Smad7 Is Highly Expressed in Human Degenerative Discs and Participates in IL-1ß-Induced Apoptosis of Rat AF Cells via the Mitochondria Pathway.

Background: Abnormal Smad7 expression can lead to apoptosis in different cell types. Previously, we found high expression of Smad7 in rat degenerative discs. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear. Methods: Degenerative and nondegenerative human lumbar intervertebral discs were collected from patients during operation. The expressions of SMAD7 mRNA and protein in the different components of these discs were measured with real-time PCR and Western blotting, respectively. Annulus fibrosus (AF) cells were isolated and cultivated from the discs of young healthy rats. Smad7 in the AF cells was overexpressed with adenovirus and knocked down with siRNA. IL-1ß was used to induce apoptosis in the AF cells. Loss-and-gain cell function experiments were performed to show the effect of Smad7 on the apoptosis of AF cells. The function recovery experiments were performed to verify whether Smad7 regulates the apoptosis of AF cells through the mitochondria-mediated pathway. Results: Both the mRNA and protein expressions of Smad7 were significantly higher in the different components of human degenerative discs than in those of the nondegenerative discs. IL-1ß stimulated apoptosis while upregulating the Smad7 expression in the AF cells in vitro. Overexpression of Smad7 in AF cells exaggerated the IL-1ß-induced apoptosis in the cells while knockdown of Smad7 expression suppressed this apoptosis. With the exaggerated apoptosis in the AF cells with Smad7 overexpression, both active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. Conclusion: Smad7 is highly expressed in human degenerative discs and participates in IL-1ß-induced apoptosis of rat AF cells via the mitochondria pathway. Smad7 may be a potential target for the prevention and treatment of degenerative disc disease.

MicroRNA-503 Targets Mothers Against Decapentaplegic Homolog 7 Enhancing Hepatic Stellate Cell Activation and Hepatic Fibrosis.

BACKGROUND: The hyper-accumulation of extracellular matrix (ECM) is the leading cause of hepatic fibrosis, and TGF-ß-induced activation of hepatic stellate cells (HSCs) is the central event of hepatic fibrosis pathogenesis. The deregulation and dysfunction of miRNAs in hepatic fibrosis have been reported previously. AIMS: To identify miRNA(s) playing a role in HSC activation and the underlying mechanism. METHODS: We analyzed online microarray expression datasets from Gene Expression Omnibus (GEO) for differentially expressed miRNAs in hepatic fibrosis-related disease liver tissues, examined the specific effects of the candidate miRNA on TGF-ß-induced HSC activation, and screened for the targets of the candidate miRNA in the TGF-ß/SMAD signaling. Then, the predicted miRNA-mRNA binding, the specific effects of the target mRNA, and the dynamic effects of miRNA and mRNA on TGF-ß-induced HSC activation were investigated. RESULTS: The miR-503 expression was upregulated in TGF-ß-activated HSCs. miR-503 overexpression enhanced, while miR-503 inhibition attenuated TGF-ß-induced HSC proliferation and ECM accumulation in HSCs. miR-503 targeted SMAD7 to inhibit SMAD7 expression. SMAD7 knockdown also aggravated TGF-ß-induced HSC proliferation and ECM accumulation in HSCs. The effects of miR-503 overexpression on TGF-ß-induced HSC activation were partially reversed by SMAD7 overexpression. In CCl4-induced hepatic fibrosis model in rats, miR-503 overexpression aggravated, whereas SMAD7 overexpression improved CCl4-induced fibrotic changes in rats' liver tissues. The effects of miR-503 overexpression on CCl4-induced fibrotic changes were partially reversed by SMAD7 overexpression. CONCLUSION: miR-503 acts on HSC activation and hepatic fibrosis through SMAD7. The miR-503/SMAD7 axis enhances HSC activation and hepatic fibrosis through the TGF-ß/SMAD pathway.

Bone Morphogenetic Proteins Inhibit Ciliogenesis of Ependymal Cells in Vitro.

Ependymal cells have an essential role in regulating the dynamics of the cerebrospinal fluid flow by the movement of their multiple cilia. Impaired generation or function of cilia could cause hydrocephalus due to the disordered dynamics of the cerebrospinal fluid flow. However, molecular bases regulating differentiation of the ependymal cells and their ciliogenesis have not been fully elucidated. We report here that bone morphogenetic proteins (BMPs), growth factors orchestrating tissue architecture throughout the body, inhibit ciliogenesis during ependymal cell differentiation in primary cell culture. Previous in vitro study has reported that ectopic expression of Smad6 and Smad7 promotes differentiation of embryonic stem cells into multi-ciliated ependymal-like cells. Since Smad6 and Smad7 have been known as the intracellular inhibitory factors of the BMP signaling pathway, the activation of the pathway could cause a deficit in ciliogenesis of ependymal cells. To examine whether activation of the pathway affects ciliogenesis, we investigated the effects of two BMPs, BMP2 and BMP4, on the ependymal differentiation of the primary cultured cells prepared from the neonatal mouse brain. Supplementation of BMP2 or BMP4 in culture media significantly reduced the number of cells with multiple cilia among the total cells, while most of the cells expressed FoxJ1, a master regulator of ciliogenesis. Activation of the pathway was confirmed by the phosphorylation of intracellular Smad1/5/8, downstream factors of the BMP receptors. These in vitro results suggest that inhibition of the BMP signaling pathway might be essential for ciliogenesis during the ependymal cell differentiation in vivo.

Gene Signatures of Early Response to Anti-TNF Drugs in Pediatric Inflammatory Bowel Disease.

Around a 20-30% of inflammatory bowel disease (IBD) patients are diagnosed before they are 18 years old. Anti-TNF drugs can induce and maintain remission in IBD, however, up to 30% of patients do not respond. The aim of the work was to identify markers that would predict an early response to anti-TNF drugs in pediatric patients with IBD. The study population included 43 patients aged <18 years with IBD who started treatment with infliximab or adalimumab. Patients were classified into primary responders (n = 27) and non-responders to anti-TNF therapy (n = 6). Response to treatment could not be analyzed in 10 patients. Response was defined as a decrease in over 15 points in the disease activity indexes from week 0 to week 10 of infliximab treatment or from week 0 to week 26 of adalimumab treatment. The expression profiles of nine genes in total RNA isolated from the whole-blood of pediatric IBD patients taken before biologic administration and after 2 weeks were analyzed using qPCR and the 2-∆∆Ct method. Before initiation and after 2 weeks of treatment the expression of SMAD7 was decreased in patients who were considered as non-responders (p value < 0.05). Changes in expression were also observed for TLR2 at T0 and T2, although that did not reach the level of statistical significance. In addition, the expression of DEFA5 decreased 1.75-fold during the first 2 weeks of anti-TNF treatment in responders, whereas no changes were observed in non-responders. Expression of the SMAD7 gene is a pharmacogenomic biomarker of early response to anti-TNF agents in pediatric IBD. TLR2 and DEFA5 need to be validated in larger studies.

MiR-503-5p functions as an oncogene in oral squamous cell carcinoma by targeting Smad7.

BACKGROND: Oral squamous cell carcinoma (OSCC) is a common oral malignancy. Previous studies indicated that the level of miR-503-5p was upregulated in OSCC tissues. However, the mechanism by which miR-503-5p regulates the proliferation and invasion of OSCC cells remains unclear. Therefore, this study aimed to investigate the role of miR-503-5p during the progression of OSCC. METHODS: The level of miR-503-5p in Tca8113 cells was detected using RT-qPCR assay. In addition, CCK-8, transwell assays and flow cytometry assays were conducted to detect cell viability, migration, invasion and apoptosis, respectively. Meanwhile, the dual luciferase reporter assay was applied to explore the interaction between miR-503-5p and Smad7 in Tca8113 cells. RESULTS: Overexpression of miR-503-5p significantly promoted the proliferation, migration and invasion of Tca8113 cells, while downregulation of miR-503-5p markedly inhibited proliferation, migration and invasion of cells. In addition, knockdown of miR-503-5p obviously induced the apoptosis of Tca8113 cells via increasing the levels of Bax and cleaved caspase 3, and decreased the expression of Bcl-2. Moreover, SMAD family member 7 (Smad7) was identified as a direct binding target of miR-503-5p in Tca8113 cells. Overexpression of miR-503-5p significantly downregulated the levels of Smad7 and E-cadherin, but upregulated the levels of N-cadherin and MMP-9 in Tca8113 cells. CONCLUSION: These results indicated that miR-503-5p might act as an oncogene in OSCC cells by targeting Smad7. Therefore, miR-503-5p might act as a novel and potential therapeutic target for the treatment of OSCC.

Upregulation of circular SMAD7 inhibits tumorigenesis of gastric cancer by reversing epithelial-to-mesenchymal transition.

OBJECTIVE: Gastric cancer (GC) is one of the most ordinary malignant tumors. Recent studies have revealed that circular RNAs (circRNAs) play an important role in the progression of tumorigenesis. In this research, circ-SMAD7 was selected to identify how it functions in the progression of GC. PATIENTS AND METHODS: Circ-SMAD7 expression in paired GC patients' tissue samples and cell lines was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The role of circ-SMAD7 in the metastasis of GC was detected through wound healing assay and transwell assay. Western blot assay and RT-qPCR were used to discover the function of circ-SMAD7 in epithelial-to-mesenchymal transition (EMT) process. Furthermore, tumor metastasis assay was also performed in vivo. RESULTS: In this study, RT-qPCR results showed that circ-SMAD7 expression was significantly lower in GC tissues compared to that in adjacent ones. Cell migration and invasion of GC were inhibited via upregulation of circ-SMAD7. Moreover, the results of further experiments revealed that the EMT-related proteins were regulated via upregulation of circ-SMAD7 in GC. Furthermore, tumor metastasis of GC was inhibited via upregulation of circ-SMAD7 in nude mice. CONCLUSIONS: These results indicate that upregulation of circ-SMAD7 inhibits GC cell migration and invasion via reversing the EMT process.

Curcumin mitigates the epithelial-to-mesenchymal transition in biliary epithelial cells through upregulating CD109 expression.

Biliary epithelial cells (BECs) can secrete bile and the epithelial-to-mesenchymal transition (EMT) of BECs can cause fibrosis or damage interlobular bile ducts, leading to chronic cholangiopathies, such as primary biliary cholangitis (PBC). Transforming growth factor-ß1 (TGF-ß1) is a potent inducer of the EMT while curcumin, a diarylheptanoid, can inhibit the EMT of hepatocytes in many liver diseases. However, the protection and underlying mechanisms of curcumin against the EMT of BECs have not been clarified. Herein, we show that curcumin treatment significantly mitigates the EMT of BECs in vitro and in vivo. Mechanistically, curcumin significantly attenuated the TGF-ß1-induced Smad and Hedgehog signaling, and upregulated CD109 expression in BECs. Collectively, these findings highlighted the therapeutic potential of curcumin to counteract the EMT process in PBC.

Up-regulation of circ-SMAD7 inhibits tumor proliferation and migration in esophageal squamous cell carcinoma.

BACKGROUND: Increasing evidences demonstrate that circular RNAs (circRNAs) play an important role in the development and progression of human cancers. Nevertheless, the functions and molecular mechanism of circRNAs in esophageal squamous cell carcinoma (ESCC) tumorigenesis are largely unknown. The purpose of this research is to investigate the expression and potential role of a new circular RNA named circ-SMAD7 on ESCC carcinogenesis. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure circ-SMAD7 expression amount in both ESCC plasmas and tissues. Then the correlation between the expression of circ-SMAD7 and clinicopathological features was analyzed. Furthermore, by loss-of-function and gain-of-function experiments in ESCC cells, a functional analysis of circ-SMAD7 on ESCC cell proliferation and migration was performed. RESULTS: The expression of circ-SMAD7 was validated to be significantly down-regulated in ESCC plasmas in comparison with normal controls, showing a high negative correlation with TNM stage (P = 0.000) and lymphatic metastasis (P = 0.000). Moreover, circ-SMAD7 was significantly down-regulated in ESCC tissues compared to adjacent normal tissues. Furthermore, Loss-of-function and gain-of-function experiments revealed that the expression level of circ-SMAD7 affected the proliferation and migration of ESCC cell lines. CONCLUSION: Our study firstly exposed that over-expressioncirc-SMAD7, an influential regulator in cancer progression, can inhibit tumor proliferation and migration in ESCC and have the potential of becoming a biomarker for the diagnosis and therapy of ESCC.

High expression levels of SMAD3 and SMAD7 at diagnosis predict poor prognosis in acute myeloid leukemia patients undergoing chemotherapy.

The SMAD family (SMAD1-9) was critically important for regulating cellular process through transforming growth factor-ß signaling pathway, and contributed to carcinogenesis; however, their prognostic roles in acute myeloid leukemia (AML) remained unclear. This study collected 84 de novo AML patients treated with chemotherapy and 71 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Kaplan-Meier survival estimate indicated that among SMAD1-9, high SMAD3 and SMAD7 expression were both associated with poor event-free survival (EFS) and overall survival (OS; all P < 0.05) in AML patients undergoing chemotherapy; and high SMAD6 expression was associated with shorter EFS and OS (all P < 0.01) in patients underwent allo-HSCT. Multivariate analysis showed that only high SMAD7 expression had adverse effect on EFS and OS (P = 0.021, 0.026) independently. Furthermore, High SMAD3 and SMAD7 expressers had significantly shorter EFS and OS than low expressers (P = 0.006, 0.001). In AML patients who went through allo-HSCT, there were no significant differences for EFS and OS between patients with high and low-expression SMAD3 or SMAD7. Our study suggested that high expression of SMAD3 and SMAD7 predicted adverse prognosis in AML patients undergoing chemotherapy and SMAD7 was a better prognostic marker than SMAD3. Their prognosis impact may be overcome by allo-HSCT.

Smad7 Ameliorates TGF-ß-Mediated Skin Inflammation and Associated Wound Healing Defects but Not Susceptibility to Experimental Skin Carcinogenesis.

We assessed the roles of Smad7 in skin inflammation and wound healing using genetic and pharmacological approaches. In K5.TGFß1/K5.Smad7 bigenic (double transgenic) mice, Smad7 transgene expression reversed transforming growth factor (TGF)-ß1 transgene-induced inflammation, fibrosis, and subsequent epidermal hyperplasia and molecularly abolished TGF-ß and NF-κB activation. Next, we produced recombinant human Smad7 protein with a Tat-tag (Tat-Smad7) that rapidly enters cells. Subcutaneous injection of Tat-Smad7 attenuated infiltration of F4/80+ and CD11b+ leukocytes and α-smooth muscle actin+ fibroblasts before attenuating epidermal hyperplasia in K5.TGFß1 skin. Furthermore, topically applied Tat-Smad7 on K5.TGFß1 skin wounds accelerated wound closure, with improved re-epithelialization and reductions in inflammation and fibrotic response. A short treatment with Tat-Smad7 was also sufficient to reduce TGF-ß and NF-κB signaling in K5.TGFß1 skin and wounds. Relevant to the clinic, we found that human diabetic wounds had elevated TGF-ß and NF-κB signaling compared with normal skin. To assess the oncogenic risk of a potential Smad7-based therapy, we exposed K5.Smad7 skin to chemical carcinogenesis and found reduced myeloid leukocyte infiltration in tumors but not accelerated carcinogenesis compared with wild-type littermates. Our study suggests the feasibility of using exogenous Smad7 below an oncogenic level to alleviate skin inflammation and wound healing defects associated with excessive activation of TGF-ß and NF-κB.

SETDB1 regulates SMAD7 expression for breast cancer metastasis.

Breast cancer (BRC) is the most invasive cancer in women. Although the survival rate of BRC is gradually increasing due to improved screening systems, development of novel therapeutic targets for inhibition of BRC proliferation, metastasis and recurrence have been constantly needed. Thus, in this study, we identified overexpression of SETDB1 (SET Domain Bifurcated 1), a histone methyltransferase, in RNA-seq data of BRC derived from TCGA portal. In Gene Ontology (GO) analysis, cell migration-related GO terms were enriched, and we confirmed down-regulation of cell migration/invasion and alteration of EMT /MET markers after knockdown of SETDB1. Moreover, gene network analysis showed that SMAD7 expression is regulated by SETDB1 levels, indicating that up-regulation of SMAD7 by SETDB1 knockdown inhibited BRC metastasis. Therefore, development of SETDB1 inhibitors and functional studies may help develop more effective clinical guidelines for BRC treatment. [BMB Reports 2019; 52(2): 139-144].

Systemic SMAD7 Gene Therapy Increases Striated Muscle Mass and Enhances Exercise Capacity in a Dose-Dependent Manner.

Striated muscle wasting occurs with a variety of disease indications, contributing to mortality and compromising life quality. Recent studies indicate that the recombinant adeno-associated virus (serotype 6) Smad7 gene therapeutic, AVGN7, enhances skeletal and cardiac muscle mass and prevents cancer-induced wasting of both tissues. This is accomplished by attenuating ActRIIb intracellular signaling and, as a result, the physiological actions of myostatin and other ActRIIb ligands. AVGN7 also enhances isolated skeletal muscle twitch force, but is unknown to improve systemic muscle function similarly, especially exercise capacity. A 2-month-long dose-escalation study was therefore conducted using 5 × 1011, 1 × 1012, and 5 × 1012 vg/mouse and different tests of systemic muscle function. Body mass, skeletal muscle mass, heart mass, and forelimb grip strength were all increased in a dose-dependent manner, as was the fiber cross-sectional area of tibialis anterior muscles. Maximal oxygen consumption (VO2max), a measure of metabolic rate, was similarly enhanced during forced treadmill running, and although the total distance traveled was only elevated by the highest dose, all doses reduced the energy expenditure rate compared to control mice injected with an empty vector. Such improvements in VO2max are consistent with physiological cardiac hypertrophy, which is highly beneficial and a normal adaptive response to exercise. This was particularly evident at the lowest dose tested, which had minimal significant effects on skeletal muscle mass and/or function, but increased heart weight and exercise capacity. These results together suggest that AVGN7 enhances striated muscle mass and systemic muscle function. They also define minimally effective and optimal doses for future preclinical trials and toxicology studies and in turn will aid in establishing dose ranges for clinical trials.

MiR-21-Mediated Suppression of Smad7 Induces TGFß1 and Can Be Inhibited by Activation of Nrf2 in Alcohol-Treated Lung Fibroblasts.

BACKGROUND: We previously demonstrated that chronic alcohol ingestion augments TGFß1 expression in the lung fibroblast and increases the risk of fibroproliferative disrepair in a mouse model of acute lung injury. The effect of alcohol on TGFß1 is mitigated by treatment with sulforaphane (SFP), which can activate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, the mechanisms by which alcohol amplifies, or SFP attenuates, TGFß1 expression in the fibroblast are not known. MicroRNA (miR)-21 has been shown to inhibit Smad7, a TGFß1 signaling inhibitor. In this study, we hypothesized that alcohol augments TGFß1 expression through up-regulation of miR-21, which subsequently inhibits Smad7. METHODS: Primary mouse lung fibroblasts were cultured ± alcohol ± SFP and assessed for gene expression of miR-21, and gene and/or protein expression of Nrf2, Nrf2-regulated antioxidant enzymes, Smad7, STAT3, and TGFß1. NIH 3T3 fibroblasts were transfected with a miR-21 inhibitor and cultured ± alcohol. α-SMA, Smad7, and TGFß1 protein expression were then assessed. In parallel, NIH 3T3 lung fibroblasts were transfected with Nrf2 silencing RNA (siRNA) and cultured ± alcohol ± SFP. Gene expression of miR-21, Nrf2, Smad7, and TGFß1 was assessed. RESULTS: MiR-21 gene expression was increased by 12-fold at 48 hours, and Smad7 gene expression and protein expression were reduced by ~30% in alcohol-treated fibroblasts. In parallel, inhibition of miR-21 attenuated alcohol-mediated decrease in Smad7 and increase in TGFß1 and α-SMA protein expression. Treatment with SFP mitigated the effect of alcohol on miR-21, Smad7 and total and phosphorylated STAT3, and restored Nrf2-regulated antioxidant gene expression. Silencing of Nrf2 prevented the effect of SFP on miR-21, Smad7, and TGFß1 gene expression in alcohol-treated NIH 3T3 fibroblasts. CONCLUSIONS: Alcohol treatment increases TGFß1 in fibroblasts, at least in part, through augmentation of miR-21, which then inhibits Smad7 expression. These effects can be attenuated by activation of Nrf2 with SFP.

Estrogen receptor ß, a regulator of androgen receptor signaling in the mouse ventral prostate.

As estrogen receptor ß-/- (ERß-/-) mice age, the ventral prostate (VP) develops increased numbers of hyperplastic, fibroplastic lesions and inflammatory cells. To identify genes involved in these changes, we used RNA sequencing and immunohistochemistry to compare gene expression profiles in the VP of young (2-mo-old) and aging (18-mo-old) ERß-/- mice and their WT littermates. We also treated young and old WT mice with an ERß-selective agonist and evaluated protein expression. The most significant findings were that ERß down-regulates androgen receptor (AR) signaling and up-regulates the tumor suppressor phosphatase and tensin homolog (PTEN). ERß agonist increased expression of the AR corepressor dachshund family (DACH1/2), T-cadherin, stromal caveolin-1, and nuclear PTEN and decreased expression of RAR-related orphan receptor c, Bcl2, inducible nitric oxide synthase, and IL-6. In the ERß-/- mouse VP, RNA sequencing revealed that the following genes were up-regulated more than fivefold: Bcl2, clusterin, the cytokines CXCL16 and -17, and a marker of basal/intermediate cells (prostate stem cell antigen) and cytokeratins 4, 5, and 17. The most down-regulated genes were the following: the antioxidant gene glutathione peroxidase 3; protease inhibitors WAP four-disulfide core domain 3 (WFDC3); the tumor-suppressive genes T-cadherin and caveolin-1; the regulator of transforming growth factor ß signaling SMAD7; and the PTEN ubiquitin ligase NEDD4. The role of ERß in opposing AR signaling, proliferation, and inflammation suggests that ERß-selective agonists may be used to prevent progression of prostate cancer, prevent fibrosis and development of benign prostatic hyperplasia, and treat prostatitis.

The distribution and potential prognostic value of SMAD protein expression in chronic lymphocytic leukemia.

The SMAD proteins are responsible for transducing signals from activated transforming growth factor-beta. This is the first study assessing the expression of SMAD-1/8, SMAD-2/3, SMAD-4, and SMAD-7 in chronic lymphocytic leukemia cells with regard to their clinical significance and potential prognostic value. Overexpression of SMAD-1/8 was observed in 160 chronic lymphocytic leukemia patients compared to 42 healthy volunteers (p = 0.023) and was associated with a more progressive course of the disease (p = 0.016). Moreover, the high expression of SMAD-1/8 correlated with other, well-established prognostic factors, including clinical stage (p = 0.010) and lymphocyte doubling time (p = 0.021). The expression of SMAD-4 was lower in chronic lymphocytic leukemia patients compared with the control group (p = 0.003). Importantly, lower SMAD-4 levels correlated with longer progression-free survival (p = 0.009), progressive course of the disease (p = 0.002), advanced clinical stage (p = 0.0004), elevated beta-2-microglobulin and lactate dehydrogenase levels (p < 0.05), shorter lymphocyte doubling time (p = 0.009), and CD38 antigen expression (p = 0.039). In addition, lower SMAD-4 expression correlated with lower apoptotic index (p = 0.0007) and lower expression of receptors for vascular endothelial growth factors VEGFR-1 and VEGFR-2. A significant association was found between the low expression of inhibitory protein SMAD-7 and both zeta-chain-associated protein kinase 70-negative cells (p = 0.04) and lower apoptotic index (p = 0.004). No differences were observed in SMAD-2/3 expression. In conclusion, our results demonstrate a significant correlation between greater SMAD-1/8 and lower SMAD-4 expression in chronic lymphocytic leukemia cells, as well as more progressive outcome and poor prognosis. These data provide supporting evidence that the expression of SMAD proteins plays an important role in disease development and may be considered as a novel, biologic prognostic factor in this disease.

Anti-hepatic fibrosis effects of a novel turtle shell decoction by inhibiting hepatic stellate cell proliferation and blocking TGF-ß1/Smad signaling pathway in rats.

Hepatic fibrosis (HF), a wound-healing response to a variety of chronic stimuli, is characterized by the excessive synthesis of extracellular matrix (ECM) proteins by hepatic stellate cells (HSC) and eventually the development of hepatic cirrhosis. Turtle shell pill (TSP) is a common traditional Chinese medicine used for preventing and treating HF and early hepatic cirrhosis, but its side-effects and the shortage of ingredients limit its clinical application. In addition, its mechanism of action is not clear. In the present study, we first improved the original formula of TSP to produce a novel turtle shell decoction (NTSD) with less toxicity and easier accessible materials. In a carbon tetrachloride (CCl4)-induced HF rat model, we observed that NTSD and TSP had similar effects on the improvement of liver functions in rats, including a decrease in serum alanine amino transferase (ALT) and aspartate amino transferase (AST) serum concentrations and increased albumin content in addition to a marked attenuation of CCl4-induced liver damage and fibrosis. NTSD containing rat serum inhibited rat liver stellate cell line HSC-T6 cell proliferation and induced cell apoptosis in vitro. Moreover, the NTSD treatment significantly decreased the transforming growth factor beta 1 (TGF-ß1) and Smad3 gene expression and increased inhibitory Smad7 gene expression in liver tissues of HF rats, suggesting that NTSD inhibited the ECM expression of HSC by downregulating the TGF-ß1/Smad signaling pathway. The results of our rat model study revealed that NTSD showed good in vitro and in vivo anti-HF effects via proliferation inhibition and the induction of apoptosis of HSCs and blocked the TGF-ß1/Smad signaling pathway.

Analysis of the spatiotemporal expression of major genes in the TGF-ß/Smad signaling pathway and correlation analysis using Hu sheep muscle tissue.

The mRNA expression levels of key genes (Smads, MSTN, and MyoG) in the TGF-ß/Smad signaling pathway in Hu sheep at different growth stages (2 days, 2 months, and 6 months of age) and in different skeletal muscles (longissimus dorsi muscle and soleus muscle) and different genders were detected; and correlation of the Smad family (Smad2, Smad3, Smad4, and Smad7), MSTN, MyoG expressions was analyzed in Hu sheep. The results showed that the expression of Smads was higher in the soleus muscle than in the longissimus dorsi muscle; the expressions of Smad2, Smad3, and Smad4 were significantly higher in 2-day-old sheep than in sheep belonging to the other age groups (P < 0.05); the expressions of Smad2, Smad4, and Smad7 were higher in rams than in 2-day-old ewes, but lower in rams than in 2-month-old and 6-month-old ewes; and the expression of Smad3 was higher in rams than in 2-day-old and 2-month-old ewes, but lower in rams than in 6-month-old ewes. In the 2 different muscle tissues, expression of Smad2 was significantly positively correlated (P < 0.01) with that of Smad3. The expression of Smad3 was significantly positively correlated (P < 0.01) with that of Smad4, which showed that the Smad family genes could have an inhibitory effect on the TGF-ß/Smad signaling pathway.

Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression.

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3) and functions as an oncogenic factor in many cancer types. However, the role of EZH2 in renal fibrogenesis remains unexplored. In this study, we found high expression of EZH2 and H3K27me3 in cultured renal fibroblasts and fibrotic kidneys from mice with unilateral ureteral obstruction and humans with CKD. Pharmacologic inhibition of EZH2 with 3-deazaneplanocin A (3-DZNeP) or GSK126 or siRNA-mediated silencing of EZH2 inhibited serum- and TGFß1-induced activation of renal interstitial fibroblasts in vitro, and 3-DZNeP administration abrogated deposition of extracellular matrix proteins and expression of α-smooth muscle actin in the obstructed kidney. Injury to the kidney enhanced Smad7 degradation, Smad3 phosphorylation, and TGFß receptor 1 expression, and 3-DZNeP administration prevented these effects. 3-DZNeP also suppressed phosphorylation of the renal EGF and PDGFß receptors and downstream signaling molecules signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 after injury. Moreover, EZH2 inhibition increased the expression of phosphatase and tensin homolog (PTEN), a protein previously associated with dephosphorylation of tyrosine kinase receptors in the injured kidney and serum-stimulated renal interstitial fibroblasts. Finally, blocking PTEN with SF1670 largely diminished the inhibitory effect of 3-DZNeP on renal myofibroblast activation. These results uncovered the important role of EZH2 in mediating the development of renal fibrosis by downregulating expression of Smad7 and PTEN, thus activating profibrotic signaling pathways. Targeted inhibition of EZH2, therefore, could be a novel therapy for treating CKD.

CXCL9 Regulates TGF-ß1-Induced Epithelial to Mesenchymal Transition in Human Alveolar Epithelial Cells.

Epithelial to mesenchymal cell transition (EMT), whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). CXCR3 and its ligands are recognized to play a protective role in pulmonary fibrosis. In this study, we investigated the presence and extent of EMT and CXCR3 expression in human IPF surgical lung biopsies and assessed whether CXCR3 and its ligand CXCL9 modulate EMT in alveolar epithelial cells. Coexpression of the epithelial marker thyroid transcription factor-1 and the mesenchymal marker α-smooth muscle actin and CXCR3 expression was examined by immunohistochemical staining of IPF surgical lung biopsies. Epithelial and mesenchymal marker expression was examined by quantitative real-time PCR, Western blotting, and immunofluorescence in human alveolar epithelial (A549) cells treated with TGF-ß1 and CXCL9, with Smad2, Smad3, and Smad7 expression and cellular localization examined by Western blotting. We found that significantly more cells were undergoing EMT in fibrotic versus normal areas of lung in IPF surgical lung biopsy samples. CXCR3 was expressed by type II pneumocytes and fibroblasts in fibrotic areas in close proximity to cells undergoing EMT. In vitro, CXCL9 abrogated TGF-ß1-induced EMT. A decrease in TGF-ß1-induced phosphorylation of Smad2 and Smad3 occurred with CXCL9 treatment. This was associated with increased shuttling of Smad7 from the nucleus to the cytoplasm where it inhibits Smad phosphorylation. This suggests a role for EMT in the pathogenesis of IPF and provides a novel mechanism for the inhibitory effects of CXCL9 on TGF-ß1-induced EMT.

miR-195 plays a role in steroid resistance of ulcerative colitis by targeting Smad7.

An imbalance in pro- and anti-inflammation is an important mechanism of steroid resistance in UC (ulcerative colitis), and miRNAs may participate in this process. The present study aimed to explore whether miRNAs play a role in the steroid resistance of UC by regulating gene expression of the inflammation signal pathway. SS (steroid-sensitive) patients, SR (steroid-resistant) patients and healthy individuals were recruited. In vivo miRNA profiles of serum samples showed that miR-195 was decreased significantly in the SR group compared with the SS group (P<0.05). This result was confirmed by qPCR (quantitative real-time PCR) and miRNA ISH (in situ hybridization) in serum and colon tissue samples. Online software was used to identify Smad7 mRNA as a potential target of miR-195. The direct interaction of miR-195 and Smad7 mRNA was investigated using a biotinylated miR-195 pull-down assay. Overexpression of a miR-195 precursor lowered cellular levels of Smad7 protein; conversely, antagonism of miR-195 enhanced Smad7 translation without disturbing Smad7 mRNA levels. A luciferase reporter assay revealed a repressive effect of miR-195 via a single Smad7 3'-UTR target site, and point mutation of this site prevented miR-195-induced repression of Smad7 translation. Furthermore, increased levels of miR-195 led to a decrease in c-Jun and p65 expression. In contrast, transfection with anti-miR-195 led to increased levels of c-Jun and p65 protein. The decrease in miR-195 led to an increase in Smad7 expression and corresponding up-regulation of p65 and the AP-1 (activator protein 1) pathway, which might explain the mechanism of steroid resistance in UC patients.