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Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from “quiescent” HSC to fibrogenic myofibroblasts. Transforming growth factor-β (TGF-β), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-β signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-β-treated cells demonstrated more focal adhesions and increased α-smooth muscle actin (α-SMA) organization in stress fibers, although all cells reached the same level of α-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-β, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.

AIM: To analyse the influence of Smad7, antagonist of transforming growth factor (TGF)-β canonical signaling pathways on hepatic stellate cell (HSC) transdifferentiation in detail.

METHODS: We systematically analysed genes regulated by TGF-β/Smad7 in activated HSCs by microarray analysis and validated the results using real time polymerase chain reaction and Western blotting analysis.

RESULTS: We identified 100 known and unknown targets underlying the regulation of Smad7 expression and delineated 8 gene ontology groups. Hk2, involved in glycolysis, was one of the most downregulated proteins, while BMP2, activator of the Smad1/5/8 pathway, was extremely upregulated by Smad7. However, BMP2 dependent Smad1 activation could be inhibited in vitro by Smad7 overexpression in HSCs.

CONCLUSION: We conclude (1) the existence of a tight crosstalk of TGF-β and BMP2 pathways in HSCs and (2) a Smad7 dependently decreased sugar metabolism ameliorates HSC activation probably by energy withdrawal.

Liver fibrosis is a major cause of liver failure, but treatment remains ineffective. In the present study, we investigated the mechanisms and anti-hepatofibrotic activities of asiatic acid (AA) in a rat model of liver fibrosis induced by carbon tetrachloride (CCl4) and in vitro in TGF-beta1-stimulated rat hepatic stellate cell line (HSC-T6). Treatment with AA significantly attenuated CCl4-induced liver fibrosis and functional impairment in a dosage-dependent manner, including blockade of the activation of HSC as determined by inhibiting de novo alpha smooth muscle actin (a-SMA) and collagen matrix expression, and an increase in ALT and AST (all p<0.01). The hepatoprotective effects of AA on fibrosis were associated with upregulation of hepatic Smad7, an inhibitor of TGF-beta signaling, thereby blocking upregulation of TGF-beta1 and CTGF and the activation of TGF-beta/Smad signaling. The anti-fibrosis activity and mechanisms of AA were further detected in vitro in HSC-T6. Addition of AA significantly induced Smad7 expression by HSC-T6 cells, thereby inhibiting TGF-beta1-induced Smad2/3 activation, myofibroblast transformation, and collagen matrix expression in a dosage-dependent manner. In contrast, knockdown of Smad7 in HSC-T6 cells prevented AA-induced inhibition of HSC-T6 cell activation and fibrosis in response to TGF-beta1, revealing an essential role for Smad7 in AA-induced anti-fibrotic activities during liver fibrosis in vivo and in vitro. In conclusion, AA may be a novel therapeutic agent for liver fibrosis. Induction of Smad7-dependent inhibition of TGF-beta/Smad-mediated fibrogenesis may be a central mechanism by which AA protects liver from injury.

AIM: To investigated if paclitaxel can attenuate hepatic fibrosis in rat hepatic stellate cells (RHSCs).

METHODS: RHSCs were cultured in vitro and randomly assigned to four groups: normal control group (treated only with Dulbecco’s Modified Eagle’s Medium), Taxol group (200 nmol/L paclitaxel was added to the cell culture), transforming growth factor (TGF)-β group (5 ng/mL recombinant human TGF-β1 was added to the cell culture), and TGF-β + Taxol group. TGF-β signaling cascade and status of various extracellular matrix proteins were evaluated by real time reverse transcriptase polymerase chain reaction and Western blotting.

RESULTS: The paclitaxel treatment markedly suppressed Smad2/3 phosphorylation. This was associated with attenuated expression of collagen I and III and fibronectin in RHSCs.

CONCLUSION: These data indicate that 200 nmol/L paclitaxel ameliorates hepatic fibrosis via modulating TGF-β signaling, and that paclitaxel may have some therapeutic value in humans with hepatic fibrosis.

Background/Aims

A recent decrease in patient survival has been reported among hepatitis C virus (HCV)-infected liver transplant recipients and this may be attributable to progression of fibrosis. We reported previously that cyclosporine suppressed the proliferation of, and collagen production in, hepatic stellate cells (HSCs). Here, we investigated the effects of NIM811, a cyclosporine analogue, on cell growth, collagen production and collagenase activity in HSCs.

Methods

Rat HSCs and human HSC-derived TWNT-4 cells were cultured for the study. The expression of collagen, matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and collagenase activity was evaluated. Cell proliferation and apoptosis were measured. Phosphorylation of mitogen-activated protein kinases (MAPKs), Smad2 and Smad3 was evaluated. The expression of the tumour growth factor-β (TGF-β)-receptor and Smad7 genes was also evaluated.

Results

NIM811, as well as cyclosporine, suppressed the transcription and synthesis of collagen and stimulated the production of MMP-1 with a concomitant enhancement of collagenase activity, although it did not change the expression of TIMP-1. NIM811 inhibited proliferation without induction of apoptosis. In the MAPKs and TGF-β signalling pathways, NIM811 enhanced the phosphorylation of JNK and p38, but not extracellular signal-regulated kinases 1 and 2, and suppressed the phosphorylation of Smad2 and Smad3, accompanied by increased Smad7 transcription and decreased TGF-β-receptor transcription.

Conclusion

These findings demonstrate that NIM811 not only suppresses collagen production and proliferation but also increases collagenase activity. These effects are accompanied by inhibition of TGF-β signalling pathways.

Hepatic stellate cells (HSCs) play a major role in the pathogenesis of liver fibrosis. Working on primary HSCs requires difficult isolation procedures; therefore we have generated and here characterize a mouse hepatic stellate cell line expressing GFP under control of the collagen 1(I) promoter/enhancer. These cells are responsive to pro-fibrogenic stimuIi, such as PDGF or TGF-β1, and are able to activate intracellular signalling pathways including Smads and MAP kinases. Nevertheless, due to the basal level of activation, TGF-β1 did not significantly induce GFP expression contrasting the TGF-β1 regulated endogenous collagen I expression. We could demonstrate that the accessory TGF-β-receptor endoglin, which is endogenously expressed at very low levels, has a differential effect on signalling of these cells when transiently overexpressed. In the presence of endoglin activation of Smad1/5/8 was drastically enhanced. Moreover, the phosphorylation of ERK1/2 was increased, and the expression of vimentin, α-smooth muscle actin and connective tissue growth factor was upregulated. Endoglin induced a slight increase in expression of the inhibitor of differentiation-2 while the amount of endogenous collagen type I was reduced. Therefore, this profibrogenic cell line with hepatic stellate cell origin is not only a promising novel experimental tool, which can be used in vivo for cell tracing experiments. Furthermore it allows investigating the impact of various regulatory proteins (e.g. endoglin) on profibrogenic signal transduction, differentiation and hepatic stellate cell biology.

Antisense Smad3 adenoviral vectors were used to transfect vascular smooth muscle cells (VSMCs) from rats with balloon injury or infused into the rat balloon-catheter injured carotid arteries, and the role of TGF-β1/Smad3 signaling pathway in the secretion of type I and III collagen by VSMCs following balloon injury was investigated. Antisense Smad3 adenoviral vectors were used to transfect these VSMCs (antisense Smad3 group). A total 90 rats were randomly assigned into blank control group, experiment group, negative control group. In the in vitro study, the expression of type I and III collagen was markedly reduced in the antisense Smad3 group when compared with the control groups (P < 0.05). In the in vivo study, the expression of type I and III collagen was significantly lower than that in the negative control group at 3 days, 1 week and 2 weeks after injury (P < 0.05). At 2 weeks and 3 months after injury, the lumen area in the antisense Smad3 group was markedly increased but the intimal area dramatically reduced when compared with the negative control (P < 0.05). We conclude that transfection of VSMCs with antisense Smad3 can reduce the secretion of type I and III collagen which then inhibit intimal hyperplasia.

Little is known about the involvement of Smad-related molecules in the regulation of the Transforming Growth Factor (TGF)-beta signaling pathway during hepatocarcinogenesis, particularly with respect to preneoplastic lesions of a rat liver. The aims of this study were to investigate the localizations and temporal expressions of TGF-beta Receptor Type 1 (TGR1) and Smads during the promotion stage of chemical hepatocarcinogenesis in rats. We investigated expressions and localizations of TGR1, Smad2, Smad4, and Smad7 by using semi-quantitative RT-PCR and immunohistochemistry in preneoplastic lesions during rat chemical hepatocarcinogenesis induced by Solt and Farber's method. The down-regulation of TGR1, Smad2, and Smad4 was evident during the later steps of the promotion stage of chemical hepatocarcinogenesis. In contrast with other Smads, increased Smad7 expression was evident during the later steps of the promotion stage. Also immunohistochemistry revealed that the main site of TGR1, Smad2, Smad4, and Smad7 expression was mainly in hepatocytes of the preneoplastic lesions of a rat liver. Dysregulation of the downstream effectors of TGF-beta such as TGR1, Smad2, Smad4 and, Smad7 might contribute to the progression of preneoplastic lesions during chemical hepatocarcinogenesis in a rat.

Salvianolic acid B (SA-B) is water-soluble component of Radix Salvia miltiorrhiza. The previous work indicated that SA-B can inhibit MAPK and Smad signaling in activated hepatic stellate cells (HSCs) to perform anti-fibrotic activity Lv et al. 2010. However, some studies have shown that there is cross-talk between MAPK and Smad in certain cell types. Thus, the anti-fibrotic action of SA-B may be through the cross-talk. In order to clarify the mechanism of SA-B further, we knocked down Smad in LX-2 cells (SRV4) via RNAi, and then added TGF-β1, and PD98059 or SB203580 and SA-B. The levels of p-MEK and p-p38 were inhibited by SA-B in SRV4 independent of TGF-β1. The expression of Col I and α-SMA in SRV4 could be reduced by SA-B independent TGF-β1. SB203580 had not significant effect on p-MEK in SRV4 stimulated by TGF-β1. The levels of p-MEK in SRV4 were not increased significantly after TGF-β1 stimulation. PD98059 had no effect on the levels of p-p38 in SRV4 irrespective of TGF-β1. In conclusion, SA-B inhibits the synthesis of Col I in LX-2 cells independent of TGF-β1 stimulation, and the anti-fibrotic effect of SA-B is due to direct inhibition of p38 signaling and inhibition the cross-talk of Smad to ERK signaling.

Transforming growth factor-beta 1 (TGF-β1) is an important growth inhibitor of epithelial cells and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. TGF-β1 signals through the TGF-β type I and type II receptors, and activates the Smad pathway via phosphorylation of Smad2 and Smad3. Since little is known about the selective activation of Smad2 versus Smad3, we set out to identify novel Smad2 and Smad3 interacting proteins in epithelial cells. A nontransformed human cell line was transduced with Myc-His6-Smad2 or Myc-His6-Smad3-expressing retrovirus and was treated with TGF-β1. Myc-His6-Smad2 or Myc-His6-Smad3 was purified by tandem affinity purification, eluates were subject to SDS-PAGE and Colloidal Blue staining, and select protein bands were digested with trypsin. The resulting tryptic peptides were analyzed by liquid chromatography and tandem mass spectrometry and the SEQUEST algorithm was employed to identify proteins in the bands. A number of proteins that are known to interact with Smad2 or Smad3 were detected in the eluates. In addition, a number of putative novel Smad2 and Smad3 associated proteins were identified that have functions in cell proliferation, apoptosis, Actin cytoskeleton regulation, cell motility, transcription, and Ras or insulin signaling. Specifically, the interaction between Smad2/3 and the Cdc42 guanine nucleotide exchange factor, Zizimin1, was validated by co-immunoprecipitation. The discovery of these novel Smad2 and/or Smad3 associated proteins may reveal how Smad2 and Smad3 are regulated and/or uncover new functions of Smad2 and Smad3 in TGF-β1 signaling.

Transforming growth factor β (TGF-β) induces the process of epithelial-mesenchymal transition (EMT) through the Smad and JNK signaling. However, it is unclear how these pathways interact in the TGF-β1-induced EMT in rat peritoneal mesothelial cells (RPMCs). Here, we show that inhibition of JNK activation by introducing the dominant-negative JNK1 gene attenuates the TGF-β1-down-regulated E-cadherin expression, and TGF-β1-up-regulated α-SMA, Collagen I, and PAI-1 expression, leading to the inhibition of EMT in primarily cultured RPMCs. Furthermore, TGF-β1 induces a bimodal JNK activation with peaks at 10 minutes and 12 hours post treatment in RPMCs. In addition, the inhibition of Smad3 activation by introducing a Smad3 mutant mitigates the TGF-β1-induced second wave, but not the first wave, of JNK1 activation in RPMCs. Moreover, the inhibition of JNK1 activation prevents the TGF-β1-induced Smad3 activation and nuclear translocation, and inhibition of the TGF-β1-induced second wave of JNK activation greatly reduced TGF-β1-induced EMT in RPMCs. These data indicate a crosstalk between the JNK1 and Samd3 pathways during the TGF-β1-induced EMT and fibrotic process in RPMCs. Therefore, our findings may provide new insights into understanding the regulation of the TGF-β1-related JNK and Smad signaling in the development of fibrosis.

Purpose

Extracellular matrix (ECM) deposits lead to elevated resistance of aqueous humor outflow which play an important role in the development of primary open angle glaucoma (POAG). The TGF-β2 (transforming growth factor β)/Smad (signaling mathers against decapentaplegic) pathway is known to regulate the ECM deposits. In this study, we determined the effect of Smad7 siRNA transfection in inhibiting the expression of ECM components.

Methods

Plasmid containing Smad7 siRNA was used to transfect cultured human trabecular meshwork cells (HTM). Protein expression of Smad7, fibronectin, and laminin was determined using western blot.

Results

Downregulation of Smad7 interrupts the effects of TGF-β2 on the expression of several ECM components. Smad7 siRNA can partially decrease the expression of Smad7, fibronectin, and laminin.

Conclusions

Smad7 plays an important role in regulating the ECM protein in the aqueous outflow pathway.

Current evidence suggests that regulation of extracellular matrix (ECM) accumulation by fibrogenic transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF) signals involves different mechanisms in acute and chronic liver injuries, even though hepatic stellate cells (HSC) are the principal effecter in both cases. As a result of chronic liver damage, HSC undergo progressive activation to become myofibroblasts (MFB)-like cells. Our current review will discuss the differential regulation of TGF-β signaling between HSC and MFB in vitro and in vivo. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad-homology (MH) 1 and MH2 domains. TGF-β type I receptor and Ras-associated kinases differentially phosphorylate Smad2 and Smad3 to create COOH-terminally (C), linker (L), or dually (L/C) phosphorylated (p) isoforms. After acute liver injury, TGF-β and PDGF synergistically promote collagen synthesis in the activated HSC via pSmad2L/C and pSmad3L/C pathways. To avoid unlimited ECM deposition, Smad7 induced by TGF-β negatively regulates the fibrogenic TGF-β signaling. In contrast, TGF-β and PDGF can transmit the fibrogenic pSmad2L/C and mitogenic pSmad3L signals in MFB throughout chronic liver injury, because Smad7 cannot be induced by the pSmad3L pathway. This lack of Smad7 induction might lead to constitutive fibrogenesis in MFB, which eventually develop into accelerated liver fibrosis.

PPARγ agonists inhibit liver fibrosis, but the mechanisms involved are uncertain. We hypothesized that PPARγ agonists inhibit transforming growth factor (TGF)β1-activation of TGFβ receptor (TGFβR)-1 signaling in quiescent stellate cells, thereby abrogating Smad3-dependent induction of extracellular matrix (ECM) genes, such as PAI-1 and collagen-1αI. To test this, human HSC were cultured to induce a quiescent phenotype, characterized by lipid accumulation and PPARγ expression and transcriptional activity. These adipocytic HSC were then treated with TGFβ1 ± a TGFβR-1 kinase inhibitor (SB431542) or a PPARγ agonist (GW7845). TGFβ1 caused dose- and time-dependent increases in Smad3 phosphorylation, followed by induction of collagen and PAI-1 expression. Like the TGFβR-1 kinase inhibitor, the PPARγ agonist caused dose-dependent inhibition of all of these responses without effecting HSC proliferation or viability. Thus, the anti-fibrotic actions of PPARγ agonists reflect their ability to inhibit TGFβ1-TGFβR1 signaling that initiates ECM gene expression in quiescent HSC.

The mechanistic relationship between the widely used monocrotaline model of primary pulmonary hypertension and altered TGFβ family signaling due to genetic defects in the Bone Morphogenetic Protein type II receptor in affected humans has not been investigated. In this study we use fluorescent microscopy to demonstrate nuclear translocation of Smad 4 in human pulmonary arterial endothelial cell (HPAEC) cultures treated with monocrotaline pyrrole (MCTP), Bone Morphogenetic Protein (BMP) and TGFβ. While MCTP induced transient nuclear accumulation of phosphorylated Smad 1 (P-Smad 1) and phosphylated Smad 2 (P-Smad 2), only expression of P-Smad 1 was significantly altered in western blots. P-Smad 1 expression significantly increased 30 minutes following treatment with MCTP correlating with P-Smad 1 and Smad 4 nuclear translocation. Although a modest, but significant decrease in P-Smad 1 expression occurred 1 hr after treatment, expression was significantly increased at 72 hr. Evaluation of components of the signal and response pathway at 72 hours showed decreased expression of the BMP type II receptor (BMPrII), no change in TGFβ Activin Receptor-like Kinase 1 (Alk 1), no change in Smad 4 but increase in the inhibitory Smad 6, decrease in the alternate BMP signaling pathway p38MAPK but no change in the psmad1 response element ID 1. Our results suggest transient activation of Smad signaling pathways in initial MCTP endothelial cell toxicity, and a persistent dysregulation of BMP signaling. Electron microscopy of cell membrane caveoli revealed a dramatic decrease in these structures after 72 hrs. Loss of these structural elements, noted for their sequestration and inhibition of receptor activity, may contribute to prolonged alterations in BMP signaling.

AIM: To investigate the role of insulin-like growth factor binding protein-7 (IGFBP-7) in the activation and transdifferentiation of hepatic stellate cells (HSC) in vitro.

METHODS: Rat HSC-T6 cells were cultured in separate dishes and treated with various concentration of transforming growth factor (TGF)-β1, IGFBP-7 or anti-IGFBP-7 antibody for 24 h. The supernatant or a cytoplasm suspension was obtained from cultured HSC, followed by transfer of cells to form cell-coated dishes. Immunocytochemistry and Western blotting were used to analyze the expression of IGFBP-7 induced by TGF-β1 and the level of fibronectin, collagen I and α-smooth muscle actin (SMA). The pro-apoptotic effect of anti-IGFBP-7 antibody was determined by flow cytometry.

RESULTS: Immunocytochemistry and Western blotting revealed that the expression of IGFBP-7 in TGF-β1 treated HSC was significantly up-regulated compared to that in the control group. In addition, fibronectin, collagen I and α-SMA also showed enhanced expression in accordance with the transdifferentiation process in a dose-dependent manner to some extent. Moreover, flow cytometry suggested that anti-IGFBP-7 antibody induced apoptosis of activated HSC, which is responsible for the development of liver fibrosis, and may represent a novel pathway and target for therapeutic intervention.

CONCLUSION: IGFBP-7 showed increased expression in activated HSC and played an important role in the activation and transdifferentiation process of HSC. Anti-IGFBP-7 antibody may ameliorate liver fibrogenesis.

Background

Hypertrophic scars are pathologic proliferations of the dermal skin layer resulting from excessive collagen deposition during the healing process of cutaneous wounds. Current research suggests that the TGF-β/Smad signaling pathway is closely associated with normal scar and hypertrophic scar formation. TRAP-1-like protein (TLP), a cytoplasmic protein, has been reported to efficiently regulate Smad2- and Smad3-dependent signal expression in the TGF-β pathway. The relationship between TLP and Type I/III collagen (Col I/III) synthesis explored in the present study provides an effective target for wound healing and gene therapy of hypertrophic scarring.

Objective

To investigate the effects of TLP on collagen synthesis in human dermal fibroblasts.

Methods

Lentiviral vectors encoding TLP was constructed to transfect fibroblasts derived from normal human skin. The expression of Col I/III and phosphorylation of Smad2 and Smad3 in fibroblasts were examined after TLP treatment. In addition, the comparison of TLP expression in normal skin tissues and in hypertrophic scar tissues was performed, and the effect of TLP on cell viability was analyzed by MTT assay.

Results

TLP expression in hypertrophic scar tissue was markedly higher than in normal skin tissue. The Real Time PCR and Western blot test results both revealed that the synthesis of Col I/III was positively correlated with the expression of TLP. TLP also facilitate Smad2 phosphorylation while, conversely, inhibiting Smad3 phosphorylation. TLP may play a cooperative role, along with the cytokine TGF-β1, in improving the overall cell viability of skin fibroblasts.

Conclusions

TLP likely acts as a molecular modulator capable of altering the balance of Smad3- and Smad2-dependent signaling through regulation of phosphorylation, thus facilitating collagen synthesis in fibroblasts. Based on genetic variation in TLP levels in different tissues, these results suggest that TLP plays a key role in the process of TGF-β1/Smad3 signaling that contributes to wound healing and genesis of pathologic scars.

Background

At present there is no effective and accepted therapy for hepatic fibrosis. Transforming growth factor (TGF)-β1 signaling pathway contributes greatly to hepatic fibrosis. Reducing TGF-β synthesis or inhibiting components of its complex signaling pathway represent important therapeutic targets. The aim of the study was to investigate the effect of curcumin on liver fibrosis and whether curcumin attenuates the TGF-β1 signaling pathway.

Methods

Sprague–Dawley rat was induced liver fibrosis by carbon tetrachloride (CCl4) for six weeks together with or without curcumin, and hepatic histopathology and collagen content were employed to quantify liver necro-inflammation and fibrosis. Moreover, the mRNA and protein expression levels of TGF-β1, Smad2, phosphorylated Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) were determined by quantitative real time-PCR, Western blot, or immunohistochemistry.

Results

Rats treated with curcumin improved liver necro-inflammation, and reduced liver fibrosis in association with decreased α-smooth muscle actin expression, and decreased collagen deposition. Furthermore, curcumin significantly attenuated expressions of TGFβ1, Smad2, phosphorylated Smad2, Smad3, and CTGF and induced expression of the Smad7.

Conclusions

Curcumin significantly attenuated the severity of CCl4-induced liver inflammation and fibrosis through inhibition of TGF-β1/Smad signalling pathway and CTGF expression. These data suggest that curcumin might be an effective antifibrotic drug in the prevention of liver disease progression.

AIM: To identify the role of herbal compound 861 (Cpd 861) in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells (HSCs).

METHODS: mRNA levels of collagen typesIand III, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), membrane type-1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase 1 (TIMP-1), and transforming growth factor β1 (TGF-β1) in cultured-activated HSCs treated with Cpd 861 or interferon-γ (IFN-γ) were determined by real-time PCR.

RESULTS: Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type III, MMP-2 and TGF-β1. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to (6.3 + 0.3)- fold compared to the control group.

CONCLUSION: The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen synthesis by inhibiting the transcription of collagen type III and TGF-β1 and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

This study was designed to investigate the potentially protective effects of glycyrrhetinic acid (GA) and the role of transcription factor nuclear factor-erythroid 2(NF-E2)-related factor 2 (Nrf2) signaling in the regulation of Carbon Tetrachloride (CCl4)-induced chronic liver fibrosis in mice. The potentially protective effects of GA on CCl4-induced chronic liver fibrosis in mice were depicted histologically and biochemically. Firstly, histopathological changes including regenerative nodules, inflammatory cell infiltration and fibrosis were induced by CCl4.Then, CCl4 administration caused a marked increase in the levels of serum aminotransferases (GOT, GPT), serum monoamine oxidase (MAO) and lipid peroxidation (MDA) as well as MAO in the mice liver homogenates. Also, decreased nuclear Nrf2 expression, mRNA levels of its target genes such as superoxide dismutase 3 (SOD3), catalase (CAT), glutathione peroxidase 2 (GPX2), and activity of cellular antioxidant enzymes were found after CCl4 exposure. All of these phenotypes were markedly reversed by the treatment of the mice with GA. In addition, GA exhibited the antioxidant effects in vitro by on FeCl2-ascorbate induced lipid peroxidation in mouse liver homogenates, and on DPPH scavenging activity. Taken together, these results suggested that GA can protect the liver from oxidative stress in mice, presumably through activating the nuclear translocation of Nrf2, enhancing the expression of its target genes and increasing the activity of the antioxidant enzymes. Therefore, GA may be an effective hepatoprotective agent and viable candidate for treating liver fibrosis and other oxidative stress-related diseases.

Introduction

We have previously demonstrated that TGF-β in the presence of elevated levels of its primary signaling protein, Smad3, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. Moreover, we have shown that the mechanism in part, is through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluate the role of the ERK mitogen-activated protein kinase (ERK MAPK) in TGF-β-induced VSMC proliferation.

Methods

Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with ansiRNA. For in vivo experiments, Male Sprague-Dawley rats underwent carotid balloon injury followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK and Proliferating Cell Nuclear Antigen (PCNA).

Results

In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time and concentration-dependent manner. Overexpression of the signaling protein, Smad3 enhanced TGF-β-induced activation of ERK MAPK whereas inhibition of Smad3 with ansiRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by PCNA.

Conclusion

Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that through a Smad3/ERK MAPK signaling pathway, TGF-β enhances VSMC proliferation. These findings explain at least in part, the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

AIM: To investigate the role of phosphatidylinositol 3-kinase (PI 3-K)/Akt signaling pathway in the balance of HSC activation and apoptosis in rat hepatic stellate cells (HSC).

METHODS: An activated HSC cell line was used in this study. LY 294002, the PI 3-K/Akt signal pathway blocker was used to investigate the molecular events on apoptosis in HSC and to interpret the role of this pathway in HSC apoptosis. Immunocytochemistry, Western blot and reverse transcription polymerase chain reaction (RT-PCR) analysis were applied to detect the expression of PI 3-K, and simultaneously phosphorylated-Akt (p-Akt) and total-Akt were determined by Western blot. The HSC apoptosis was examined by annexin-V/propidium iodide double-labelled flow cytometry and transmission electron microscopy.

RESULTS: The apoptosis rates in LY 294002 (30.82% ± 2.90%) and LY 294002 + PDGF-BB (28.16% ± 2.58%) groups were significantly increased compared with those of control (9.02% ± 1.81%) and PDGF-BB (4.35% ± 1.18%). PDGF-BB augmented PI 3-K and p-Akt expression. LY 294002 significantly reduced the contents of PI 3-K and p-Akt. mRNA transcription evaluated by RT-PCR showed similar tendencies as protein expression.

CONCLUSION: Inhibition of PI 3-K/Akt signaling pathway induces apoptosis in HSC.

Background

Transforming growth factor-β (TGF-β) plays an important role in vascular homeostasis through effects on vascular smooth muscle cells (SMC). Fine-tuning of TGF-β signaling occurs at the level of ALK receptors or Smads, and is regulated with cell type specificity.

Methods

Our goal was to understand TGF-β signaling in regulating SMC differentiation marker expression in human SMC. Activation of Smads was characterized, and loss- and gain-of-function reagents used to define ALK pathways. In addition, Smad-independent mechanisms were determined.

Results

TGF-β type I receptors, ALK1 and ALK5, are expressed in human SMC, and TGF-β1 phosphorylates Smad1/5/8 and Smad2/3 in a time- and dosage-dependent pattern. ALK5 activity, not bone morphogenetic protein type I receptors, is required for Smad phosphorylation. Endoglin, a TGF-β type III receptor, is a TGF-β1 target in SMC, yet endoglin does not modify TGF-β1 responsiveness. ALK5, not ALK1, is required for TGF-β1-induction of SMC differentiation markers, and ALK5 signals through an ALK5/Smad3- and MAP kinase-dependent pathway.

Conclusion

The definition of the specific signaling downstream of TGF-β regulating SMC differentiation markers will contribute to a better understanding of vascular disorders involving changes in SMC phenotype.

AIM: To investigate whether Notch signaling is involved in liver fibrosis by regulating the activation of hepatic stellate cells (HSCs).

METHODS: Immunohistochemistry was used to detect the expression of Notch3 in fibrotic liver tissues of patients with chronic active hepatitis. The expression of Notch3 in HSC-T6 cells treated or not with transforming growth factor (TGF)-β1 was analyzed by immunofluorescence staining. The expression of Notch3 and myofibroblastic marker α-smooth muscle actin (α-SMA) and collagen I in HSC-T6 cells transfected with pcDNA3.1-N3ICD or control vector were detected by Western blotting and immunofluorescence staining. Moreover, effects of Notch3 knockdown in HSC-T6 by Notch3 siRNA were investigated by Western blotting and immunofluorescence staining.

RESULTS: The expression of Notch3 was significantly up-regulated in fibrotic liver tissues of patients with chronic active hepatitis, but not detected in normal liver tissues. Active Notch signaling was found in HSC-T6 cells. TGF-β1 treatment led to up-regulation of Notch3 expression in HSC-T6 cells, and over-expression of Notch3 increased the expression of α-SMA and collagen I in HSC-T6 without TGF-β1 treatment. Interestingly, transient knockdown of Notch3 decreased the expression of myofibroblastic marker and antagonized TGF-β1-induced expression of α-SMA and collagen I in HSC-T6.

CONCLUSION: Notch3 may regulate the activation of HSCs, and the selective interruption of Notch3 may provide an anti-fibrotic strategy in hepatic fibrosis.

Previously, we found that high doses of genistein show an inhibitory effect on uterine leiomyoma (UtLM) cell proliferation. In this study, using microarray analysis and Ingenuity Pathways Analysis™, we identified genes (up- or down-regulated, ≥ 1.5 fold, P ≤ 0.001), functions and signaling pathways that were altered following treatment with an inhibitory concentration of genistein (50 µg/ml) in UtLM cells. Downregulation of TGF-β signaling pathway genes, activin A, activin B, Smad3, TGF-β2 and genes related to cell cycle regulation, with the exception of the upregulation of the CDK inhibitor P15, were identified and validated by real-time RT-PCR studies. Western blot analysis further demonstrated decreased protein expression of activin A and Smad3 in genistein-treated UtLM cells. Moreover, we found that activin A stimulated the growth of UtLM cells, and the inhibitory effect of genistein was partially abrogated in the presence of activin A. Overexpression of activin A and Smad3 were found in tissue samples of leiomyoma compared to matched myometrium, supporting the contribution of activin A and Smad3 in promoting the growth of UtLM cells. Taken together, these results suggest that down-regulation of activin A and Smad3, both members of the TGF-β pathway, may offer a mechanistic explanation for the inhibitory effect of a high-dose of genistein on UtLM cells, and might be potential therapeutic targets for treatment of clinical cases of uterine leiomyomas.