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1.  MODULATION OF TGF-β SIGNALING BY PROINFLAMMATORY CYTOKINES IN ARTICULAR CHONDROCYTES 
Objective
The normal structure and function of articular cartilage is the result of a precisely balanced interaction between anabolic and catabolic processes. The TGF-β family of growth factors generally exerts an anabolic or repair response; in contrast, proinflammatory cytokines such as IL-1β and TNF-α exert a strong catabolic effect. Recent evidence has shown that IL-1β, and TNF-α, and the TGF-β signaling pathways share an antagonistic relationship. The aim of this study was to determine whether the modulation of the response of articular chondrocytes to TGF-β by IL-1β or TNF-α signaling pathways occurs through regulation of activity and availability of Smad proteins.
Methods
Human articular chondrocytes isolated from knee joints from patients with osteoarthritis (OA) or normal bovine chondrocytes were cultured in suspension in polyHEMA-coated dishes with either 10% FBS media or serum-deprived media six hours before treatment with IL-1β alone, TNF-α alone or IL-1β followed by TGF-β. Nuclear extracts were examined by electrophoretic mobility shift assays (EMSA) for NF-κB and Smad3/4 DNA binding. Nuclear extracts were also subjected to the TranSignal Protein/DNA array (Panomics, Redwood City, CA) enabling the simultaneous semiquantitative assessment of DNA-binding activity of 54 different transcription factors. Nuclear phospho-Smad2/3 and total Smad7 protein expression in whole cell lysates were studied by Western blot. Cytoplasmic Smad7, COL2A1, aggrecan and SOX9 mRNA expression was measured by real-time PCR.
Results
The DNA-binding activity of Smad3/4 in the TranSignal Protein/DNA array was down-regulated by TNF-α (46%) or IL-1β treatment (42%). EMSA analysis showed a consistent reduction in Smad 3/4 DNA-binding activity in human articular chondrocytes treated with IL-1β or TNF-α. TGF-β-induced Smad3/4 DNA-binding activity and Smad2/3 phosphorylation were also reduced following pre-treatment with IL-1β in human OA and bovine chondrocytes. Real-Time PCR and Western blot analysis showed that IL-1β partially reversed the TGF-β stimulation of Smad7 mRNA and protein levels in TGF-β-treated human OA cells. In contrast, TGF-β-stimulated COL2A1, aggrecan, and SOX9 mRNA levels were abrogated by IL-1β.
Conclusions
IL-1β or TNF-α exerted a suppressive effect on Smad3/4 DNA-binding activity in human articular chondrocytes, as well, as on TGF-β-induced stimulation of Smad3/4 DNA-binding activity and Smad 2/3 phosphorylation in human OA and bovine articular chondrocytes. IL-1β partially reversed the increase in TGF-β-stimulated Smad7 mRNA or protein levels suggesting that Smad7 may not be involved in the suppression of TGF-β signaling induced by IL-1β or TNF-α in articular chondrocytes. The balance between the IL-1β or TNF-α and the TGF-β signaling pathways is crucial for maintenance of articular cartilage homeostasis and its disruption likely plays a substantial role in the pathogenesis of OA.
doi:10.1016/j.joca.2007.04.011
PMCID: PMC2153443  PMID: 17604656
2.  Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling 
AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro.
METHODS: HSCs were isolated from rats by in situ perfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 h with DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1, 2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscle-α-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type III procollagen (PCIII) in supernatants were determined by radioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively.
RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCIII expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2% (control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P<0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCIII to 84.6%, 81.5%, 75.7% or 80.7% respectively (P<0.05 vs control), with no significant difference among tetrandrine groups. RT-PCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r = -0.755, P<0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P<0.01; r = 0.938, P<0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L) was confirmed by Western blotting as well.
CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.
doi:10.3748/wjg.v11.i19.2922
PMCID: PMC4305660  PMID: 15902729
Tetrandrine; Hepatic stellate cell; Transforming growth factor-β; Smad 7; Liver fibrosis; Signal transduction
3.  NIM811, a nonimmunosuppressive cyclosporine analogue, suppresses collagen production and enhances collagenase activity in hepatic stellate cells 
Liver International   2007;27(9):1273-1281.
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.
doi:10.1111/j.1478-3231.2007.01560.x
PMCID: PMC2156109  PMID: 17919240
cyclosporine analogue; hepatic fibrosis; hepatic stellate cell; NIM811; TGF-β
4.  Smad2 and Smad3 Play Different Roles in Rat Hepatic Stellate Cell Function and α-Smooth Muscle Actin OrganizationD⃞V⃞ 
Molecular Biology of the Cell  2005;16(9):4214-4224.
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.
doi:10.1091/mbc.E05-02-0149
PMCID: PMC1196331  PMID: 15987742
5.  Differential Regulation of TGF-β/Smad Signaling in Hepatic Stellate Cells between Acute and Chronic Liver Injuries 
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.
doi:10.3389/fphys.2012.00053
PMCID: PMC3307138  PMID: 22457652
HSC; MFB; TGF-β; Smad; liver fibrosis
6.  Asiatic Acid Inhibits Liver Fibrosis by Blocking TGF-beta/Smad Signaling In Vivo and In Vitro 
PLoS ONE  2012;7(2):e31350.
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.
doi:10.1371/journal.pone.0031350
PMCID: PMC3280334  PMID: 22363627
7.  Effect of aldosterone and its antagonist on the expression of PAI-1 and TGF-β1 in rat hepatic stellate cells 
Background: Aldosterone has been implicated in a variety of organ fibroses, but its role and mechanism in liver fibrosis remain unclear. Methods: Rat primary hepatic stellate cells (HSCs) were isolated, cultured, and characterized. HSCs were incubated with aldosterone (10-6 M) for 4 h, 8 h, 12 h, 24 h, and 48 h, after which TGF-β1 (transforming growth factor beta 1) expression was measured by real-time PCR. Rat HSCs were treated with different concentrations of aldosterone (10-6 M, 10-7 M, 10-8 M, and 10-9 M), and the expressions of PAI-1 (plasminogen activator inhibitor-1) and TGF-β1 were determined by measuring mRNA and protein. HSCs were incubated in groups containing aldosterone (10-6 M), spironolactone (10-5 M), both aldosterone and spironolactone, or neither aldosterone nor spironolactone (control), after which mRNA and protein expression of PAI-1 and TGF-β1 were measured. Collagen I expression was detected by immunohistochemical analysis of supernatants of the aldosterone (10-6 M), TGF-β1, and aldosterone plus TGF-β1 groups. SMAD expression was detected in rat HSC control, HSC plus aldosterone (10-6 M), HSC plus TGF-β1, and HSC plus aldosterone plus TGF-β1 groups. Results: HSCs were incubated with aldosterone for 4 h, 8 h, 12 h, 24 h, and 48 h after which TGF-β1 expression was measured. We found that TGF-β1 expression increased in a time dependent manner and reached a peak at 24 h. The expression of TGF-β1 in groups treated with aldosterone for 4 h, 8 h, 12 h, and 24 h was significantly different from the control group (P < 0.01). No significant difference was seen in TGF-β1 expression between the groups treated with aldosterone for 24 h and 48 h (P > 0.05). Compared with the control group, TGF-β1 expression was significantly increased after incubation with different concentrations of aldosterone (10-6 M, 10-7 M, 10-8 M, and 10-9 M) (P < 0.01). There were significant differences in the expression of TGF-β1 between 10-6 M and 10-7 M aldosterone treatment groups (P < 0.01). Compared with the control group, the expression of PAI-1 was significantly increased after incubation with different concentrations of aldosterone (10-6 M, 10-7 M, 10-8 M, and 10-9 M) (P < 0.01). PAI-1 expression was increased in the aldosterone, spironolactone, and aldosterone plus spironolactone groups. The expression of PAI-1 was significantly enhanced in the aldosterone and aldosterone plus spironolactone groups compared with the control group (P < 0.01). There was a marked enhancement of collagen I expression in the aldosterone, TGF-β1, and aldosterone plus TGF-β1 groups (P < 0.05). Collagen I expressions in the aldosterone and TGF-β1 groups were significantly different from the aldosterone plus TGF-β1 group (P < 0.01). Compared with the control group, SMAD expression was markedly elevated in the aldosterone, TGF-β1, and aldosterone plus TGF-β1 groups (P < 0.05). The expression of SMAD was significantly increased in the aldosterone plus TGF-β1 group compared with the aldosterone group (P < 0.01). Conclusion: This study demonstrated that aldosterone promoted HSC activation and the expression of TGF-β1, PAI-1, and collagen in hepatic fibrosis progression and that spironolactone administration partially reversed the effects. The aldosterone promotional effect on hepatic fibrosis was partially mediated by TGF-β1.
PMCID: PMC4307412  PMID: 25663965
Aldosterone; hepatic stellate cells; PAI-1; TGF-β1; spironolactone
8.  IGFBPrP1 induces liver fibrosis by inducing hepatic stellate cell activation and hepatocyte apoptosis via Smad2/3 signaling 
AIM: To investigate the role and mechanism of insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) in the development of liver fibrosis.
METHODS: An in vitro model using hepatic stellate cell (HSC)-T6 cells and an in vivo model of rat liver overexpressing IGFBPrP1 were established using an IGFBPrP1-expressing recombinant adenovirus. The expression of IGFBPrP1 was examined by immunofluorescence, and the expression of collagen I and fibronectin was measured by real-time reverse transcription-polymerase chain reaction and Western blot analysis. The expression of Smad2/3 and p-Smad2/3 was examined by Western blot and immunohistochemistry. A shSmad3-expressing recombinant adenovirus (AdshSmad3) was designed and used to knockdown the Smad3 gene in HSC-T6 cells and rat liver fibrosis transfected with IGFBPrP1. The expression of collagen I, fibronectin, and α-smooth muscle actin (α-SMA) was determined by Western blot analysis and immunohistochemistry. Hepatocyte apoptosis was assessed using TUNEL assay.
RESULTS: IGFBPrP1 overexpression induced collagen deposition and up-regulated the expression of α-SMA and p-Smad2/3, and AdshSmad3 inhibited IGFBPrP1-stimulated p-Smad2/3 activation and the expression of α-SMA, collagen I and fibronectin in HSC-T6 cells. Similarly, increased hepatocyte apoptosis (38.56% ± 3.42% vs 0.24% ± 0.03%, P < 0.05), α-SMA positive stained cells (29.84% ± 1.36% vs 5.83% ± 1.47%, P < 0.05), and increased numbers of Smad3 (35.88% ± 2.15% vs 10.24% ± 1.31%, P < 0.05) and p-Smad2/3 positive cells (28.87% ± 2.73% vs 8.23% ± 0.98%, P < 0.05) were detected in the livers of IGFBPrP1-overexpressing rats compared with the control group. Moreover, AdshSmad3 reduced IGFBPrP1-stimulated Smad3 expression and attenuated α-SMA expression (29.84% ± 1.36% vs 8.23% ± 1.28%, P < 0.05), hepatocyte apoptosis (38.56% ± 3.42% vs 6.75% ± 0.52%, P < 0.05), and both collagen I and fibronectin deposition in the livers of AdIGFBPrP1-treated rats.
CONCLUSION: IGFBPrP1 induces liver fibrosis by mediating the activation of hepatic stellate cells and hepatocyte apoptosis in a Smad3-dependent mechanism.
doi:10.3748/wjg.v20.i21.6523
PMCID: PMC4047337  PMID: 24914373
Insulin-like growth factor binding protein-related protein 1; Liver fibrosis; Hepatic stellate cells; Hepatocyte apoptosis; Smad pathway
9.  Poly(ADP-ribose) Polymerase 1 Is Indispensable for Transforming Growth Factor-β Induced Smad3 Activation in Vascular Smooth Muscle Cell 
PLoS ONE  2011;6(10):e27123.
Background
Transforming growth factor type-β (TGF-β)/Smad pathway plays an essential role in vascular fibrosis. Reactive oxygen species (ROS) generation also mediates TGF-β signaling-induced vascular fibrosis, suggesting that some sort of interaction exists between Smad and redox pathways. However, the underlying molecular mechanism is largely unknown. This study aims to investigate the influence of poly(ADP-ribose) polymerase 1 (PARP1), a downstream effector of ROS, on TGF-β signaling transduction through Smad3 pathway in rat vascular smooth muscle cells (VSMCs).
Methods and Results
TGF-β1 treatment promoted PARP1 activation through induction of ROS generation in rat VSMCs. TGF-β1-induced phosphorylation and nuclear accumulation of Smad3 was prevented by treatment of cells with PARP inhibitor, 3-aminobenzamide (3AB) or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino)acetami (PJ34), or PARP1 siRNA. TGF-β1 treatment promoted poly(ADP-ribosy)lation of Smad3 via activation of PARP1 in the nucleus. Poly(ADP-ribosy)lation enhanced Smad-Smad binding element (SBE) complex formation in nuclear extracts and increased DNA binding activity of Smad3. Pretreatment with 3AB, PJ34, or PARP1 siRNA prevented TGF-β1-induced Smad3 transactivation and expression of Smad3 target genes, including collagen Iα1, collagen IIIα1 and tissue inhibitor of metalloproteinase 1, in rat VSMCs.
Conclusions
PARP1 is indispensable for TGF-β1 induced Smad3 activation in rat VSMCs. Targeting PARP1 may be a promising therapeutic approach against vascular diseases induced by dysregulation of TGF-β/Smad3 pathway.
doi:10.1371/journal.pone.0027123
PMCID: PMC3205050  PMID: 22073128
10.  Inhibitory Effects of microRNA 19b in Hepatic Stellate Cell-Mediated Fibrogenesis 
Hepatology (Baltimore, Md.)  2012;56(1):300-310.
Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGFβ). microRNAs (miRs), small non-coding RNAs modulating mRNA and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGFβ signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Since miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGFβ signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGFβ signaling components demonstrated by decreased TGFβ receptor II (TGFβRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3’UTR of TGFβRII was validated by luciferase reporter assay. Inhibition of TGFβ signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGFβ-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased αSMA expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared to normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared to normal human livers.
CONCLUSIONS
miR 19b is a novel regulator of TGFβ signaling in HSCs suggesting a potential therapeutic approach for hepatic fibrosis.
doi:10.1002/hep.25613
PMCID: PMC3342471  PMID: 22278637
Transforming growth factor β; fibrosis; miR 19b; biomarker; hepatic stellate cell
11.  Quantitative analysis of transient and sustained transforming growth factor-β signaling dynamics 
Mathematical modeling and experimental analyses reveal that TGF-β ligand depletion has an important role in converting short-term graded signaling responses to long-term switch-like responses.
Cells respond in real time to the absolute number of TGF-β molecules in their environment.A single pulse of TGF-β stimulation results in transient SMAD activation whereas repeated short pulses of stimulation result in sustained SMAD activation.Ligand-induced short-term TGF-β/SMAD signaling activation is graded while long-term signaling response is switch-like or ultrasensitive.TGF-β ligand depletion is a major cause of conversion from graded short-term responses to ultrasensitive long-term responses.
The transforming growth factor-β (TGF-β) pathway is a prominent signaling pathway that regulates diverse aspects of cellular homeostasis, including proliferation, differentiation, migration, and death (Massague, 1998). Remarkably, the pleiotropic biological effects of TGF-β are mediated by a relatively simple signaling module (Clarke and Liu, 2008). An interesting question is how such an apparently straightforward and simple cascade can generate a wide array of biological responses depending on the cellular context.
Members of the TGF-β superfamily are frequently used as morphogens in early embryo development (Green, 2002). The best-studied examples include Dpp in Drosophila and Activin in Xenopus (Gurdon and Bourillot, 2001; Lander, 2007). In the developmental context, cells can respond to a graded ligand concentration and produce discrete biological responses (e.g., transcription of certain genes, proliferation, or differentiation; Green, 2002). To convert continuous morphogen stimulation into discrete responses, mechanisms must exist to provide a threshold for the cellular response. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remains poorly understood.
We have previously reported that ligand molecules per cell is the input variable to which the cells respond, and ligand number per cell is the best predictor of signaling responses (Zi and Klipp, 2007a; Clarke et al, 2009). Here, we developed an improved mathematical model to predict TGF-β signaling responses by calibrating the model with various experimental data sets from different TGF-β stimulations. Using a combined experimental and mathematical modeling approach, we showed that TGF-β pulse stimulation results in transient activation of the pathway while repeated short pulses at short time intervals lead to a sustained activation similar to persistent ligand exposure.
We next investigate the system response to variable doses of TGF-β in HaCaT cells. Our mathematical model predicts that the short-term Smad2 phosphorylation (after 45 min of TGF-β stimulation) is a graded response, while long-term Smad2 activation (after 24 h of TGF-β stimulation) is a switch-like response (Figure 5A and B). As shown in Figure 5A–D, both short- and long-term Smad2 phosphorylation can be saturated but doses of TGF-β that cause maximum response are quite different. Additionally, the shapes of response curves were different. The short-term Smad2 activation was a graded (Michaelis–Menten-like) response with a very low apparent Hill coefficient of about 0.8 (Figure 5A and C) while the long-term Smad2 activation (P-Smad2 at 24 h) yielded a switch-like response with an apparent Hill coefficient of about 4.5 (Figure 5B and D). Thus, the Smad2 response is initially graded and sharpens over time to become ultrasensitive. To address whether TGF-β-inducible gene expression responses are graded or switch-like in the short and long term, we measured mRNA levels of Smad7, an early responsive gene of TGF-β and protein levels of p21 and PAI-1 whose inductions are delayed and late, respectively. The experimental data show that Smad7 induction exhibits a graded response with corresponding Hill coefficients of about 1.3 (Figure 5E), which is consistent with the graded P-Smad2 response at 45 min (Figure 5A and C). PAI-1 induction in response to variable doses of TGF-β for 24 h is highly ultrasensitive with an apparent Hill coefficient of ∼5.3. Compared with Smad7 and PAI-1, p21 induction is only modest ultrasensitive (nHill≈2) (Figure 5G). These results suggest short-term gene induction by TGF-β appears to be graded while long-term targets are more switch-like. Finally, we measured the growth inhibitory response of HaCaT cells to variable doses of TGF-β. The level of BrdU incorporation is also ultrasensitive with an apparent Hill coefficient of about 4.3 (Figure 5H). Therefore, the long-term TGF-β growth inhibitory response also shows a switch-like behavior. Finally, we show that TGF-β depletion affects long-term Smad phosphorylation and switch-like response of TGF-β signaling system. These findings shed new light on how continuous ligand doses are translated into discontinuous cell fate decisions in biological systems.
In summary, we have shown that the dose and time course of TGF-β stimulation have profound effects on Smad signaling dynamics. The rate of ligand depletion controls the duration of Smad2 phosphorylation. Cells can respond to a short pulse of TGF-β stimulation, and periodic short ligand exposures are sufficient to generate long-term signaling responses. Short-term TGF-β stimulation causes only transient pathway activation and can be terminated by ligand depletion. TGF-β-induced Smad2 phosphorylation is graded in the short-term but ultrasensitive (switch-like) in the long-term (Figure 7). Additionally, cell growth arrest in response to TGF-β shows switch-like rather than graded behavior. Our modeling and experimental analyses suggest that ligand depletion is likely to be involved in sharpening a graded response into a switch-like response.
Mammalian cells can decode the concentration of extracellular transforming growth factor-β (TGF-β) and transduce this cue into appropriate cell fate decisions. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remain poorly understood. Using a combined experimental and mathematical modeling approach, we discovered that cells respond differently to continuous and pulsating TGF-β stimulation. The TGF-β pathway elicits a transient signaling response to a single pulse of TGF-β stimulation, whereas it is capable of integrating repeated pulses of ligand stimulation at short time interval, resulting in sustained phospho-Smad2 and transcriptional responses. Additionally, the TGF-β pathway displays different sensitivities to ligand doses at different time scales. While ligand-induced short-term Smad2 phosphorylation is graded, long-term Smad2 phosphorylation is switch-like to a small change in TGF-β levels. Correspondingly, the short-term Smad7 gene expression is graded, while long-term PAI-1 gene expression is switch-like, as is the long-term growth inhibitory response. Our results suggest that long-term switch-like signaling responses in the TGF-β pathway might be critical for cell fate determination.
doi:10.1038/msb.2011.22
PMCID: PMC3130555  PMID: 21613981
mathematical model; Smad; TGF-β; ultrasensitivity
12.  Effects of TRAP-1-Like Protein (TLP) Gene on Collagen Synthesis Induced by TGF-β/Smad Signaling in Human Dermal Fibroblasts 
PLoS ONE  2013;8(2):e55899.
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.
doi:10.1371/journal.pone.0055899
PMCID: PMC3572169  PMID: 23418473
13.  Identification of Novel Smad2 and Smad3 Associated Proteins in Response to TGF-β1 
Journal of cellular biochemistry  2008;105(2):596-611.
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.
doi:10.1002/jcb.21860
PMCID: PMC2700048  PMID: 18729074
TGF-β1; Smad; Mass spectrometry; Proteomics; Zizimin1
14.  Aberrant hypertrophy in Smad3-deficient chondrocytes is rescued by restoring TAK1-ATF-2 signaling: a potential clinical implication for osteoarthritis 
Arthritis and rheumatism  2010;62(8):2359-2369.
Objective
To investigate the biological significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and ATF-2 in the TGF-β signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation.
Methods
Joint disease in Smad3 knockout (Smad3−/−) mice was examined by micro-CT and histology. Numerous in vitro methods including immunostaining, real-time PCR, Western blotting, an ATF-2 DNA-binding assay and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3−/− mice.
Results
Smad3−/− mice gradually developed an end-stage OA phenotype. TGF-β-induced TAK1-ATF-2 signaling was disrupted in Smad3−/− chondrocytes at the level of p38 MAP kinase activation resulting in reduced ATF-2 phosphorylation and transcriptional activity. Re-introduction of Smad3 into the Smad3−/− cells restored the normal p38 response to TGF-β. Phospho-p38 formed a complex with Smad3 by binding to the Smad3 MH1-linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAP kinase phosphatase-1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild type and Smad3−/− chondrocytes. p38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms α, β and γ, but not δ.
Conclusions
Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1-ATF-2 signaling, most likely by disrupting the Smad3-phospho-p38 complex and, thereby, promoting p38 dephosphorylation and inactivation by MKP-1. p38 and ATF-2 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA.
doi:10.1002/art.27537
PMCID: PMC2921996  PMID: 20506210
15.  BRCA1 Interacts with Smad3 and Regulates Smad3-Mediated TGF-β Signaling during Oxidative Stress Responses 
PLoS ONE  2009;4(9):e7091.
Background
BRCA1 is a key regulatory protein participating in cell cycle checkpoint and DNA damage repair networks. BRCA1 plays important roles in protecting numerous cellular processes in response to cell damaging signals. Transforming growth factor-beta (TGF-β) is a potent regulator of growth, apoptosis and invasiveness of tumor cells. TFG-β activates Smad signaling via its two cell surface receptors, the TbetaRII and ALK5/TbetaRI, leading to Smad-mediated transcriptional regulation.
Methodology/Principal Findings
Here, we report an important role of BRCA1 in modulating TGF-β signaling during oxidative stress responses. Wild-type (WT) BRCA1, but not mutated BRCA1 failed to activate TGF-β mediated transactivation of the TGF-β responsive reporter, p3TP-Lux. Further, WT-BRCA1, but not mutated BRCA1 increased the expression of Smad3 protein in a dose-dependent manner, while silencing of WT-BRCA1 by siRNA decreased Smad3 and Smad4 interaction induced by TGF-β in MCF-7 breast cancer cells. BRCA1 interacted with Smad3 upon TGF-β1 stimulation in MCF-7 cells and this interaction was mediated via the domain of 298–436aa of BRCA1 and Smad3 domain of 207–426aa. In addition, H2O2 increased the colocalization and the interaction of Smad3 with WT-BRCA1. Interestingly, TGF-β1 induced Smad3 and Smad4 interaction was increased in the presence of H2O2 in cells expressing WT-BRCA1, while the TGF-β1 induced interaction between Smad3 and Smad4 was decreased upon H2O2 treatment in a dose-dependent manner in HCC1937 breast cancer cells, deficient for endogenous BRCA1. This interaction between Smad3 and Smad4 was increased in reconstituted HCC1937 cells expressing WT-BRCA1 (HCC1937/BRCA1). Further, loss of BRCA1 resulted in H2O2 induced nuclear export of phosphor-Smad3 protein to the cytoplasm, resulting decreased of Smad3 and Smad4 interaction induced by TGF-β and in significant decrease in Smad3 and Smad4 transcriptional activities.
Conclusions/Significance
These results strongly suggest that loss or reduction of BRCA1 alters TGF-β growth inhibiting activity via Smad3 during oxidative stress responses.
doi:10.1371/journal.pone.0007091
PMCID: PMC2740868  PMID: 19768112
16.  Effects of Chinese traditional compound, JinSanE, on expression of TGF-β1 and TGF-β1 type II receptor mRNA, Smad3 and Smad7 on experimental hepatic fibrosis in vivo 
AIM: The transforming growth factor-beta (TGF-β)/Smad signaling pathway system plays a prominent role in the control of cell growth and extracellular matrix formation in the progression of liver fibrogenesis. Smad proteins can either positively or negatively regulate TGF-β responses. In this study, the therapeutic effects of Chinese traditional compound decoction, JinSanE, and the changes of TGF-β/Smad signaling pathway system in carbon tetrachloride (CCl4)-induced rat experimental liver fibrosis were examined.
METHODS: Seventy-two healthy Wistar rats were assigned to groups including normal control group, CCl4 model group, JinSanE treatment group I and JinSanE treatment group II. Each group contained 18 rats. All groups, except the normal control group, received CCl4 subcutaneous injection for 8 wk. Rats in JinSanE groups I and II were orally treated with JinSanE daily at the 1st and 5th wk, respectively, after exposure to CCl4. The expression of TGF-β1 and TGF-β1 type II receptor (TRII) mRNA in the liver was determined by reverse transcription polymerase chain reaction, and the expression of TGF-β1, Smad3 and Smad7 by immunohistochemistry. The liver histopathology was also examined by HE staining and observed under electron microscope. The activities of several serum fibrosis-associated enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), the levels of serum hyaluronic acid (HA) were assayed.
RESULTS: Hepatic fibrosis caused by CCl4 was significantly inhibited in the JinSanE-treated groups. The degrees of necrosis/degeneration and fibrosis scores were significantly lower in the JinSanE-treated groups than in the model control group. The expression of TGF-β1, TRII and Smad3 was significantly higher in the model group than that in the JinSanE-treated groups, and the active/total TGF-β1 ratio in the JinSanE groups was suppressed. Expression of TRII mRNA and Smad3 proteins showed a distribution pattern similar to that of TGF-β1 with a direct correlation in terms of the degree of hepatic fibrosis. The amount of positive staining Smad7 cells was significantly less in the model group than in the JinSanE-treated groups and the normal group. The contents of ALT, AST and HA were significantly lower in the JinSanE-treated groups than those in the model group.
CONCLUSION: Traditional Chinese medicine, JinSanE, prevents the progression of hepatic damage and fibrosis through the inhibition of TGF-β1, TRII and Smad3 signal proteins, and increases expression of Smad7 signal protein in vivo.
doi:10.3748/wjg.v11.i15.2269
PMCID: PMC4305811  PMID: 15818738
TGF-β; Liver fibrogenesis
17.  Emodin protects rat liver from CCl4-induced fibrogenesis via inhibition of hepatic stellate cells activation 
AIM: To investigate the role of emodin in protecting the liver against fibrogenesis caused by carbon tetrachloride (CCl4) in rats and to further explore the underlying mechanisms.
METHODS: Rat models of experimental hepatic fibrosis were established by injection with CCl4; the treated rats received emodin via oral administration at a dosage of 20 mg/kg twice a week at the same time. Rats injected with olive oil served as a normal group. Histopathological changes were observed by hematoxylin and eosin staining. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and hepatic hydroxyproline content were assayed by biochemical analyses. The mRNA and protein relevant to hepatic stellate cell (HSC) activation in the liver were assessed using real-time reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, western blotting and enzyme-linked immunosorbent assay.
RESULTS: The degree of hepatic fibrosis increased markedly in the CCl4 group compared to the normal group (P < 0.01), and decreased markedly in the emodin group compared to the CCl4 group according to METAVIR scale (P < 0.01) compared with those in the normal control group (51.02 ± 10.64 IU/L and 132.28 ± 18.14 IU/L). The activities of serum ALT and AST were significantly higher in rats injected with CCl4 (289.25 ± 68.84 IU/L and 423.89 ± 35.67 IU/L, both P < 0.05). The activities of serum ALT and AST were significantly reduced by administration of emodin (176.34 ± 47.29 IU/L and 226.1 ± 44.52 IU/L, both P < 0.05). Compared with the normal controls (54.53 ± 13.46 mg/g), hepatic hydroxyproline content was significantly higher in rats injected with CCl4 (120.27 ± 28.47 mg/g, P < 0.05). Hepatic hydroxyproline content was significantly reduced in the rats treated with emodin at 20 mg/kg (71.25 ± 17.02 mg/g, P < 0.05). Emodin significantly protected the liver from injury by reducing serum AST and ALT activities and reducing hepatic hydroxyproline content. The mRNA levels of transforming growth factor-β1 (TGF-β1), Smad4 and α-SMA in liver tissues were significantly down-regulated in SD rats that received emodin treatment. Furthermore, significant down-regulation of serum TGF-β1 protein levels and protein expression of Smad4 and α-SMA in liver tissues was also observed in the rats. Emodin inhibited HSC activation by reducing the abundance of TGF-β1 and Smad4.
CONCLUSION: Emodin protects the rat liver from CCl4-induced fibrogenesis by inhibiting HSC activation. Emodin might be a therapeutic antifibrotic agent for the treatment of hepatic fibrosis.
doi:10.3748/wjg.15.4753
PMCID: PMC2761551  PMID: 19824107
Emodin; Hepatic fibrosis; Transforming growth factor-β1; Smad4; Hepatic stellate cell; α-smooth muscle actin
18.  Serine-204 in the Linker Region of Smad3 Mediates the Collagen-I Response to TGF-β in a Cell Phenotype-Specific Manner 
Experimental cell research  2013;319(19):2928-2937.
Regulation of TGF-β1/Smad3 signaling in fibrogenesis is complex. Previous work by our lab suggests that ERK MAP kinase phosphorylates the linker region (LR) of Smad3 to enhance TGF-β-induced collagen-I accumulation. However the roles of the individual Smad3LR phosphorylation sites (T179, S204, S208 and S213) in the collagen-I response to TGF-β are not clear. To address this issue, we tested the ability of Smad3 constructs expressing wild-type Smad3 or Smad3 with mutated LR phosphorylation sites to reconstitute TGF-β-stimulated COL1A2 promoter activity in Smad3-null or -knockdown cells. Blocking ERK in fibroblasts and renal mesangial cells inhibited both S204 phosphorylation and Smad3-mediated COL1A2 promoter activity. Mutations replacing serine at S204 or S208 in the linker region decreased Smad3-mediated COL1A2 promoter activity, whereas mutating T179 enhanced basal COL1A2 promoter activity and did not prevent TGF-β stimulation. Interestingly, mutation of all four Smad3LR sites (T179, S204, S208 and S213) was not inhibitory, suggesting primacy of the two inhibitory sites. These results suggest that in these mesenchymal cells, phosphorylation of the T179 and possibly S213 sites may act as a brake on the signal, whereas S204 phosphorylation by ERK in some manner releases that brake. Renal epithelial cells (HKC) respond differently from MEF or mesangial cells; blocking ERK neither changed TGF-β-stimulated S204 phosphorylation nor prevented Smad3-mediated COL1A2 promoter activity in HKC. Furthermore, re-expression of wild type-Smad3 or the S204A-Smad3 mutant in Smad3-knockdown HKC reconstituted Smad3-mediated COL1A2 promoter activity. Collectively, these data suggest that Serine-204 phosphorylation in the Smad3LR is a critical event by which ERK enhances Smad3-mediated COL1A2 promoter activity in mesenchymal cells.
doi:10.1016/j.yexcr.2013.07.013
PMCID: PMC3900285  PMID: 24080014
Fibrosis; Extracellular Matrix; Fibroblast; Epithelial; mesenchymal; ERK MAP kinase
19.  Transforming growth factor-beta inhibits aromatase gene transcription in human trophoblast cells via the Smad2 signaling pathway 
Background
Transforming growth factor-beta (TGF-beta) is known to exert multiple regulatory functions in the human placenta, including inhibition of estrodial production. We have previously reported that TGF-beta1 decreased aromatase mRNA levels in human trophoblast cells. The objective of this study was to investigate the molecular mechanisms underlying the regulatory effect of TGF-beta1 on aromatase expression.
Methods
To determine if TGF-beta regulates aromatase gene transcription, several reporter constructs containing different lengths of the placental specific promoter of the human aromatase gene were generated. JEG-3 cells were transiently transfected with a promoter construct and treated with or without TGF-beta1. The promoter activity was measured by luciferase assays. To examine the downstream signaling molecule mediating the effect of TGF-beta on aromatase transcription, cells were transiently transfected with dominant negative mutants of TGF-beta type II (TbetaRII) and type I receptor (ALK5) receptors before TGF-beta treatment. Smad2 activation was assessed by measuring phophorylated Smad2 protein levels in cytosolic and nuclear fractions. Smad2 expression was silenced using a siRNA expression construct. Finally, aromatase mRNA half-life was determined by treating cells with actinomycin D together with TGF-beta1 and measuring aromatase mRNA levels at various time points after treatment.
Results and Discussion
TGF-beta1 inhibited the aromatase promoter activity in a time- and dose-dependent manner. Deletion analysis suggests that the TGF-β1 response element resides between -422 and -117 nucleotides upstream from the transcription start site where a Smad binding element was found. The inhibitory effect of TGF-beta1 was blocked by dominant negative mutants of TbetaRII and ALK5. TGF-beta1 treatment induced Smad2 phosphorylation and translocation into the nucleus. On the other hand, knockdown of Smad2 expression reversed the inhibitory effect of TGF-beta1 on aroamtase transcription. Furthermore, TGF-beta1 accelerated the degradation of aromatase mRNA.
Conclusion
Our results demonstrate that TGF-beta1 exerts regulatory effects on aromatase gene at both transcriptional and post-transcriptional levels. The transcriptional regulation of aromatase gene by TGF-beta1 is mediated by the canonical TGF-beta pathway involving TbetaRII, ALK5 and Smad2. These findings further support the role of TGF-beta1 in regulating human placental functions and pregnancy.
doi:10.1186/1477-7827-7-146
PMCID: PMC2797513  PMID: 20003198
20.  ULTRAVIOLET IRRADIATION INDUCES SMAD7 VIA INDUCTION OF TRANSCRIPTION FACTOR AP-1 IN HUMAN SKIN FIBROBLASTS 
The Journal of biological chemistry  2004;280(9):8079-8085.
SUMMARY
Smad7 functions as an endogenous negative regulator of TGF-β/Smad signaling. TGF-β/Smad pathway is a major regulator of collagen production in connective tissue. Reduced expression of Smad7 has been reported in TGF-β-mediated fibrotic diseases, characterized by over production of collagen. Solar ultraviolet (UV) irradiation reduces collagen production by fibroblasts in human skin in vivo. We have investigated regulation of Smad7 gene expression by UV irradiation in human skin fibroblasts. UV irradiation transiently increased Smad7 mRNA and protein levels. Induction of Smad7 mRNA and protein was maximal within five hours, and returned to initial basal levels 24 hours post UV. UV irradiation induced Smad7 promoter reporter activity 3-fold. Smad7 promoter contains functional enhancer sequences that bind transcription factors Smad3 and activator protein-1 (AP-1). UV irradiation reduced protein binding to the Smad3 enhancer, and increased binding to the AP-1 enhancer. Deletion of AP-1 binding site in the Smad7 promoter completely abolished UV stimulation of Smad7 transcription. Deletion of Smad3 element had no effect on UV-induced promoter activity. UV irradiation increased mRNA and protein expression of AP-1 family members, c-Jun and c-Fos, which bound to the AP-1 element in the Smad7 promoter. Furthermore, over-expression of dominant negative c-Jun substantially reduced UV induction of Smad7 transcription. These data demonstrate that induction of Smad7 gene expression by UV irradiation is mediated via induction of transcription factor AP-1 in human skin fibroblasts.
doi:10.1074/jbc.M409647200
PMCID: PMC3738262  PMID: 15579469
Smad7; Ultraviolet radiation; AP-1
21.  The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-β2 in human retinal pigment epithelial cells 
Purpose
Transforming growth factor (TGF)-β is a key mediator of proliferative vitreoretinopathy, but the cellular mechanisms by which TGF-β induces extracellular matrix protein (ECM) synthesis are not fully understood. This study examined whether the PI3K/Akt pathway is involved in TGF-β2-induced collagen expression in human retinal pigment epithelial cells.
Methods
Human retinal pigment epithelial cells ARPE-19 were cultured and stimulated with TGF-β2. The role of the PI3K/Akt pathway was evaluated using the biochemical inhibitor, wortmannin. The effect of wortmannin on the expression of type I collagen mRNA (COL1A1, COL1A2) induced by TGF-β2 was evaluated by real-time RT-PCR. The effect of wortmannin on the synthesis of type I collagen induced by TGF-β2 was assessed by an immunocytochemical analysis with anti-type I collagen antibody. Luciferase reporter assays were performed to examine the effect of wortmannin on the transcriptional activities of COL1A2. A luciferase assay using a mutation construct of the Smad binding site in COL1A2 promoter (Smad-mut/Luc) was also performed to examine the crosstalk between the Smad pathway and the PI3K/Akt pathway. The effects of wortmannin on the transcriptional activity of Smad3 were also examined using CAGA12-Luc. Moreover, the effect of wortmannin on TGF-β2-induced Smad7 mRNA expression was evaluated.
Results
The biochemical blockade of PI3K/Akt activation inhibited TGF-β2-induced type I collagen mRNA expression and type I collagen synthesis. The blockade of PI3K/Akt pathway inhibited the increase in COL1A2 promoter activities when induced by TGF-β2 and reduced TGF-β2 induction of Smad-mut/Luc promoter activity and CAGA12-Luc activity. Moreover, wortmannin increased the TGF-β2-induced Smad7 mRNA expression levels.
Conclusions
The PI3K/Akt pathway plays a role in relaying the TGF-β2 signal to induce type I collagen synthesis in the retinal pigment epithelium through Smad-dependent and Smad-independent pathways.
doi:10.1007/s00417-011-1766-x
PMCID: PMC3262137  PMID: 21858467
Retinal pigment epithelium; TGF-β; Collagen; PI3K/Akt; Vitreoretinopathy
22.  Withagulatin A inhibits hepatic stellate cell viability and procollagen I production through Akt and Smad signaling pathways 
Acta Pharmacologica Sinica  2010;31(8):944-952.
Aim:
To investigate the effects of the natural product Withagulatin A on hepatic stellate cell (HSC) viability and type I procollagen production. The potential mechanism underlying the pharmacological actions was also explored.
Methods:
The effect of Withagulatin A on cell viability was evaluated in HSC and LX-2 cells using a sulforhodamine B (SRB) assay. Cell cycle distribution was analyzed using flow cytometry. Type I procollagen gene expression was determined using real-time PCR. Regulation of signaling molecules by Withagulatin A was detected using Western blotting.
Results:
Primary rat HSCs and the human hepatic stellate cell line LX-2 treated with Withagulatin A (0.625-20 μmol/L) underwent a dose-dependent decrease in cell viability, which was associated with S phase arrest and the induction of cell apoptosis. In addition, the natural product decreased phosphorylation of the Akt/mTOR/p70S6K pathway that controls cell proliferation and survival. Furthermore, Withagulatin A (1, 2 μmol/L) inhibited transforming growth factor-β (TGF-β) stimulated type I procollagen gene expression, which was attributable to the suppression of TGF-β stimulated Smad2 and Smad3 phosphorylation.
Conclusion:
Our results demonstrated that Withagulatin A potently inhibited HSC viability and type I procollagen production, thereby implying that this natural product has potential use in the development of anti-fibrogenic reagents for the treatment of hepatic fibrosis.
doi:10.1038/aps.2010.72
PMCID: PMC4007817  PMID: 20644552
liver fibrosis; collagen; hepatic stellate cell; Akt; Smad
23.  Transforming Growth Factor β-Independent Shuttling of Smad4 between the Cytoplasm and Nucleus 
Molecular and Cellular Biology  2000;20(23):9041-9054.
Smad4 plays a pivotal role in all transforming growth factor β (TGF-β) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-β signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-β signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-β signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm.
PMCID: PMC86557  PMID: 11074002
24.  INHIBITION OF TRANSFORMING GROWTH FACTOR-β/SMAD SIGNALING BY PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY 
Cancer letters  2006;242(2):207-214.
SUMMARY
Gastrin-releasing peptide (GRP) activates phosphatidylinositol 3-kinase (PI3-K)/Akt, an important cell survival signaling pathway, to stimulate growth of various cell types. Transforming growth factor (TGF) superfamily ligands activate intracellular Smad signaling to regulate cell growth, differentiation and apoptosis; dysregulation of the TGF-β/Smad pathway has been noted in cancer cells. Therefore, we sought to determine whether a potential cross-talk exists between the TGF-β/Smad and PI3-K pathways in the regulation of neuroblastoma cell growth. Increased Smad DNA binding was noted in SK-N-SH human neuroblastoma cells when treated with LY294002, an inhibitor of PI3-K, by transcription factor/DNA array analysis and electrophoretic mobility shift assay. LY294002 treatment resulted in Smad2 accumulation in the nuclei and an increased Smad binding element (SBE)-luciferase activity. These findings were corroborated by co-transfection with pCGNN-Δp85 plasmid, which expresses a PI3-K mutant p85 subunit. In contrast, GRP treatment decreased Smad binding activity in neuroblastoma cells. Our findings demonstrate that the PI3-K pathway negatively regulates TGF-β/Smad signaling in neuroblastoma cells. GRP-induced activation of PI3-K, resulting in neuroblastoma cell growth promotion, is potentiated by down-regulation of TGF-β/Smad signaling.
doi:10.1016/j.canlet.2005.11.007
PMCID: PMC2614268  PMID: 16412560
TGF-β; Smad; PI3-K; GRP; Neuroblastoma
25.  Overexpression of Endoglin Modulates TGF-β1-Signalling Pathways in a Novel Immortalized Mouse Hepatic Stellate Cell Line 
PLoS ONE  2013;8(2):e56116.
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.
doi:10.1371/journal.pone.0056116
PMCID: PMC3577806  PMID: 23437087

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