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1.
Adv Biol Regul ; 88: 100957, 2023 05.
Article in English | MEDLINE | ID: mdl-36739740

ABSTRACT

Colorectal cancer (CRC) remains a challenging disease to treat due to several factors including stemness and epithelial to mesenchymal transition (EMT). Dysfunctional signaling pathways such as Notch and TGF-ß contribute to these phenomena. We previously found that cells expressing constitutively active Notch1 also had increased expression of Smad3, an important member of the TGF-ß signaling pathway. We hypothesized that Smad3, mediates the Notch-induced stemness and EMT observed in CRC cells. The human colorectal carcinoma cell line HCT-116, stably transduced with constitutively active Notch-1 (ICN) or a GFP-vector control was treated with different combinations of TGF-ß1, DAPT (a Notch inhibitor), or SIS3 (a Smad3 inhibitor). Western blot analysis was performed to determine the effects of Smad3 stimulation and inhibition on Notch and potential downstream EMT-related targets, CD44, Slug and Snail. Smad3 inhibition induced a decrease in Notch1 and Notch3 receptor expression and effectively inhibited CD44, Slug, and Snail expression. Colosphere forming ability was also reduced in cells with inhibited Smad3. These results indicate a key role of TGF-ß signaling in Notch1-induced tumorigenesis, and suggest a potential use for Smad3 inhibitors in combination with Notch1 inhibitors that are already in use for CRC treatments.


Subject(s)
Colonic Neoplasms , Epithelial-Mesenchymal Transition , Humans , Phosphorylation , Cell Movement/physiology , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta1/pharmacology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Colonic Neoplasms/genetics , Smad3 Protein/genetics , Smad3 Protein/metabolism , Smad3 Protein/pharmacology
2.
Burns ; 49(4): 889-900, 2023 06.
Article in English | MEDLINE | ID: mdl-35850880

ABSTRACT

OBJECTIVE: Curcumin, a natural extract from the rhizomes of Curcuma longa, is also known as a curcuminoid. Curcumin has been studied as a therapeutic drug for wound healing because of its anti-inflammatory, anti-oxidant, and anti-bacterial activities. However, the detailed mechanism of curcumin in wound healing is not clear. It is well-known that the skin is the largest organ in humans and prevents tissues from damage, including infection, radiation, and mechanical damage. Wound healing of the skin is a complex physiological regulation process requiring various cell types and cytokines; hence, wound healing, including surgery and care, incurs a huge expenditure each year. Transient receptor potential cation channel subfamily M member 7 (TRPM7) regulates multiple physiological and pharmacological processes through its channel and kinase activities. In addition, TRPM7 regulates cell adhesion, migration, and anti-oxidative activity, thereby playing a regulatory role in the wound healing process. This study aimed to explore the function of curcumin in the wound healing process. METHODS: We first established TRPM7 overexpression and knockdown models in fibroblasts using lentivirus. CCK-8 and wound healing assays were used to clarify whether overexpression of TRPM7 promoted proliferation and migration in fibroblasts. Expression of target genes and proteins was detected using qPCR and western blotting. Concentrations of migration-related cytokines were measured using ELISA. RESULTS: Proliferation and migration of fibroblasts increased after curcumin treatment and was further enhanced after overexpression of TRPM7. In addition, expression of proliferation-related genes and proteins was elevated after TRPM7 overexpression. Further, the secretion of migration-related cytokines was elevated after TRPM7 overexpression. CONCLUSION: Curcumin treatment promoted proliferation and migration of fibroblasts, and these effects were mediated by the signal transducer and activator of transcription 3 (STAT3)/SMAD family member 3/hypoxia-inducible factor 1 subunit alpha signaling pathway. Thus, we conclude that overexpression of TRPM7 might contribute to wound healing.


Subject(s)
Burns , Curcumin , TRPM Cation Channels , Humans , Burns/metabolism , Curcumin/metabolism , Curcumin/pharmacology , Cytokines/metabolism , Fibroblasts/metabolism , Protein Serine-Threonine Kinases , Signal Transduction , Smad3 Protein/metabolism , Smad3 Protein/pharmacology , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/pharmacology , TRPM Cation Channels/metabolism , Wound Healing
3.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 34(11): 1161-1166, 2022 Nov.
Article in Chinese | MEDLINE | ID: mdl-36567559

ABSTRACT

OBJECTIVE: To investigate the effect of digoxin on bleomycin-induced pulmonary fibrosis in mice, and investigate its possible mechanism through in vitro and in vivo experiments. METHODS: (1) In vivo experiment: 60 C57/BL6J mice were randomly divided into control group, pulmonary fibrosis model group (model group), pirfenidone (300 mg/kg) group, digoxin 1.0 mg/kg and 0.2 mg/kg groups, with 12 mice in each group. The pulmonary fibrosis model of mice was reproduced by single intratracheal infusion of bleomycin (5 mg/kg). The control group was given the same amount of sterile normal saline. From the next day after modeling, each group was received corresponding drugs by intragastric administration once a day for 28 days. Control group and model group were given the same amount of normal saline. The mice were sacrificed and the lung tissue was collected to detect the lung coefficient. After hematoxylin-eosin (HE) and Masson staining, the lung tissue morphology and collagen changes were observed under light microscope. Immunohistochemistry was used to detect the positive expressions of α-smooth muscle actin (α-SMA) and extracellular matrix (ECM) collagen (COL-I and COL-III) in lung tissue. The protein expressions of ECM fibronectin (FN), transforming growth factor-ß (TGF-ß) and phosphorylation of Smad3 (p-Smad3) in lung tissue were detected by Western blotting. (2) In vitro experiment: human embryonic lung fibroblast-1 (HFL-1) cells were cultured and divided into blank control group, fibroblast activation model group (model group), pirfenidone (2.5 mmol/L) group and digoxin 100 nmol/L and 50 nmol/L groups when cell density reached 70%-90%. After 3-hour treatment with corresponding drugs, except blank control group, the other groups were treated with TGF-ß for 48 hours to establish fibroblast activation model. The expressions of α-SMA, FN and p-Smad3 proteins and the phosphorylations of phosphatidylinositol-3-kinase (PI3K)/Akt pathway proteins PI3K and Akt (p-PI3K, p-Akt) were detected by Western blotting. RESULTS: (1) In vivo, compared with the control group, the alveolar structure of mice in the model group was significantly damaged, a large number of inflammatory cells infiltrated, collagen deposition in the lung interstitium was increased, the deposition of ECM in the lung tissue was also increased, and the expressions of α-SMA, FN, TGF-ß and p-Smad3 protein were increased, indicating that the model of bleomycin-induced pulmonary fibrosis in mice was successfully prepared. Compared with the model group, digoxin significantly inhibited airway inflammation and collagen fiber deposition, reduced ECM deposition, and decreased the protein expressions of α-SMA, FN, TGF-ß and p-Smad3, while the effect was better than that of the pirfenidone group, and the digoxin 1.0 mg/kg group had a better effect except FN [α-SMA (A value): 5.37±1.10 vs. 9.51±1.66, TGF-ß protein (TGF-ß/GAPDH): 0.09±0.04 vs. 0.33±0.23, p-Smad3 protein (p-Smad3/GAPDH): 0.05±0.01 vs. 0.20±0.07, all P < 0.01]. (2) In vitro, compared with the blank control group, the expressions of FN, α-SMA, p-Smad3 and PI3K/Akt signaling proteins in the model group were increased, indicating that the fibroblast activation model induced by TGF-ß was successfully reproduced. Compared with the model group, digoxin significantly inhibited fibroblast activation, and decreased the expressions of FN, α-SMA, p-Smad3, and PI3K/Akt pathway proteins, moreover, the effect was better than that of the pirfenidone group, and decreased FN, SMA and p-Akt protein expressions were more obvious in digoxin 100 nmol/L group [FN protein (FN/GAPDH): 0.21±0.15 vs. 0.88±0.22, α-SMA protein (α-SMA/GAPDH): 0.20±0.01 vs. 0.50±0.08, p-Akt protein (p-Akt/GAPDH): 0.30±0.01 vs. 0.65±0.10, all P < 0.01]. CONCLUSIONS: Digoxin could suppress the pulmonary fibrosis in mice induced by bleomycin, which might be associated with the regulation of fibroblast activation via suppressing PI3K/Akt signaling pathway in a dose-dependent manner.


Subject(s)
Pulmonary Fibrosis , Mice , Humans , Animals , Pulmonary Fibrosis/drug therapy , Pulmonary Fibrosis/chemically induced , Proto-Oncogene Proteins c-akt/metabolism , Smad3 Protein/metabolism , Smad3 Protein/pharmacology , Digoxin/metabolism , Digoxin/pharmacology , Digoxin/therapeutic use , Phosphatidylinositol 3-Kinases/metabolism , Saline Solution/therapeutic use , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta/therapeutic use , Fibroblasts/metabolism , Fibroblasts/pathology , Signal Transduction , Bleomycin/metabolism , Bleomycin/pharmacology , Bleomycin/therapeutic use , Collagen/metabolism , Collagen/pharmacology , Collagen/therapeutic use , Phosphatidylinositols/metabolism , Phosphatidylinositols/pharmacology , Phosphatidylinositols/therapeutic use , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta1/pharmacology
4.
Int Urol Nephrol ; 54(11): 2855-2866, 2022 Nov.
Article in English | MEDLINE | ID: mdl-35922702

ABSTRACT

BACKGROUND: Ureteral stricture (US) is a fibrotic process that leads to urinary tract obstruction and even kidney damage, with the characteristic of reduced extracellular matrix (ECM) degradation and increased collagen synthesis. Verapamil, as a calcium channel blocker, was reported to prevent scar formation. Our work aimed to investigate the biological effects and mechanism of verapamil in US. METHODS: Fibroblasts were subjected to transforming growth factor-beta 1 (TGF-ß1) to stimulate collagen synthesis, and the messenger ribonucleic acid (mRNA) and protein expressions in fibroblasts were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The location of phosphorylation-signal transducer and activator of transcription 3 (p-STAT3) and Jund proto-oncogene subunit (JunD) in fibroblasts were determined by immunofluorescence (IF). The binding relationship between signal transducer and activator of transcription 3 (STAT3) and collagen type I alpha1 (COL1A1)/collagen type III alpha 1 chain (COL3A1) and the binding relationship between JunD and tissue inhibitor of metalloproteinases-1 (TIMP-1) were verified by dual luciferase reporter gene and chromatin Immunoprecipitation (ChIP) assays. RESULTS: Herein, we found that verapamil could inhibit TGF-ß1/Ca2 + /calmodulin-dependent protein kinase II (CaMK II)-mediated STAT3 activation in fibroblasts, and STAT3 inhibition repressed collagen production. In addition, verapamil could inhibit TGF-ß1/CaMK II-mediated Mothers against DPP homolog 3 (Smad3)/JunD pathway activation in fibroblasts, and JunD silencing inhibited TIMP1 (a matrix metalloproteinase inhibitor) expression. Our subsequent experiments revealed that STAT3 bound with COL1A1 promoter and COL3A1 promoter and activated their transcription, and JunD bound with TIMP1 promoter and activated its transcription. Moreover, as expected, STAT3 activation could eliminate the inhibitory effect of verapamil treatment on TGF-ß1-induced collagen production in fibroblasts, and JunD overexpression reversed the inhibitory effect of verapamil treatment on TGF-ß1-induced TIMP1 expression in fibroblasts. CONCLUSION: Verapamil inhibited collagen production and TIMP-1 expression in US by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.


Subject(s)
Transforming Growth Factor beta1 , Ureteral Obstruction , Calcium Channel Blockers/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/pharmacology , Collagen/metabolism , Collagen Type I/genetics , Collagen Type III , Constriction, Pathologic , Fibroblasts/metabolism , Humans , Luciferases/metabolism , Matrix Metalloproteinase Inhibitors/metabolism , Matrix Metalloproteinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-jun , RNA , RNA, Messenger/metabolism , STAT3 Transcription Factor , Signal Transduction , Smad3 Protein/metabolism , Smad3 Protein/pharmacology , Tissue Inhibitor of Metalloproteinase-1/metabolism , Transforming Growth Factor beta1/metabolism , Transforming Growth Factors/metabolism , Ureteral Obstruction/metabolism , Verapamil/metabolism , Verapamil/pharmacology
5.
BMC Gastroenterol ; 22(1): 378, 2022 Aug 08.
Article in English | MEDLINE | ID: mdl-35941537

ABSTRACT

BACKGROUND: The stromal antigen 3 (STAG3) gene encodes an adhesion complex subunit that can regulate sister chromatid cohesion during cell division. Chromosome instability caused by STAG3 gene mutation may potentially promote tumor progression, but the effect of STAG3 on hepatocellular carcinoma (HCC) and the related molecular mechanism are not reported in the literature. The mechanism of the occurrence and development of HCC is not adequately understood. Therefore, the biological role of STAG3 in HCC remains to be studied, and whether STAG3 might be a sensitive therapeutic target in HCC remains to be determined. METHODS: The expression and clinical significance of STAG3 in HCC tissues and cell lines were determined by RT-qPCR and immunohistochemistry analyses. The biological functions of STAG3 in HCC were determined through in vitro and in vivo cell function tests. The molecular mechanism of STAG3 in HCC cells was then investigated by western blot assay. RESULTS: The mRNA expression of STAG3 was lower in most HCC cells than in normal cells. Subsequently, an immunohistochemical analysis of STAG3 was performed with 126 samples, and lower STAG3 expression was associated with worse overall survival in HCC patients. Moreover, cytofunctional tests revealed that the lentivirus-mediated overexpression of STAG3 in HCC cells inhibited cell proliferation, migration, and invasion; promoted apoptosis; induced G1/S phase arrest in vitro; and inhibited tumor growth in vivo. Furthermore, studies of the molecular mechanism suggested that the overexpression of STAG3 increased Smad3 expression and decreased CDK4, CDK6, cyclin D1, CXCR4 and RhoA expression. CONCLUSION: STAG3 exhibits anticancer effects against HCC, and these effects involve the Smad3-CDK4/CDK6-cyclin D1 and CXCR4/RhoA pathways. STAG3 is a tumor-suppressor gene that may serve as a potential target for molecular therapy, which provides a new idea for the treatment of HCC.


Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Carcinoma, Hepatocellular/pathology , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6 , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/pathology , Receptors, CXCR4 , Smad3 Protein/genetics , Smad3 Protein/metabolism , Smad3 Protein/pharmacology , Up-Regulation , rhoA GTP-Binding Protein/genetics
6.
BMC Mol Cell Biol ; 23(1): 18, 2022 Apr 12.
Article in English | MEDLINE | ID: mdl-35413833

ABSTRACT

BACKGROUND: Triple-negative breast cancer (TNBC) is the most severe type of breast cancer owing to its high heterogeneity, aggressiveness and lack of treatment. Studies have reported that uncarboxylated osteocalcin (GluOC) promotes the development of prostate and other cancers. Studies have also found elevated levels of serum osteocalcin in breast cancer patients with bone metastasis, and serum osteocalcin can be a marker of bone metastasis. However, whether GluOC promotes the development of TNBC and the related mechanisms need to be further clarified. RESULTS: Our results revealed that GluOC is associated with the proliferation and metastasis of MDA-MB-231 cells. GluOC increased the viability and proliferation of MDA-MB-231 cells. In addition, GluOC enhanced the metastatic ability of MDA-MB-231 cells by promoting the expression of matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-13 (MMP13), and vascular endothelial growth factor (VEGF) and inducing epithelial-mesenchymal transition (EMT). We also found that GluOC upregulated the expression of interleukin-8 (IL-8) and parathyroid hormone-related protein (PTHrP) genes in MDA-MB-231 breast cancer cells. Moreover, the promoting effect of GluOC was reversed in MDA-MB-231 breast cancer cells treated with specific inhibitor of SMAD3 (SIS3), a SMAD3 phosphorylation inhibitor. CONCLUSION: Our research proved for the first time that GluOC facilitates the proliferation and metastasis of MDA-MB-231 cells by accelerating the transforming growth factor-ß (TGF-ß)/SMAD3 signaling pathway. Moreover, GluOC also promotes the gene expression of IL-8 and PTHrP. Both IL-8 and PTHrP can act as osteolytic factors in breast cancer cells. This study indicates that GluOC may be a useful target for preventing TNBC bone metastasis.


Subject(s)
Osteocalcin , Parathyroid Hormone-Related Protein , Transforming Growth Factor beta , Triple Negative Breast Neoplasms , Cell Proliferation , Humans , Interleukin-8/metabolism , Matrix Metalloproteinase 2/metabolism , Osteocalcin/metabolism , Parathyroid Hormone-Related Protein/metabolism , Parathyroid Hormone-Related Protein/pharmacology , Signal Transduction , Smad3 Protein/metabolism , Smad3 Protein/pharmacology , Transforming Growth Factor beta/metabolism , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Vascular Endothelial Growth Factor A/metabolism
7.
BMC Cardiovasc Disord ; 22(1): 97, 2022 03 12.
Article in English | MEDLINE | ID: mdl-35279096

ABSTRACT

PURPOSE: The aim of the present study was to investigate the efficacy of recombinant human endostatin (ES) (rh-ES) combined with radiation on rat cardiomyocyte apoptosis and the regulatory mechanism of transforming growth factor beta1 (TGF-ß1)/Sma and Mad-related protein 3 (Smad3)/connective tissue growth factor (CTGF) signaling. METHOD: The primary cardiomyocytes were isolated from neonatal Sprague-Dawley rats for culture in vitro and divided into blank control group (without treatment), 10 Gy radiation + siTGF-ß1 siRNA (gene silencing) group, ES + siTGF-ß1 siRNA group, and 10 Gy radiation + ES + siTGF-ß1 siRNA group. Methyl thiazolyl tetrazolium assay was used to calculate the half-maximal inhibitory concentration (IC50) of rh-ES on cardiomyocytes. Adenoviral vector was constructed for virus packaging to silence TGF-ß1 expression in cardiomyocytes. Quantitative real-time polymerase chain reaction and Western blot were carried out to analyze TGF-ß1, Smad2, Smad3 and CTGF expression at both gene and protein levels. Flow cytometry and electron microscope were used to examine cell apoptosis. RESULTS: ES had a dose-dependent inhibitory effect on the proliferation of primary rat cardiomyocytes. ES combined with radiotherapy significantly inhibited cardiomyocyte proliferation and promoted cell apoptosis (P < 0.01). The gene and protein expression of TGF-ß1, Smad2, Smad3 and CTGF were significantly up-regulated in primary cardiomyocytes transfected with TGF-ß1 gene (P < 0.05). CONCLUSION: The combination therapy with rh-ES and radiation can promote cardiomyocyte apoptosis and aggravate myocardial cell damage via TGF-ß1/Smad3/CTGF signaling pathway.


Subject(s)
Myocytes, Cardiac , Transforming Growth Factor beta1 , Animals , Apoptosis , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Connective Tissue Growth Factor/pharmacology , Endostatins/genetics , Endostatins/metabolism , Endostatins/pharmacology , Humans , Myocytes, Cardiac/metabolism , RNA, Small Interfering/pharmacology , Rats , Rats, Sprague-Dawley , Signal Transduction , Smad3 Protein/genetics , Smad3 Protein/metabolism , Smad3 Protein/pharmacology , Transforming Growth Factor beta1/metabolism
8.
Biomed Res Int ; 2019: 8483765, 2019.
Article in English | MEDLINE | ID: mdl-31467913

ABSTRACT

Intimal hyperplasia is a complex process which contributes to several clinical problems such as atherosclerosis and postangioplasty restenosis. Inhibition of Smad3 expression inhibits intimal thickening. Our previous study has modified biscarbamate cross-linked polyethylenimine derivative (PEI-Et) through PEGylation thus obtained polyethylene glycol-graft-polyethylenimine derivative (PEG-Et 1:1), which has lower cytotoxicity and higher gene transfection efficiency compared with PEI-Et. In this study, PEG-Et 1:1 was employed in Smad3 shRNA (shSmad3) delivery for preventing intimal hyperplasia after vascular injury. It was observed that PEG-Et 1:1 could condense shSmad3 gene into nanoparticles with particle size of 115-168 nm and zeta potential of 3-6 mV. PEG-Et 1:1 displayed remarkably lower cytotoxicity, higher transfection efficiency, and shRNA silencing efficiency than PEI-Et and PEI 25 kDa in vascular smooth muscle cells (VSMCs). Moreover, PEG-Et 1:1/shSmad3 polyplex treatment significantly inhibited collagen, matrix metalloproteinase 1 (MMP1), MMP2 and MMP9 expression, and upregulated tissue inhibitor of metalloproteinase 1 (TIMP1) expression both in vitro and in vivo. Furthermore, intravascular delivery of shSmad3 with PEG-Et 1:1 polyplex efficiently reduced Smad3 expression and inhibited intimal thickening 14 days after vascular injury. Ultimately, this study indicated that PEG-Et 1:1-mediated local delivery of shSmad3 is a promising strategy for preventing intimal thickening.


Subject(s)
Carotid Intima-Media Thickness , Polyethyleneimine/pharmacology , Smad3 Protein/genetics , Vascular System Injuries/genetics , Animals , Gene Expression Regulation/drug effects , Humans , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Nanoparticles , Plasmids , Polyethylene Glycols/chemistry , Polyethyleneimine/chemistry , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Rabbits , Smad3 Protein/pharmacology , Transfection , Vascular System Injuries/pathology , Vascular System Injuries/therapy
9.
Am J Pathol ; 186(5): 1114-27, 2016 05.
Article in English | MEDLINE | ID: mdl-26948424

ABSTRACT

The ß-galactoside-binding animal lectin galectin-3 is predominantly expressed by activated macrophages and is a promising biomarker for patients with heart failure. Galectin-3 regulates inflammatory and fibrotic responses; however, its role in cardiac remodeling remains unclear. We hypothesized that galectin-3 may be up-regulated in the pressure-overloaded myocardium and regulate hypertrophy and fibrosis. In normal mouse myocardium, galectin-3 was constitutively expressed in macrophages and was localized in atrial but not ventricular cardiomyocytes. In a mouse model of transverse aortic constriction, galectin-3 expression was markedly up-regulated in the pressure-overloaded myocardium. Early up-regulation of galectin-3 was localized in subpopulations of macrophages and myofibroblasts; however, after 7 to 28 days of transverse aortic constriction, a subset of cardiomyocytes in fibrotic areas contained large amounts of galectin-3. In vitro, cytokine stimulation suppressed galectin-3 synthesis by macrophages and cardiac fibroblasts. Correlation studies revealed that cardiomyocyte- but not macrophage-specific galectin-3 localization was associated with adverse remodeling and dysfunction. Galectin-3 knockout mice exhibited accelerated cardiac hypertrophy after 7 days of pressure overload, whereas female galectin-3 knockouts had delayed dilation after 28 days of transverse aortic constriction. However, galectin-3 loss did not affect survival, systolic and diastolic dysfunction, cardiac fibrosis, and cardiomyocyte hypertrophy in the pressure-overloaded heart. Despite its potential role as a prognostic biomarker, galectin-3 is not a critical modulator of cardiac fibrosis but may delay the hypertrophic response.


Subject(s)
Cardiomegaly/physiopathology , Galectin 3/metabolism , Myocardium/metabolism , Ventricular Remodeling/physiology , Animals , Biomarkers/metabolism , Blood Pressure/physiology , Cardiomegaly/diagnostic imaging , Echocardiography, Doppler , Endomyocardial Fibrosis/diagnostic imaging , Endomyocardial Fibrosis/physiopathology , Female , Interleukin-1beta/pharmacology , Macrophages/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myofibroblasts/metabolism , Myofibroblasts/physiology , Smad3 Protein/pharmacology , Transforming Growth Factor beta/pharmacology , Up-Regulation/physiology , Ventricular Dysfunction, Left/diagnostic imaging , Ventricular Dysfunction, Left/physiopathology
10.
J Lipid Res ; 49(9): 1981-9, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18511845

ABSTRACT

The TGFbeta1/Smad pathway plays a critical role in cholestasis and liver fibrosis. Previous studies show that TGFbeta1, TNFalpha, and insulin inhibit cholesterol 7alpha-hydroxylase (CYP7A1) gene transcription and bile acid synthesis in human hepatocytes. In this study, we investigated insulin, TGFbeta1, and TNFalpha regulation of rat Cyp7a1 gene transcription. In contrast to inhibition of human CYP7A1 gene transcription, TGFbeta1 stimulates rat Cyp7a1 reporter activity. Smad3, FoxO1, and HNF4alpha synergistically stimulated rat Cyp7a1 gene transcription. Mutations of the Smad3, FoxO1, or HNF4alpha binding site attenuated the rat Cyp7a1 promoter activity. Furthermore, TNFalpha and cJun attenuated TGFbeta1 stimulation of rat Cyp7a1. Insulin or adenovirus-mediated expression of constitutively active AKT1 inhibited FoxO1 and Smad3 synergy. In streptozotocin-induced diabetic rats, Cyp7a1 mRNA expression levels were induced and insulin attenuated CYP7A1 mRNA levels. Chromatin immunoprecipitation assay showed that FoxO1 binding to Cyp7a1 chromatin was increased in diabetic rat livers and insulin reduced FoxO1 binding. These results suggest a mechanistic basis for induction of Cyp7a1 activity and bile acid synthesis in cholestatic rats and in diabetic rats. The crosstalk of insulin, TGFbeta and TNFalpha signaling pathways may regulate bile acid synthesis and lipid homeostasis in diabetes, fatty liver disease, and liver fibrosis.


Subject(s)
Cholesterol 7-alpha-Hydroxylase/genetics , Gene Expression Regulation, Enzymologic , Insulin/physiology , Transforming Growth Factor beta1/physiology , Tumor Necrosis Factor-alpha/physiology , Animals , Cell Line, Tumor , Forkhead Transcription Factors/physiology , Humans , Male , Nerve Tissue Proteins/physiology , Rats , Rats, Sprague-Dawley , Signal Transduction , Smad3 Protein/antagonists & inhibitors , Smad3 Protein/pharmacology
11.
Am J Physiol Cell Physiol ; 292(1): C259-68, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16837651

ABSTRACT

Cyclooxygenase-2 (COX-2) mediates various inflammatory responses and is expressed in pancreatic tissue from patients with chronic pancreatitis. To examine the role of COX-2 in chronic pancreatitis, we investigated its participation in regulating functions of pancreatic stellate cells (PSCs), using isolated rat PSCs. COX-2 was expressed in culture-activated PSCs but not in freshly isolated quiescent PSCs. TGF-beta1, IL-1beta, and IL-6 enhanced COX-2 expression in activated PSCs, concomitantly increasing the expression of alpha-smooth muscle actin (alpha-SMA), a parameter of PSC activation. The COX-2 inhibitor NS-398 blocked culture activation of freshly isolated quiescent PSCs. NS-398 also inhibited the enhancement of alpha-SMA expression by TGF-beta1, IL-1beta, and IL-6 in activated PSCs. These data indicate that COX-2 is required for the initiation and promotion of PSC activation. We further investigated the mechanism by which cytokines enhance COX-2 expression in PSCs. Adenovirus-mediated expression of dominant negative Smad2/3 inhibited the increase in expression of COX-2, alpha-SMA, and collagen-1 mediated by TGF-beta1 in activated PSCs. Moreover, dominant negative Smad2/3 expression attenuated the expression of COX-2 and alpha-SMA enhanced by IL-1beta and IL-6. Anti-TGF-beta neutralizing antibody also attenuated the increase in COX-2 and alpha-SMA expression caused by IL-1beta and IL-6. IL-6 as well as IL-1beta enhanced TGF-beta1 secretion from PSCs. These data indicate that Smad2/3-dependent pathway plays a central role in COX-2 induction by TGF-beta1, IL-1beta, and IL-6. Furthermore, IL-1beta and IL-6 promote PSC activation by enhancing COX-2 expression indirectly through Smad2/3-dependent pathway by increasing TGF-beta1 secretion from PSCs.


Subject(s)
Cyclooxygenase 2/metabolism , Cytokines/physiology , Inflammation Mediators/physiology , Pancreas/physiology , Actins/antagonists & inhibitors , Actins/metabolism , Animals , Antibodies/pharmacology , Cells, Cultured , Cyclooxygenase Inhibitors/pharmacology , Genes, Dominant , Interleukin-1beta/physiology , Interleukin-6/physiology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Muscle, Smooth/metabolism , Nitrobenzenes/pharmacology , Pancreas/cytology , Pancreas/drug effects , Pancreas/enzymology , Protein Kinase Inhibitors/pharmacology , Rats , Smad2 Protein/genetics , Smad2 Protein/pharmacology , Smad3 Protein/genetics , Smad3 Protein/pharmacology , Sulfonamides/pharmacology , Transforming Growth Factor beta1/immunology , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta1/physiology
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