Specific Smad2/3 Linker Phosphorylation Indicates Esophageal Non-neoplastic and Neoplastic Stem-Like Cells and Neoplastic Development.

BACKGROUND: There is little known about stem cells in human non-neoplastic and neoplastic esophageal epithelia. We have demonstrated expression of linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr), suggesting presence of stem-like cells in mouse esophageal epithelium, and identified presence of pSmad2/3L-Thr-positive cells that might function as cancer stem cells in mouse model of colorectal carcinoma. AIMS: We explore whether pSmad2/3L-Thr can be used as a biomarker for stem cells of human esophageal epithelia and/or neoplasms. METHODS: We have used esophageal tissues from inpatients undergoing endoscopic submucosal dissection and performed double immunofluorescent staining of pSmad2/3L-Thr and Ki67, CDK4, p63, Sox2, CK14, p53, ALDH1, CD44 or D2-40 after which the sections were stained with hematoxylin and eosin. RESULTS: pSmad2/3L-Thr-positive cells showed immunohistochemical co-localization with CDK4, p63, CD44 and Sox2 in the basal and parabasal layers of non-neoplastic esophageal epithelia. In esophageal neoplasms, they showed immunohistochemical co-localization with p53, CDK4, ALDH1 and CD44. There was a significant increase in the percentage of pSmad2/3L-Thr-positive cells in the p53-positive neoplastic cell population with development of esophageal neoplasia. pSmad2/3L-Thr-positive cells localized to the lower section of low-grade intraepithelial neoplasia and were observed up to the upper section in carcinoma in situ. In invasive squamous cell carcinoma, they were scattered throughout the tumor with disappearance of polarity and were found in intraepithelial primary lesions and sites of submucosal and vessel invasion. CONCLUSIONS: We determined significant expression of pSmad2/3L-Thr in human esophageal non-neoplastic and neoplastic epithelia, indicating that these are epithelial stem-like cells and cancer stem cells, respectively, that correlate with developing esophageal neoplasms.

Effect of miR-10a on sepsis-induced liver injury in rats through TGF-ß1/Smad signaling pathway.

OBJECTIVE: To observe the effect of the micro ribonucleic acid (miR)-10a on sepsis-induced liver injury in rats through the transforming growth factor-b1 (TGF-b1)/Smad signaling pathway. MATERIALS AND METHODS: The rat model of sepsis was established via cecal ligation and puncture, in which miR-10a was overexpressed and silenced using liposome transfection. The rats were randomly divided into miR-10a mimics group (Mimics group, n=10) and miR-10a inhibitors group (Inhibitors group, n=10), and the sham operation group (Sham group, n=10) was also set up. The transfection efficiency of miR-10a in liver tissues in each group was detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), the serum liver function indexes aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were determined. Moreover, the content of the serum reactive oxygen species (ROS), glutathione (GSH), and GSH peroxidase (GSH-Px) was determined using enzyme-linked immunosorbent assay (ELISA). The content of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and myeloperoxidase (MPO) in liver tissues was detected, and the pathological changes in liver tissues were observed through hematoxylin-eosin (HE) staining. Finally, the expression levels of cytochrome P4502E1 (CYP2E1) and TGF-b1/Smad signaling pathway genes and proteins in liver tissues were detected via qRT-PCR and Western blotting. RESULTS: The expression of miR-10a was significantly increased in Mimics group (p<0.05) and extremely low in the Inhibitors group (p<0.05). In Mimics group, the levels of serum AST, ALT, and LDH were significantly increased (p<0.05), the content of ROS, TNF-α, IL-6, and MPO was substantially increased (p<0.05), while that of GSH and GSH-Px notably declined (p<0.05). According to the HE staining results, the liver cells were orderly arranged in the Inhibitors group, and they were disorderly arranged with more inflammatory cells in the Mimics group. The results of the gene and protein assays showed that the expression levels of CYP2E1, TGF-b1, and Smad2 in Mimics group were markedly higher than those in the Sham group (p<0.05), while they displayed the opposite trends in the Inhibitors group (p<0.05). CONCLUSIONS: Silencing miR-10a can inhibit the occurrence of sepsis-induced liver injury in rats by downregulating the TGF-ß1/Smad pathway.

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.

Prostaglandin E2 inhibits profibrotic function of human pulmonary fibroblasts by disrupting Ca2+ signaling.

We have shown that calcium (Ca2+) oscillations in human pulmonary fibroblasts (HPFs) contribute to profibrotic effects of transforming growth factor-ß (TGF-ß) and that disruption of these oscillations blunts features of pulmonary fibrosis. Prostaglandin E2 (PGE2) exerts antifibrotic effects in the lung, but the mechanisms for this action are not well defined. We thus sought to explore interactions between PGE2 and the profibrotic agent TGF-ß in pulmonary fibroblasts (PFs) isolated from patients with or without idiopathic pulmonary fibrosis (IPF). PGE2 inhibited TGF-ß-promoted [Ca2+] oscillations and prevented the activation of Akt and Ca2+/calmodulin-dependent protein kinase-II (CaMK-II) but did not prevent activation of Smad-2 or ERK. PGE2 also eliminated TGF-ß-stimulated expression of collagen A1, fibronectin, and α-smooth muscle actin and reduced stress fiber formation in the HPFs. RNA sequencing revealed that HPFs preferentially express EP2 receptors relative to other prostanoid receptor subtypes: EP2 expression is ~10-fold higher than that of EP4 receptors; EP1 and EP3 receptors are barely detectable; and EP2-receptor expression is ~3.5-fold lower in PFs from IPF patients than in normal HPFs. The inhibitory effects of PGE2 on synthetic function and stress fiber formation were blocked by selective EP2 or EP4 antagonists and mimicked by selective EP2 or EP4 agonists, the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin, all of which elevate cellular cAMP concentrations. We conclude that PGE2, likely predominantly via EP2 receptors, interferes with Ca2+ signaling, CaMK-II activation, and Akt activation in IPF-HPFs and HPFs treated with TGF-ß. Moreover, a decreased expression of EP2 receptors in pulmonary fibroblasts from IPF patients may contribute to the pathophysiology of this disease.

TRPM7 regulates angiotensin II-induced sinoatrial node fibrosis in sick sinus syndrome rats by mediating Smad signaling.

Sinoatrial node fibrosis is involved in the pathogenesis of sinus sick syndrome (SSS). Transient receptor potential (TRP) subfamily M member 7 (TRPM7) is implicated in cardiac fibrosis. However, the mechanisms underlying the regulation of sinoatrial node (SAN) fibrosis in SSS by TRPM7 remain unknown. The aim of this study was to investigate the role of angiotensin II (Ang II)/TRPM7/Smad pathway in the SAN fibrosis in rats with SSS. The rat SSS model was established with sodium hydroxide pinpoint pressing permeation. Forty-eight rats were randomly divided into six groups: normal control (ctrl), sham operation (sham), postoperative 1-, 2-, 3-, and 4-week SSS, respectively. The tissue explant culture method was used to culture cardiac fibroblasts (CFs) from rat SAN tissues. TRPM7 siRNA or encoding plasmids were used to knock down or overexpress TRPM7. Collagen (Col) distribution in SAN and atria was assessed using PASM-Masson staining. Ang II, Col I, and Col III levels in serum and tissues or in CFs were determined by ELISA. TRPM7, smad2 and p-smad2 levels were evaluated by real-time PCR, and/or western blot and immunohistochemistry. SAN and atria in rats of the SSS groups had more fibers and higher levels of Ang II, Col I and III than the sham rats. Similar findings were obtained for TRPM7 and pSmad2 expression. In vitro, Ang II promoted CFs collagen synthesis in a dose-dependent manner, and potentiated TRPM7 and p-Smad2 expression. TRPM7 depletion inhibited Ang II-induced p-Smad2 expression and collagen synthesis in CFs, whereas increased TRPM7 expression did the opposite. SAN fibrosis is regulated by the Ang II/TRPM7/Smad pathway in SSS, indicating that TRPM7 is a potential target for SAN fibrosis therapy in SSS.

Immunolocalization of connective tissue growth factor, transforming growth factor-beta1 and phosphorylated-SMAD2/3 during the postnatal tooth development and formation of junctional epithelium.

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta 1 (TGF-ß1) and TGF-ß1-induced CTGF expression is regulated through SMAD pathway. However, there is no literature showing the expression of TGF-ß1-SMAD2/3-CTGF signaling pathway during postnatal tooth development and the formation of junctional epithelium (JE). Hence, we aimed to analyze the localization of TGF-ß1, CTGF and phosphorylated SMAD2/3 (p-SMAD2/3) in the developing postnatal rat molars. Wistar rats were killed at postnatal (PN) 0.5, 3.5, 7, 14 and 21days and the upper jaws were processed for immunohistochemistry. At PN0.5 and PN3.5, weak staining for TGF-ß1 and CTGF was evident in preameloblasts (PA), while moderate to strong staining was seen in odontoblasts (OD), dental papilla (DPL), secretary ameloblasts (SA), preodontoblasts (PO) and polarized odontoblasts (PoO). There was no staining for p-SMAD2/3 in PA, SA, PO and PoO, although strong staining was localized in DPL. OD was initially moderately positive and then negative for p-SMAD2/3. At PN7, intense staining for TGF-ß1 and CTGF was observed in SA, OD, dental pulp (DP) and predentin respectively. p-SMAD2/3 was strongly expressed in DP and moderately expressed in SA and OD. At PN14 and PN21, both reduced enamel epithelium (REE) and JE showed a strong reaction for TGF-ß1 and CTGF. p-SMAD2/3 was intensely and weakly expressed in REE and JE respectively. These data demonstrate that the expression of CTGF, TGF-ß1 and p-SNAD2/3 is tissue-specific and stage-specific, and indicate a regulatory role for a TGF-ß1-SMAD2/3-CTGF signaling pathway in amelogenesis, dentinogenesis and formation of JE.

Myostatin signaling is up-regulated in female patients with advanced heart failure.

BACKGROUND: Myostatin, a negative regulator of skeletal muscle mass, is up-regulated in the myocardium of heart failure (HF) and increased myostatin is associated with weight loss in animal models with HF. Although there are disparities in pathophysiology and epidemiology between male and female patients with HF, it remains unclear whether there is gender difference in myostatin expression and whether it is associated with weight loss in HF patients. METHODS: Heart tissue samples were collected from patients with advanced heart failure (n=31, female n=5) as well as healthy control donors (n=14, female n=6). Expression levels of myostatin and its related proteins in the heart were evaluated by western blotting analysis. RESULTS: Body mass index was significantly lower in female HF patients than in male counterparts (20.0±4.2 in female vs 25.2±3.8 in male, p=0.04). In female HF patients, both mature myostatin and pSmad2 were significantly up-regulated by 1.9 fold (p=0.05) and 2.5 fold (p<0.01) respectively compared to female donors, while expression of pSmad2 was increased by 2.8 times in male HF patients compared to male healthy subjects, but that of myostatin was not. There was no significant difference in protein expression related to myostatin signaling between male and female patients. CONCLUSION: In this study, myostatin and pSmad2 were significantly up-regulated in the failing heart of female patients, but not male patients, and female patients displayed lower body mass index. Enhanced myostatin signaling in female failing heart may causally contribute to pathogenesis of HF and cardiac cachexia.

Downregulation of HIF1-α, Smad-2, AKT, and Bax gene expression in sodium nitrite-induced lung injury via some antioxidants.

The aim of the current study is to evaluate the efficacy of pretreatment with either l-arginine (L-arg) or Carnosine (Car) and their combination in ameliorating some of the biochemical indices induced in the lung of sodium nitrite (NaNO2 )-intoxicated rats. The results revealed that NaNO2 significantly increased serum tumor necrosis factor-α, C-reactive protein, heat shock proteins-70, vascular endothelial growth factor, and Interleukin 6. Moreover, transforming growth factor-ß, hypoxia-inducible factor, Smad-2, Protein Kinase B (AKT), and Bax were overexpressed, whereas Bcl2 protein was downregulated compared with the normoxic group. The administration of the fore mentioned antioxidants, either alone or in combination, markedly downregulated the previously mentioned inflammatory, apoptotic, as well as the fibrotic markers in lung tissue compared with the NaNO2 -intoxicated rats. The histopathological examination reinforced the previous results. In conclusion, the current data revealed the efficacy of l-arg and Car in ameliorating the pulmonary damage via suppression of the inflammatory markers in response to NaNO2 -intoxication. Interestingly the combination regimen showed the most significant effect.

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.

All-Trans Retinoic Acid supplementation prevents cardiac fibrosis and cytokines induced by Methylglyoxal.

Methylglyoxal (MG), a metabolic intermediate of glycolysis is a precursor for endogeneous production of advanced glycation end-products. The increased production of MG have negative influence over the structure and function of different biomolecules and thus plays an important role in the pathogenesis of diabetic cardiac complications. Retinoic acid (RA), an active metabolite of vitamin A, has a major role in preventing cardiac remodeling and ventricular fibrosis. Hence, the objective of the present study was to determine whether rats administered with all-trans retinoic acid (RA) could attenuate MG induced pathological effects. Wistar rats were divided into 4 groups. Group 1 rats were kept as control; Group 2 rats were administrated with MG (75 mg/kg/day) for 8 weeks. Group 3 rats were given RA (Orally, 1.0 mg/kg/day) along with MG; Group 4 rats received RA alone. Cardiac antioxidant status, induction of fibrosis, AGE receptor (RAGE) and cytokines expression was evaluated in the heart tissues. Administration of MG led to depletion of antioxidant enzymes, induction of fibrosis (p < 0.001), up-regulated expression of RAGE (3.5 fold), TGF-ß (4.4 fold), SMAD2 (3.7 fold), SMAD3 (6.0 fold), IL-6 (4.3 fold) and TNF-α (5.5 fold) in the heart tissues compared to control rats. Moreover, the exogenous administration of MG caused significant (p < 0.001) increase in the circulating CML levels. Whereas, RA treatment prevented the induction of fibrosis and restored the levels of cytokines and RAGE expression. Methylglyoxal-induced fibrosis can lead to pathological effects in the heart tissues. RA attenuates the effects of MG in the heart, suggesting that it can be of added value to usual diabetic therapy.

Triazole RGD antagonist reverts TGFß1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor ß1 (TGFß1) play a central role. When exposed to TGFß1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFß1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvß3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFß1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFß1, and reduces both ALK5/TGFß1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

Osthole inhibits the expressions of collagen I and III through Smad signaling pathway after treatment with TGF-ß1 in mouse cardiac fibroblasts.

BACKGROUND: Osthole, a natural coumarin and bioactive compound isolated from the fruit of Cnidium monnieri (L.) Cusson, was reported to prevent isoprenaline-induced myocardial fibrosis in mice by inhibiting the transforming growth factor-ß1 (TGF-ß1) expression, but the underlying mechanism is still unclear. The aim of this study is to illuminate whether the mechanism of osthole inhibiting collagen I and III expressions is associated with Smad signaling pathway in mouse cardiac fibroblasts (CFs) treated with TGF-ß1. METHODS: The mouse CFs stimulated with TGF-ß1 were cultured and treated with osthole 1.25-5µg/ml for 24h. The expressions of α-SMA, collagen I, collagen III, TGF-ß receptor I (TßRI), Smad2/3, phospho-Smad2/3 (P-Smad2/3), Smad4 and Smad7 were detected by real-time PCR method and western blot method, respectively. RESULTS: After treatment with TGF-ß1 and osthole in CFs, the levels of α-SMA expression and collagen I and III were reduced by osthole treatment. Accordingly, the ratio of collagen I/III had a similar changing trend. Besides, the levels of TßRI, Smad2/3, P-Smad2/3 and Smad4 expressions were decreased, while the level of Smad7 expression was increased after treatment with osthole. CONCLUSION: The present results demonstrated that osthole could inhibit the collagen I and III expressions and their ratio in CFs treated with TGF-ß1 via Smad signaling pathway, which might be one of its anti-fibrotic action mechanisms.

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.

Transforming Growth Factor ß Mediates Drug Resistance by Regulating the Expression of Pyruvate Dehydrogenase Kinase 4 in Colorectal Cancer.

Drug resistance is one of the main causes of colon cancer recurrence. However, our understanding of the underlying mechanisms and availability of therapeutic options remains limited. Here we show that expression of pyruvate dehydrogenase kinase 4 (PDK4) is positively correlated with drug resistance of colon cancer cells and induced by 5-fluorouracil (5-FU) treatment in drug-resistant but not drug-sensitive cells. Knockdown of PDK4 expression sensitizes colon cancer cells to 5-FU or oxaliplatin-induced apoptosis in vitro and increases the effectiveness of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo In addition, we demonstrate for the first time that TGFß mediates drug resistance by regulating PDK4 expression and that 5-FU induces PDK4 expression in a TGFß signaling-dependent manner. Mechanistically, knockdown or inhibition of PDK4 significantly increases the inhibitory effect of 5-FU on expression of the anti-apoptotic factors Bcl-2 and survivin. Importantly, studies of patient samples indicate that expression of PDK4 and phosphorylation of Smad2, an indicator of TGFß pathway activation, show a strong correlation and that both positively associate with chemoresistance in colorectal cancer. These findings indicate that the TGFß/PDK4 signaling axis plays an important role in the response of colorectal cancer to chemotherapy. A major implication of our studies is that inhibition of PDK4 may have considerable therapeutic potential to overcome drug resistance in colorectal cancer patients, which warrants the development of PDK4-specific inhibitors.

The role of TGF-ß1 in osteoarthritis of the temporomandibular joint in two genetic mouse models.

OBJECTIVE: The objective of this study is to elucidate osteoarthritis (OA) progression in the temporomandibular joint (TMJ) in two genetic mouse models by assessing the expression of an identified inflammatory marker associated with OA, viz., Tgf-ß1. This study provides mechanistic insight into disease progression based on the temporal expression of Tgf-ß1 in the TMJ. DESIGN: The two models included the heterozygous chondrodysplasia mutation (cho/+), a Coll11a1 mutation, and the autosomal semidominant disproportionate micromelia mutation (Dmm/+), a Col2a1 mutation. To determine OA status histologically, TMJs from each mutant were fixed, sectioned and stained with Safranin O to identify proteoglycans in condylar cartilage and counterstained with Fast Green. The extent of staining and onset of OA-like changes were quantified using the Modified Mankin scoring system. Using immunofluorescence, selected tissue sections of each genotype were stained for the presence of Tgf-ß1, HtrA1, and p-Smad2. RESULTS: The results revealed Mankin scores of the condylar cartilage of both mutants that are consistent with established histopathological changes of OA. Immunofluorescence indicated increased expression of all three molecular markers and their co-localization within condylar chondrocytes of both mutants. CONCLUSIONS: Elevated Tgf-ß1 expression in mutant condylar cartilage supports the hypothesis that this inflammatory mediator is mechanistically involved in the pathogenesis of TMJ OA. Compared to basal expression in control TMJs, the positive co-localized staining for Tgf-ß1, HtrA1, and p-Smad2 in both mutants demonstrates involvement of these molecules in the degradative pathway of OA. Tgf-ß1 therefore is a potential target for further study for the diagnosis and treatment of TMJ OA.

One week's holiday sun exposure induces expression of photoaging biomarkers.

INTRODUCTION: Skin aging is accompanied by the upregulation of the expression of various matrix metalloproteinases (MMPs). It was shown that exposure to ultraviolet radiation (UVR) may induce skin expression of MMPs and dysregulation of the transforming growth factor beta (TGF-ß)/Smad pathway. The aim of our study was to compare the effects of short holiday UVR exposure and lifetime UVR exposure, on the expression of MMP-8, TGF-ß1, and Smad2 in human skin biopsies. MATERIAL AND METHODS: Skin biopsies were taken from the outer upper arm of 15 elderly people with significant photoaging (mean age 64.1 years) (Group 1) and from 15 healthy young adult volunteers (mean age 24.1 y) who participated in a six-day sun holiday. Biopsies were taken twice: 24 hours before leaving for holiday (Group 2a) and 24 hours after returning (Group 2b). The expression of TGF-ß1, Smad2, and MMP-8 was examined by immunochemistry and measured semiquantitatively by two independent pathologists. RESULTS: The mean expression of TGF-ß1 in dermal fibroblasts and keratinocytes in Group 1 and Group 2b was significantly lower than in Group 2a (0.54% ± 0.44% and 0.48% ± 0.51% vs. 1.48% ± 0.72%, respectively). The percentage of Smad2 (+) cells in Group 1 and Group 2b was lower than in Group 2a (2.13% ± 1.39% and 1.81% ± 1.16% vs. 4.13% ± 1.58%, respectively). The MMP-8 expression in Group 2b was 1.36% ± 0.68% and was significantly higher than in Group 1 (0.34% ± 0.42%) and Group 2a in which the protein was not detected (p < 0.001). CONCLUSIONS: We conclude that the decrease in the expression of TGF-ß1 and Smad2 is a persistent biomarker of skin photoaging, while the increased expression of MMP-8 in keratinocytes can be regarded as a marker of acute sun exposure.

Inhibition of Migration and Invasion by Tet-1 Overexpression in Human Lung Carcinoma H460 Cells.

In the present study, we found that lung cancer cell line (H460 cells) expressing Tet1 showed higher levels of adhesion, and Tet1 inhibited H460 cell proliferation. In addition, these cells showed a significantly reduced ability of collagen degradation and Smad2/3 phosphorylation compared to controls. Furthermore, vimentin was found to be highly expressed in larger metastatic cancer area. Tet1 overexpression was reduced in the epithelial marker E-cadherin. Moreover, Tet1 repressed cancer cell metastasis in nude mice. Collectively, these findings suggest that Tet1 expression plays a critical role in metastasis of lung cancer cells by suppression of invasion and epithelial-mesenchymal transition (EMT).

Identification of high-grade serous ovarian cancer miRNA species associated with survival and drug response in patients receiving neoadjuvant chemotherapy: a retrospective longitudinal analysis using matched tumor biopsies.

BACKGROUND: Neoadjuvant chemotherapy (NACT) has been recognized as a reliable therapeutic strategy in patients with unresectable advanced epithelial ovarian cancer (EOC). The molecular events leading to platinum (Pt) response in NACT settings have hitherto not been explored. In the present work, longitudinal changes of miRNA expression profile were investigated to identify miRNA families with prognostic role in high-grade serous EOC patients who received the NACT regimen. PATIENTS AND METHODS: One hundred sixty-four matched tumor biopsies taken at initial laparoscopic evaluation and at interval-debulking surgery (IDS) after four courses of Pt-based therapy were selected from 82 stage IIIC-IV high-grade serous-EOC patients that were judged unsuitable for complete primary debulking and subjected the NACT protocol. miRNA profiling by microarray, real-time PCR and immuno-histochemical staining for Smad2 phosphorylation (P-Smad2) were used for data analysis. RESULTS: Analysis revealed that 369 miRNAs were differentially expressed in matched biopsies (referred to as DEMs). DEMs were not scattered across the genome, but clustered into families: miR-199, let-7, miR-30, miR-181 and miR-29. Multivariate analysis showed that miR-199a-3p, miR-199a-5p, miR-181a-5p and let-7g-5p associated with overall and progression-free survival (P < 0.05); miR-199a-3p, miR-199a-5p and miR-181a-5p associated with residual tumor volume and Pt-free interval (P < 0.05). Immuno-histochemical staining confirmed an enrichment of P-Smad2, a marker of transforming growth factor-ß activation, in tumors from patients with shorter PFS and OS, and with high levels of expression of miR-181a-5p (P < 0.05). Kaplan-Meier curves plotting concomitant expression of P-Smad2 and miR-181a-5p show significant differences in PFS and OS compared with those depicting the expression of each biomarker alone (P < 0.001). CONCLUSIONS: This study describes several miRNA families with a prognostic role in the NACT setting. It also confirms that concomitant analysis of P-Smad2 and miR-181a-5p in surgical samples may be capable of identifying those ovarian cancer patients with poor outcome and little chance of response to Pt-based NACT.

The downregulation of SnoN expression in human renal proximal tubule epithelial cells under high-glucose conditions is mediated by an increase in Smurf2 expression through TGF-ß1 signaling.

Transforming growth factor (TGF)-ß1 is a profibrotic cytokine that plays a critical role in the progression of diabetic nephropathy (DN). Previous studies have demonstrated that the Smad transcriptional co-repressor, Ski-related novel protein N (SnoN), an antagonizer of TGF-ß1/Smad signaling, is downregulated in the kidneys of diabetic rats; however, the underlying molecular mechanisms remain elusive. In the present study, we demonstrated that the upregulation of Smad ubiquitination regulatory factor-2 (Smurf2), through TGF-ß1/Smad signaling, contributes to the downregulation of SnoN under high-glucose conditions in primary human renal proximal tubule epithelial cells (hRPTECs). The hRPTECs were cultured in high-glucose (30 mmol/l D-glucose) medium in the presence or absence of either the proteasome inhibitor, MG132, or the TGF-ß type I receptor kinase inhibitor, SB-431542. Small interfering RNA (siRNA) was used to silence Smurf2. The expression levels of SnoN, Smurf2, Smad2 and phosphorylated (p-)Smad2 were measured by western blot analysis and RT-qPCR. The protein levels of SnoN were markedly downregulated, while its mRNA levels were increased in the hRPTECs cultured under high-glucose conditions. The protein and mRNA levels of Smurf2 were significantly increased under high-glucose conditions. The knockdown of Smurf2 increased SnoN expression in the hRPTECs cultured in high-glucose medium. Moreover, MG132 partially inhibited SnoN degradation in the hRPTECs under high-glucose conditions and SB-431542 decreased the phosphorylation of Smad2 and the expression of Smurf2 induced under high-glucose conditions. Taken together, the findings of this study demonstrate that the downregulation of SnoN expression in hRPTECs under high-glucose conditions is mediated by the increased expression of Smurf2 through the TGF-ß1/Smad signaling pathway.

Pathobiological mechanisms of peritoneal adhesions: The mesenchymal transition of rat peritoneal mesothelial cells induced by TGF-ß1 and IL-6 requires activation of Erk1/2 and Smad2 linker region phosphorylation.

Peritoneal adhesions, primarily caused by surgical procedures, are the leading cause of pelvic pain, bowel obstruction, and infertility. TGF-ß1 and IL-6 have been found to be elevated in the peritoneal fluid of patients during/after abdominal surgery. However, it remains to be determined whether these cytokines interact and facilitate adhesion formation by promoting mesothelial to mesenchymal transition (MMT). In the present study, isolated rat peritoneal mesothelial cells were treated with TGF-ß1 and/or IL-6 which elicited MMT as determined by morphologic and biochemical techniques. During this transition, cellular morphology changed from that of cobblestone polygonal cells to elongated/spindle-shaped fibroblast-like cells. There was decreased expression of genes characteristic of mesothelial cells, such as E-cadherin, and increased expression of genes characteristic of the myofibroblast phenotype, including α-smooth muscle actin and the EDA form of fibronectin, both of which appear to mediate the transfer of force to the extracellular matrix. Partial characterization of relevant signaling pathways identified Erk1/2 activation, which was enhanced by combined TGF-ß1/IL-6 administration, as a crucial necessary factor in the transition. Erk1/2 activation as well as the phosphorylation of the linker region of Smad2 and MMT could be blocked by the MEK inhibitor, U0126, suggesting that such activation may be a potential pharmaceutical target to prevent MMT. In addition, the phenotypic transition could be prevented by hydrocortisone.