Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.
Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; physiology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line, Tumor ; Connective Tissue Growth Factor ; genetics ; metabolism ; pharmacology ; Cytochalasin D ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; HEK293 Cells ; Humans ; Immunohistochemistry ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; Palmitic Acid ; pharmacology ; Phosphoproteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Rats ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; Thiazolidines ; pharmacology

The aim of this research is to evaluate the effect of tetramethylpyrazine (TMP) and connective tissue growth factor (CTGF) miRNA plasmids on the expressive levels of CTGF, transforming growth factor-beta (TGFbeta) and type I collagen of rat hepatic stellate cells (HSC) which are stimulated by high glucose. The rat HSCs which were successfully transfected rat CTGF miRNA plasmids and the rat HSCs which were successfully transfected negative plasmids were cultured in vitro. After stimulus of the TMP and the high glucose, the protein levels and gene expressive levels of CTGF, TGF-beta and type I collagen were tested. The results indicated that high glucose increased the expression of CTGF mRNA, CTGF protein, TGF-beta mRNA,TGF-beta protein and type I collagen (P < 0.05). The expressive levels of CTGF mRNA, CTGF protein, TGF-beta mRNA, TGF-beta and type I collagen in TMP group were lower than those in high glucose group and showed statistically significant differences (P < 0.05). Compared with high glucose group, the expressive levels of CTGF mRNA, CTGF protein, TGF-beta mRNA, TGF-beta and type I collagen in rat CTGF miRNA plasmid interference group were significantly lower (P < 0.05). However, no statistically significant difference was found in CTGF mRNA and CTGF protein levels between TMP group and CTGF miRNA group (P > 0.05), while type I collagen levels showed statistically significant differences (P < 0.05). It is concluded that high glucose could promote the expressions of CTGF, TGF-beta and type I collagen, and TMP and rat CTGF miRNA plasmids could reduce the expressions of CTGF, TGF-beta, type I collagen.
Animals ; Cells, Cultured ; Collagen Type I ; metabolism ; Connective Tissue Growth Factor ; genetics ; Culture Media ; pharmacology ; Glucose ; pharmacology ; Hepatic Stellate Cells ; drug effects ; metabolism ; MicroRNAs ; genetics ; Plasmids ; Pyrazines ; pharmacology ; RNA, Messenger ; Rats ; Transfection ; Transforming Growth Factor beta ; metabolism

Long-term treatment with an agonist of peroxisome proliferator-activated receptor (PPAR)-γ is associated with bone fractures in the clinical practice. However, the mechanisms underlying the fractures are not fully understood. This study was aimed to examine the effect of rosiglitazone (an agonist of PPAR-γ) of different doses on the proliferation, differentiation, and transforming growth factor beta 1 (TGF-β1)-induced expression of connective tissue growth factor (CTGF) in primary rat osteoblasts in vitro. Osteoblasts were isolated from newly born SD rats and treated with different doses of rosiglitazone (0-20 μmol/L). The proliferation and differentiation of osteoblasts were measured by MTT assay and NPP assay, respectively. The expression of CTGF was determined by RT-PCR and Western blotting. The results showed that most isolated osteoblasts displayed strong alkaline phosphatase (ALP) activity and treatment with different doses of rosiglitazone did not affect their proliferation, but significantly inhibited the differentiation of osteoblasts in a dose-dependent manner. Moreover, treatment with different doses of rosiglitazone significantly reduced the TGF-β1-induced CTGF mRNA transcription and protein expression in a dose-dependent manner in rat osteoblasts. It was concluded that the activation of PPAR-γ may inhibit the differentiation of osteoblasts by reducing the TGF-β1-induced CTGF expression in vitro.
Animals ; Animals, Newborn ; Blotting, Western ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Gene Expression ; drug effects ; Hypoglycemic Agents ; pharmacology ; Osteoblasts ; cytology ; drug effects ; metabolism ; PPAR gamma ; agonists ; metabolism ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Thiazolidinediones ; pharmacology ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVE: To explore the inhibitory effect of calcitonin gene-related peptide (CGRP) on cardiac fibroblast proliferation and collagen synthesis induced by isoprenaline and the underlying mechanism. METHODS: The primary cultured cardiac fibroblasts were incubated with isoprenaline (10(-5) mol/L) for 48 h after pretreatment with CGRP (10(-8) or 10(-7) mol/L) for 1 h. Cell activity was detected by MTT. The mRNA expression of collagen (types I and III) and connective tissue growth factor (CTGF) was determined by RT-PCR, and the levels of intracellular ROS were determined by DCFH-DA fluorescent probe. RESULTS: Isoprenaline significantly promoted fibroblast proliferation and up-regulated collagen (types I and III) and CTGF mRNA expression concomitantly with an increase in ROS production, which were attenuated by CGRP. The effect of CGRP on cardiac fibroblasts was inhibited by CGRP8-37, a selective antagonist of CGRP receptor. CONCLUSION CGRP is able to protect cardiac fibroblasts against isoprenaline-induced proliferation and collagen expression, which might be related to the down-regulation of CTGF expression through inhibition of ROS production.
Animals ; Animals, Newborn ; Calcitonin Gene-Related Peptide ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Connective Tissue Growth Factor ; genetics ; metabolism ; Fibroblasts ; cytology ; Isoproterenol ; antagonists & inhibitors ; pharmacology ; Myocytes, Cardiac ; cytology ; Primary Cell Culture ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism

To investigate the effect of short hairpin RNA (shRNA)-mediated silencing of CTGF and TIMP-1 in hepatic stellate cells (HSCs) on mRNA expression of TIMP-1, CTGF, and procollagen type-I (PC I), as well as secretion of extracellular matrix (ECM) proteins. Two recombinant expression plasmids harboring shRNAs against CTGF and TIMP-1 (psiRNA-GFP-CTGF and psiRNA-GFP-TIMP-1) were transfected alone or together into TGFb1-activated HSC-T6 cells. The mRNA expression levels of CTGF, TIMP-1, and PC I were detected by fluorescence quantitative PCR (FQ-PCR). The concentrations of secreted PC type-III, hyaluronate (HA), and laminin (LN) were measured by radioimmunoassay (RIA) of culture supernatants. FQ-PCR analysis showed that CTGFshRNA and TIMP-1shRNA specifically inhibited the expression of CTGF, TIMP-1, and PC I mRNA in activated HSC-T6 cells. The concentrations of secreted PC III, HA, and LN were decreased significantly in HSC-T6 cells with shRNA-silenced CTGF or TIMP-1 (P less than 0.01 or P less than 0.05). Moreover, HSC-T6 cells with shRNA-silenced CTGF and TIMP-1 showed a more robust decrease in synthesis of PC III, HA and LN (all, P less than 0.01), as well as in mRNA expression of PC I (P less than 0.05). CTGFshRNA and TIMP-1shRNA effectively inhibit expression of the respective target genes, as well as of PC I, and decrease secretion of ECM components from HSC-T6 cells. Silencing of both CTGF and TIMP-1 produces more robust effects than either in isolation. These data suggest that CTGF and TIMP-1 may be effective targets of shRNA-based gene therapy to treat liver fibrosis.
Animals ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Connective Tissue Growth Factor ; genetics ; metabolism ; Down-Regulation ; Extracellular Matrix ; metabolism ; Gene Expression Regulation ; Gene Silencing ; Hepatic Stellate Cells ; drug effects ; metabolism ; Hyaluronic Acid ; metabolism ; Laminin ; metabolism ; Liver Cirrhosis ; metabolism ; pathology ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transfection ; Transforming Growth Factor beta ; metabolism

To explore the angiotensin peptide [Ang (1-7)]-mediated inhibition of Ang II in human hepatic stellate cells (HSCs) and determine the involvement of the ACE2-Ang (1-7)-Mas axis. The human HSC line, LX2, was used in all experiments, and divided into control (unstimulated) and Ang II-stimulated (10-6 mol/L) groups. The Ang II-stimulated cells were further divided among several pre-treatment (prior to Ang II) groups: ROCK-inhibited (Y27632 blocking agent, 10-6 mol/L); irbesartan-inhibited (AT-1 receptor antagonist, 10-6 mol/L); and Mas receptor-inhibited (A779 Mas receptor antagonist, 10-6 mol/L). To explore the potential inhibitory effects of various Ang family members, the Ang II-stimulated and pre-treated LX2 cells were exposed to Ang (1-7) (10-6 mol/L) for 24 h. Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and QuantiGene assay were used to assess changes in protein and mRNA expression levels of RhoA, ROCK, and connective tissue growth factor (CTGF). Compared with the control group, Ang II-stimulated cells showed significantly increased levels of RhoA protein (0.337+/-0.074 vs. 0.870+/-0.093), ROCK2 mRNA (0.747+/-0.061 vs. 0.368+/-0.023), and CTGF mRNA (0.262+/-0.007 vs. 0.578+/-0.028) (all, P less than 0.01). Pre-treatment with irbesartan or Y27632 eliminated these responses. Ang (1-7) inhibited the Ang II-stimulated up-regulation of RhoA, ROCK, and CTGF. Ang (1-7) can inhibit the Ang II-stimulated up-regulation of RhoA, ROCK and CTGF in hepatic stellate cells, indicating that the ACE2-Ang (1-7)-Mas axis, an important branch of the renin-Ang-aldosterone system is involved in the occurrence and development of liver fibrosis.
Angiotensin I ; pharmacology ; Angiotensin II ; pharmacology ; Cells, Cultured ; Connective Tissue Growth Factor ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Humans ; Peptide Fragments ; pharmacology ; RNA, Messenger ; genetics ; Signal Transduction ; rho-Associated Kinases ; metabolism ; rhoA GTP-Binding Protein ; metabolism

OBJECTIVETo detect the effect of connective tissue growth factor (CTGF) on podocalyxin expression in mouse podocytes exposed to high glucose in vitro and explore the possible pathway involved.

METHODSThe expression vector carrying a small interfering RNA (siRNA) targeting CTGF was transfected into mouse podocytes cultured in the presence of 1 g/L glucose (normal control), 4.5 g/L glucose (high glucose group), 1 g/L glucose + 3.5 g/L mannitol (iso-osmolar control group). The changes in the protein expression levels of podocalyxin, CTGF and ERK1/2 in the cells in response to the treatments were investigated using Western blotting.

RESULTSHigh glucose exposure for 24 and 48 h resulted in significantly decreased expression of podocalyxin and increased CTGF in the podocytes (P<0.05). Phosphorylation of ERK1/2 occurred as early as 30 min after the exposure, and the activation was maintained till 24 h. Transfection of the cells with siRNA targeting CTGF significantly inhibited these changes.

CONCLUSIONCTGF is an important mediator of high glucose-induced podocyte damage and decreases the protein level of podocalyxin by the ERK1/2 pathway. CTGF-specific siRNA can alleviate high glucose-induced podocyte injury, suggesting its potential value in treatment of diabetic nephropathy.

Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; metabolism ; Diabetic Nephropathies ; Glucose ; adverse effects ; MAP Kinase Signaling System ; drug effects ; Mice ; Podocytes ; cytology ; drug effects ; metabolism ; RNA, Small Interfering ; genetics ; Sialoglycoproteins ; metabolism

OBJECTIVETo explore the effects of integrin β3 pathway on the proliferation, migration and extracellular matrix deposition of pulmonary arterial smooth muscle cells (PASMCs) induced by connective tissue growth factor (CTGF).

METHODSPASMCs of SD Rats were cultured in M199 culture system in vitro and the 3rd-7th passages of PASMCs were used in the experiments. The cells were randomly divided into three groups: (1) CONTROL GROUP: culture system contained no any stimulation factor; (2) CTGF group: culture system was added into 50 ng/ml CTGF; (3) CTGF+ anti-integrin β3 antibody group:culture system was added with 50 ng/ml CTGF and 10 mg/L anti-integrin β3 antibody. The PASMCs were cultured with 50 ng/ml CTGF and anti-integrin β3 antibody (0, 5, 10, 15, 20 mg/L) for 24, 48, 72 and 96 h, the proliferation of PASMCs was detected by WST-1 Cell Proliferation Assay Kit. The migration of PASMCs was observed by Transwell cell test under the phase contrast microscope. RT-PCR assay was applied to detect the mRNA expression of collagenI-α1, collagen III-α1 and fibronectin-1 gene of PASMCs. The expression of fibronectin protein was examined by Western blotting and immunohistochemistry.

RESULTSThe results of WST-1 test showed that the anti-integrin β3 antibody inhibited significantly the proliferation of PASMCs induced by CTGF (P < 0.05), among which the inhibition rate of anti-integrin β3 antibody (10 mg/L) was the most significant. Transwell test results showed that CTGF group of PASMCs migration numbers (25 ± 1.57) were higher than that of the control group (11 ± 2.08, P < 0.01); PASMCs migration numbers of CTGF+ integrin β3 antibody group (17 ± 4.16) were less than that of the CTGF group (P < 0.05). Compared with the control group, the mRNA expression of collagen typeI-α1 (4.28 ± 0.33), collagen typeIII-α1 (4.41 ± 0.35), fibronectin-1 (4.05 ± 0.33) of PASMCs was increased in CTGF group, with a time-dependence (P < 0.01); Compared with the CTGF group, the mRNA expression of collagen typeI-α1 (3.38 ± 0.30), collagen typeIII-α1 (3.40 ± 0.30), fibronectin-1 (3.12 ± 0.29) of PASMCs was reduced in CTGF+ anti-integrin β3 antibody group (P < 0.05), which was higher than that of the control group (P < 0.05); Western blot and immunohistochemical tests showed that compared with the control group, CTGF group could stimulate the expression of fibronectin protein of PASMCs (P < 0.01); the anti-integrin β3 antibody could inhibit the expression of fibronectin protein induced by CTGF(P < 0.01), which was more remarkable than that in the control group (P < 0.01).

CONCLUSIONIntegrin β3 pathway can mediate CTGF-induced proliferation, migration and extracellular matrix deposition of PASMCs, CTGF-integrin β3 signaling pathway may play an important role in pulmonary vascular remodeling.

Animals ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; pharmacology ; Extracellular Matrix ; metabolism ; Integrin beta3 ; metabolism ; Male ; Muscle Cells ; cytology ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Pulmonary Artery ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

BACKGROUNDConnective tissue growth factor (CTGF) is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. CTGF plays an important role in extracellular matrix production by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. We herein evaluated whether CTGF was required for extracellular matrix synthesis of meniscal fibrochondrocytes and/or angiogenesis during the repair of meniscal tears.

METHODSMeniscal fibrochondrocytes were isolated from the inner-1/2 of rabbit meniscus by trypsin collagenase treatment and further treated with 100 ng/ml CTGF in vitro. Characterization of fibrochondrocytes was identified by flow cytometry analyzing CD31, CD44, CD45 and CD105, and was further tested by type II collagen immunocytochemistry. Changes in gene expression of meniscal fibrochondrocytes were monitored by quantitative real-time polymerase chain reaction. Histological sections prepared from a 3-mm portion of a longitudinal tearing defect in the middle of the rabbit meniscus were subjected to fluorescence-immunohistochemistry analysis at 1, 4 and 10 weeks following surgical treatment with 1.5 µg of CTGF/fibrin-glue composites.

RESULTSQuantitative RT-PCR assay showed that types I and II collagen and vascular endothelial growth factor mRNA expression in the 100 ng/ml CTGF group were remarkably enhanced as compared to levels in the no-dose group at 14 days ((2.38 ± 0.63) fold, (2.96 ± 0.87) fold, (2.14 ± 0.56) fold, respectively). Likewise, fluorescence-immunohistochemical analysis revealed that in the group implanted with CTGF-fibrin glue, types I and II collagen, as well as the capillaries, completely filled the defect by 10 weeks, postoperatively. In contrast, only soft tissue repair occurred when PBS-fibrin glue was implanted.

CONCLUSIONSThese findings suggest that CTGF can significantly promote extracellular matrix deposition (types I and II collagen) within the meniscal avascular zone; CTGF can greatly heighten the expression of vascular endothelial growth factor activity simultaneously in vivo, further enhancing the repair of meniscal tears in the avascular zone.

Animals ; Cells, Cultured ; Chondrocytes ; cytology ; Collagen Type I ; metabolism ; Collagen Type II ; metabolism ; Connective Tissue Growth Factor ; pharmacology ; Flow Cytometry ; Immunohistochemistry ; Male ; Menisci, Tibial ; cytology ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; Tibial Meniscus Injuries ; Vascular Endothelial Growth Factor A ; metabolism ; Wound Healing ; drug effects

OBJECTIVE: To explore the curative effect and the underlying mechanism of interferon-α (IFN-α) on rat liver fibrosis induced by CCl(4). METHODS: Forty-five Wistar male rats were randomly divided into 3 groups: a normal control group (n=15), a liver fibrosis group (n=15) and a IFN-α treatment group (n=15). The rats of the control group and the liver fibrosis group received peanut oil (0.2 mL/100 g body weight), twice a week for 8 weeks. The rats of the liver fibrosis group and the IFN-α treatment group were received intraperitoneal injection of 50% CCl(4) (CCl(4):peanut oil=1:1, 0.2 mL/100 g body weight, ip) or IFN-α (CCl(4):peanut oil=1:1, 0.2 mL/100 g body weight, ip), twice a week for 8 weeks. In the 9th week, the rats of IFN-α treatment group were switch to receive IFN-α at 100 000 units (s.c.) per day for 3 weeks. The rats were all sacrificed in the 11th week. Pathological changes of liver, semi-quantitative scoring of rat liver was observed. Tissue hydroxyproline, the mRNA expression of Collagen I, Collagen III, transforming growth factor-beta 1 (TGF-β1), connective tissue growth factor (CTGF), a-smooth muscle actin (α-SMA), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) of rat liver was detected. The protein expression of MMP-9 and TIMP-1 also was detected. RESULTS: Semi-quantitative scoring of inflammation,semi-quantitative scoring of liver fibrosis, hydroxyproline and collagen in the IFN-α treatment group were significantly lower than those in the liver fibrosis group (all P<0.05). The mRNA expression of TGF-β1, CTGF, α-SMA and TIMP-1, and the protein expression of TIMP-1 in the IFN-α treatment group were significantly lower than those in the liver fibrosis group (all P<0.05). But there was no significant difference in MMP-9 between the IFN-α treatment group and the liver fibrosis group. CONCLUSION IFN-α can decrease the liver fibrogenesis induced by CCl(4) in rats and reduce liver inflammation response. The anti-fibrosis effect of IFN-α may be related to decrease in TGF-β1, CTGF and TIMP-1 expression and to inhibiting of the hepatic satellite cells' activation and extracellular matrix synthesis.
Animals ; Carbon Tetrachloride ; toxicity ; Connective Tissue Growth Factor ; genetics ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Interferon-alpha ; therapeutic use ; Liver Cirrhosis, Experimental ; chemically induced ; drug therapy ; metabolism ; Male ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism