Transforming Growth Factor-β1 Induced Epithelial-Mesenchymal Transition in Hepatic Fibrosis

The epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are crucial for the regulation of cellular plasticity during liver fibrosis. Transforming growth factor (TGF)-β1 is an important cytokine for the induction of the EMT in liver fibrosis. TGF-β1 signaling induces the EMT through various signaling mechanisms and is the predominant agent mediating these fibrotic changes. Chronic exposure to TGF-β1 induces the transition of hepatocytes to collagen-producing mesenchymal cells, prolonged exposure of hepatocytes to TGF-β1 increases the expression of collagen and induces cytoskeletal rearrangement that resembles the EMT. These morphological and molecular alterations may provide the foundation for liver fibrosis. This review discussed the relation and mechanisms between EMT and liver fibrosis and ulteriorly elaborated on TGF-β1 induced EMT and each of their roles in liver fibrosis. Better understanding of the cellular and molecular characteristics of the cirrhotic hepatocyte may enable the development of chemo-preventative agents for liver fibrosis.     

Increased Expression of Transforming Growth Factor-β1 During Gastric Ulcer Healing in Rats

This study was done to investigate theexpression and localization of transforming growthfactor-₁ (TGF-₁) inthe gastric ulcerated tissues produced by acetic-acidduring the healing process, by northern blot analysis and immunohistochemicaltechnique. Ulcerated TGF-₁ mRNA levelswere significantly increased from days 3 to 18, in asimilar manner to extracellular matrix proteins, andreturned to control levels at the scarred phase.Immunoreactive TGF-₁ was localized inepithelial cells beneath proliferative zone in intacttissues. 1 In ulcerated tissues, TGF-₁was localized in macrophages in the ulcer bed and in fibroblasts ormyofibroblasts in the granulation tissues. Treatmentwith prostaglandin E₁ (PGE₁)further stimulated ulcerated TGF-₁expression, being associated with the acceleration of gastric ulcer healing, while treatment withindomethacin reduced TGF-₁ expression,being accompanied by the delayed ulcer healing. Thecombination of PGE₁ and indomethacin reversedthe indomethacin-induced decrease in ulcerated TGF-₁.Thus, TGF-₁ may be implicated in theacceleration of gastric ulcer healing.     

Expression of Transforming Growth Factor β1 in Bronchial Biopsies in Asthma and COPD

The role of transforming growth factor β₁ (TGF β₁) in airway remodeling in asthma and chronic obstructive pulmonary disease (COPD) has not been fully described. To evaluate the possible pathogenetic role of TGF β₁ in asthma and COPD, immunohistochemical expression of TGF β₁ was described in bronchial biopsies from patients with asthma and COPD compared with healthy individuals. Twelve subjects with asthma, 13 subjects with COPD, and 10 healthy individuals enrolled in the study. Bronchial biopsies were stained with hematoxylin and eosin and anti‐TGF β₁ antibody. As a result, immunoreactive TGF β₁ was mainly localized in association with connective tissue in all groups. The staining intensity was not statistically different among the groups in bronchial epithelium, whereas it was significantly higher in the group of asthma in the submucosa. Because there is evidence showing a significant increase of staining intensity in the submucosa from asthmatics but not from subjects with COPD, we may conclude that TGF β₁ may play a significant role in pathogenesis of asthma but not in COPD.     

The Role of Tumor Necrosis Factor-α and Interferon-γ in Regulating Angiomotin-Like Protein 1 Expression in Lung Microvascular Endothelial Cells

BackgroundAngiogenesis in the alveolar septa is thought be a critical factor in pulmonary emphysema. Angiomotin-like protein 1 (AmotL1) is involved in angiogenesis via regulating endothelial cell function. However, the role of AmotL1 in the pathogenesis of pulmonary emphysema has not been elucidated. The objective of this study is to evaluate the expression of AmotL1 in lung tissues from a murine model with emphysema, as well as from patients with chronic obstructive pulmonary disease (COPD). Furthermore, we analyzed the regulation of AmotL1 expression by TNF-α and IFN-γ in endothelial cells in vitro.MethodsNrf2 knockout mice were exposed to cigarette smoke (CS) for 4 weeks, and the down-regulated genes affecting vascularity in the whole lung were identified by microarray analysis. This analysis revealed that the mRNA expression of AmotL1 decreased in response to CS when compared with air exposure. To confirm the protein levels that were indicated in the microarray data, we determined the expression of AmotL1 in lung tissues obtained from patients with COPD and also determined the expression of AmotL1, NFκB and IκBα in cultured normal human lung microvascular endothelial cells (HLMVECs) that were stimulated by TNF-α and IFN-γ.ResultsWe found that the number of AmotL1-positive vessels decreased in the emphysema lungs compared with the normal and bronchial asthmatic lungs. IFN-γ pretreatment diminished the TNF-α-induced AmotL1 in the cultured HLMVECs by blocking the degradation of IκBα.ConclusionsThese results suggested that IFN-γ exhibits anti-angiogenesis effects by regulating the expression of TNF-α-induced AmotL1 via NFκB in emphysema lungs.     

ASPP2 Inhibits the Profibrotic Effects of Transforming Growth Factor-β1 in Hepatic Stellate Cells by Reducing Autophagy

Background

Apoptosis-stimulating protein of p53-2 (ASPP2) is a damage-inducible P53-binding protein that enhances damage-induced apoptosis. Fibrosis is a wound-healing response, and hepatic stellate cells (HSCs) are key players in liver fibrogenesis. However, little is known about the relationship between ASPP2 and hepatic fibrosis.

Aims

We investigated the effects of ASPP2 overexpression in HSCs and the role of ASPP2 in mouse liver fibrogenesis.

Methods

Human HSCs (LX-2 cells) were pre-incubated with GFP adenovirus (Ad) or ASPP2 adenovirus (AdASPP2) for 24 h and then treated with or without TGF-β1. ASPP2^+/? and ASPP2+/+ Balb/c mice were used to examine the effects of ASPP2 on liver fibrosis in vivo. ASPP2+/+ Balb/c mice were generated by injecting AdASPP2 into the tail vein of ASPP2 WT Balb/c mice; all mice received intraperitoneal injections of carbon tetrachloride.

Results

In this study, ASPP2 was found to markedly inhibit TGF-β1-induced fibrogenic activation of LX-2 cells. Further experiments using an autophagic flux assay confirmed that ASPP2 reduced the fibrogenic activation of LX-2 cells by inhibiting autophagy. Moreover, we found that ASPP2 overexpression attenuated the anti-apoptotic effects of TGF-β1 in LX-2 cells. The extent of liver fibrosis was markedly reduced in ASPP2+/+ mouse liver tissue compared with control mice; however, in ASPP2^+/? mice, hepatic collagen deposition was significantly increased.

Conclusion

These results suggest that TGF-β1-induced autophagy is required for the fibrogenic response in LX-2 cells and that ASPP2 may both inhibit TGF-β1-induced autophagy and decrease liver fibrosis.

     

Inhibitory Effects of Atorvastatin on the mRNA Expression of ICAM-1 and VCAM-1 Activated by TNF-α in Cultured Human Umbilical Vein Endothelial Cells

Objectives To investigate the effects of atorvastatin on the mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) activated by TNF-α in cultured human umbilical vein endothelial cells (HUVEC). Methods and Results Lactic dehydrogenase (LDH) activity in the culture media increased when HUVEC were incubated with TNF-α, suggesting a cytotoxic effect of TNF-α on HUVEC. The mRNA expression of ICAM-1 and VCAM-1 increased in HUVEC incubated with 10μg/L TNF-α and reached peak in HUVEC incubated with 30μg/L TNF-α. The mRNA expression of ICAM-1 and VCAM-1 in HUVEC incubated with 30μg/L TNF-α began to increase at 6 h, reached peak at 48 h, and kept a plateau until 72 h. Atorvastatin dose-dependently inhibited the mRNA expressions of ICAM-1 and VCAM-1 activated by incubating HUVEC with 30μg/L TNF-α for 48 hours. Conclusions Atorvastatin might stabilize plaque and decelerate the process of AS by inhibiting the mRNA expressions of ICAM-1 and VCAM-1.     

High-Expression of Transforming Growth Factor β1 and Phosphorylation of Extracellular Signal-Regulated Protein Kinase in Vascular Smooth Muscle Cells from Aorta and Renal Arterioles of Spontaneous Hypertension Rats

To further elucidate the molecular mechanisms involved in hypertensive vascular remodeling, an immunohistochemical technique and Western blot were applied to study phospho-extracellular signal-regulated kinase (ERK1/2) and transforming growth factor β₁ (TGF-β₁) expression in endothelial and vascular smooth muscle cell (VSMC) of the thoracic aorta and renal arterioles from SHR of different ages. Results of both the immunohistochemistry and Western blot assays showed that either the phospho-ERK1/2 at endothelium or VSMC of renal small arteries from SHR8, SHR16, and SHR20 groups and of the aorta from SHR16 and SHR20 were higher than that from control group. Comparing with that in the small arteries of the kidney, the phospho-ERK1/2 in the endothelium and in VSMC was markedly increased in the aorta, and high expression of TGF-β1 was detected in the aorta and kidney from SHR16 and SHR20 by Western blot. These results suggested that ERK 1/2 could be activated by phosphorylation with over-expression of TGF-β₁ in the endothelium and in VSMC of aorta and renal arterioles from SHR, which might play an important role in VSMC proliferation under hypertension.     

Role of Extracellular Signal-Regulated Kinase, p38 Kinase, and Activator Protein-1 in Transforming Growth Factor-β1–Induced Alpha Smooth Muscle Actin Expression in Human Fetal Lung Fibroblasts In Vitro

Myofibroblasts characterized by alpha smooth muscle actin(α-SMA) expression play a key role in pulmonary fibrosis. Transforming growth factor-beta1 (TGF-β1) is likely to be involved in the emergence of myofibroblasts, but the intracellular signal pathways for this process have not been well determined. The aim of the present study was to investigate the role of mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) signaling pathways in TGF-β1–induced α-SMA expression in human fetal lung fibroblasts (HLF-02). We found that TGF-β1 treatment activated p38 kinase and extracellular signal-regulated kinase (Erk) in HLF-02 cells. The induction of α-SMA by TGF-β1 was suppressed by p38 kinase inhibitor (SB203580) and Erk inhibitor (PD98059). AP-1 inhibitor curcumin also inhibited TGF-β1–induced α-SMA expression. In addition, dominant negative mutant c-Jun (TAM67) downregulated TGF-β1–induced AP-1 transactivation and α-SMA expression. In additional, PD98059 but not SB203580 inhibited the AP-1 DNA binding activity induced by TGF-β1. Based on these findings, we conclude that p38 kinase, Erk, and AP-1 are responsible for the α-SMA expression induced by TGF-β1 in human fetal lung fibroblasts. Erk is involved in inducing α-SMA expression via AP-1 activation.     

Interleukin-1β and Tumor Necrosis Factor-α Induce Chemokine and Matrix Metalloproteinase Gene Expression in Human Colonic Subepithelial Myofibroblasts

Background: Colonic subepithelial myofibroblasts may play a role in the inflammatory responses and in extracellular matrix (ECM) metabolism. In this study, we investigated the effects of interleukin (IL)-1 β and tumor necrosis factor (TNF)- α on chemokine (IL-8 and monocyte chemoattractant protein (MCP)-1) and ECM turnover (proliferation of subepithelial myofibroblasts, and secretion of ECM and matrix metalloproteinases (MMPs) in colonic subepithelial myofibroblasts. Methods: Human colonic sub-epithelial myofibroblasts were isolated using the method described by Mahida et al. (4). Chemokine and MMP expressions were determined by ELISA and Northern blotting. Nuclear factor (NF)- &#115 B and NF-IL6 DNA binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA). Results: IL-1 β and TNF- α did not affect the proliferation of subepithelial myofibroblasts, but stimulated the secretion of types I and IV collagens weakly. Unstimulated subepithelial myofibroblasts secreted a large amount of MMP-2, but a small amount of IL-8, MCP-1 and MMP-1. IL-1 β and TNF- α both induced a dose- and time-dependent increase in IL-8, MCP-1 and MMP-1 secretion, and weakly stimulated MMP-2 secretion. IL-1 β and TNF- α both rapidly evoked NF- &#115 B DNA-binding activity. The inhibition of NF- &#115 B activation markedly blocked both IL-1 β - and TNF- α -induced IL-8 and MCP-1 mRNA expression, but did not affect MMP-1 mRNA expression. Conclusions: These observations indicate that chemokine secretion and ECM metabolism are collectively regulated by the proinflammatory cytokines, IL-1 β and TNF- α , in colonic subepithelial myofibroblasts. Thus, colonic subepithelial myofibroblasts may play an important role in the pathophysiology of inflammation in the colon.     

Enhanced Expression of Transforming Growth Factor (TGF) -α Precursor and TGF-β1 During Paneth Cell Regeneration

An intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induces selective killing and rapid regeneration of Paneth cells, which have a large amount of zinc in their cytoplasmic granules. We examined the expression pattern of transforming growth factor (TGF) - and TGF-₁ in this regenerative process. Messenger RNA expression of TGF- and TGF-₁ reached their peaks at 12 and 24 hr after dithizone injection, respectively. Protein expression of TGF- precursor and TGF-₁ increased to a maximum at 24 and 72 hr, respectively. Their immunoreactivities were localized in the epithelial cells in the vicinity of Paneth cells, whereas they were prominent in the upper half of the crypts in control rats. In conclusion, destruction of Paneth cells induced TGF- precursor expression, followed by an increase of TGF-₁ especially in the crypt bases. This unique expression pattern of two growth factors may be involved in rapid regeneration of Paneth cells.     

Store-Operated Ca2+ Entry is involved in Transforming Growth Factor-β1 Facilitated Proliferation of Rat Airway Smooth Muscle Cells

Objective. To investigate the role and underlying mechanisms of store-operated Ca²⁺ entry (SOCE) in mediating the promoting effect of transforming growth factor (TGF)-β1 on the proliferation of airway smooth muscle cells (ASMCs). Methods. Rat bronchial smooth muscle cells were cultured as we described previously. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) of ASMCs was measured by laser confocal microscope Ca²⁺ fluorescence imaging with Fluo-3/AM. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and p27 expression assay were used to determine the proliferation rate of ASMCs. Results. We demonstrated that TGF-β1 (10 ng/ml) increased basal (Ca²⁺]_i) level, [Ca²⁺]_i rise induced by thapsigargin-induced Ca²⁺ release and SOCE in rat ASMCs. This effect of TGF-β1 on SOCE was not inhibited by glucocorticoid dexamethasone (DXM, 100 nM), antioxidant α-tocopherol (100 μM), and intermediate-conductance Ca²⁺-activated K⁺ channels (IK_Ca) inhibitor charybdotoxin (100 nM), suggesting that reactive oxygen species and IK_Ca channels might not mediate the effect of TGF-β1. TGF-β1 slightly increased the expression of Orai1 and STIM1, two important molecules involved in the molecule component and regulation of SOC channels, in the presence of 10% fetal bovine serum (FBS). The proliferation of ASMC stimulated with 2.5% FBS was promoted by TGF-β1, and partly inhibited by non-specific Ca²⁺ channel blocker SKF-96365 (10 μM) and Ni²⁺ (100 μM). DXM, α-tocopherol, and charybdotoxin had no effect on the proliferation promoted by TGF-β1. Conclusion. TGF-β1 promotes ASMC proliferation partly through increasing the expression and activity of SOC channels.     

Transforming Growth Factor-β1 and Tumor Necrosis Factor-α are Associated with Clinical Severity and Airflow Limitation of COPD in an Additive Manner

Background

The role of tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) in chronic obstructive pulmonary disease (COPD) is controversial. The purpose of this study was to assess the relationships among polymorphisms, clinical phenotypes, and the serum levels of TNF-α and TGF-β1.

Methods

Polymorphisms of promoters of TNF-α (rs 361525 and rs 1800629) and TGF-β1 (rs 1800469) in 110 COPD patients, 110 nonsmoker health controls without COPD, and 34 smokers were evaluated. Pulmonary functions, chest computed tomography, TGF-β1, and TNF-α were assessed.

Results

The genetic polymorphism of TNF-α (rs 361525) was associated with COPD. More severe COPD patients had higher serum levels of TNF-α and TGF-β1; moreover, serum levels of TGF-β1of mild COPD patients were higher than normal controls. All of the studied subjects were divided into four groups by the 95th percentile value of control as cutoff serum value of TGF-β1 (224.35 ρg/ml) or TNF-α (17.56 ρg/ml) to define the high value of TGF-β1 or TNF-α, which are higher than those cutoff of values (>224.35 or 17.56 ρg/ml). The FEV₁ of the group with high TGF-β1 + low TNF-α or low TGF-β1 + high TNF-α or high TNF-α + high TGF-β1 was lower than the group with low TGF-β1 + low TNF-α group. Moreover, the lowest value of FEV₁ was in the group with high TNF-α + high TGF-β1.

Conclusions

The genetic polymorphism of the TNF-α is associated with COPD. Both TGF-β1 and TNF-α modulate clinical severity and airflow limitation in an additive manner.