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Journal of Digestive Diseases 2015; 16; 408–415
doi: 10.1111/1751-2980.12253
Original article
Expression of Smad7 and Smad ubiquitin regulatory factor 2 in a rat model of chronic pancreatitis Xiao Jia HOU,1 Zhen Dong JIN, Fei JIANG, Jian Wei ZHU & Zhao Shen LI Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai, China
OBJECTIVE: To quantify the expressions of Smad7 and Smad ubiquitin regulatory factor 2 (Smurf2) in the pancreas in rats with chronic pancreatitis (CP). METHODS: A total of 16 male Wistar rats were randomly divided into the control group and the CP group, with 8 rats in each group. CP was induced in vivo with dibutyltin dichloride (DBTC). Four weeks after DBTC administration, histological assessment and the measurement of hydroxyproline content in the pancreatic tissues were performed to assess the inflammation and fibrosis of the pancreas. Immunohistochemisty and reverse transcription polymerase chain reaction (RT-PCR) for transforming growth factor (TGF)-β1 and α-smooth muscle actin (α-SMA) were applied to assess activated pancreatic stellate cells (PSC) and TGF-β1 KEY WORDS:
RESULTS: Typical histopathological characteristics of DBTC-induced CP in the rats with extensively activated PSC. Compared with the control group, the expressions of TGF-β1, α-SMA and hydroxyproline content in the pancreatic tissues in the CP group were significantly increased. Meanwhile, the mRNA and protein expressions of Smad7 and Smurf2 were significant increased in the fibrotic pancreas, in which the expressions of Smad7 proteins showed an obvious reduction compared with controls. CONCLUSION: The dysregulation of Smad7 and Smurf2 may be associated with the pathogenesis of pancreatic fibrosis through the TGF-β signaling pathway.
chronic pancreatitis, expression, pancreatic fibrosis, Smad ubiquitin regulatory factor 2, Smad7.
INTRODUCTION Chronic pancreatitis (CP) is a progressive inflammatory process caused by a variety of pathogenic factors, leading eventually to irreversible pancreatic damage Correspondence to: Zhen Dong JIN, Department of Gastroenterology, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai 200433, China. Email: [email protected] Conflict of interest: None. 1
Present address: Department of Gastroenterology, The 210th Hospital of PLA, 80 Shengli Road, Dalian, Liaoning Province 161021, China. © 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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expression. Smad7 and Smurf2 expressions in the pancreas were measured using Western blot and RT-PCR.
and fibrosis.1 Clinical manifestations of CP include abdominal pain, steatorrhea, weight loss and diabetes. Although the molecular mechanisms of pancreatic fibrosis have not been fully explicated, recent advances in the investigation of the pathogenesis of this disease have been attributed largely to the isolation and characterization of pancreatic stellate cells (PSC).2,3 Pancreatic injury induces the release of growth factors and cytokines which, in turn, transform quiescent PSC into an activated state. With characteristic expression of α-smooth muscle actin (α-SMA), activated PSC synthesize and secrete abundant collagen. The activation of PSC and subsequent collagen production is persistently increased if the injury or inflammation repeats or continues, resulted in the progression of pancreatic fibrosis.4
Journal of Digestive Diseases 2015; 16; 408–415 Among the cytokines involved in pancreatic fibrosis, transforming growth factor (TGF)-β1 has been shown to be critical in the activation of PSC and the synthesis of extracellular matrix (ECM).5,6 The intracellular transduction and modulation of TGF-β1 signal pathway is mainly regulated by Smad. First of all, after bound to its receptors, TGF-β1 combines and phosphorylates Smad2 and Smad3, and then binds to Smad4 to form a multimeric complex. Subsequently, the complex enters the nucleus and activates downstream target genes to complete signal transduction. Smad7 inhibits TGF-β1 signaling transduction by directly targeting TGF-β1 gene and interacting with TGF-β1 receptor type I (TβRI) through negative feedback loops.7–9 The ubiquitin–proteasome pathway (UPP) is a principal mechanism of protein degradation and plays an important role in maintaining the normal physiological function of organisms. There are three kinds of catalytic enzymes involved in UPP protein decomposition: the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E2 and ubiquitin ligase E3 (E3). E3 plays a central role in the ubiquitination, functioning as a specific recognition of substrates that promote or directly catalyze the target proteins for degradation.10 Smurf2 is a recently reported homologue to the E6-accessory protein C-terminus (HECT)-domain E3 ubiquitin ligase that negatively regulates TGF-β signals by directly targeting its positive signaling components for degradation via UPP. Meanwhile, Smurf2 binds to Smad2 in the nucleus and then translocates to the cytoplasm, and subsequently associates with activated TβRI, resulting in TβRI turnover.11 It has been reported that the dysregulation of Smad7– Smurf2-mediated negative regulation of TGF-β signals could contribute to liver or kidney fibrosis, myocardial fibrosis and scleroderma in both human beings and animal models.12–16 However, to our knowledge, there have hitherto been no reports on Smurf2/Smad7 expression in pancreatic fibrosis. Therefore, in this study we aimed to clarify Smurf2 and Smad7 expressions in a rat model of pancreatic fibrosis.
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into the control group and the CP group, with 8 rats in each group. The rats were kept under a 12-h day-night cycle and had free access to standard laboratory chow and water. The study protocol was approved by the Animal Care Committee of the Second Military Medical University. Induction of CP The experimental CP model was induced by dibutylin dichloride (DBTC) (Sigma-Aldrich, St. Louis, MO, USA) infusion via the tail vein (8 mg/kg), while the rats in the control group were treated with an equal volume of glycerol and ethanol mixture, as described previously.17 The rats were observed daily, while the changes in their body weight at 0, 1, 2, 3 and 4 weeks after DBTC or glycerol and ethanol mixture administration were recorded. Four weeks later, all the rats were sacrificed by exsanguination after anesthesia with pentobarbital. The pancreas was quickly removed and the head of the pancreas was fixed immediately in 10% formalin. The remaining part of the pancreas was directly frozen and stored in liquid nitrogen for further analysis. Histological examinations and Masson stain The head of the pancreas was embedded in paraffin, cut into 4-μm-thick sections and was then stained with hematoxylin and eosin (HE). According to Niina et al.’ study,18 histological scores were estimated by a single pathologist who was blinded to the grouping of the rats. Fibrosis and edema were evaluated according to the following scores: 0, no lesion; 1, with lesion; 2, light lesions in lobule or intralobular areas; and 3, lesions over lobule areas or the destruction of lobular architecture. Pancreatic inflammation was evaluated by counting the number of inflammatory cells under five different fields of vision at original magnification (×200): 0, 0–10/high-power field (HPF); 1, 11–30/HPF; 2, 31–100/HPF; and 3, ≥101/HPF.
MATERIALS AND METHODS
Collagen production in the pancreatic tissues was assessed using a Masson staining kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China) according to the manufacturer’s instructions.
Animals
Immunohistochemistry
A total of 16 male Wistar rats (6-week-old) weighing 180–200 g were obtained from the Experimental Animal Center of the Second Military Medical University (Shanghai, China) and were randomly divided
After having been deparaffinized, rehydrated and rinsed in phosphate-buffered saline (PBS) thrice for 3 min successfully, the tissue sections were then subjected to heat-mediated antigen retrieval. For
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immunohistochemical analysis, the UltraSensitive S-P Kit (Fuzhou Maixin Biotech. Co., Ltd., Fuzhou, Fujian Province, China) was used according to the manufacturer’s protocol. Briefly, the tissue slices were treated with a methanol solution containing 3% hydrogen peroxide for 15 min at room temperature to block endogenous peroxidase. Then the sections were washed with PBS and incubated in goat serum for 60 min at room temperature to prevent nonspecific reactions. The immunohistochemical analysis was performed using mouse monoclonal antibody against α-SMA (1:200; Abcam, Cambridge, UK) or TGF-β1 (1:200; Abcam) at 4°C overnight. Subsequently, the sections were washed with PBS thrice for 3 min and treated with biotinylated secondary antibody (rabbit anti-mouse, 1:100; Fuzhou Maixin Biotech. Co., Ltd.) at room temperature for 30 min. After being washed with PBS thrice for 3 min, the sections were incubated with streptavidin-conjugated horseradish peroxidase (HRP) (Fuzhou Maixin Biotech. Co., Ltd.). The specific detections were then developed with 3,3′diaminobenzidine (Fuzhou Maixin Biotech. Co., Ltd.) and counterstained with HE for 5 min. Immunochemical scores were evaluated according to the staining intensity and the percentage of positive cells in five random HPF. The staining intensity was scored as follows: 0, no staining; 1, straw yellow; 2, brown; and 3, dark brown. The percentage of positive cells was scored as follows: 0, 0%; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The positive index (PI) of immunohistochemistry was calculated as the multiple of staining intensity and the percentage of positive cells.19 Detection of intrapancreatic hydroxyproline As a primary outcome to reflect collagen depositon in the pancreatic tissues, hydroxyproline content was measured using detection kits (Nanjing Jiancheng Bioengineering Institute) following the manufacturer’s protocol and was expressed as micrograms per gram of pancreatic tissue. Real-time polymerase chain reaction (PCR) Extraction of total RNA of intrapancreatic hydroxyproline was performed with frozen pancreatic tissues using Trizol reagent (TaKaRa Biotechnology [Dalian] Co., Ltd., Dalian, Liaoning Province, China) and the first-strand complementary DNA was reversely transcribed from total RNA with a reverse transcription kit (TaKaRa Biotechnology [Dalian] Co., Ltd.). Reverse transcription (RT)-PCR for each target gene expression
Journal of Digestive Diseases 2015; 16; 408–415 was performed with an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the SYBR Premix Ex Taq II (TaKaRa Biotechnology [Dalian] Co., Ltd.). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. A comparative 2−ΔΔCT method was used to calculate the relative changes of target mRNA expression to that in the control group. The primers were designed and synthesized by a business company (Shanghai Biological Engineering Co., Ltd., Shanghai, China), and the sequences are as follows: TGF-β1, forward: 5′-ATTCCTGGCGTTACCTTGG-3′, reverse: 5′AGCCCTGTATTCCGTCTCCT-3′; Smad7, forward: 5′-AT GTCGTCCATCTTGCCATT-3′, reverse: 5′-TCTGTTCTCC ACCACCTGCT-3′; Smurf2, forward: 5′-CCATCAATCGC CTCAAAGAC-3′, reverse: 5′-CTTGTCCTCCCGTGCCT AT-3′; α-SMA, forward:5′-CTTTGCTGGTGATGATGC TC-3′, reverse: 5′-TCGGATACTTCAGGGTCAGG-3′; GAPDH, forward: 5′-ACAGCAACAGGGTGGTGGAC3′, reverse: 5′-TTTGAGGGTGCAGCGAACTT-3′. Western blot In the liquid nitrogen, the rat pancreatic tissues were quickly grounded into powder. The resulting powder was treated with an ice-cold lysis buffer (SigmaAldrich) for 30 min and then centrifuged for 20 min at 12 000 ×g at 4°C to extract the total protein. A total protein assay kit (Nanjing Jiancheng Bioengineering Institute) was used to measure the concentrations of the proteins. An equal amount of protein (40 μg/lane) was electrophoresed on sodium dodecyl sulfatepolyacrylamide gels and subsequently transferred to membranes (Bio-Rad, Hercules, CA, USA). To block the non-specific binding site, the membranes were incubated with 5% non-fat milk in Tris-buffered saline and Tween 20 (TBST) for 2 h at room temperature. Thereafter, the membranes were washed with TBST thrice for 5 min and were incubated with rabbit monoclonal antibodies against Smurf2 (1:500; Abcam), Smad7 (1:1000; Abcam) or GAPDH (1:10000; Abcam) at 4°C overnight. The membranes were then washed with TBST thrice for 5 min and incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5000; Proteintech Group, Inc., Hangzhou, Zhejiang Province, China) for 2 h at room temperature and washed with TBST thrice for 5 min. The target proteins were then visualized by an enhanced chemiluminescence detection system (Thermo Scientific, Tewksbury, MA, USA) and the images were analyzed with Image J software (Wayne Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA).
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2015; 16; 408–415 Statistical analysis Statistical analyses were performed using SPSS 18.0 (IBM, Armonk, NY, USA). Continuous variables were expressed as mean ± standard error, whereas discrete variables were expressed as numbers and percentages. Student’s t-test was used to determine the differences in continuous variables and the Mann–Whitney U-test was used to evaluate the differences in histological
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scores between the CP and control groups. P < 0.05 was considered as statistically significant. RESULTS Changes in body weight of the rats The body weight of the rats in the control group was increased normally. However, after the induction of DBTC that of the rats in the CP model group was barely increased (Fig. 1). Histopathological changes Compared with the control group, significant inflammatory cell infiltration, edema, glandular atrophy and fibrosis were found in the rats treated with DBTC (Fig. 2a,e). The histological scores for inflammation, edema and fibrosis were significantly higher in the CP group than in the control group (all P < 0.05, Table 1). Masson stain also revealed obvious extracellular matrix deposition in the pancreatic tissues in the CP group while there was no collagen deposition except in the duct and vessels in the control group (Fig. 2b,f). α-SMA and TGF-β1 expressions
Figure 1. Changes in body weight of the rats in the control and chronic pancreatitis groups (n = 8 per group). , Control group; , chronic pancreatitis group.
There were significant differences between the CP and the control groups in terms of the PI for α-SMA (4.71 ± 0.40 vs 0.09 ± 0.07, P < 0.05, Fig. 2c,g) and TGF-β1 (4.39 ± 0.84 vs 0.50 ± 0.17, P < 0.05, Fig. 2d,h) (Table 1).
Figure 2. Representative histological appearance of pancreas tissue in (a–d) the control and (e–h) the chronic pancreatitis groups (magnification, ×400). (a,e) HE stain. (b,f) Masson stain, collagen fibers stained blue. Immunohistochemistry showing (c,g) α-smooth muscle actin-positive cells and (d,h) transforming growth factor-β1-positive cells in the two groups.
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Table 1. Histological scores, immunochemical positive index for α-smooth muscle actin (α-SMA) and transforming growth factor (TGF)-β1 in the pancreas in two groups (n = 8 per group) Histological score
DBTC Control
Positive index
Inflammation
Edema
Fibrosis
α-SMA
TGF-β1
2.72 ± 0.22* 0.17 ± 0.11
1.44 ± 0.25* 0.22 ± 0.16
2.61 ± 0.16* 0.11 ± 0.07
4.71 ± 0.40* 0.09 ± 0.07
4.39 ± 0.84* 0.50 ± 0.17
*P < 0.05. Date are expressed as mean ± standard error (SE). DBTC, dibutyltin dichloride.
Figure 3. The mRNA expression of α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β1, Smad7 and Smad ubiquitin regulatory factor 2 (Smurf2) in the pancreas in two groups (n = 8 per group). , control group; , dibutyltin dichloride (DBTC) group.
Compared with the control group, the mRNA expressions of α-SMA and TGF-β1 were significantly elevated in the pancreatic tissues of CP rats (P < 0.05, Fig. 3). Changes in pancreatic hydroxyproline The hydroxyproline of pancreatic tissue in the CP group was increased remarkably compared with the control group (615.0 ± 16.1 μg/g vs 184.5 ± 3.2 μg/g, P < 0.05; Fig. 4). Expression of Smad7 and Smurf2 Compared with the control group, mRNA expressions of Smad7 and Smurf2 in the pancreatic tissues of the CP rats were increased by 3-fold and 2.5-fold, respectively (all P < 0.05, Fig. 3). Smurf2 protein expression, which was determined by Western blot, was correspondingly increased in CP rats compared with the control group (P < 0.05, Fig. 5). However, the protein expression of Smad7 in the CP group was significantly decreased (P < 0.05, Fig. 5) and the intensities of the bands in the two groups were analyzed and normalized by internal control (Fig. 6).
Figure 4. Pancreatic hydroxyproline in the control and dibutyltin dichloride (DBTC)-treated groups (n = 8 per group). *P < 0.05.
DISCUSSION Smurf2 and Smad7 play an important role in the pathogenesis of fibrosis in multiple organs, functioning as anti-fibrosis genes.13–16,20 However, the expressions and the roles of Smurf2 and Smad7 in the pathogenesis of pancreatic fibrosis remain unclear. In this study abnormal expression of the Smad7– Smurf2-mediated negative regulation of TGF-β signal pathway was observed in the CP rats. A previous study21 has shown that the development of CP model induced by DBTC can simulate the whole pathological process from acute pancreatitis to CP. After intravenous injection of DBTC, a rapid excretion of organotin can cause bile duct epithelial necrosis, bile duct obstruction and cholestasis, leading to acute biliary pancreatitis and chronic inflammation.17 Meanwhile, DBTC can affect the pancreatic parenchyma through blood, and has a direct toxic effect on the acinar cells. Although the mechanism of pancreatic fibrosis has not been fully interpreted, the discovery of PSC and the separation and cultivation of PSC
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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endogenous TGF-β1 mRNA expression, and raises ECM formation via Smad2- and Smad3-dependent pathways or the extracellular signal-regulated kinasesdependent pathway, respectively.3 In the present study, TGF-β1 expressions and the hydroxyproline contents in the pancreas of DBTC-treated rats were significantly increased, accompanied by extensively activated PSC and typical histomorphological changes of fibrosis. These results indicate that the CP model induced by DBTC is suitable for the study of pancreatic fibrosis in vivo.
Figure 5. Western blot analysis for Smad7 and Smad ubiquitin regulatory factor 2 (Smurf2) in the pancreatic tissues in the chronic pancreatitis (CP) and the control group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 6. Quantification of Smad7 and Smad ubiquitin regulatory factor 2 (Smurf2) expression (n = 8 per group). The relative expressions of Smad7 and Smurf2 are determined by the ratio to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. *P < 0.05. , Control group; , dibutyltin dichloride (DBTC) group.
has made a significant contribution to the investigation of the pathogenesis of pancreatic fibrosis. TGF-β1 is a fundamental signaling protein that regulates multiple physiological processes, such as the cell cycle, differentiation, apoptosis and ECM synthesis.22 TGF-β1 is crucial in regulating of PSC activation and ECM formation in the pancreas through autocrine or paracrine loops. Moreover, exogenous TGF-β1 induces PSC activation from a quiescent state, stimulates
Several molecules have been identified in the regulation of TGF-β signals, and Smad7 is one of these molecules which inhibits the signal transduction of TGFβ.7–9 Smad7 binds to activated TGF-β receptors and prevents them from interacting with Smad2 or Smad3, thereby inhibiting the phosphorylated activation of Smad2 and Smad3, showing that the cellular level of Smad7 is a main factor in determining TGF-β responses, which could regulate the intensity and duration of TGF-β signals.14 The aberrant regulation of Smad7 is related to varied human diseases or organic fibrosis.23,24 Ubiquitin is a highly conserved polypeptide that can ubiquitinate and degrade target proteins with high selectivity and efficiency. The UPP is involved in the regulation of numerous physiological processes, such as the cell cycle, apoptosis, signal transduction or transcription.25,26 Both Smurf1 and Smurf2 are E3 ubiquitin ligases of the C2-WW-HECT domain family, which exhibits an overall sequence identity of 83% with each other.27 Smurf1 is indispensable for the regulation of intracellular mediators of bone morphogenetic proteins (BMP) signaling by specifically targeting Smad1 and Smad5 for degradation.28 In contrast, Smurf2 plays an important role in the degradation of many components of the TGF-β signal pathway. Smurf1 and Smurf2 differentially control BMP and TGF-β signaling, respectively, by selectively targeting different Smad for destruction. In normal physiological conditions, both positive and negative components of TGF-β signaling pathway get together to keep a dynamic balance and a delicate regulation, ensuring the homeostasis of TGF-β signal outputs. The dysregulation of Smurf2 expression could lead to an imbalance of TGF-β signal outputs, contributing to the progression of fibrotic diseases.12–16 Smurf2 recruits Smad7 by associating with intranuclear Smad7 and inducing the nuclear export of Smurf2 to Smad7 complexes. The complexes then interact with TβRI and increase its degradation. Moreover, a previous study
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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has shown that Smurf2 can associate with Smad2 and induce its degradation.27 These findings suggest that Smurf2 is a negative regulator of TGF-β signaling by targeting TGF-β-positive signaling proteins. However, the expressions and physiological roles of Smurf2 and Smad7 in pancreatic diseases remain to be clarified and, to our knowledge, they have not been investigated in a CP model with progressive pancreatic fibrosis. In this study we found that mRNA expressions of TGFβ1, Smurf2 and Smad7 as well as Smurf2 protein expression were significantly increased in fibrotic pancreas, while Smad7 protein expression showed an opposite trend to that of Smurf2. The increased Smad7 mRNA level might be a negative feedback mechanism to control the enhanced TGF-β1 signals and prevent the production of ECM. Ohashi et al.29 have suggested that Smurf2 expression enhanced by TGF-β1 is possibly a negative feedback mechanism, and thereby inhibits the TGF-β signaling pathway. Meanwhile, Fukasawa et al.14 have demonstrated that Smad7 is regulated dynamically by Smurf2 via the UPP. Kavsak et al.11 have confirmed that binding to Smad7 induces the export and recruitment of Smurf2 to the activated TGF-β receptor, where it causes the degradation of receptors and Smad7 via the UPP. Therefore, in this study, we hypothesized that increased expression of Smurf2 might be the reaction to enhanced TGF-β signaling and might induce the decrease in Smad7 protein expressions in the fibrotic pancreas. However, the possibility of the decreased synthesis of Smad7 protein cannot be ruled out, and further studies are needed to investigate the molecular mechanisms of these abnormal expressions in pancreatic fibrosis. In conclusion, we demonstrated that the expressions of Smurf2 and Smad7 mRNA were dramatically increased in the pancreas of CP rats compared with those in control group while the expression of Smad7 proteins was significantly decreased. These results suggest that the dysregulation of Smad7 and Smurf2 may be associated with the pathogenesis of pancreatic fibrosis through the TGF-β signaling pathway. ACKNOWLEDGMENT This study was supported by Changhai Hospital, Second Military Medical University (No. CH125510306).
Journal of Digestive Diseases 2015; 16; 408–415 REFERENCES 1 Witt H, Apte MV, Keim V, Wilson JS. Chronic pancreatitis: challenges and advances in pathogenesis, genetics, diagnosis, and therapy. Gastroenterology 2007; 132: 1557–73. 2 Erkan M, Adler G, Apte MV et al. StellaTUM: current consensus and discussion on pancreatic stellate cell research. Gut 2012; 61: 172–8. 3 Shimizu K. Mechanisms of pancreatic fibrosis and applications to the treatment of chronic pancreatitis. J Gastroenterol 2008; 43: 823–32. 4 Masamune A, Watanabe T, Kikuta K, Shimosegawa T. Roles of pancreatic stellate cells in pancreatic inflammation and fibrosis. Clin Gastroenterol Hepatol 2009; 7: S48–54. 5 Schneider E, Schmid-Kotsas A, Zhao J et al. Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells. Am J Physiol Cell Physiol 2001; 281: C532–43. 6 Mews P, Phillips P, Fahmy R et al. Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis. Gut 2002; 50: 535–41. 7 Miyazawa K, Shinozaki M, Hara T, Furuya T, Miyazono K. Two major Smad pathways in TGF-β superfamily signalling. Genes Cells 2002; 7: 1191–204. 8 Hayashi H, Abdollah S, Qiu Y et al. The MAD-related protein Smad7 associates with the TGFβ receptor and functions as an antagonist of TGFβ signaling. Cell 1997; 89: 1165–73. 9 Itoh S, ten Dijke P. Negative regulation of TGF-β receptor/Smad signal transduction. Curr Opin Cell Biol 2007; 19: 176–84. 10 Inoue Y, Imamura T. Regulation of TGF-β family signaling by E3 ubiquitin ligases. Cancer Sci 2008; 99: 2107–12. 11 Kavsak P, Rasmussen RK, Causing CG et al. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol Cell 2000; 6: 1365–75. 12 Cai Y, Shen XZ, Zhou CH, Wang JY. Abnormal expression of Smurf2 during the process of rat liver fibrosis. Chin J Dig Dis 2006; 7: 237–45. 13 Cai Y, Zhou CH, Fu D, Shen XZ. Overexpression of Smad ubiquitin regulatory factor 2 suppresses transforming growth factor-β mediated liver fibrosis. J Dig Dis 2012; 13: 327–34. 14 Fukasawa H, Yamamoto T, Togawa A et al. Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice. Proc Natl Acad Sci USA 2004; 101: 8687–92. 15 Liu FY, Li XZ, Peng YM, Liu H, Liu YH. Arkadia-Smad7-mediated positive regulation of TGF-beta signaling in a rat model of tubulointerstitial fibrosis. Am J Nephrol 2007; 27: 176–83. 16 Cunnington RH, Nazari M, Dixon IM. c-Ski, Smurf2, and Arkadia as regulators of TGF-β signaling: new targets for managing myofibroblast function and cardiac fibrosis. Can J Physiol Pharmacol 2009; 87: 764–72. 17 Jiang F, Liao Z, Hu LH et al. Comparison of antioxidative and antifibrotic effects of α-tocopherol with those of tocotrienol-rich fraction in a rat model of chronic pancreatitis. Pancreas 2011; 40: 1091–6. 18 Niina Y, Ito T, Oono T et al. A sustained prostacyclin analog, ONO-1301, attenuates pancreatic fibrosis in experimental chronic pancreatitis induced by dibutyltin dichloride in rats. Pancreatology 2014; 14: 201–10. 19 Merkord J, Jonas L, Henninghansen G. Morphological lesions of pancreas and bile ducts in rats induced by dibutyltin dichloride. Arch Toxicol Suppl 1991; 14: 75–9.
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Journal of Digestive Diseases 2015; 16; 408–415 20 Asano Y, Ihn H, Yamane K, Kubo M, Tamaki K. Impaired Smad7-Smurf–mediated negative regulation of TGF-β signaling in scleroderma fibroblasts. J Clin Invest 2004; 113: 253–64. 21 Merkord J, Jonas L, Weber H, Kröning G, Nizze H, Hennighausen G. Acute interstitial pancreatitis in rats induced by dibutylin dichloride (DBTC): pathogenesis and natural course of lesions. Pancreas 1997; 15: 392–401. 22 Siegel PM, Massagué J. Cytostatic and apoptotic actions of TGF-β in homeostasis and cancer. Nat Rev Cancer 2003; 3: 807–21. 23 Briones-Orta MA, Tecalco-Cruz AC, Sosa-Garrocho M, Caligaris C, Macías-Silva M. Inhibitory Smad7: emerging roles in health and disease. Curr Mol Pharmacol 2011; 4: 141–53. 24 Ghosh AK, Quaggin SE, Vaughan DE. Molecular basis of organ fibrosis: potential therapeutic approaches. Exp Biol Med (Maywood) 2013; 238: 461–81.
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25 Mani A, Gelmann EP. The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol 2005; 23: 4776–89. 26 David D, Nair SA, Pillai MR. Smurf E3 ubiquitin ligases at the cross roads of oncogenesis and tumor suppression. Biochim Biophys Acta 2013; 1835: 119–28. 27 Lin X, Liang M, Feng XH. Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-β signaling. J Biol Chem 2000; 275: 36818–22. 28 Zhu H, Kavsak P, Abdollah S, Wrana JL, Thomsen GH. A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature 1999; 400: 687–93. 29 Ohashi N, Yamamoto T, Uchida C et al. Transcriptional induction of Smurf2 ubiquitin ligase by TGF-β. FEBS Lett 2005; 579: 2557–63.
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Journal of Digestive Diseases 2015; 16; 408–415
doi: 10.1111/1751-2980.12253
Original article
Expression of Smad7 and Smad ubiquitin regulatory factor 2 in a rat model of chronic pancreatitis Xiao Jia HOU,1 Zhen Dong JIN, Fei JIANG, Jian Wei ZHU & Zhao Shen LI Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai, China
OBJECTIVE: To quantify the expressions of Smad7 and Smad ubiquitin regulatory factor 2 (Smurf2) in the pancreas in rats with chronic pancreatitis (CP). METHODS: A total of 16 male Wistar rats were randomly divided into the control group and the CP group, with 8 rats in each group. CP was induced in vivo with dibutyltin dichloride (DBTC). Four weeks after DBTC administration, histological assessment and the measurement of hydroxyproline content in the pancreatic tissues were performed to assess the inflammation and fibrosis of the pancreas. Immunohistochemisty and reverse transcription polymerase chain reaction (RT-PCR) for transforming growth factor (TGF)-β1 and α-smooth muscle actin (α-SMA) were applied to assess activated pancreatic stellate cells (PSC) and TGF-β1 KEY WORDS:
RESULTS: Typical histopathological characteristics of DBTC-induced CP in the rats with extensively activated PSC. Compared with the control group, the expressions of TGF-β1, α-SMA and hydroxyproline content in the pancreatic tissues in the CP group were significantly increased. Meanwhile, the mRNA and protein expressions of Smad7 and Smurf2 were significant increased in the fibrotic pancreas, in which the expressions of Smad7 proteins showed an obvious reduction compared with controls. CONCLUSION: The dysregulation of Smad7 and Smurf2 may be associated with the pathogenesis of pancreatic fibrosis through the TGF-β signaling pathway.
chronic pancreatitis, expression, pancreatic fibrosis, Smad ubiquitin regulatory factor 2, Smad7.
INTRODUCTION Chronic pancreatitis (CP) is a progressive inflammatory process caused by a variety of pathogenic factors, leading eventually to irreversible pancreatic damage Correspondence to: Zhen Dong JIN, Department of Gastroenterology, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai 200433, China. Email: [email protected] Conflict of interest: None. 1
Present address: Department of Gastroenterology, The 210th Hospital of PLA, 80 Shengli Road, Dalian, Liaoning Province 161021, China. © 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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expression. Smad7 and Smurf2 expressions in the pancreas were measured using Western blot and RT-PCR.
and fibrosis.1 Clinical manifestations of CP include abdominal pain, steatorrhea, weight loss and diabetes. Although the molecular mechanisms of pancreatic fibrosis have not been fully explicated, recent advances in the investigation of the pathogenesis of this disease have been attributed largely to the isolation and characterization of pancreatic stellate cells (PSC).2,3 Pancreatic injury induces the release of growth factors and cytokines which, in turn, transform quiescent PSC into an activated state. With characteristic expression of α-smooth muscle actin (α-SMA), activated PSC synthesize and secrete abundant collagen. The activation of PSC and subsequent collagen production is persistently increased if the injury or inflammation repeats or continues, resulted in the progression of pancreatic fibrosis.4
Journal of Digestive Diseases 2015; 16; 408–415 Among the cytokines involved in pancreatic fibrosis, transforming growth factor (TGF)-β1 has been shown to be critical in the activation of PSC and the synthesis of extracellular matrix (ECM).5,6 The intracellular transduction and modulation of TGF-β1 signal pathway is mainly regulated by Smad. First of all, after bound to its receptors, TGF-β1 combines and phosphorylates Smad2 and Smad3, and then binds to Smad4 to form a multimeric complex. Subsequently, the complex enters the nucleus and activates downstream target genes to complete signal transduction. Smad7 inhibits TGF-β1 signaling transduction by directly targeting TGF-β1 gene and interacting with TGF-β1 receptor type I (TβRI) through negative feedback loops.7–9 The ubiquitin–proteasome pathway (UPP) is a principal mechanism of protein degradation and plays an important role in maintaining the normal physiological function of organisms. There are three kinds of catalytic enzymes involved in UPP protein decomposition: the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E2 and ubiquitin ligase E3 (E3). E3 plays a central role in the ubiquitination, functioning as a specific recognition of substrates that promote or directly catalyze the target proteins for degradation.10 Smurf2 is a recently reported homologue to the E6-accessory protein C-terminus (HECT)-domain E3 ubiquitin ligase that negatively regulates TGF-β signals by directly targeting its positive signaling components for degradation via UPP. Meanwhile, Smurf2 binds to Smad2 in the nucleus and then translocates to the cytoplasm, and subsequently associates with activated TβRI, resulting in TβRI turnover.11 It has been reported that the dysregulation of Smad7– Smurf2-mediated negative regulation of TGF-β signals could contribute to liver or kidney fibrosis, myocardial fibrosis and scleroderma in both human beings and animal models.12–16 However, to our knowledge, there have hitherto been no reports on Smurf2/Smad7 expression in pancreatic fibrosis. Therefore, in this study we aimed to clarify Smurf2 and Smad7 expressions in a rat model of pancreatic fibrosis.
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into the control group and the CP group, with 8 rats in each group. The rats were kept under a 12-h day-night cycle and had free access to standard laboratory chow and water. The study protocol was approved by the Animal Care Committee of the Second Military Medical University. Induction of CP The experimental CP model was induced by dibutylin dichloride (DBTC) (Sigma-Aldrich, St. Louis, MO, USA) infusion via the tail vein (8 mg/kg), while the rats in the control group were treated with an equal volume of glycerol and ethanol mixture, as described previously.17 The rats were observed daily, while the changes in their body weight at 0, 1, 2, 3 and 4 weeks after DBTC or glycerol and ethanol mixture administration were recorded. Four weeks later, all the rats were sacrificed by exsanguination after anesthesia with pentobarbital. The pancreas was quickly removed and the head of the pancreas was fixed immediately in 10% formalin. The remaining part of the pancreas was directly frozen and stored in liquid nitrogen for further analysis. Histological examinations and Masson stain The head of the pancreas was embedded in paraffin, cut into 4-μm-thick sections and was then stained with hematoxylin and eosin (HE). According to Niina et al.’ study,18 histological scores were estimated by a single pathologist who was blinded to the grouping of the rats. Fibrosis and edema were evaluated according to the following scores: 0, no lesion; 1, with lesion; 2, light lesions in lobule or intralobular areas; and 3, lesions over lobule areas or the destruction of lobular architecture. Pancreatic inflammation was evaluated by counting the number of inflammatory cells under five different fields of vision at original magnification (×200): 0, 0–10/high-power field (HPF); 1, 11–30/HPF; 2, 31–100/HPF; and 3, ≥101/HPF.
MATERIALS AND METHODS
Collagen production in the pancreatic tissues was assessed using a Masson staining kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China) according to the manufacturer’s instructions.
Animals
Immunohistochemistry
A total of 16 male Wistar rats (6-week-old) weighing 180–200 g were obtained from the Experimental Animal Center of the Second Military Medical University (Shanghai, China) and were randomly divided
After having been deparaffinized, rehydrated and rinsed in phosphate-buffered saline (PBS) thrice for 3 min successfully, the tissue sections were then subjected to heat-mediated antigen retrieval. For
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immunohistochemical analysis, the UltraSensitive S-P Kit (Fuzhou Maixin Biotech. Co., Ltd., Fuzhou, Fujian Province, China) was used according to the manufacturer’s protocol. Briefly, the tissue slices were treated with a methanol solution containing 3% hydrogen peroxide for 15 min at room temperature to block endogenous peroxidase. Then the sections were washed with PBS and incubated in goat serum for 60 min at room temperature to prevent nonspecific reactions. The immunohistochemical analysis was performed using mouse monoclonal antibody against α-SMA (1:200; Abcam, Cambridge, UK) or TGF-β1 (1:200; Abcam) at 4°C overnight. Subsequently, the sections were washed with PBS thrice for 3 min and treated with biotinylated secondary antibody (rabbit anti-mouse, 1:100; Fuzhou Maixin Biotech. Co., Ltd.) at room temperature for 30 min. After being washed with PBS thrice for 3 min, the sections were incubated with streptavidin-conjugated horseradish peroxidase (HRP) (Fuzhou Maixin Biotech. Co., Ltd.). The specific detections were then developed with 3,3′diaminobenzidine (Fuzhou Maixin Biotech. Co., Ltd.) and counterstained with HE for 5 min. Immunochemical scores were evaluated according to the staining intensity and the percentage of positive cells in five random HPF. The staining intensity was scored as follows: 0, no staining; 1, straw yellow; 2, brown; and 3, dark brown. The percentage of positive cells was scored as follows: 0, 0%; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The positive index (PI) of immunohistochemistry was calculated as the multiple of staining intensity and the percentage of positive cells.19 Detection of intrapancreatic hydroxyproline As a primary outcome to reflect collagen depositon in the pancreatic tissues, hydroxyproline content was measured using detection kits (Nanjing Jiancheng Bioengineering Institute) following the manufacturer’s protocol and was expressed as micrograms per gram of pancreatic tissue. Real-time polymerase chain reaction (PCR) Extraction of total RNA of intrapancreatic hydroxyproline was performed with frozen pancreatic tissues using Trizol reagent (TaKaRa Biotechnology [Dalian] Co., Ltd., Dalian, Liaoning Province, China) and the first-strand complementary DNA was reversely transcribed from total RNA with a reverse transcription kit (TaKaRa Biotechnology [Dalian] Co., Ltd.). Reverse transcription (RT)-PCR for each target gene expression
Journal of Digestive Diseases 2015; 16; 408–415 was performed with an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the SYBR Premix Ex Taq II (TaKaRa Biotechnology [Dalian] Co., Ltd.). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. A comparative 2−ΔΔCT method was used to calculate the relative changes of target mRNA expression to that in the control group. The primers were designed and synthesized by a business company (Shanghai Biological Engineering Co., Ltd., Shanghai, China), and the sequences are as follows: TGF-β1, forward: 5′-ATTCCTGGCGTTACCTTGG-3′, reverse: 5′AGCCCTGTATTCCGTCTCCT-3′; Smad7, forward: 5′-AT GTCGTCCATCTTGCCATT-3′, reverse: 5′-TCTGTTCTCC ACCACCTGCT-3′; Smurf2, forward: 5′-CCATCAATCGC CTCAAAGAC-3′, reverse: 5′-CTTGTCCTCCCGTGCCT AT-3′; α-SMA, forward:5′-CTTTGCTGGTGATGATGC TC-3′, reverse: 5′-TCGGATACTTCAGGGTCAGG-3′; GAPDH, forward: 5′-ACAGCAACAGGGTGGTGGAC3′, reverse: 5′-TTTGAGGGTGCAGCGAACTT-3′. Western blot In the liquid nitrogen, the rat pancreatic tissues were quickly grounded into powder. The resulting powder was treated with an ice-cold lysis buffer (SigmaAldrich) for 30 min and then centrifuged for 20 min at 12 000 ×g at 4°C to extract the total protein. A total protein assay kit (Nanjing Jiancheng Bioengineering Institute) was used to measure the concentrations of the proteins. An equal amount of protein (40 μg/lane) was electrophoresed on sodium dodecyl sulfatepolyacrylamide gels and subsequently transferred to membranes (Bio-Rad, Hercules, CA, USA). To block the non-specific binding site, the membranes were incubated with 5% non-fat milk in Tris-buffered saline and Tween 20 (TBST) for 2 h at room temperature. Thereafter, the membranes were washed with TBST thrice for 5 min and were incubated with rabbit monoclonal antibodies against Smurf2 (1:500; Abcam), Smad7 (1:1000; Abcam) or GAPDH (1:10000; Abcam) at 4°C overnight. The membranes were then washed with TBST thrice for 5 min and incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5000; Proteintech Group, Inc., Hangzhou, Zhejiang Province, China) for 2 h at room temperature and washed with TBST thrice for 5 min. The target proteins were then visualized by an enhanced chemiluminescence detection system (Thermo Scientific, Tewksbury, MA, USA) and the images were analyzed with Image J software (Wayne Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA).
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2015; 16; 408–415 Statistical analysis Statistical analyses were performed using SPSS 18.0 (IBM, Armonk, NY, USA). Continuous variables were expressed as mean ± standard error, whereas discrete variables were expressed as numbers and percentages. Student’s t-test was used to determine the differences in continuous variables and the Mann–Whitney U-test was used to evaluate the differences in histological
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scores between the CP and control groups. P < 0.05 was considered as statistically significant. RESULTS Changes in body weight of the rats The body weight of the rats in the control group was increased normally. However, after the induction of DBTC that of the rats in the CP model group was barely increased (Fig. 1). Histopathological changes Compared with the control group, significant inflammatory cell infiltration, edema, glandular atrophy and fibrosis were found in the rats treated with DBTC (Fig. 2a,e). The histological scores for inflammation, edema and fibrosis were significantly higher in the CP group than in the control group (all P < 0.05, Table 1). Masson stain also revealed obvious extracellular matrix deposition in the pancreatic tissues in the CP group while there was no collagen deposition except in the duct and vessels in the control group (Fig. 2b,f). α-SMA and TGF-β1 expressions
Figure 1. Changes in body weight of the rats in the control and chronic pancreatitis groups (n = 8 per group). , Control group; , chronic pancreatitis group.
There were significant differences between the CP and the control groups in terms of the PI for α-SMA (4.71 ± 0.40 vs 0.09 ± 0.07, P < 0.05, Fig. 2c,g) and TGF-β1 (4.39 ± 0.84 vs 0.50 ± 0.17, P < 0.05, Fig. 2d,h) (Table 1).
Figure 2. Representative histological appearance of pancreas tissue in (a–d) the control and (e–h) the chronic pancreatitis groups (magnification, ×400). (a,e) HE stain. (b,f) Masson stain, collagen fibers stained blue. Immunohistochemistry showing (c,g) α-smooth muscle actin-positive cells and (d,h) transforming growth factor-β1-positive cells in the two groups.
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Table 1. Histological scores, immunochemical positive index for α-smooth muscle actin (α-SMA) and transforming growth factor (TGF)-β1 in the pancreas in two groups (n = 8 per group) Histological score
DBTC Control
Positive index
Inflammation
Edema
Fibrosis
α-SMA
TGF-β1
2.72 ± 0.22* 0.17 ± 0.11
1.44 ± 0.25* 0.22 ± 0.16
2.61 ± 0.16* 0.11 ± 0.07
4.71 ± 0.40* 0.09 ± 0.07
4.39 ± 0.84* 0.50 ± 0.17
*P < 0.05. Date are expressed as mean ± standard error (SE). DBTC, dibutyltin dichloride.
Figure 3. The mRNA expression of α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β1, Smad7 and Smad ubiquitin regulatory factor 2 (Smurf2) in the pancreas in two groups (n = 8 per group). , control group; , dibutyltin dichloride (DBTC) group.
Compared with the control group, the mRNA expressions of α-SMA and TGF-β1 were significantly elevated in the pancreatic tissues of CP rats (P < 0.05, Fig. 3). Changes in pancreatic hydroxyproline The hydroxyproline of pancreatic tissue in the CP group was increased remarkably compared with the control group (615.0 ± 16.1 μg/g vs 184.5 ± 3.2 μg/g, P < 0.05; Fig. 4). Expression of Smad7 and Smurf2 Compared with the control group, mRNA expressions of Smad7 and Smurf2 in the pancreatic tissues of the CP rats were increased by 3-fold and 2.5-fold, respectively (all P < 0.05, Fig. 3). Smurf2 protein expression, which was determined by Western blot, was correspondingly increased in CP rats compared with the control group (P < 0.05, Fig. 5). However, the protein expression of Smad7 in the CP group was significantly decreased (P < 0.05, Fig. 5) and the intensities of the bands in the two groups were analyzed and normalized by internal control (Fig. 6).
Figure 4. Pancreatic hydroxyproline in the control and dibutyltin dichloride (DBTC)-treated groups (n = 8 per group). *P < 0.05.
DISCUSSION Smurf2 and Smad7 play an important role in the pathogenesis of fibrosis in multiple organs, functioning as anti-fibrosis genes.13–16,20 However, the expressions and the roles of Smurf2 and Smad7 in the pathogenesis of pancreatic fibrosis remain unclear. In this study abnormal expression of the Smad7– Smurf2-mediated negative regulation of TGF-β signal pathway was observed in the CP rats. A previous study21 has shown that the development of CP model induced by DBTC can simulate the whole pathological process from acute pancreatitis to CP. After intravenous injection of DBTC, a rapid excretion of organotin can cause bile duct epithelial necrosis, bile duct obstruction and cholestasis, leading to acute biliary pancreatitis and chronic inflammation.17 Meanwhile, DBTC can affect the pancreatic parenchyma through blood, and has a direct toxic effect on the acinar cells. Although the mechanism of pancreatic fibrosis has not been fully interpreted, the discovery of PSC and the separation and cultivation of PSC
© 2015 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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endogenous TGF-β1 mRNA expression, and raises ECM formation via Smad2- and Smad3-dependent pathways or the extracellular signal-regulated kinasesdependent pathway, respectively.3 In the present study, TGF-β1 expressions and the hydroxyproline contents in the pancreas of DBTC-treated rats were significantly increased, accompanied by extensively activated PSC and typical histomorphological changes of fibrosis. These results indicate that the CP model induced by DBTC is suitable for the study of pancreatic fibrosis in vivo.
Figure 5. Western blot analysis for Smad7 and Smad ubiquitin regulatory factor 2 (Smurf2) in the pancreatic tissues in the chronic pancreatitis (CP) and the control group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 6. Quantification of Smad7 and Smad ubiquitin regulatory factor 2 (Smurf2) expression (n = 8 per group). The relative expressions of Smad7 and Smurf2 are determined by the ratio to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. *P < 0.05. , Control group; , dibutyltin dichloride (DBTC) group.
has made a significant contribution to the investigation of the pathogenesis of pancreatic fibrosis. TGF-β1 is a fundamental signaling protein that regulates multiple physiological processes, such as the cell cycle, differentiation, apoptosis and ECM synthesis.22 TGF-β1 is crucial in regulating of PSC activation and ECM formation in the pancreas through autocrine or paracrine loops. Moreover, exogenous TGF-β1 induces PSC activation from a quiescent state, stimulates
Several molecules have been identified in the regulation of TGF-β signals, and Smad7 is one of these molecules which inhibits the signal transduction of TGFβ.7–9 Smad7 binds to activated TGF-β receptors and prevents them from interacting with Smad2 or Smad3, thereby inhibiting the phosphorylated activation of Smad2 and Smad3, showing that the cellular level of Smad7 is a main factor in determining TGF-β responses, which could regulate the intensity and duration of TGF-β signals.14 The aberrant regulation of Smad7 is related to varied human diseases or organic fibrosis.23,24 Ubiquitin is a highly conserved polypeptide that can ubiquitinate and degrade target proteins with high selectivity and efficiency. The UPP is involved in the regulation of numerous physiological processes, such as the cell cycle, apoptosis, signal transduction or transcription.25,26 Both Smurf1 and Smurf2 are E3 ubiquitin ligases of the C2-WW-HECT domain family, which exhibits an overall sequence identity of 83% with each other.27 Smurf1 is indispensable for the regulation of intracellular mediators of bone morphogenetic proteins (BMP) signaling by specifically targeting Smad1 and Smad5 for degradation.28 In contrast, Smurf2 plays an important role in the degradation of many components of the TGF-β signal pathway. Smurf1 and Smurf2 differentially control BMP and TGF-β signaling, respectively, by selectively targeting different Smad for destruction. In normal physiological conditions, both positive and negative components of TGF-β signaling pathway get together to keep a dynamic balance and a delicate regulation, ensuring the homeostasis of TGF-β signal outputs. The dysregulation of Smurf2 expression could lead to an imbalance of TGF-β signal outputs, contributing to the progression of fibrotic diseases.12–16 Smurf2 recruits Smad7 by associating with intranuclear Smad7 and inducing the nuclear export of Smurf2 to Smad7 complexes. The complexes then interact with TβRI and increase its degradation. Moreover, a previous study
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has shown that Smurf2 can associate with Smad2 and induce its degradation.27 These findings suggest that Smurf2 is a negative regulator of TGF-β signaling by targeting TGF-β-positive signaling proteins. However, the expressions and physiological roles of Smurf2 and Smad7 in pancreatic diseases remain to be clarified and, to our knowledge, they have not been investigated in a CP model with progressive pancreatic fibrosis. In this study we found that mRNA expressions of TGFβ1, Smurf2 and Smad7 as well as Smurf2 protein expression were significantly increased in fibrotic pancreas, while Smad7 protein expression showed an opposite trend to that of Smurf2. The increased Smad7 mRNA level might be a negative feedback mechanism to control the enhanced TGF-β1 signals and prevent the production of ECM. Ohashi et al.29 have suggested that Smurf2 expression enhanced by TGF-β1 is possibly a negative feedback mechanism, and thereby inhibits the TGF-β signaling pathway. Meanwhile, Fukasawa et al.14 have demonstrated that Smad7 is regulated dynamically by Smurf2 via the UPP. Kavsak et al.11 have confirmed that binding to Smad7 induces the export and recruitment of Smurf2 to the activated TGF-β receptor, where it causes the degradation of receptors and Smad7 via the UPP. Therefore, in this study, we hypothesized that increased expression of Smurf2 might be the reaction to enhanced TGF-β signaling and might induce the decrease in Smad7 protein expressions in the fibrotic pancreas. However, the possibility of the decreased synthesis of Smad7 protein cannot be ruled out, and further studies are needed to investigate the molecular mechanisms of these abnormal expressions in pancreatic fibrosis. In conclusion, we demonstrated that the expressions of Smurf2 and Smad7 mRNA were dramatically increased in the pancreas of CP rats compared with those in control group while the expression of Smad7 proteins was significantly decreased. These results suggest that the dysregulation of Smad7 and Smurf2 may be associated with the pathogenesis of pancreatic fibrosis through the TGF-β signaling pathway. ACKNOWLEDGMENT This study was supported by Changhai Hospital, Second Military Medical University (No. CH125510306).
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25 Mani A, Gelmann EP. The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol 2005; 23: 4776–89. 26 David D, Nair SA, Pillai MR. Smurf E3 ubiquitin ligases at the cross roads of oncogenesis and tumor suppression. Biochim Biophys Acta 2013; 1835: 119–28. 27 Lin X, Liang M, Feng XH. Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-β signaling. J Biol Chem 2000; 275: 36818–22. 28 Zhu H, Kavsak P, Abdollah S, Wrana JL, Thomsen GH. A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature 1999; 400: 687–93. 29 Ohashi N, Yamamoto T, Uchida C et al. Transcriptional induction of Smurf2 ubiquitin ligase by TGF-β. FEBS Lett 2005; 579: 2557–63.
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