BMP signaling in rats with TNBS-induced colitis following BMP7

Am J Physiol Gastrointest Liver Physiol 302: G1151–G1162, 2012.
First published February 23, 2012; doi:10.1152/ajpgi.00244.2011.
BMP signaling in rats with TNBS-induced colitis following BMP7 therapy
Ivana Maric,1 Natalia Kucic,2 Tamara Turk Wensveen,3 Ivana Smoljan,4 Blazenka Grahovac,5
Sanja Zoricic Cvek,1 Tanja Celic,1 Dragica Bobinac,1 and Slobodan Vukicevic6
1
Department of Anatomy and 2Department of Physiology and Immunology, Faculty of Medicine, University of Rijeka,
and 3Department of Internal Medicine, Clinical Hospital Rijeka, Rijeka; 4Psychiatric Hospital Rab, Rab; 5Department
of Pathology, Faculty of Medicine, University of Rijeka, Rijeka; and 6Laboratory of Mineralized Tissues, Center
for Translational and Clinical Research, School of Medicine, University of Zagreb, Zagreb, Croatia
Submitted 22 June 2011; accepted in final form 20 February 2012
bone morphogenetic proteins; inflammatory bowel disease; Smad
proteins
(BMPs) comprise the largest
subgroup of the transforming growth factor (TGF-␤) superfamily of molecules. They were discovered and named based
on their osteoinductive ability. Today we know that BMPs
have an essential role in embryonic development and tissue
differentiation, regulation of cell survival, and apoptosis, as
well as in the organogenesis of skeletal and nonskeletal tissues
(14, 45, 60, 67, 68, 69, 74).
BMPs transduce their signals by binding to two different
serine/threonine kinase receptors, type I (BMPRIA and BMPRIB) and type II (BMPRII), triggering the phosphorylation
and activation of the BMPRI by the BMPRII and activating the
downstream Smad signaling molecules through Smad-depenBONE MORPHOGENETIC PROTEINS
Address for reprint requests and other correspondence: I. Maric, Dept. of
Anatomy, Faculty of Medicine, Univ. of Rijeka, B. Branchetta 20, 51000
Rijeka, Croatia (e-mail: [email protected]).
http://www.ajpgi.org
dent and Smad-independent pathways (36, 47, 48, 59). The
activated type I receptor phosphorylates R-Smads and this
permits their association with Smad4. The R-Smad family
includes the BMP-specific receptor-regulated Smad subfamily
(BR-Smads, Smad1/5/8) and activin/TGF-␤-specific receptorregulated Smad subfamily (AR-Smads, Smad2/3). R-Smad
forms a complex with Co-Smad (Smad4) and transduces to the
nucleus, where the transcription of TGF-␤/BMP target genes is
determined (41).
The specific expression pattern of BMPs and the components of the BMP signaling pathway that were observed in all
three germ layers pointed to the essential role of BMPs in
normal gastrointestinal tract development (4, 18, 31, 37).
Germline mutations in BMPRI and Smad4 have been identified
in patients with juvenile polyposis (JP), a rare autosomal
dominant syndrome characterized by the development of multiple hamartomatous polyps in the gastrointestinal tract (11,
32). Additionally, several molecular pathways have been identified that control crypt fission, an important element underlying the clonal expansion of intestinal tumors (27, 30). Target
inactivation of BMPRIA in epithelial cells results in the expansion of the intestinal stem cell population and growth of
polyps in the small intestine (32). A phenotype similar to
human JP has been created in villin-noggin mice by the
inhibition of BMP activity in the intestinal epithelium via
transgenic expression of noggin (4, 27). There is also increasing evidence that BMPs and their signaling pathway have an
important role in intestinal carcinoma, particularly in tumor
progression, invasion, and metastasis, as described for TGF-␤
and BMP7 (52). Recently, researches have shown that BMPs
act mainly as tumor suppressor factors in intestine tumorigenesis, as evidenced by the loss of BMP signaling pathway in the
colorectal cancer (28, 29, 35). Smad5 gene downregulation
was observed in human inflammatory bowel disease (IBD),
which leads to higher susceptibility to experimental colitis (2).
Conditional mutations of the BMPRII in mice lead to appearance of epithelial hyperplasia and hamartomatous polyps (5).
BMPs are important in enteric gliogenesis with enhancement
of the glial differentiation and expression of ErbB3 receptor
and their ligands in enteric crest-derived cells (12).
In our previous study we showed the expression profile of
proinflammatory cytokines, BMP7, and its receptors in the
colon during different stages of experimental IBD. BMP7
when applied prophylactically or therapeutically reduced the
inflammation due to a decrement of proinflammatory cytokine,
especially IL-6 (44). To further characterize the role of BMPs
in the intestine, in this study we examined the expression of
various BMPs, their receptors, and downstream molecules of
BMP signaling (Smad family members) in rats with IBD. We
0193-1857/12 Copyright © 2012 the American Physiological Society
G1151
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Maric I, Kucic N, Turk Wensveen T, Smoljan I, Grahovac B,
Zoricic Cvek S, Celic T, Bobinac D, Vukicevic S. BMP signaling in
rats with TNBS-induced colitis following BMP7 therapy. Am J
Physiol Gastrointest Liver Physiol 302: G1151–G1162, 2012. First
published February 23, 2012; doi:10.1152/ajpgi.00244.2011.—Beyond stimulating bone formation, bone morphogenetic proteins
(BMPs) are important in development, inflammation, and malignancy
of the gut. We have previously shown that BMP7 has a regenerative,
anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the
BMP signaling pathway we monitored the effect of BMP7 therapy on
the BMP signaling components in the rat colon during different stages
of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic
acid (TNBS). The results showed a significantly decreased BMP7
expression in the acute phase, followed by a significantly increased
BMP2 and decreased BMP6 expression during the chronic phase of
colitis. BMP7 therapy influenced the expression of several BMPs with
the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the
acute stage and significantly decreased upon BMP7 therapy. BMP
receptor I expression was unchanged, whereas BMP receptor II was
decreased at day 2 and increased at days 14 and 30 of TNBS
inflammation. However, an opposite pattern of expression following
BMP7 therapy has been observed. BMP7 increased the expression of
BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy
at later stages of the disease. We suggest that BMP signaling was
altered during TNBS-induced colitis and was recovered with BMP7
administration, suggesting that IBD is a reversible process.
G1152
BMP SIGNALING IN TNBS COLITIS
showed that BMPs and BMP pathway components are preserved in the colon of diseased animals and the alternations in
their expression during IBD are recovered by the BMP7
therapy.
MATERIALS AND METHODS
Table 1. Criteria for macroscopic scoring of colonic lesions
(49, 77)
Score
0
1
0
1
2
3
4
5
6–10
0
1
2
Criteria
Diarrhea
No
Yes
Ulceration
No damage
Hyperemia, no ulcers
Ulceration without hyperemia or bowel wall thicknening
Ulceration with inflammation at one site
Ulceration/inflammation at two or more site
Major sites of damage extending ⬎1 cm along the length
of the colon
When an area of damage extended ⬎2 cm along the length
of the colon, the score is increased by 1 each additional
centimeter of involvement
Adhesions
No adhesions
Minor adhesions (colon can be separated from other tissue
with little effort)
Major adhesions
Score
Criteria
0
1
2
3
4
Normal tissue appearance
Damage of surface epithelium
Focal ulceration limited to mucosa
Focal, transmural inflammation, and ulceration
Extensive transmural ulceration and inflammation bordered by
normal mucosa
Extensive transmural ulceration and inflammation involving
entire section
5
of bleeding and diarrhea, ulcer with/without inflammation at one or
more sites, as well as the size of extending ulceration and the presence
of adhesions. After the completion of the macroscopic scoring, three
colon samples with visible inflammation were taken for microscopic
analyses. In the absence of inflammation the samples were taken 2, 4,
and 6 cm proximal from the anus. Tissue sections were examined by
two independent observers (T. Turk Wensveen and I. Maric) and
scored on a scale from 0 to 5, including extension of the focal
ulceration and the presence of transmural inflammation (Table 2) (44,
62). The scored indexes are expressed as a median (range).
Subsequently, the samples of colon tissue were stored in the liquid
nitrogen for further analyses.
Immunohistochemistry. For immunohistochemistry, colons were
fixed in 4% buffered formalin, embedded in paraffin and sectioned in
serial 4-␮m slices. Immunostaining was performed by standard procedures. Briefly, sections were incubated for 30 min in methanol and
3% hydrogen peroxide. Antigen retrieval was performed in citrate
buffer (pH ⫽ 6) at 100°C for 15 min and cooled for 10 –20 min at
room temperature. After blocking in 3% BSA-PBS Tween, the sections were incubated overnight at 4°C with a polyclonal antibody to
pSmad1/5/8, which recognizes the doubly phosphorylated forms of
Smad1 (Ser463/465), Smad5 (Ser463/465), and Smad8 (Ser426/428)
at a concentration 1:100 (Cell Signaling Technology, Beverly, MA).
Dako EnVision⫹System-HRP detection system was used as a secondary reagent following the manufacturer’s instruction. Liquid
Dab⫹ substrate (Dako, Carpinteria, CA) was used as a chromogen.
Slides were counterstained with hematoxylin. To determine pSmad1/
5/8 expression in colon samples, the positive cells were counted on 10
high-power fields (⫻400 magnification) in the mucosa and submucosa
of each animal (n ⫽ 5 per experimental group). Cell counting was
performed by use of an image analyzer system equipped with a
software package (Issa, VAMS, Zagreb, Croatia) by two independent
observers (I. Smoljan and T. Celic). The staining score of counted
cells revealed a significant interobserver concordance (Pearson’s r ⫽
0.683, P ⬍ 0.05). The results are expressed as a mean ⫾ SD of
pSmad1/5/8-positive cells per high-powered field.
Western blot analysis. The colon proteins were extracted from the
rat colons by homogenization in RIPA buffer containing 1% protease
inhibitor cocktail (Sigma-Aldrich, Germany) and phosphatase inhibitors (Roche Diagnostics, Mannheim, Germany). Tissue homogenates
were centrifuged and prepared in the SDS sample buffer. The samples
(50 ␮g protein) were subjected to electrophoresis on 10% SDS-PAGE
and blotted onto a PVDF Western blotting membrane (Roche Diagnostics). To detect pSmad1/5/8 immunoreactivity, the membrane was
incubated with a rabbit polyclonal antibody to pSmad1/5/8 (Cell
Signaling Technology) diluted 1:500 overnight at 4°C with agitation.
After washing with TBS-T buffer (TBS with 0·1% Tween 20), the
membrane was incubated with anti-rabbit IgG-horseradish peroxidase
(HRP)-linked antibody (Cell Signaling Technology) diluted 1:1,000
for 1 h at room temperature. Blots were then stripped and reblotted
with a rabbit ␤-actin antibody (Sigma-Aldrich) diluted 1:200 for 1 h
at room temperature. Blots were visualized by using SuperSignal
West Pico Chemiluminescent Substrate; the bands were intensified
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Animals. Male Sprague-Dawley rats weighing 230 –250 g were
used. The rats were housed in standard cages and fed with standard
laboratory chow and tap water ad libitum. During the experiments the
fundamental ethical principles of working with animals were respected. All animal procedures were approved by the Animal Ethics
Committee of the Faculty of Medicine, University of Rijeka, Croatia.
Experimental colitis and study design. We used the experimental
model of IBD as described (51). Animals were deprived of food for 24
h before the induction of colitis. Under light ether anesthesia, colitis
was induced by intracolonic installation of a solution containing
hapten 2,4,6-trinitrobenzenesulfonic acid (TNBS; 30 mg; SigmaAldrich, Taufkirchen, Germany) in 50% ethanol. TNBS-solution was
introduced into the colon through a plastic catheter at a distance of ⬃8
cm from the anus. The animals were maintained in a head-down
position for some time to prevent the leakage of TNBS solution.
During the study the animals were monitored for signs of colon
inflammation, including diarrhea and rectal bleeding. The rats were
randomized into four experimental groups, as follows: control animals
without treatment (normal group; n ⫽ 10); rats receiving 50% vol/vol
ethanol alone (ethanol control; n ⫽ 40); rats receiving TNBS enema
(TNBS group; n ⫽ 40), and rats receiving TNBS enema plus BMP7
(TNBS/BMP7 group; n ⫽ 40). BMP7 was applied intravenously in a
dose of 100 ␮g/kg at 1, 2, 3, 5, 7, 14, 20, and 25 days after colitis
induction. BMP7 was administered systemically according to the
results of Grgurevic et al. (26), who showed the presence of intact
protein in serum 30 min after its administration. For the time-course
study, we euthanized 10 animals on days 2, 5, 14, and 30 by colitis in
TNBS and TNBS/BMP7-treated rats, respectively.
Assessment of colonic damage. The animals were weighed at the
beginning of the experiment as well as on the day of termination to
determine alternations in body weight. The distal 8 cm of the rat colon
was dissected, rinsed with saline, and reviewed for the existence of
colitis. After these procedures were completed, the colon was examined in a blind fashion and scored for macroscopically visible damage
according to criteria described previously (Table 1) (10, 44, 72).
Briefly, the scoring on a scale from 0 to 14 included the appearance
Table 2. Criteria for microscopic scoring of colonic lesions
(49, 67)
G1153
BMP SIGNALING IN TNBS COLITIS
Mannheim, Germany) and 0.1 mg/ml DNase I (Roche Diagnostics)
for 12 h at 37° in a shaking incubator. After the mixture was filtered
through a nylon mesh, the cells were washed in complete RPMI
medium and finally purified by discontinuous Percoll gradient at the
interface 40/70% (39). The number of viable cells was counted by
Trypan blue dye exclusion. After the cell isolation, LPMC culture was
prepared in RPMI 1640 supplement with 10% fetal bovine serum,
L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100
␮g/ml) (Sigma). Cells were cultured in 6-cm dishes under standard
conditions. After 24 h preculture, BMP7 was added to the culture
media (100 ng/ml). After 24 h of stimulation with BMP7, the cells
were collected and processed for immunofluorescence and RT-PCR
analysis.
Immunofluorescent cell staining. LPMC cells, from a suspension
containing 5 ⫻ 105 cells, were centrifuged on a glass slide in a
cytospin centrifuge. The attached cells were first washed with PBS
and then fixed in 2% paraformaldehyde and permeabilized with 0.1%
Triton X-100 at room temperature. After blocking in 2% bovine serum
albumin in PBS, the slides were incubated at 4°C overnight with a
primary antibody against pSmad1/5/8 in dilution 1:100. The bound
antibodies were visualized by incubation with a FITC-conjugated goat
anti-rabbit IgG as secondary antibody (dilution 1:300) (Invitrogen). At
the same time, the cells were stained by Evans blue and analyzed by
fluorescent microscopy (Olympus BX51).
Statistical methods. Results were analyzed by use of the Statistica
10 software package (StatSoft, Tulsa, OK). The data were expressed
as means ⫾ SD or medians (range). Comparison of parametric data
was performed by Student’s t-test for unpaired observations and
Table 3. Oligonucleotide primer sequences used in the study
Primer
BMP2
BMP4
BMP6
BMP7
CTGF
Noggin
BMPRIA
BMPRIB
BMPRII
Smad1
Smad2
Smad3
Smad4
Smad5
Smad6
Smad7
Smad8
GAPDH
Sequence
5=-GAG
5=-TGA
5=-GAG
5=-AAA
5=-CCT
5=-GAC
5=-GCA
5=-AAG
5=-TAG
5=-TTC
5=-CCA
5=-CAC
5=-GAT
5=-CCC
5=-CTC
5=-TCA
5=-GCT
5=-GTG
5=-TTG
5=-ACA
5=-CAG
5=-TAC
5=-TGA
5=-CGA
5=-TGT
5=-AAT
5=-CAG
5=-TCG
5=-ATG
5=-ATC
5=-GCT
5=-GCT
5=-ACT
5=-TGG
5=-TCC
5=-AGA
TTT
GTC
CCT
CGA
TGG
ATT
CCT
CCC
CAA
ACT
TGC
GGT
TCA
ATC
TGG
TAA
TCG
GAC
TTT
GTT
CTT
TCT
CAG
TCC
CTC
CCA
ATG
TTT
ACC
TGT
GTG
GTA
TCC
GGA
CTC
TCC
GAG
ACT
TTC
CCA
TCC
GGA
CCA
AGA
GAG
TGC
CGA
GAG
CCA
CAT
GAG
GCT
CAG
TGA
AGA
AGA
CTC
GTG
TGC
CTT
ACC
TTC
GGC
ACA
AGG
GGT
TTG
GGC
GGC
TCT
AAG
ACA
TTG
AAC
CAG
TCA
ACC
GTT
GGG
TGG
CTG
CAC
GCG
GTG
AAA
ACT
ATT
TCC
AAT
GTG
AAT
GGA
TGA
GCT
TAT
TAC
TGG
TGC
TCT
ATA
CTG
TGT
CTG
CTT
CAG
TGC
ATT
ACG
AGG
CTG
CAA
GCA
TTA
GTC
AAA
TAC
GGT
AAG
AGA
CAC
GCC
TCT
GCA
CCA
CAA
GTG
GAA
ATT
ACA
CAA
TTT
TCC
AAT
TGC
CCG
CTT
TCA
TGA
TGA
TTC
TTT
AGA
GTC
GAT
Product Size (bp)
CTG CTC-3=
GTG TCC-3=
GTT TG-3=
TTC G-3=
TGA ATC C-3=
GTC GAA-3=
AC-3=
GG-3=
GTG TG-3=
CTG TC-3=
TCA
AGA CTT-3=
CAG-3=
CCA TA-3=
AGG AG-3=
TTT GC-3=
GAA CG-3=
TTG TG-3=
TGG GTT-3=
AAC CAG CTG-3=
AAC CAG G-3=
TTC CTG CTG-3=
CGT CCA GTC T-3=
CAG TGT CT-3=
TGA TCT CTC TG-3=
TGT CCT GGC TG-3=
CTG AAC C-3=
TTG AAA G-3=
GCA TCT TCT A-3=
GGA GGA TCT-3=
ATC TTA C-3=
ATA GT-3=
GCT AC-3=
CAG TG-3=
AGC AA-3=
ACA TT-3=
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00244.2011 • www.ajpgi.org
440
117
104
422
156
263
419
193
348
410
338
375
351
252
472
601
351
308
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and quantified with a calibrated imaging densitometer (Kodak Image
Station 440, LabImage, Halle, Germany).
RT-PCR analysis. Total RNA extraction from the colons was
performed by homogenization with QIAzol lysis reagent (1 mg/100
mg of tissues; Qiagen) by the acid guanidinium thiocyanate phenolchloroform method as described (18). The total RNA from the cells
was extracted with NucleoSpinRNA II kit (Machnery-Nagel, Düren,
Germany) according to the manufacturer’s instructions. The integrity
of each sample was verified by electrophoresis in a 1% denatured
agarose gel. Reverse transcription was performed by use of a Takara
RNA PCR kit (AMV) ver.3.0. (Takara Bio, Shiga, Japan). The
resulting cDNA was used for PCR amplification on Mastercycler
personal (Eppendorf, Hamburg, Germany). The primer sequences and
product size are shown in Table 3. PCR products were separated by
1.5% agarose gel electrophoresis with ethidium bromide. Using a
100-bp DNA ladder, we confirmed the predicted sizes of the obtained
PCR products. The intensity of bands was quantified by a calibrated
imaging densitometer (Kodak Image Station 440, LabImage).
LPMC isolation and cell culture. Lamina propria mononuclear
cells (LPMC) were isolated from the freshly dissected colons as
described (25, 71). Briefly, the colon samples were flushed with
ice-cold PBS and the Peyer’s patches were removed. The colon was
opened longitudinally, cut into 5-mm pieces, and incubated in HBSS
free of calcium and magnesium (HBSS-CMF) containing 1 mmol/l of
DTT for 30 min at room temperature. Subsequently, to remove
epithelial cells from the samples, pieces of colons were incubated
three times for 1 h in HBSS-CMF containing 0.75 mmol/l of EDTA
at room temperature. Furthermore, the tissue was digested in RPMI
medium containing 0.15 mg/ml Collagenase D (Roche Diagnostics,
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BMP SIGNALING IN TNBS COLITIS
Mann-Whitney U-test or Kruskal-Wallis ANOVA for nonparametric
data. In all analyses P ⬍ 0.05 was considered to be significant.
RESULTS
Table 4. Effects of BMP7 treatment on macroscopic and
microscopic signs of colitis
Day of TNBS Colitis
Experimental Group
2
5
14
30
TNBS group
TNBS/BMP7 group
Ethanol control
TNBS group
TNBS/BMP7 group
Ethanol control
9 (5–12)
7 (5–10)
1(0–1)
4,5 (3–5)
3 (3–5)
1 (0-1)
7 (4–9)
6 (4–8)
1(0–1)
3 (2–4)
3 (2–4)
0
6 (4–12)
4 (3–6)#
1 (0–1)
3,5 (2–5)
2 (1–3)*
0
4.5 (3–7)
3 (3–4)#
1(0–1)
2.5 (2–4)
1,5 (1–2)*
0
Data are expressed as median (range); n ⫽ 10. aMacroscopic damage score
from 0 to 14; bmicroscopic damage score from 0 to 5; cethanol control is
significantlly different from both groups; *P ⬍ 0.01; #P ⬍ 0.05.
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Macroscopic and microscopic signs of colitis in experimental groups. Animals receiving the TNBS/ethanol enema had
clinical signs of colitis characterized by rectal bleeding and
diarrhea. During the acute phase of TNBS colitis the body
weight was lower by 7.4 –13%, whereas during the chronic
phase of the disease animals gained weight (0.92– 8.14%), with
the exception of ethanol-treated rats, which did not show body
weight changes during the acute phase. Also, BMP7 therapy
did not influence animals’ weight (data not shown). Macroscopic observations of colons isolated from the rats with TNBS
colitis more frequently revealed the presence of cobblestonelike lesions, larger ulcers, and adhesions with scored index 9
(5–12), 7 (4 –9), 6 (4 –12), and 4.5 (3–7) on days 2, 5, 14, and
30, respectively (Table 4). The TNBS/BMP7-treated group
showed lower index scoring for the same data points, which
were 7 (5–10), 6 (4 – 8), 4 (3– 6), and 3 (3– 4), with significant
difference on days 14 and 30. Microscopic analyses showed
diminished surface epithelium and colon crypts with transmural infiltration at the acute phase of TNBS colitis with scored
index 4.5 (3–5) and 3 (2– 4) for TNBS group on days 2 and 5,
respectively; and 3 (3–5) for TNBS/BMP7 group at both time
points. On days 14 and 30, the scored microscopic index was
3.5 (2–5) and 2.5 (2– 4) for TNBS group, and 2 (1–3) and 1.5
(1–2) for TNBS/BMP7 group, which was significantly different (Table 4). The colons of ethanol-treated animals had no
macroscopic changes, with minor damage of the surface epithelium during the acute phase of colitis (data not shown).
Expression of BMPs in the TNBS-induced colonic inflammation.
We determined the expression of BMP2, 4, 6, and 7 in colon
during acute (2nd and 5th day of colitis) and chronic phase
(14th and 30th day of colitis) of TNBS colitis by RT-PCR.
Expression of all BMPs was detected with a significantly
decreased expression of BMP7 in the acute phase, and BMP6
in the chronic phase of colitis. The expression of BMP4 was
equal at early and late stages of colitis, whereas the BMP2
expression significantly increased in the chronic stage (Fig. 1).
Systemic administration of BMP7 resulted in significantly
reduced BMP2 and increased expression of BMP4 in the
chronic phase of colitis. In parallel, the expression of other
investigated BMPs was also present in the normal rat colon and
the ethanol control rats. The expression of connective tissue
growth factor (CTGF), a major profibrotic molecule, was
increased in colons of TNBS rats, especially on the day 2 after
induction of colitis, whereas the BMP7 treatment significantly
reduced its expression (Fig. 1). The basal level of CTGF was
also present in the normal colon and ethanol control. The
expression of the BMP antagonist noggin was increased in
colons of TNBS rats, showing a significant increment compared with controls and was also reduced upon BMP7 therapy.
BMP signaling pathway is preserved in TNBS rat model of
IBD. BMPRs were expressed in all examined samples with a
constitutive expression of BMPRIA, whereas the expression of
BMPRIB was significantly decreased during TNBS colitis and
following BMP7 therapy, compared with the ethanol-treated
rats (Fig. 2). Expression of BMPRII showed an opposite
pattern in BMP7-treated rats with a significantly higher receptor expression level in the chronic stage of colitis.
Analysis of the BMP/Smad signaling system including RSmads (Smad1/5/8) and Co-Smad (Smad4) showed the strongest expression of Smad1, whereas the expression of Smad5
was low (Fig. 3). BMP7 therapy significantly increased the
expression of R-Smads, particularly Smad1 in the acute and
Smad8 in the chronic stage of colitis. R-Smads were significantly decreased in the ethanol-treated rats compared with
TNBS/BMP7-treated animals. Expression of Smad4 was
mostly unchanged throughout the whole observation period,
with the exception of day 5 where a significant decrement in
the TNBS colitis group was found. The TGF-␤-responsive
Smad2 and -3 were increased in the colon of BMP7-treated rats
with significantly increased Smad3 expression on days 5 and
30 (Fig. 3). During TNBS-induced colitis, the expression of
Smad2 and Smad3 showed concurrent enhancement on days 2
and 14 of colitis, which represents the beginning of the acute or
chronic stage of colon inflammation. It was also noticed that
the ethanol control rats showed a significantly lower expression
of Smad2, compared with BMP7-treated rats. Regarding the
inhibitor Smads, BMP7 treatment markedly decreased the
expression level of Smad6 on day 30 and Smad7 on day 14 of
TNBS colitis.
Shift of BMP signaling in the regenerating colon. To further
investigate the activity of BMP signaling pathway in TNBS
colitis, we determined the presence of phosphorylated Smad1,
5, and 8 (pSmad1/5/8). Immunohistochemical staining showed
the presence of pSmad1/5/8 in colons taken at days 2 and 5
after the colitis induction in both diseased and BMP7-treated
colons with different staining patterns (Fig. 4A). On day 2 of
TNBS colitis, pSmad1/5/8-positive staining was present in the
crypt epithelium located in the deeper parts of the mucosa and
was almost absent in the surface epithelium, whereas in BMP7treated animals, pSmad1/5/8-positive staining was observed in
the glandular epithelium of the superficial crypts. Ethanoltreated rats showed a positive staining in the glandular epithelium of colon crypts and in the lamina propria. On day 5,
pSmad1/5/8-positive staining was present in irregularly shaped
crypts and in the superficial epithelium. The BMP7 therapy
resulted in the recovery of crypts, characterized by the formation of numerous crypts with a regular shape. Besides the crypt
and surface epithelium, pSmad1/5/8-positive staining was additionally detected in the lamina propria. The same staining
pattern was observed in the ethanol-treated rats. There was no
difference in the number of pSmad1/5/8-positive cells between
the groups during the acute stage of TNBS colitis. On day 14
of TNBS colitis, the nuclear pSmad1/5/8 staining was localized
G1155
BMP SIGNALING IN TNBS COLITIS
in the crypt epithelium, lamina propria, and submucosa, which
led to significantly higher number of pSmad1/5/8-positive cells
in BMP7-treated rats (95.2 ⫾ 21.2 vs. 55.23 ⫾ 19.12 in TNBS
colitis; P ⬍ 0.001). At a later stage of colitis, pSmad1/5/8
staining was slightly increased in the lining epithelium, probably because of its recovery on day 30. In addition, pSmad1/
5/8 staining was also present in the crypt epithelium and the
lamina propria; however, at this time point it was almost absent
in the submucosa. The ethanol group showed an intense staining in the crypt epithelium and in the lamina propria (56.13 ⫾
19.68 vs. 28.66 ⫾ 10.31 in TNBS control and 32.86 ⫾ 12.31
in TNBS/BMP7 group). In all colon sections, a strong pSmad1/
5/8 staining was present in the enteric plexus located between
the muscle layers (Fig. 4A, arrowhead).
To quantify pSmad1/5/8 in the colon samples on different
days of TNBS colitis, we performed Western blot analysis
from whole colon lysates. The expression of pSmad1/5/8 was
significantly higher in the samples from BMP7-treated rats on
days 5 and 30 from the TNBS group and ethanol control rats
with the exception of day 14 of colitis (Fig. 4B). The recovery
of the BMP signaling activity was observed from day 14 of
TNBS colitis or ethanol-treated rats.
BMP signaling pathway is active in LPMC. To validate the
importance of LPMC in the BMP signal transduction we
examined the expression of pSmad1/5/8 in LPMC isolated
from the colon at different time points of colitis. pSmad1/5/8
were present in all examined samples as a punctuate pattern
localized in the cytoplasm by green immunofluorescence (Fig.
3C). Next, LPMCs extracted from normal and inflamed colons
were stimulated with BMP7 and analyzed for BMP-responsive
Smads by RT-PCR. Smad1/5/8 were barely detectable in
LPMCs in untreated as well as in BMP7-treated cells from
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Fig. 1. Expression of bone morphogenetic proteins (BMPs) and BMP antagonists in the rat
colon following 2,4,6-trinitrobenzene sulfonic
acid (TNBS)-induced colitis and BMP7 treatment. A: RT-PCR analyses of BMP ligands,
CTGF and noggin in BMP7-treated and control
rats on 2nd, 5th, 14th and 30th day following
induction of colitis. B: BMPs and BMP antagonists levels were measured by densitometry from
day 2 to day 30. Data represent mean values ⫾
SD; net intensity is expressed as no. ⫻ 104; n ⫽
6 or 7 rats at each time point. *P ⬍ 0.05 vs.
TNBS group and TNBS/BMP7 group, #P ⬍ 0.05
vs. TNBS group and ethanol control, §P ⬍ 0.05
vs. TNBS/BMP7 group and ethanol control,
†P ⬍ 0.05 vs. TNBS group and normal group,
⫹P ⬍ 0.05 vs. TNBS/BMP7 group and normal
group, ‡P ⬍ 0.05 vs. ethanol control and normal
rats.
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BMP SIGNALING IN TNBS COLITIS
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Fig. 2. Expression of BMP receptors in the rat
colon during TNBS colitis and BMP7 therapy.
A: RT-PCR analyses of BMP receptors type I
(IA and IB; BMPRIA and BMPRIB) and type
II (BMPRII) in the colon from diseased,
BMP7-treated and control rats on 2nd, 5th,
14th, and 30th day following induction of colitis. B: densitometric analysis of expression intensity in colons was measured at different
time point. Data represent values ⫾ SD, net
intensity is expressed as no. ⫻ 104; n ⫽ 6 or 7
rats at each time point. *P ⬍ 0.05 vs. TNBS
group and TNBS/BMP7 group, #P ⬍ 0.05 vs.
TNBS group and ethanol control, §P ⬍ 0.05
vs. TNBS/BMP7 group and ethanol control.
normal colons. As shown in Fig. 1C, Smad1/8 were marginally
detected in unstimulated LPMCs from TNBS animals, whereas
after BMP7 treatment their expression was increased. Smad5
was undetectable in both normal and inflamed colons. In
addition, LPMCs isolated from the colons of both normal and
TNBS rats demonstrated a three times higher cell number 24 h
following BMP7 therapy. This indicates that BMP7 influenced
the proliferation of LPMCs (Fig. 4C).
DISCUSSION
In the present study we showed that BMP-Smad signaling is
altered in the colon during TNBS-induced colonic inflammation and is characterized by a decreased expression of BMPs,
BMPRs, and Smads, except BMP2, BMPRII, and Smad6.
Exogenously applied BMP7 restored the components of the
BMP signaling pathway and decreased the expression of CTGF
and noggin. The activity of the BMP pathway shown by the
presence of pSmad1/5/8 was observed mostly in the crypt
epithelium and the LPMC of the colon.
Several studies have indicated the importance of BMPs in
gastrointestinal development and diseases (27, 28, 48). During intestinal morphogenesis, BMPs are important mediators of numerous epithelial-mesenchymal interactions (57).
Analysis of BMP gene expression in the colon revealed the
presence of Bmp1, 2, 5, 7, and SMAD7 and BmprII in the top
part of the colon crypt (37). Recent studies showed the
perturbance of BMP signaling pathway in hereditary gastrointestinal diseases and the efficacy of growth factor
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BMP SIGNALING IN TNBS COLITIS
therapy along with the conventional treatment of IBD (27,
38). Downregulation of BMP2 and BMP4 because of the
mutation of Fkh/FoxL1 or disruption of Nkx2–3 transcription factors affects the villus morphology because of increased epithelial proliferation (32, 54). Karlsson et al. (34)
observed BMP2 and BMP4 expression in the villus cluster
and subsequent epithelial proliferation limited to the intervillus and prospective crypt regions, whereas our results
show that BMP2 and BMP4 are expressed in the colon of
both, normal and TNBS rats. It was also shown that BMP2
is produced in the gut by epithelial surface and its secretion
is increased by colonic myofibroblasts in the response to the
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Fig. 3. Expression of Smad molecules in rat
the colon during TNBS colitis and BMP7
therapy. A: RT-PCR analyses of Smads
(Smad1, Smad2, Smad3, Smad4, Smad5,
Smad6, Smad7, Smad8) in colon samples
from diseased, BMP7-treated, and control
rats (ethanol control and normal colon) on
2nd, 5th, 14th, and 30th day following induction of colitis. B: densitometric analysis of
the expression intensity were performed.
Data represent mean values (SD), net intensity is expressed as no. ⫻ 104, n ⫽ 6 or 7 rats
at each time point. *P ⬍ 0.05 vs. TNBS
group and TNBS/BMP7 group, #P ⬍ 0.05
vs. TNBS group and ethanol control, §P ⬍
0.05 vs. TNBS/BMP7 group and ethanol
control. ⫹P ⬍ 0.05 vs. TNBS/BMP7 group
and normal group.
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BMP SIGNALING IN TNBS COLITIS
calcium-sensing receptor activation (29, 55). We showed a
marked increase of BMP2 expression in TNBS colitis and
its reduction following BMP7 treatment. Increased expression of BMP2 in TNBS-induced colonic inflammation could
be associated with a reduced sensitivity or loss of inhibitory
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Fig. 4. Expression of pSmad1/5/8 in colon at
different stages of TNBS colitis and BMP7
treatment. A: immunohistochemical analyze of
pSmad1/5/8 in rat colons from normal, TNBS
colitis (a, b, c, d), TNBS/BMP7-treated rats (e,
f, g, h) and ethanol control (i, j, k, l) at different
time points. Dark brown dots indicate the positive pSmad1/5/8 staining in the basal part of
colonic crypts and in myenteric plexus (arrowhead; inset). On the 2nd and 5th day of TNBS
inflammation pSmad1/5/8 staining was limited
to mucosa whereas on the 14th day it was also in
submucosa. On day 30 the pSmad1/5/8 staining
was limited to the lining epithelium. Magnification ⫻10 (a, f) and ⫻20 (b, c, d, e, g, h, i, j,
k, l). B: immunoblot and densitometric analyses
of pSmad1/5/8 in colon during different stages
of IBD. Arrow indicates pSmad1/5/8. Actin
blot shows the relative amount of loaded protein. Results are given as means ⫾ SD (*P ⬍
0.05 vs. TNBS group and TNBS/BMP7 group,
§P ⬍ 0.05 vs. TNBS/BMP7 group and ethanol
control, †P ⬍ 0.05 vs. TNBS group and normal
group, ⫹P ⬍ 0.05 vs. TNBS/BMP7 group and
normal group, ‡P ⬍ 0.05 vs. ethanol control
and normal group). C: expression of BMPspecific receptor-regulated Smads (Smad 1/5/8)
in lamina propria nuclear cells (LPMC) of rat
inflamed colon. RT-PCR analysis was performed on LPMC isolates from colon samples
with TNBS colitis followed by cultivation with
BMP7 (100 ng/ml). Immunofluorescent pattern
of intracellular localization of pSmad1/5/8 in
LPMC was shown; cell cytoplasm was stained
with Evans blue (red fluorescence). Magnification ⫻1,000.
factors, resulting in an increased epithelial cell proliferation.
BMP7 decreased the BMP4 expression in MC3T3-E1 cells
but maintained the capacity for promoting the osteoblastic
phenotype (45), whereas we showed here that BMP7 treatment restored the colonic BMP4 expression in TNBS colitis.
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BMP SIGNALING IN TNBS COLITIS
as its expression is significantly decreased in human
Crohn’s disease and dextran sodium sulfate colitis (2). We
found a decreased expression of Smad5 during colitis.
Additionally, in LPMC isolated from inflamed colons, the
expression of BR-Smads following BMP7 therapy was more
pronounced except for the Smad5. In the TNBS colitis we
found a preserved but altered activity of BMP signaling
(pSmad1/5/8), which was improved in the chronic stage and
recovered by BMP7 therapy. Fiocchi (20) described the
intestinal inflammation as an interaction between immune
and nonimmune cells including epithelial, endothelial, mesenchymal, and nerve cells, with nonimmune cells as an
important factor of the inflammatory response. The presence
of pSmad 1/5/8 in the crypt epithelium and the lamina
propria cells as well as in isolated LPMC revealed the
preserved activity of BMP pathway in both the nonimmune
and immune intestinal cells during the TNBS colitis. The
findings of pSmad 1/5/8 in the enteric plexus pointed to
another possible effect of preserved BMP pathway to arrest
the smooth muscle dysfunction, which was observed in
TNBS colitis as the outcome of both oxidative stress and
proinflammatory cytokines, specifically IGF-1 and TGF-␤
(58). In vivo regulation of Smad molecules is dependent not
only on the ligand application but also on the BMP pathway
modulation by RAS/ERK, TLR-IL-1R, or Wnt pathways (8,
15, 30, 50). It was also shown that Dragon, repulsive
guidance molecule (RGM) family member, enhanced the
BMP signaling activity and decreased the IL-6 expression in
a BMP-ligand-dependent manner via a non-Smad pathway,
like p38MAPK and Erk1/2 (70). Our data show that Smad4
is continuously present during the experimental colitis,
suggesting that Smad4 is essential for the transcriptional
Smad complex although the TGF-␤ signaling independently
of Smad4 might be possible (17).
The importance of TGF-␤1 is well known in the pathogenesis of IBD and its lack is characterized by an inadequate
immune response. We have previously reported that TGF-␤
is upregulated in TNBS model of colitis (44) although
TGF-␤ as a negative regulator of T cell response should
have reduced the intestinal inflammation in IBD (7, 9, 49,
50). TNBS inflammation is characterized by hypertrophy of
smooth muscle cells in muscularis externa induced by an
enhanced level of TGF-␤ and IGF1 (58). The disruption of
TGF-␤/Smad signaling pathway in IBD is characterized by
high expression of Smad7 and reduced Smad3 phosphorylation, leading to an inadequate inflammatory cell response
to TGF-␤, and a persisting inflammation, whereas Smad7
inhibition leads to its restoration (7, 49, 50). In Smad3
knockout mice there is an increased staining of TGF-␤1 and
Smad7 in the intestine with an absence of macroscopic
lesions (75) and a reduction of fibrosis (24, 40, 63). We
found reduced expression of TGF-␤ R-Smads in TNBS
colitis, especially Smad3, which is significantly increased
following BMP7 therapy. Smad7 expression was significantly
increased in colons from TNBS rats on day 14 with a prominent
decrease after BMP7 therapy. Therefore, we suggest that BMP7
affects healing by inhibition of TGF-␤1, rather than via a reduction of the TGF-␤/Smad signaling pathway (23, 44). In addition,
BMP7 downregulates the expression level of inhibitory Smads,
which supports the observation that blocking Smad7 is the key
event in the regulation of inflammation in IBD (49).
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BMP6 shares some identical features with BMP7, like the
affinity to BMP receptors and mechanisms for osteoblastic
differentiation of human mesenchymal stem cells (41, 48,
66). BMP6 is present in the gastrointestinal smooth muscle
and in the pancreas during development and its misexpression leads to agenesis of the pancreas and reduced stomach
size (19, 56). The liver is probably the source of endogenous
BMP6, which regulates the expression of hepcidin and iron
absorption in the gut regulating its concentration in the
serum (3, 66). Also, BMP-hepcidin axis is a potential
therapeutic target in IBD because it is upregulated during
the disease (73). Blocking hepcidin expression by antiBMP6 reagents leads to restoring IBD-associated anemia
and to decreased IL-17 expression. Overexpression of
BMP7 accompanied with several profibrotic growth factors
was found in strictured segments of human Crohn’s disease
intestine, whereas in the experimental model of IBD the
antifibrotic property of BMP7 had been observed (9, 23).
Our results showed that both BMP6 and BMP7 were expressed in the normal colon. Expression of BMP7 in the
acute stage and BMP6 in the chronic stage of colitis are
lower in these respective stages of colitis and were improved by BMP7 therapy. This finding suggested a synergistic and/or complementary effect of BMP6 and -7 during
healing. It is known that BMP6 has 20 times higher affinity
to BMPRIA and is more resistant to noggin inhibition than
BMP7 (61).
CTGF was identified as being frequently upregulated in IBD
and diabetic nephropathy, which are accompanied by fibrosis
(9, 53). It was shown that upregulated CTGF inhibits BMP4
and BMP7 activity leading to deficiency of BMP signaling (1,
53, 64). Also, noggin has a reciprocal relationship with BMP2
in colonic myofibroblasts and coexpression with BMP4 in
atherosclerotic lesions of human coronary arteries (13, 55).
Overexpression of noggin caused the absence of Smad 1/5/8
signaling and a disease similar to JP, whereas its downregulation showed opposite effect on different types of arthritis (27,
43). We found significantly increased CTGF and noggin expression during the acute stage of IBD colitis and their reduction by BMP7. CTGF expression was concomitant with BMP4
expression, whereas a correlation of noggin and examined
BMPs was not observed. A balance between different BMPs
and their antagonists is present in the normal gut but is also
subject to change upon inflammation.
JP is an another disease associated with the dysfunction of
BMP signaling, primarily Smad4 and BMPRIA (11, 32).
The inhibition of BMP signaling by transgenic expression of
noggin or BMPRII in mice resulted in a disease similar to JP
(5, 27). We found preserved expression of BMP receptors in
colon samples, especially BMPRIA, whose expression is
unchanged and uniform during the TNBS colitis, and upon
BMP7 treatment. The BMPRIB and BMPRII expression is
significantly decreased during the acute phase of colitis and
normalized following BMP7 treatment. Persistence of
BMPR expression in TNBS-induced colon inflammation
suggested that the BMP signaling variations in IBD are
reversible. BR-Smads were present in the colon during
TNBS colitis, showing lower expression, and were maintained by the BMP7 therapy, which is in accordance with the
results of Li et al. (42), showing that the loss of Smad5 leads
to an increased susceptibility to experimental colitis, where-
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BMP SIGNALING IN TNBS COLITIS
ACKNOWLEDGMENTS
We thank Lara Baticic, PhD, and Kristina Pilipovic, MD, for assistance
with the Western blot and densitometric analysis, Andrica Lekic for statistical
analysis, as well as Jelena Djirlic for assistance with the immunofluorescenece.
We also thank Snjezana Simac, Marija Mrsic, Djurdja Car, and Mirjana-Marija
Palcic for excellent technical help.
AUTHOR CONTRIBUTIONS
I.M. designed and performed the research and wrote the first draft of the
manuscript; N.K. performed experiments and contributed to the analysis of
data; T.T.W. and I.S performed experiments and assisted in writing of the
manuscript; B.G. and S.Z.C. analyzed and interpreted the data; T.C. performed
experiments; D.B. helped in designing experiments and wrote parts of the
manuscript; S.V. initiated the project, critically reviewed and revised the
manuscript for important intellectual content, and finally approved the manuscript.
GRANTS
This work was supported by grant from the Ministry of Science, Education
and Sports of the Republic of Croatia (062-0620226-0209).
DISCLOSURES
The authors declare that they have no conflict of interest, financial or
otherwise.
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We have previously demonstrated that BMP7 has an
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