl A : Alcohol soluble components of soybeans seem to reduce digestibility in fish-meal-based diets for Atlantic salmon , Salmo salar L. Aquac Res 1995 , 26 : 831 - 835 .
10. Krogdahl A , Bakke-McKellep AM , Roed KH , Baeverfjord G : Feeding Atlantic salmon Salmo salar L. soybean products: effects on disease resistance (furunculosis), and lysozyme and IgM levels in the intestinal mucosa . Aquacult Nutr 2000 , 6 : 77 - 84 .
11. Lilleeng E , Froystad MK , Ostby GC , Valen EC , Krogdahl A : Effects of diets containing soybean meal on trypsin mRNA expression and activity in Atlantic salmon (Salmo salar L) . Comp Biochem Phys A 2007 , 147 : 25 - 36 .
12. Refstie S , Glencross B , Landsverk T , Sorensen M , Lilleeng E , Hawkins W , Krogdahl A : Digestive function and intestinal integrity in Atlantic salmon (Salmo salar) fed kernel meals and protein concentrates made from yellow or narrow-leafed lupins . Aquaculture 2006 , 261 : 1382 - 1395 .
13. Ingh TSGA van den, Olli JJ , Krogdahl A : Alcohol-soluble components in soybeans cause morphological changes in the distal intestine of Atlantic salmon , Salmo salar L. J Fish Dis 1996 , 19 : 47 - 53 .
14. Bureau DP , Harris AM , Cho CY : The effects of purified alcohol extracts from soy products on feed intake and growth of chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss) . Aquaculture 1998 , 161 : 27 - 43 .
15. Knudsen D , Uran P , Arnous A , Koppe W , Frokiaer H : Saponin-containing subfractions of soybean molasses induce enteritis in the distal intestine of Atlantic salmon . J Agric Food Chem 2007 , 55 : 2261 - 2267 .
16. Johnson IT , Gee JM , Price K , Curl C , Fenwick GR : Influence of Saponins on Gut Permeability and Active Nutrient Transport Invitro . J Nutr 1986 , 116 : 2270 - 2277 .
17. Cenac N , Coelho AM , Nguyen C , Compton S , ndrade-Gordon P , MacNaughton WK , Wallace JL , Hollenberg MD , Bunnett NW , Garcia-Villar R , Bueno L , Vergnolle N : Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2 . Am J Pathol 2002 , 161 : 1903 - 1915 .
18. Cenac N , Garcia-Villar R , Ferrier L , Larauche M , Vergnolle N , Bunnett NW , Coelho AM , Fioramonti J , Bueno L : Proteinase-activated receptor-2-induced colonic inflammation in mice: Possible involvement of afferent neurons, nitric oxide, and paracellular permeability . J Immunol 2003 , 170 : 4296 - 4300 .
19. Fiorucci S , Mencarelli A , Palazzetti B , Distrutti E , Vergnolle N , Hollenberg MD , Wallace JL , Morelli A , Cirino G : Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis . Proc Natl Acad Sci USA 2001 , 98 : 13936 - 13941 .
20. Kim JA , Choi SC , Yun KJ , Kim DK , Han MK , Seo GS , Yeom JJ , Kim TH , Nah YH , Lee YM : Expression of protease-activated receptor 2 in ulcerative colitis . Inflamm Bowel Dis 2003 , 9 : 224 - 229 .
21. Nystedt S , Ramakrishnan V , Sundelin J : The proteinase-activated receptor 2 is induced by inflammatory mediators in human endothelial cells - Comparison with the thrombin receptor . J Biol Chem 1996 , 271 : 14910 - 14915 .
22. Roka R , Demaude J , Cenac N , Ferrier L , Salvador-Cartier C , GarciaVillar R , Fioramonti J , Bueno L : Colonic luminal proteases activate colonocyte proteinase-activated receptor-2 and regulate paracellular permeability in mice . Neurogastroenterol Motil 2007 , 19 : 57 - 65 .
23. Schmidlin F , Amadesi S , Dabbagh K , Lewis DE , Knott P , Bunnett NW , Gater PR , Geppetti P , Bertrand C , Stevens ME : Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway . J Immunol 2002 , 169 : 5315 - 5321 .
24. Ishihara H , Connolly AJ , Zeng DW , Kahn ML , Zheng YW , Timmons C , Tram T , Coughlin SR : Protease-activated receptor 3 is a second thrombin receptor in humans . Nature 1997 , 386 : 502 - 506 .
25. Nystedt S , Emilsson K , Larsson AK , Strombeck B , Sundelin J : Molecular-Cloning and Functional Expression of the Gene Encoding the Human Proteinase-Activated Receptor-2 . Eur J Biochem 1995 , 232 : 84 - 89 .
26. Vu TKH , Hung DT , Wheaton VI , Coughlin SR : Molecular-Cloning of A Functional Thrombin Receptor Reveals A Novel Proteolytic Mechanism of Receptor Activation . Cell 1991 , 64 : 1057 - 1068 .
27. Xu WF , Andersen H , Whitmore TE , Presnell SR , Yee DP , Ching A , Gilbert T , Davie EW , Foster DC : Cloning and characterization of human protease-activated receptor 4 . Proc Natl Acad Sci USA 1998 , 95 : 6642 - 6646 .
28. Nystedt S , Emilsson IE , Wahlestedt C , Sundelin J : Molecular-Cloning of A Potential Proteinase Activated Receptor . Proc Natl Acad Sci USA 1994 , 91 : 9208 - 9212 .
29. Nystedt S , Larsson AK , Aberg H , Sundelin J : The Mouse Proteinase-Activated Receptor-2 Cdna and Gene - Molecular-Cloning and Functional Expression . J Biol Chem 1995 , 270 : 5950 - 5955 .
30. Bohm SK , Khitin LM , Grady EF , Aponte G , Payan DG , Bunnett NW : Mechanisms of desensitization and resensitization of proteinase-activated receptor-2 . J Biol Chem 1996 , 271 : 22003 - 22016 .
31. Jacob C , Cottrell GS , Gehringer D , Schmidlin F , Grady EF , Bunnett NW: c-Cbl mediates ubiquitination, degradation, and downregulation of human protease-activated receptor 2 . J Biol Chem 2005 , 280 : 16076 - 16087 .
32. Macfarlane SR , Seatter MJ , Kanke T , Hunter GD , Plevin R : Proteinase-activated receptors. Pharmacol Rev 2001 , 53 : 245 - 282 .
33. Steinhoff M , Buddenkotte J , Shpacovitch V , Rattenholl A , Moormann C , Vergnolle N , Luger TA , Hollenberg MD : Proteinase-activated receptors: Transducers of proteinase-mediated signaling in inflammation and immune response . Endocr Rev 2005 , 26 : 1 - 43 .
34. Lindner JR , Kahn ML , Coughlin SR , Sambrano GR , Schauble E , Bernstein D , Foy D , Hafezi-Moghadam A , Ley K : Delayed onset of inflammation in protease-activated receptor-2-deficient mice . J Immunol 2000 , 165 : 6504 - 6510 .
35. Vergnolle N : Proteinase-activated receptor-2-activating peptides induce leukocyte rolling, adhesion, and extravasation in vivo . J Immunol 1999 , 163 : 5064 - 5069 .
36. Fields RC , Schoenecker JG , Hart JP , Hoffman MR , Pizzo SV , Lawson JH : Protease-activated receptor-2 signaling triggers dendritic cell development . Am J Pathol 2003 , 162 : 1817 - 1822 .
37. Gecse K , Roka R , Ferrier L , Leveque M , Eutamene H , Cartier C , itBelgnaoui A , Rosztoczy A , Izbeki F , Fioramonti J , Wittmann T , Bueno L : Increased faecal serine protease activity in diarrhoeic IBS patients: a colonic lumenal factor impairing colonic permeability and sensitivity . Gut 2008 , 57 : 591 - 598 .
38. Kraugerud OF , Penn M , Storebakken T , Refstie S , Krogdahl A , Svihus B : Nutrient digestibilities and gut function in Atlantic salmon (Salmo salar) fed diets with cellulose or non-starch polysaccharides from soy . Aquaculture 2007 , 273 : 96 - 107 .
39. Tatusova TA , Madden TL : BLAST 2 SEQUENCES, a new tool for comparing protein and nucleotide sequences . FEMS Microbiol Lett 1999 , 174 : 247 - 250 .
40. FastPCR [http://www.biocenter.helsinki.fi/bi/programs/fast pcr.htm]
41. Rozen S , Skaletsky HJ : Primer3 on the WWW for general users and for biologist programmers . In Methods Mol Biol Volume 132. Edited by: Krawetz S, Misener S. Totowa , NJ: Humana Press ; 2000 : 365 - 386 .
42. Vandesompele J , De Preter K , Pattyn F , Poppe B , Van Roy N , De Paepe A , Speleman F : Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes . Genome Biol 2002 , 3 : RESEARCH0034 .
43. Tamura K , Dudley J , Nei M , Kumar S : MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4 .0. Mol Biol Evol 2007 , 24 : 1596 - 1599 .
44. Clamp M , Cuff J , Searle SM , Barton GJ : The Jalview Java alignment editor . Bioinformatics 2004 , 20 : 426 - 427 .
45. Allendorf FW , Thorgaard GH : Tetraploidy and the evolution of Salmonid fishes . In Evolutionary genetics of fishes Edited by: Turner BJ. New York : Plenum Publishing Corporation ; 1984 : 1 - 53 .
46. Haugland O , Mercy IS , Romoren K , Torgersen J , Evensen O : Differential expression profiles and gene structure of two tumor necrosis factor-alpha variants in Atlantic salmon ( Salmo salar L.). Mol Immunol 2007 , 44 : 1652 - 1663 .
47. Ng SHS , Artieri CG , Bosdet IE , Chiu R , Danzmann RG , Davidson WS , Ferguson MM , Fjell CD , Hoyheim B , Jones SJM , de Jong PJ , Koop BF , Krzywinski MI , Lubieniecki K , Marra MA , Mitchell LA , Mathewson C , Osoegawa K , Parisotto SE , Phillips RB , Rise ML , von Schalburg KR , Schein JE , Shin HS , Siddiqui A , Thorsen J , Wye N , Yang G , Zhu BL : A physical map of the genome of Atlantic salmon, Salmo salar . Genomics 2005 , 86 : 396 - 404 .
48. Thorsen J , Zhu BL , Frengen E , Osoegawa K , de Jong PJ , Koop BF , Davidson WS , Hyheim B : A highly redundant BAC library of Atlantic salmon (Salmo salar): an important tool for salmon projects . BMC Genomics 2005 , 6 :.
49. Thorsen J , Hoyheim B , Koppang EO : Isolation of the Atlantic salmon tyrosinase gene family reveals heterogenous transcripts in a leukocyte cell line . Pigment Cell Res 2006 , 19 : 327 - 336 .
50. Bohm SK , Kong WY , Bromme D , Smeekens SP , Anderson DC , Connolly A , Kahn M , Nelken NA , Coughlin SR , Payan DG , Bunnett NW : Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2 . Biochem J 1996 , 314 : 1009 - 1016 .
51. Tremaine WJ , Brzezinski A , Katz JA , Wolf DC , Fleming TJ , Mordenti J , Strenkoski-Nix LC , Kurth MC : Treatment of mildly to moderately active ulcerative colitis with a tryptase inhibitor (APC 2059): an open-label pilot study . Aliment Pharmacol Ther 2002 , 16 : 407 - 413 .
52. Bakke-McKellep AM , Penn MH , Salas PM , Refstie S , Sperstad S , Landsverk T , Ringo E , Krogdahl A : Effects of dietary soyabean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon ( Salmo salar L.). Br J Nutr 2007 , 97 : 699 - 713 .
53. Lupp C , Robertson ML , Wickham ME , Sekirov I , Champion OL , Gaynor EC , Finlay BB : Host-mediated inflammation disrupts the intestinal microbiota and promotes the Overgrowth of Enterobacteriaceae . Cell Host & Microbe 2007 , 2 : 119 - 129 .
54. Stecher B , Robbiani R , Walker AW , Westendorf AM , Barthel M , Kremer M , Chaffron S , Macpherson AJ , Buer J , Parkhill J , Dougan G , von Mering C , Hardt WD : Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota . Plos Biology 2007 , 5 : 2177 - 2189 .