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ESPS Manuscript NO: 7356
Columns: ORIGINAL ARTICLE
Resveratrol inhibits collagen I synthesis by suppressing IGF-1R activation in intestinal fibroblasts
Li P et al. Resveratrol effect on collagen I synthesis
Ping Li, Mei-Lan Liang, Ying Zhu, Yao-Yao Gong, Yun Wang, Ding Heng, Lin Lin
Ping Li, Ying Zhu, Yao-Yao Gong, Yun Wang, Ding Heng, Lin Lin, Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Mei-Lan Liang, Nanping First Hospital Affiliated to Fujian Medical University, Nanping 353000, Fujian Province, China
Author contributions: Li P and Lin L designed research; Li P, Liang ML, Zhu Y performed research; Gong YY, Wang Y, Heng D contributed new reagents or analytic tools; Li P analyzed data; Li P and Lin L wrote the paper.
Supported by the National Natural Science Foundation of China, No. 81270462; The International Cooperation Project of Jiangsu Province Department of Health, SBZ201100103
Correspondence to: Lin Lin, MD, PhD, Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing 210029, Jiangsu Province, China. lin9100@aliyun.com
Telephone: +86-25-83718836-6920 Fax: +86-25-83674636
Received: November 14, 2014 Revised: January 13, 2014
Accepted: February 20, 2014
Published online:
Abstract
AIM: To investigate whether resveratrol (3,4,5-trihydroxy-trans-stilbene) inhibits collagen I synthesis induced by insulin growth factor-1 (IGF-1) in intestinal fibroblasts, and to explore the possible molecular mechanisms.
METHODS: Male Sprague-Dawley rats were randomly divided into two groups: control group and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis group. After 21d of TNBS administration, the degree of inflammation and fibrosis in colon were measured by HE staining and Massons trichrome staining. First, we used western blot to examine collagen I, IGF-1 and silent information regulator 1 (SIRT1) protein expressions in colitis tissues. Then, Western blot and quantitative real-time polymerase chain reaction were used to characterize collagen I protein and col1a2 mRNA expression in mouse intestinal fibroblasts and CCD-18Co cells treated with IGF-1. MEK inhibitor (UO126) was used to determine whether IGF-1-induced collagen I expression was mediated by extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent mechanism. Effects of resveratrol on collagen I protein level, insulin growth factor-1 receptor (IGF-1R) and ERK1/2 phosphorylation levels were also examined after IGF-1 treatment in fibroblasts. To evaluate whether SIRT1 was necessary for the anti-fibrosis effect of resveratrol, cells were transfected with SIRT1 small interfering RNA, wild-type SIRT1, deacetylase-inactive mutant SIRT1.
RESULTS: Collagen I and IGF-1 expression was increased, SIRT1 expression is decreased (0.67 EMBED Equation.3 0.04 vs 1.05 EMBED Equation.3 0.07, P < 0.001) in TNBS-induced Crohns disease intestinal fibrosis rats compared with the control group. In vitro, IGF-1 could induce collagen I expression, mainly through the ERK 1/2 signal pathway. Resveratrol reduced basal and IGF-1-induced collagen I gene and protein expression in intestinal fibroblasts. Overexpression of wild-type SIRT1, not deacetylase-inactive mutant SIRT1 decreased expression of collagen I induced by IGF-1. Moreover, silencing SIRT1 restored collagen I expression in fibroblasts challenged with resveratrol. However, disruption of SIRT1 did not influence the anti-fibrotic effects of resveratrol in IGF-1-induced collagen I expression. Further analysis revealed that resveratrol significantly decreased phosphorylation of IGF-1R and its downstream signaling molecules by inhibiting IGF-1 binding to its receptor.
CONCLUSION: Our data suggest that resveratrol effectively inhibits collagen I synthesis in IGF-1-stimulated fibroblasts, partly by inhibiting IGF-1R activation and SIRT1 is also responsible for the process.
2014 Baishideng Publishing Group Co., Limited. All rights reserved.
Key words: Intestinal fibrosis; Insulin-like growth factor-1; Resveratrol; Silent information regulator 1; Fibroblasts
Core tip: This study showed that the expression of silent information regulator 1 (SIRT1) was decreased in 2,4,6-trinitrobenzenesulfonic acid-induced colitis tissues, and resveratrol down-regulated insulin growth factor (IGF)-1-induced collagen I synthesis by inhibiting IGF-1 receptor (IGF-1R) phosphorylation and its downstream extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway in intestinal fibroblasts. Resveratrol alone suppressed collagen I synthesis through up-regulating activity of SIRT1. Our data highlight a previously resveratrol unknown function on IGF-1R activation and provide novel insight of resveratrol as a therapeutic role in intestinal fibrosis.
Li P, Liang ML, Zhu Y, Gong YY, Wang Y, Heng D, Lin L. Resveratrol inhibits collagen I synthesis by suppressing IGF-1R activation in intestinal fibroblasts. World J Gastroenterol 2014;
Available from: URL: http://www.wjgnet.com/esps/
DOI: DOI: 10.3748/wjg.v20.i0.0000
INTRODUCTION
Intestinal fibrosis is a common and serious complication of Crohns disease (CD) and increases the risk of intestinal stenosis or obstruction, ultimately leads to surgical intervention at substantial personal and economic cost ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_1" \o "Cosnes, 2002 #10054" 1-3]. Until now, no effective therapy exists for averting such fibrogenic events, because the fibrotic process becomes autopropagative and fails to respond to antiinflammatory interventions ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_4" \o "Froehlich, 2007 #6204" 4, HYPERLINK \l "_ENREF_5" \o "Van Assche, 2004 #6265" 5].
The pathological process of intestinal fibrosis is characterized by mesenchymal cell proliferation and extracellular matrix (ECM) accumulation in interstitial space ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_6" \o "Rieder, 2008 #405" 6, HYPERLINK \l "_ENREF_7" \o "Pucilowska, 2000 #6298" 7]. Collagens I is one of the major matrix molecules involved in intestinal fibrogenesis. The activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is one of the major downstream signaling events that participate in collagen I synthesis ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_8" \o "Svegliati-Baroni, 1999 #13673" 8, HYPERLINK \l "_ENREF_9" \o "Papakrivopoulou, 2004 #13674" 9]. Insulin-like growth factor 1 (IGF-1) is a potent profibrogenic agent involved in intestinal remodeling ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_10" \o "Lawrance, 2001 #5925" 10, HYPERLINK \l "_ENREF_11" \o "Szabo, 2010 #6181" 11]. Laboratory- and population-based studies have shown that expression of IGF-1 in Crohns disease patients is significantly increased and IGF-1 receptor (IGF-1R) is also altered in the intestines of ulcerative colitis and Crohns disease patients A D D I N E N . C I T E A D D I N E N . C I T E . D A T A [ H Y P E R L I N K \ l " _ E N R E F _ 1 2 " \ o " P u c i l o w s k a , 2 0 0 0 # 1 2 7 1 6 " 1 2 - 1 4 ] . I G F - 1 b i n d i n g t o I G F - 1 R a l l o w s t h e - s u b u n i t s o f I G F - 1 R t o d i s p l a y i n t r i n s i c t y r o s i n e k i n a s e a c t i v i t y a n d a c t i v a t e s d o w n s t r e a m s i g n a l s v i a p h o s p h o rylation of key proteins including phosphatidylinositol 3-kinase and mitogen-activated protein (MAP) kinase (MAPK) ADDIN EN.CITE Kuemmerle201210058100581005817Kuemmerle, J. F.Department of Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0341, USA. john.kuemmerle@vcu.eduInsulin-like growth factors in the gastrointestinal tract and liverEndocrinol Metab Clin North AmEndocrinology and metabolism clinics of North AmericaEndocrinol Metab Clin North Am409-23, vii412AnimalsApoptosisCell ProliferationCell SurvivalGastrointestinal Neoplasms/metabolismGastrointestinal Tract/*metabolism/pathologyHumansInflammation/metabolismInsulin-Like Growth Factor Binding Proteins/geneticsLiver/*metabolismMiceRatsReceptor, IGF Type 1/genetics/metabolismSomatomedins/genetics/*metabolism2012Jun1558-4410 (Electronic)
0889-8529 (Linking)22682638http://www.ncbi.nlm.nih.gov/pubmed/22682638337286810.1016/j.ecl.2012.04.018[ HYPERLINK \l "_ENREF_15" \o "Kuemmerle, 2012 #4266" 15]. IGF-1 not only stimulates proliferation and inhibits apoptosis of fibroblasts and myofibroblasts, but also increases collagen expression and production in intestinal smooth muscle cells ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_16" \o "Xin, 2004 #1859" 16, HYPERLINK \l "_ENREF_17" \o "Kuemmerle, 2005 #10059" 17]. However, collagen-producing fibroblasts and myofibroblasts are central cell types in intestinal fibrogenesis. There is no data show the effect of IGF-1 on collagen I in intestinal fibroblasts.
Resveratrol (3,4,5-trihydroxy-trans-stilbene) is a polyphenol naturally occurring in grapes and red wine that exhibits beneficial health effects such as extending the life span, regulating tumor growth and oxidation. Resveratrol activates silent information regulator-1 (SIRT1), a nicotinamide adenine dinucleotide-dependent deacetylase, which has many biological functions by deacetyla t i n g a n u m b e r o f k e y t r a n s c r i p t i o n f a c t o r s , i n c l u d i n g p 5 3 , n u c l e a r f a c t o r - B ( N F - B ) , a n d p e r o x i s o m e p r o l i f e r a t o r - a c t i v a t e d r e c e p t o r g a m m a c o - a c t i v a t o r - 1 A D D I N E N . C I T E A D D I N E N . C I T E . D A T A [ H Y P E R L I N K \ l " _ E N R E F _ 1 8 " \ o " C h u n g , 2 0 1 0 # 4 5 0 5 " 1 8 ] . I n addition, resveratrol also has dramatic antifibrotic effect in rodent models of renal fibrosis ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_19" \o "Li, 2010 #12731" 19, HYPERLINK \l "_ENREF_20" \o "Kurus, 2009 #1260" 20], cardiac fibrosis ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_21" \o "Sutra, 2008 #1264" 21], and hepatic fibrosis ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_22" \o "Lee, 2010 #1253" 22, HYPERLINK \l "_ENREF_23" \o "Chavez, 2008 #1274" 23], but the molecular mechanism(s) are currently unknown.
The protective role of resveratrol in colitis has been demonstrated in model of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulphonic acid (TNBS) ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_24" \o "Youn, 2009 #291" 24-26]. Resveratrol may protect against colitis through up-regulation of SIRT1 in immune cells, which functioning as an inverse regulator of NF- 8B a c t i v a t i o n a n d i n f l a m m a t i o n i n t h e c o l o n A D D I N E N . C I T E A D D I N E N . C I T E . D A T A [ H Y P E R L I N K \ l " _ E N R E F _ 2 5 " \ o " S i n g h , 2 0 1 0 # 2 9 3 " 2 5 ] . M o u n t i n g e v i d e n c e s u g g e s t s r e s v e r a t r o l a l s o h a s a n t i f i b r o t i c e f f e c t i n t h e p e p t i d o g l y c a n - p o l y s a c c h a r i d e r a t m o d e l of Crohn's disease and can diminish IGF-1-stimulated collagen production in intestinal smooth muscle cells ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_27" \o "Garcia, 2012 #1242" 27, HYPERLINK \l "_ENREF_28" \o "Rahal, 2012 #182" 28]. However, the mechanism of resveratrol inhibiting collagen synthesis and IGF-1-induced collagen production has not been established, nor whether resveratrol inhibiting collagen expression through augmentation of SIRT1 activity in intestinal fibroblasts. The aim of this study was to investigate the effect of resveratrol on collagen I synthesis in intestinal fibroblasts and to explore the possible molecular mechanisms.
MATERIALS AND METHODS
Animals and induction of colitis
The technique for induction of colitis with 2,4,6 trinitrobenzene sulphonic acid (TNBS; Sigma) was as described previously ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_29" \o "Hazelgrove, 2009 #13947" 29]. Male SD rats (200-250 g) were purchased from and maintained in the Animal Center of Nanjing Medical University (Nanjing, China). To induce chronic fibrotic colitis, rats were fasted for 24 h, lightly anesthetized with diethyl ether, and TNBS solution [2.5% in 50 % ethanol (v/v)] was injected via a catheter advanced to 8 cm proximal to the anus. In order to distribute the TNBS within the colon, the rat was kept in a vertical position with the head downwards for 3 min after the injection. All rats were checked daily for loss of body weight, stool consistency, and the presence of gross bleeding. The disease activity index (DAI) was calculated as a sum of the scores of the three parameters according to the scoring criteria as described above. Animals were sacrificed after 21 d and body weight, colon weight, colon length were recorded. HE staining and Massons trichrome staining were used to measure the degree of inflammation and fibrosis in colon by microscopy.
Fibroblast isolation and culture
Mouse intestinal fibroblasts (MIFs) were isolated and cultured as described previously ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_30" \o "Leeb, 2002 #12747" 30, HYPERLINK \l "_ENREF_31" \o "Leeb, 2003 #12748" 31]. The intestine tissue isolation from Balb/c mice (7 d) was cut into 1-mm pieces. Epithelial cells were removed in Hanks Balanced Salt Solution without Ca2+and Mg2+ with 2 mmol/L EDTA. The remaining tissue was rinsed and then digested for 30 minutes at 37C with 1 mg/mL collagenase II, 0.3 mg/mL DNase I in PBS. The isolated cells were cultured i n 2 5 - c m 2 c u l t u r e f l a s k s ( C o r n i n g ) w i t h D u l b e c c o ' s M o d i f i e d E a g l e ' s M e d i u m ( D M E M ) c o n t a i n i n g 1 0 % f e t a l b o v i n e s e r u m ( F B S , H y c l o n e ) , 1 0 g p e n i c i l l i n / s t r e p t o m y c i n ( G i b c o ) . N o n a d h e r e n t c e l l s w e r e r e m o v e d b y s u b s e q u e n t c h a n g e s o f m e d i u m a f t e r 2 h . T h e r e m a i n i n g c e l l s w e r e c h a r a c t e r i z e d b y i m m u n o c y t o c h e m i s t r y s t a i n i n g p r o p e r t i e s f o r v i m e n t i n ( 1 : 2 0 0 ) a n d - s m o o t h m u s c l e a c t i o n ( 1 : 2 0 0 ) a s p r e v i o u s l y d e s c r i b e d A D D I N E N . C I T E A D D I N E N . C I T E . D A T A [ H Y P E R L I N K \ l " _ E N R E F _ 3 1 " \ o " L e e b , 2 0 0 3 # 1 2 7 4 8 " 3 1 ] . F o r a l l e x p e r i m e n t s , f i b r o b l a s t s w e r e u s e d b e t w e e n p a s s a g e s 3 a n d 8 , a n d w e r e t r e a t e d w i t h r e c o m b i n a n t m o u s e I G F - 1 ( 1 0 0 n g / m L , R D ) o r R e s v e r a t r o l ( 5 0 , 1 0 0 m o l / L , s i g m a ) .
C e l l c u l t u r e a n d t r e a t m e n t
C C D - 1 8 C o c e l l s ( C R L 1 4 5 9 ) a t p a s s a g e 6 w e r e o b t a i n e d f r o m A m e r i c a n T y p e C u l t u r e C o l l e c t i o n a n d u s e d b e t w e e n p a s s a g e s 8 a n d 1 5 . C e l l s w e r e g r o w n i n D M E M s u p p l e m e n t e d w i t h 1 0 % F B S , a n d 1 0 g p e n i c i l l i n / s t r e p t o m y c i n . C e l l s w e r e m a i n t a i n e d a t 3 7 C i n a 5 % C O 2 i n c u b a t o r . C e l l s w e r e t r e a t e d w i t h r e c o m b i n a n t h u m a n I G F - 1 ( 1 0 0 n g / m L , R D ) o r r e s v e r a t r o l ( 5 0 , 1 0 0 m o l / L , S i g m a ) f o r 2 4 h a s i n d i c a t e d .
P l a s m i d s a n d t r a n s i e n t t r a n s f e c t i o n
W T a n d d e a c e t y l a s e - i n a c t i v e m u t a n t S I R T 1 ( H 3 6 3 Y ) c o n s t r u c t s w e r e a g i f t f r o m D r . Y o n g X u ( N a n j i n g M e d i c a l U n i v e r s i t y , N a n j i n g , C h i n a ) a n d h a v e b een described previously ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_32" \o "Wu, 2011 #16705" 32]. Silencing of SIRT1 were mediated by small interfering RNA (siRNA) using the following sequences: for human SIRT1, 1: 5-CGGGAAUCCAAAGG AUAAUTT-3, 2: 5-CCAUCUCUCUGUCACAAAUTT-3 and 3: 5-CCAAGCAGCUA AGAGUAAUTT-3. CCD-18Co cells were transfected at 30%-40% confluency using either Lipofectamine 2000 or Lipofectamine RNAiMAX (Invitrogen). At 24 h post-transfection, cells were treated with I G F - 1 f o l l o w e d b y t r e a t m e n t w i t h s e r u m - f r e e m e d i u m f o r 1 2 h . F o r s i S I R T 1 e x p e r i m e n t s , c e l l s a t 4 8 h p o s t - t r a n s f e c t i o n w e r e t r e a t e d f o r a n a d d i t i o n a l 2 4 h w i t h e i t h e r 1 0 0 m o l / L r e s v e r a t r o l o r 1 0 0 n g / m L I G F - 1 .
W h o l e - c e l l p r o t e i n e x t r a c t i o n a n d W e s t e r n b l o t
Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1%Triton X-100) with freshly added protease inhibitor and phosphatase inhibitor tablet (Roche). Cells lysates were subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore) using a Semi-Phor system (Bio-Rad). After blocking in PBS containing 5% non-fat dry milk, blots were incubated with primary antisera for overnight at 4 C, washed in PBS containing 0.05% Tween, and then incubated with peroxidase conjugated secondary antibodies for 30 min at RT. Immunoreactive proteins were identified using the ECL detection system. Antibodies against collagen type I was obtained from Rockland. IGF-1R, phosphorylated IGF-IR, ERK1/2, phosphorylated ERK1/2 and MEK inhibitor (UO126) were purchased from Cell Signaling Technology. - a c t i n ( 1 : 1 0 0 0 ) a n d G A P D H w a s p u r c h a s e d f r o m b i o w o r l d e , a n d S I R T 1 m o n o c l o n a l a n t i b o d y ( 1 : 1 0 0 0 ) w a s f r o m a b c a m .
R N A i s o l a t i o n a n d r e a l - t i m e p o l y m e r a s e c h a i n r e a c t i o n
T o t a l R N A w a s e x t r a c t e d f r o m c e l l s g r o w n i n 6 0 - m m t i s s u e c u l t u r e d i s h e s ( C o r n i n g ) u s i n g the TRIzol Reagent (Gibco), according to manufacturers instructions. Reverse transcriptase reactions were performed using PrimeScript RT Master Mix and (Takara). Real-time PCR reactions were performed using SYBR-Green PREMIX EX TAQ (Takara) on an ABI Prism 7500 system. The primers used for real-time reactions were presented. (1) mouse col1a2, forward primer (5'-GGAGGGAACGGTCCACGAT-3') and reverse primer (5'-GAGTCCGCGTATCCACAA-3'); (2) mouse col1a1, forward primer (5'-CCGGCTCCTGCTCCTCTTA-3') and reverse prim e r ( 5 ' - C C A T T G T G T A T G C A G C T G A C T T C - 3 ' ) ; ( 3 ) m o u s e - a c t i n , f o r w a r d p r i m e r ( 5 ' - C A T C G T G G G C C G C T C T A - 3 ' ) a n d r e v e r s e p r i m e r ( 5 ' - C A C C C A C A C A T A G G A G T C C T T C T G - 3 ' ) ; ( 4 ) h u m a n c o l 1 a 2 , f o r w a r d p r i m e r ( 5 ' - G C C C C C C A G G C A G A G A - 3 ' ) a n d r e v e r s e p r i m e r ( 5 ' - C C A A C T C C T T T T C C A T C A T A C T G A-3'); (5) human col1a1, forward primer (5'-ACGAAGACATCCCACCA ATC-3') and reverse primer (5'- GCACCATCCAAACCAC TGA-3'); (6) human sirt1, forward primer (5'-TGAGGCACTTCATGGGGTATGG-3') and reverse primer (5'-TCCTAGGTTGCCCAGCTGATGAA-3'); and (7) human GAPDH, forward primer (5'-GAAATCCCATCACCATCTTCCAGG-3') and reverse primer (5'-GAGCCCCAGCCT TCTCCATG-3').
Statistical analysis
The results are expressed as the mean SD. The SPSS statistical package (version 14.0; SPSS Inc, Chicago, IL, United States) was used for statistical analysis. The differences between the two groups were analyzed using Students t test. Unless otherwise specified, P values smaller than 0.05 were considered statistically significant.
RESULTS
SIRT1 expression is decreased in TNBS-induced CD intestinal fibrosis
We initially tested the expression of collagen I and SIRT1 in CD fibrosis rat colon induced by TNBS. Compared with control normal rats, the DAI, body weight and colon weight showed significant increase, and colon length decrease in TNBS-treated rats (Figure 1A). Paraffin-embedded colonic tissue samples from TNBS-treated and control rats were assessed for inflammation and fibrosis after labeling with HE and Massons trichrome staining (MTS). TNBS-treated rats showed significant colitis marked by submucosa thickening, epithelial layers destruction, lymphocyte infiltration on HE (Figure 1B). The sections labeled with MTS displayed diffuse extracellular matrix deposition and fibrosis in the mucosa and submucosa (Figure 1B). Histologic scores for both inflammation and fibrosis were greater in TNBS-induced colitis than normal rats (Figure 1B).
Furthermore, western blotting showed that collagen I protein level was increased in CD rats compared to normal rats (Figure 1C). IGF-1 protein expression was also increased in CD rats, whereas SIRT1 was decreased (Figure 1C). These data indicate that resveratrol may protect against intestinal fibrosis through stimulating SIRT1 expression.
IGF-1-induced expression of collagen I is dependent on MAPK in intestinal fibroblasts
Since earlier studies document that IGF-1 attenuates inflammation and exacerbates intestinal fibrosis, we chose to investigate the possible mechanism underlying IGF-1 induced collagen I synthesis in intestinal fibroblasts.
We first examined the expression of collagen I in both MIFs and CCD-18Co cells with increasing concentrations (50, 100 or 150 ng/mL) of IGF-1 treatment for 24 h. IGF-1 potently increased both col1a2 mRNA levels (Figure 2A) and protein levels (Figure 2B) of collagen I in a dose-dependent manner. IGF-1 treatment for 24 h showed a maximal effect on collagen I expression. In addition, IGF-1 also induced col1a1 mRNA expression (Figure 2A and B).
To investigate the molecular mechanism underlying the induction of collagen I expression by IGF-1, we measured the phosphorylation of IGF-1R and ERK1/2 in response to IGF-1 treatment. IGF-1 significantly increased levels of phospho-IGF-1R and ERK1/2 in a time-dependent manner in both MIFs and CCD-18Co cells, the most prominent was a t 3 0 m i n ( F i g u r e 2 C ) . F i b r o b l a s t s w e r e p r e t r e a t e d w i t h U 0 1 2 6 ( 5 0 m o l / L ) f o r 1 h t o b l o c k M E K 1 / 2 p h o s p h o r y l a t i o n a n d t h e n c o i n c u b a t e d w i t h I G F - 1 ( 1 0 0 n g / m L ) a n o t h e r 2 4 h . T h e a b i l i t y o f I G F - 1 t o i n c r e a s e c o l l a g e n I e x p r e s s i o n w a s s i g n i f i c a n t l y i n h i b i t e d by MAP-kinase inhibitor (Figure 2D). Taken together, these data suggest that IGF-1 inhibited collagen I synthesis in fibroblasts through the IGF-1/IGF-1R/MAP-kinase pathway.
Resveratrol treatment abrogated collagen I synthesis partly through SIRT1
Since we observed an alteration in the decreased expression of SIRT1 versus IGF-1 increasing compared with deposition of collagen I in colonic tissues of CD rats, we evaluated the effect of SIRT1 activator, resveratrol on collagen I synthesis in intestinal fibrob l a s t s a n d e l u c i d a t e d t h e u n d e r l y i n g m o l e c u l a r m e c h a n i s m s .
A s s h o w n i n F i g u r e 3 A a n d B , r e s v e r a t r o l m a r k e d l y d e c r e a s e d c o l l a g e n I p r o t e i n a n d m R N A l e v e l s i n d u c e d b y I G F - 1 a t 1 0 0 m o l / L c o n c e n t r a t i o n . I n a d d i t i o n , r e s v e r a t r o l a l o n e a l s o i n h i b i t e d c o l l a g e n I synthesis (Figure 3B).
Next, to verify whether SIRT1 is required for resveratrol induced repression of collagen I, we performed the following experiments. First, fibroblasts were transfected with SIRT1 expression constructs (WT or HY) followed by IGF-1 treatment, collagen I protein level was assessed. Overexpression of WT, but not enzyme deficient (HY) SIRT1, markedly decreased IGF-1-induced collagen I synthesis (Figure 3C). Overexpression of SIRT1 WT also led to the reduction of collagen I (Figure 3D). Second, we also used a specific siRNA targeting SIRT1 transfected into fibroblasts. To confirm the efficiency of SIRT1 siRNA knockdown, 293T cells were transiently transfected with the SIRT1 siRNA (siSIRT1 1, 2, 3) or scrambled siRNA by transfection reagent, and total cell lysates were prepared 48 h after transfection and used for Western blotting using anti-SIRT1 antibody or control anti-GAPDH antibody (Figure 3E). As shown in Figure 3F, depletion of SIRT1 by siRNA (1 and 3) in CCD-18Co cells blocked the reduction of collagen I expression induced by resveratrol. However, there was no effect of resveratrol on collagen I induced by IGF-1 (Figure 3F). Collectively, these results clearly documented that SIRT1 partly involved in the resveratrol-dependent repression of collagen I.
Resveratrol inhibits IGF-1-induced phosphorylation of IGF-1R and ERK1/2 independent on activating SIRT1
Our data so far demonstrate that IGF-1 promotes collagen I synthesis through MAPK pathway, and resveratrol down-regulates collagen I expression induced by IGF-1. To investigate whether resveratrol suppressed the IGF-1/IGF-1R/ERK1/2 pathway, we next probed the effect of resveratrol on IGF-1R expression and phosphorylation with or without IGF-1 in both MIFs and CCD-18Co cells.
Resveratrol remarkably inhibited the phosphorylation of IGF-1R and ERK1/2 stimulated by IGF-1 for 30 min, and had no effect on the expression of total IGF-1R and ERK1/2 (Figure 4A), suggesting that resveratrol reduces IGF-1R activity and the intracellular ERK signaling cascade and thereby enhances the expression of collagen I. In addition, overexpression of WT or HY SIRT1 had no effect on phosphorylation of IGF1R (Figure 4B), and we did not observe any significant difference between SIRT1 WT and HY-transfected cells (Figure 4B). Since binding of IGF-1 to the IGF-1 receptor results i n a u t o p h o s p h o r y l a t i o n o f t h e r e c e p t o r s u b u n i t s , a n d i n c r e a s e d r e c e p t o r t y r o s i n e k i n a s e a c t i v i t y , w e f u r t h e r e x a m i n e d w h e t h e r r e s v e r a t r o l a f f e c t s t h e b i n d i n g o f I G F - 1 t o t h e I G F - 1 R . C C D - 1 8 C o c e l l s w e r e p r e t r e a t e d w i t h r e s v e r a t r o l ( 1 0 0 m o l / L ) f o r 2 4 h t o induce SIRT1 expression, then removed and incubated with IGF-1 alone for another 30 min (Figure 4C, Line 4) or IGF-1 and resveratrol for 30 min (Figure 4C, Line 5), the phosphorylation of IGF-1R and ERK1/2 were tested by immunoblot analysis. The results showed that phosphorylation levels of IGF-1R were down-regulated only after treatment with IGF-1 plus resveratrol for 30 min (Line 5), compared with Line 4. In other words, resveratrol inhibited IGF-1R phosphorylation only when resveratrol was incubated together with IGF-1. Combined, these data suggest that the repression of collagen I synthesis mediated by resveratrol can be partly attributed to the inhibition of IGF-1R/ERK1/2 signaling in a SIRT1 independent mechanism, and resveratrol may inhibited IGF-1 binding to its receptor.
DISCUSSION
Intestinal fibrosis in the form of fibrotic strictures is a well described complication of longstanding Crohns disease ADDIN EN.CITE Fiocchi199814844148441484417Fiocchi, C.Division of Gastroenterology, University Hospitals of Cleveland, Case Western Reserve University School of Medicine, Ohio, USA.Inflammatory bowel disease: etiology and pathogenesisGastroenterologyGastroenterologyGastroenterologyGastroenterology182-2051151Antibodies, Antineutrophil Cytoplasmic/physiologyCytokines/physiologyHumansInflammatory Bowel Diseases/*etiology/genetics/immunologyIntestines/metabolism/microbiologyLymphocyte ActivationNeutrophils/physiologyPermeabilityReceptors, Antigen, T-Cell/physiologySmoking/adverse effectsT-Lymphocytes/immunology1998Jul0016-5085 (Print)
0016-5085 (Linking)9649475http://www.ncbi.nlm.nih.gov/pubmed/9649475[ HYPERLINK \l "_ENREF_33" \o "Fiocchi, 1998 #14844" 33]. Currently, there are no inflammatory bowel disease therapies that have been shown to effectively decrease fibrosis, leaving surgery as the only treatment for symptomatic strictures ADDIN EN.CITE Froehlich200762046204620417Froehlich, F.Juillerat, P.Mottet, C.Pittet, V.Felley, C.Vader, J. P.Gonvers, J. J.Michetti, P.Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland. florian.froehlich@bluewin.chFibrostenotic Crohn's diseaseDigestionDigestionDigestionDigestion113-5762Catheterization/methodsCrohn Disease/*complications/surgeryDigestive System Surgical Procedures/methodsEndoscopy, Digestive SystemFibrosis/etiology/pathology/surgeryHumansIleitis/*complications/therapyIntestinal Obstruction/*etiology/pathology/surgeryTreatment Outcome20071421-9867 (Electronic)
0012-2823 (Linking)18239402http://www.ncbi.nlm.nih.gov/pubmed/1823940210.1159/000111025[ HYPERLINK \l "_ENREF_4" \o "Froehlich, 2007 #6204" 4]. It has long been documented that the accumulation of ECM in the intestinal wall contributes to intestinal remodeling and stricture formation. Since collagen I is one of the major matrix molecules involved in intestinal fibrogenesis, reduction and degradation of collagen I is a prominent treatment for CD intestinal fibrosis.
Resveratrol, a naturally occurring phytochemical, also known as SIRT1 activator, possess anti-inflammatory and antioxidative effects ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_34" \o "Harikumar, 2008 #1303" 34, HYPERLINK \l "_ENREF_35" \o "de la Lastra, 2005 #1300" 35]. In vivo studies have demonstrated the antifibrosis role of resveratrol in experimental colitis ADDIN EN.CITE Rahal201218218218217Rahal, K.Schmiedlin-Ren, P.Adler, J.Dhanani, M.Sultani, V.Rittershaus, A. C.Reingold, L.Zhu, J.McKenna, B. J.Christman, G. M.Zimmermann, E. M.Department of Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor, Michigan 48109, USA.Resveratrol has antiinflammatory and antifibrotic effects in the peptidoglycan-polysaccharide rat model of Crohn's diseaseInflamm Bowel DisInflamm Bowel Dis613-231842012/03/212012Apr1536-4844 (Electronic)
1078-0998 (Linking)22431488http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2243148810.1002/ibd.21843eng[ HYPERLINK \l "_ENREF_28" \o "Rahal, 2012 #182" 28]. Increasing evidence has implicated the role of resveratrol in the regulation of inflammatory cytokines, profibrotic factors and procollagen ADDIN EN.CITE Rahal201228128128117Rahal, K.Schmiedlin-Ren, P.Adler, J.Dhanani, M.Sultani, V.Rittershaus, A. C.Reingold, L.Zhu, J.McKenna, B. J.Christman, G. M.Zimmermann, E. M.Department of Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor, Michigan 48109, USA.Resveratrol has antiinflammatory and antifibrotic effects in the peptidoglycan-polysaccharide rat model of Crohn's diseaseInflamm Bowel DisInflamm Bowel Dis613-231842012/03/212012Apr1536-4844 (Electronic)
1078-0998 (Linking)22431488http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2243148810.1002/ibd.21843eng[ HYPERLINK \l "_ENREF_28" \o "Rahal, 2012 #182" 28]. These facts suggest that resveratrol could be utilized as a therapeutic against intestinal fibrosis. Results obtained by Susana Snchez-Fidalgo et al ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_36" \o "Sanchez-Fidalgo, 2010 #292" 36] showed that dietary supplementation of resveratrol exerted a significant beneficial effect in chronic DSS-induced colitis. Larrosa et al ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_37" \o "Larrosa, 2010 #288" 37] found that resveratrol pro-prodrugs prevented the rapid metabolism of resveratrol and delivered higher quantities of resveratrol to the colon in DSS-induced colitis. However, there are no preclinical and clinical studies have shown that resveratrol can be used clinically in patients with intestinal fibrosis. Moreover, the rapid metabolism of resveratrol diminishes its effectiveness in the colon. According to previous researches, long-term epidemiologic studies and controlled clinical trials are also necessary for developing resveratrol to become a standard clinical agent.
According to previous studies, detailed investigations of the underlying mechanisms are limited. In this study, we reported several new findings that reveal the effect of resveratrol regulation on collagen I synthesis and the mechanisms in intestinal fibroblasts. As a preliminary test, we initially performed CD intestinal fibrosis in rats induced by TNBS, and identified that collagen I expression was significantly increased, as well as IGF-1, but protein level of SIRT1 was decreased in colitis tissues. These data suggest that low expression of SIRT1 is related to intestinal fibrosis, and may explain the anti-fibrosis effect of resveratrol.
Based on these findings in vivo, we further researched the functions of resveratrol in vitro. We used intestinal fibroblasts for our subsequent experiments. Evidences suggest that IGF system, including IGF-1, IGF-2, IGF-1R, and the IGF-binding proteins (IGFBPs, IGFBP1-6) play a crucial role in the gastrointestinal tract ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_15" \o "Kuemmerle, 2012 #4266" 15, HYPERLINK \l "_ENREF_38" \o "Zimmermann, 1997 #17095" 38]. Since IGF-1 is regarded as a principal mediator of intestinal fibrosis, we investigated the effect of IGF-1 on collagen I synthesis in intestinal fibroblasts. Here, we reported that IGF-1 increased the protein and mRNA expression of collagen I in intestinal fibroblasts and that this upregulation was inhibited by pre-treatment with MEK1/2 inhibitor U0126. And the phosphorylation of IGF-1R and ERK1/2 then gradually increased in a time-dependent manner after the incubation with IGF-1. Thus, these data suggests that IGF-1 stimulates collagen I synthesis in intestinal fibroblasts and that its action mechanism could be attributed to the IGF-1/IGF-1R/ERK1/2 pathway.
Previous in vitro studies have shown that resveratrol causes cell cycle arrest, decreased collagen synthesis, and apoptosis in rat intestinal smooth muscle cells ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_27" \o "Garcia, 2012 #1242" 27]. We next explored the molecular mechanisms of anti-fibrosis effect of resveratrol in intestinal fibroblasts. Our experiments suggested that resveratrol significantly decreased collagen I expression induced by IGF-1 and resveratrol alone also inhibited collagen I synthesis. Resveratrol is known to activate deacetylase SIRT1, and this compound can also inhibit a number of other signaling pathways ADDIN EN.CITE AD D I N E N . C I T E . D A T A [ H Y P E R L I N K \ l " _ E N R E F _ 3 9 " \ o " P a c h o l e c , 2 0 1 0 # 6 0 0 6 " 3 9 - 4 1 ] . S e v e r a l l i n e s o f e v i d e n c e i n d i c a t e t h a t S I R T 1 m a y p l a y a n i m p o r t a n t r o l e i n o r g a n f i b r o s i s . S I R T 1 h a s b e e n d o c u m e n t e d t o i n h i b i t t u m o r n e c r o s i s f a c t o r - - i n d u c e d i n f l a m m a t i o n i n N I H / 3 T 3 f i b r o b l a s t c e l l l i n e A D D I N E N . C I T E A D D I N E N . C I T E . D A T A [ H Y P E R L I N K \ l " _ E N R E F _ 4 2 " \ o " Z h u , 2 0 1 1 # 5 9 7 7 " 4 2 ] . S I R T 1 d e a c e t y l a t e s s m a d 3 a n d s u p p r e s s e s t h e t r a n s f o r m i n g g r o w t h f a c t o r - b1 - d r i v e n r e n a l f i b r o s i s A D D I N E N . C I T E A D D I N E N . CITE.DATA [ HYPERLINK \l "_ENREF_19" \o "Li, 2010 #12731" 19]. Therefore, we used several approaches to examine whether the ability of resveratrol to inhibit collagen I operated via SIRT1. First, overexpression of WT but not HY SIRT1 provided a protective effect against IGF-1-induced collagen I synthesis in CCD-18Co. Second, knockdown of SIRT1 protein reversed the effect of resveratrol. These results indicate that resveratrol may protect against fibrosis through up-regulation of SIRT1 and enzymatic activity of SIRT1 may be responsible for its inhibitory effect on collagen I synthesis. Whether the mechanism underlying the effects is deacetylase activity of SIRT1 remains to be determined.
Since the IGF-1/IGF-1R/ERK1/2 signaling pathway has been previously described to be necessary and sufficient for the induction of collagen by IGF-1, we focused our investigation on the effect of resveratrol on IGF-1 signaling. Whether the expression or (and) activity of IGF-1R were influenced by resveratrol? Our data demonstrated that treatment with resveratrol plus IGF-1 for 30 min inhibited the activation of IGF-1R and its downstream signaling molecules such as MAP-kinase (ERK1/2) in intestinal fibroblasts. Treatment with resveratrol for 24 h or overexpression of SIRT1 WT resulted in the down-regulation of collagen I expression in fibroblasts. However, overexpression of SIRT1 WT or HY had no effect on activation of IGF-1R. Thus, our findings suggest that resveratrol exerts its negative effect on the phosphorylation of IGF-1R independent of activating SIRT1, and resveratrol may regulate IGF-1R activity by directly inhibiting IGF-1 binding to its receptor. Further studies are needed to confirm the findings and elucidate the exact molecular mechanism underlying resveratrol -inhibited activation of IGF-1R.
In summary, our findings (Figure 5) allude to a scheme wherein upon challenge with IGF-1, collagen I expression increased in intestinal fibroblasts, is repressed by resveratrol through either activating SIRT1 or inhibiting activation of IGF-1R. Collectively, these observations provide a new mechanistic framework to better understand the effects of SIRT1 activators on intestinal fibrosis, and will allow us to examine the possibility that dysregulation of the IGF-1/IGF-1R/ERK1/2 axis is involved in the progress driving in fibroblasts.
COMMENTS
Background
Intestinal fibrosis is an incurable complication of Crohns disease (CD) involving mesenchymal cell proliferation and extracellular matrix deposition. Until now, no effective therapy exists for averting such fibrogenic events. Insulin-like growth factor-1 (IGF-1), a potent profibrotic mediator, has been reported to be involved in gastrointestinal tract growth and tissue repair. Resveratrol is a polyphenol naturally occurring in grapes and its putative antifibrotic actions has been demonstrated in models of colitis.
Research frontiers
Mounting evidence suggests resveratrol has anti-inflammatory and antifibrotic effects in the animal models of colitis. Resveratrol diminishes IGF-1-stimulated collagen production in intestinal smooth muscle cells. However, the mechanism of resveratrol on collagen I synthesis in intestinal fibroblasts remains unclear.
Innovations and breakthroughs
In this study, the authors found that resveratrol down-regulated IGF-1-induced collagen I synthesis in intestinal fibroblasts by inhibiting IGF-1 receptor (IGF-1R) phosphorylation and its downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinas signaling pathway. In addition, resveratrol alone suppressed collagen I synthesis through up-regulating activity of silent information regulator 1 (SIRT1).
Applications
This findings highlight a previously resveratrol unknown function on IGF-1R activation and provide novel insight of resveratrol as a therapeutic role in intestinal fibrosis.
Terminology
SIRT1 is a nicotinamide adenine dinucleotide-dependent deacetylase which modulates metabolic homeostasis, stress resistance, cellular survival, cellular senescence/aging, inflammation-immune function, endothelial functions by deacetylating a number of key transcription factors.
Peer review
The authors investigated the effect of resveratrol on collagen I synthesis in intestinal fibroblasts and explored the mechanism. Resveratrol effectively decreased collagen I expression in IGF-1-stimulated fibroblasts by inhibiting of IGF-IR/ERK1/2 signaling in a SIRT1 independent mechanism. However, resveratrol alone inhibited collagen I synthesis by activating SIRT1. Overall, this manuscript is highly relevant and interesting.
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