mRNA Expression Levels Of TGF-β1 Research Articles

Protective effect of adiponectin on paraquat-induced pulmonary fibrosis in mice

Pulmonary fibrosis (PF) is the most common complication of paraquat (PQ) toxicity, which lacks an effective treatment. This study aimed to investigate whether exogenous administration of globular adiponectin (APN) isoform provided protection against PQ-induced PF, and examined the possible mechanisms underlying these effects. Eighty BALB/C mice were randomly divided into control, PQ, low-dose APN, and high-dose APN groups. Human lung WI-38 fibroblasts were similarly divided into control, PQ, and APN groups and examined at 24 h, 48 h, and 72 h after PQ exposure. Hematoxylin and eosin (HE) and Masson trichrome staining were used to compare the histopathologic changes in the mouse lung tissues. Western blot and real-time quantitative-PCR (RT-PCR) were used to measure the protein and mRNA expression of transforming growth factor β1 (TGF-β1) in mice lung tissues and α-SMA, type III collagen, and NF-κB p65 in lung fibroblasts. Dihydroethidium (DHE) was used to detect intracellular superoxide anion (O2 −) in fibroblasts. APN administration significantly ameliorated PQ-mediated fibrosis histologically and reduced the protein and mRNA expression levels of TGF-β1 in mouse lung tissues in a dose-dependent manner (P<0.05). When fibroblasts were pretreated with APN, the expression of aα-SMA and NF-κB p65 were down-regulated, and O2 − decreased. Expression of the anti-inflammatory factor IL-1Ra was upregulated following PQ exposure (P<0.05). This study revealed which may have occurred through suppression of NF-κB dependent inflammatory and TGF-β1 mediated fibrotic events.

Quercetin promotes the osteogenic differentiation of rat mesenchymal stem cells via mitogen-activated protein kinase signaling.

The aim of the present study was to investigate the effects of quercetin on the mitogen-activated protein kinase (MAPK) signaling pathway in the osteogenic differentiation of rat mesenchymal stem cells (MSCs). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and an alkaline phosphatase (ALP) assay were used to determine the effects of quercetin (concentrations of 0.1, 1 and 10 µmol/l) on the proliferation and differentiation of MSCs and the expression of ALP, respectively. In addition, through the introduction of inhibitors of p38 MAPK, extracellular signal-regulated kinase (ERK)1/2 and c-Jun NH2-terminal kinase (JNK), the effects of quercetin on the proteins, ALP, collagen type I (COL I) and bone γ-carboxyglutamate protein (BGP), which are indicators of osteogenic differentiation, were investigated. Immunoblotting was performed to determine the phosphorylation levels of p38 MAPK, ERK1/2 and JNK, while fluorescent quantitative polymerase chain reaction was used to determine the mRNA expression levels of transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP)-2 and core binding factor (CBF)α1. At all the concentrations tested, the concentrations of 10, 1 and 0.1 µmol/l quercetin were shown to promote the differentiation of MSCs and the expression of ALP, in which the concentration of 10 µmol/l was optimal. When compared with the control group, the phosphorylation levels of p38 MAPK, ERK1/2 and JNK, the protein expression levels of ALP, COL I and BGP, and the mNRA expression levels of TGF-β1, BMP-2 and Cbfα1 were increased in the quercetin-treated group. However, with the introduction of inhibitors, the levels of phosphorylated p38 MAPK, ERK1/2 and JNK, and the protein expression levels of ALP, COL I and BGP decreased. Furthermore, the mRNA expression levels of TGF-β1, BMP-2 and CBFα1 decreased in the quercetin + SP600125 (inhibitor of JNK) and quercetin + PD98059 (inhibitor of ERK1/2) groups. Therefore, quercetin was demonstrated to promote the osteogenic differentiation of MSCs by activating the MAPK signaling pathway. The ERK1/2 and JNK signaling pathways regulate the expression of TGF-β1, BMP-2 and CBFα1. Thus, activation of the ERK1/2 and JNK signaling pathways may play a leading role in the quercetin-promoted osteogenic proliferation and differentiation of MSCs.

Effect of dietary arginine on the immune response and gene expression in head kidney and spleen following infection of Jian carp with Aeromonas hydrophila

This study was conducted to test the hypothesis that elevated dietary arginine enhances immunity of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed graded levels of dietary arginine for 9 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila. Head kidney and spleen weights, as well as erythrocyte and leukocyte counts were significantly influenced by dietary arginine levels. A similar trend was also observed for hemagglutination titre, serum lysozyme activity, IgM concentration, C3 and C4 content. The highest survival rates following A. hydrophila infection were obtained in fish fed the diets containing arginine at 16.1–21.9 g/kg diet. Phagocytic activity of leukocytes was significantly enhanced by dietary arginine supplementation. In contrast, acid phosphatase activity significantly decreased with dietary arginine levels. Dietary arginine levels did not have a significant effect on the total iron-binding capacity. Gene expression of TNF-α and TGF-β in head kidney significantly increased with dietary arginine levels up to 21.9 g/kg diet, and decreased thereafter. Fish fed the basal diet exhibited the highest IL-10 mRNA expression level. Gene expression of IL-1β and TOR increased with dietary arginine addition, reaching a plateau at 18.5 and 21.9 g arginine/kg diet, respectively. In spleen, higher IL-1β and TNF-α gene expressions were obtained in fish fed the diets containing 24.5 g arginine/kg diet than in fish fed the other dietary treatments. TGF-β mRNA expression levels were significantly lower in fish fed the diets containing ≤21.9 g arginine/kg diet. IL-10 and TOR mRNA expression levels were lower in fish fed 16.1 g arginine/kg diet, while 4E-BP mRNA expression levels increased with dietary arginine levels up to 12.7 g/kg diet and decreased thereafter. Our results indicate that arginine has beneficial effects on regulating mRNA expression of inflammatory cytokines, as well as TOR and 4E-BP and improving humoral and cellular immunity, therefore enhancing disease resistance of fish.

Liver X receptor β delays transformation of radial glial cells into astrocytes during mouse cerebral cortical development

Radial glial (RG) cells serve as stem cells to produce new born neurons and scaffolds for neuronal migration during corticogenesis. After neurogenesis and migration are completed, most RG cells transform into astrocytes. However, the mechanisms that determine how RG cells are transformed into astrocytes are not well understood. Using nestin as a specific marker for both RG cells and astrocytes, we found that loss of LXRβ caused a reduction in the level of RG fibers and increase in the astrocytes. At the same time, we showed that the level of brain lipid–binding protein (BLBP), a RG-specific protein, was lower in the LXRβ knockout (LXRβ−/−) mice than in the wild type (WT) littermates from E18.5 to P14, a time period when most of RG cells are transformed into astrocytes. However, loss of LXRβ induced significant increase in the number of GFAP labeled astrocytes in the cerebral cortex. An increase of the transformation of RG cells into astrocytes in LXRβ−/− mice was further confirmed by the increased percentage of BLBP and GFAP double stained cells in the total BLBP positive cells of the Layer I and Layers V–VI. TGF-β1 and Smad4 are thought to be involved in the transformation of RG cells into astrocytes. The expression levels of TGF-β1mRNA and Smad4 mRNA were significantly higher in the cerebral cortex of LXRβ−/− mice than that in the WT littermates at P2 and P7, but by P10 and P14, mRNA levels had normalized and no differences were observed between WT and LXRβ−/− mice. Taken together, our findings suggest that loss of LXRβ accelerates the transformation of RG cells into astrocytes and that this acceleration may be correlated to higher levels TGF-β1 and Smad4 in the cerebral cortex between P2 and P7.

Effect of transforming growth factor-β1 on acute lung injury caused by paraquat

In China, and other Asian countries, numerous patients have succumbed to pulmonary fibrosis induced by paquarat poisoning, but the early pathogenesis remains unclear. In this study the effect of cytokine transforming growth factor (TGF)-β1 was observed in early acute paraquat poisoning and examined the mechanism by which paraquat caused early acute lung injury. It was discovered that the rat serum TGF-β1 levels in the paraquat groups were significant higher than that in the control group (P<0.05) and the rat pulmonary TGF-β1 mRNA expression levels were also higher than that in the control group (P<0.05). Histological examination indicated that the rat lung tissue was broad and congested, and had been infiltrated by inflammatory cells. Masson's trichrome staining for collagen showed that the lung tissue appeared fibrotic following paraquat poisoning. Ultramicrostructure observation found that macrophages, red blood cells, lymphocytes and granulocytes infiltrated the alveolar space and there were cytolysosomes in the macrophages. The shape of the type II alveolar epithelial cell nuclei were irregular with karyopyknosis. The heterochromatin migrated to the cell edge and lamellar body vacuolization was also observed. Type I alveolar epithelial cells shrank. In conclusion, the effect of cytokine TGF-β1 on paraquat-induced acute lung tissue injury may be important.

Protective role of naringenin against doxorubicin-induced cardiotoxicity in a rat model: histopathology and mRNA expression profile studies.

Doxorubicin (DOX) is one of the most effective and widely used chemotherapeutic agents. Its efficacy has been proven in various malignancies alone and combined with other cytocidal agents. However, the clinical usefulness of DOX is restricted by the risk of developing congestive heart failure. Formation of free radicals and oxidative stress during DOX treatment may result in adverse side effects. Naringenin (NAR) is one of the potential bioflavonoids with excellent antioxidant properties and free-radical scavenging capability. This study was designed to evaluate whether NAR exerts a protective role against DOX-induced cardiotoxicity in rats. Male Wistar rats were administered DOX (3 mg/kg) intravenously for 10 consecutive weeks along with oral treatment with NAR (50 mg/kg/day). DOX-induced cardiac toxicity was characterized by the marked biochemical alterations of lactic acid dehydrogenase (LDH), troponin T, malondialdehyde (MDA), reduced cardiac enzymatic activities (SOD, GPx, CAT) and histopathological observations. Administration of NAR to DOX-challenged rats ameliorated alterations in biochemical markers. Indeed, DOX increased the mRNA expression levels of TGF-β1, TNF-α, IL-6, and IL-10 compared with the control group. However, cotreatment with NAR attenuated the mRNA expression levels of these inflammatory markers and improved histological cardiac damage and cardiac functions. Thus, supplementation of NAR may be beneficial in reducing DOX toxicity.

Protective Effects of Bu-Shen-Huo-Xue Formula against 5/6 Nephrectomy-Induced Chronic Renal Failure in Rats.

Chronic renal failure (CRF) is a serious disease related to increasing incidence and prevalence as well as decline in quality of life. Bu-Shen-Huo-Xue formula (BSHX), one of traditional herbal formulations, has been clinically employed to treat CRF for decades, but the mechanisms involved have not been investigated. In the present study, we investigated the effects of BSHX on some closely related parameters in 5/6 nephrectomy CRF rats. Rats with CRF were divided into five groups, namely, one control group, one enalapril group, and three BSHX treatment groups (0.25, 0.5, and 1 g/kg·d). The rats subjected to sham operation were used as a normal control. After eight weeks of treatment, BSHX significantly decreased the levels of Scr and BUN, downregulated the mRNA expression levels of TGF-β 1, CTGF, NF-κB, TNF-α, and OPN, upregulated the mRNA expression of PPARγ, and reduced in situ expression of fibronectin and laminins. Histological findings also showed significant amelioration of the damaged renal tissue. BSHX protects 5/6 nephrectomy rats against chronic renal failure probably via regulating the expression of TNF-α, NF-κB, TGF-β 1, CTGF, PPARγ, OPN, fibronectin, and laminins and is useful for therapy of CRF.

Expression of TGF-β1, SNAI1 and MMP-9 is associated with lymph node metastasis in papillary thyroid carcinoma

TGF-β1, SNAI1 and MMP-9 are implicated in tumor invasion and metastasis. The purpose of this study was to examine TGF-β1, SNAI1 and MMP-9 expression in papillary thyroid carcinoma (PTC), and to assess association of TGF-β1, SNAI1 and MMP-9 expression with several clinicopathological indicators of PTC. TGF-β1, SNAI1 and MMP-9 protein expression in 83 PTCs and their matched normal thyroid specimens were analyzed using immunohistochemistry. The mRNA expression levels of TGF-β1, SNAI1 and MMP-9 in 12 fresh PTC specimens with lymph node metastasis (LNM), 12 fresh PTC specimens without LNM and their matched normal thyroid specimens were assessed by real-time RT-PCR. The results showed that the mRNA and protein expression levels of TGF-β1, SNAI1 and MMP-9 were significantly higher in PTCs than in their matched normal thyroid tissues. There were not significant differences in TGF-β1, SNAI1 and MMP-9 protein expression relative to age, gender, tumor size and TNM stage, except for MMP-9 whose protein expression correlated with tumor size. However, high mRNA and protein expression levels of TGF-β1, SNAI1 and MMP-9 were significantly correlated with LNM. Furthermore, TGF-β1, SNAI1 and MMP-9 protein expression were significantly correlated with one another. Concomitant expression of any two or all of the three molecules had stronger correlation with LNM than did each alone. Collectively, the present results indicate that immunohistochemical and real-time RT-PCR evaluation of TGF-β1, SNAI1 and MMP-9 expression in PTC may be useful to predict the risk of LNM in PTC patients.

Effect of the protease inhibitor MG132 on the transforming growth factor-β/Smad signaling pathway in HSC-T6 cells

The activation of hepatic stellate cells (HSCs) and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-β (TGFβ)/Smad pathway is involved in the proliferation and collagen synthesis of HSCs. This study aimed to examine the effect of the protease inhibitor MG132 on the signaling pathway of TGFβ/Smad in HSC-T6 cells and seek a novel therapeutic approach for liver fibrosis. The HSC-T6 cells were treated with MG132 at different concentrations (0-10 μmol/L). Cell proliferation was detected by MTT method. The mRNA and protein expression levels of TGFβ1, Smad3 and Smad7 were determined in HSC-T6 cells by real-time PCR and Western blotting, respectively, after treatment with MG132 at different concentrations (1, 2, 3 μmol/L) or RPMI1640 alone (serving as control). The results showed that MG132 could inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the IC(50) of MG132 was 6.84 μmol/L. After treatment with MG132 at 1, 2 or 3 μmol/L for 24 h, the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased (P<0.05), but the Smad7 mRNA expression had no significant change (P>0.05). There was also a significant decrease in the protein expression level of TGF-β1 and Smad3 (P<0.05). However, the expression of Smad7 protein was substantially increased when compared with the control group (P<0.05). It was concluded that the inhibition of TGFβ/Smad pathway in HSC-T6 cells by MG132 can reduce the production of profibrosis factors (TGFβ1, Smad3) and promote the expression of anti-fibrosis factor (Smad7), suggesting that MG132 may become a potential therapeutic alternative for liver fibrosis.

Hypoxia Stimulates the EMT of Gastric Cancer Cells through Autocrine TGFβ Signaling

Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor β1 (TGFβ1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFβ1 and TGFβR was evaluated by real time RT-PCR. The TGFβ1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFβ1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFβR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFβR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFβ1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFβ/TGFβR signaling.

Suppressive Effects of Oral Administration of Heat-Killed &lt;i&gt;Lactobacillus acidophilus&lt;/i&gt; on T Helper-17 Immune Responses in a Bovine β-Lactoglobulin-Sensitized Mice Model

The present study was planned to explore the effect of Lactobacillus (L.) acidophilus on the T helper-17 (Th17) immune response in a mouse model of β-lactoglobulin (β-lg) allergy. Bovine β-lg sensitised BALB/c mice were orally administered with different doses of heat-killed L. acidophilus (low, 5×10(7) colony forming unit (CFU); medium, 5×10(8) CFU; high, 5×10(9) CFU) in 200 µL of phosphate buffered saline (PBS) three times a week, starting from 1 week before β-lg sensitisation for 4 weeks. After the allergen challenge, the numbers of blood eosinophils and neutrophils were examined by light microscope; the levels of cytokine (interleukin (IL)-12, IL-4, tumor growth factor (TGF)-β, IL-10, IL-6 and IL-17A), total immunoglobulin E (IgE) and β-lg-specific IgE contents in the serum were measured with enzyme-linked immunosorbent assay (ELISA); The mRNA expression levels of TGF-β, IL-17A,CD25, Foxp3, retinoic acid-related orphan receptor γt (RORγt) and IL-10 were analyzed using real-time polymerase chain reaction (PCR). The results showed that oral administration of L. acidophilus suppressed hypersensitivity responses, attenuated the numbers of inflammatory cells and inhibited IgE production. We found up-regulation of TGF-β and down-regulation of IL-17A in the serum of L. acidophilus-treated group, along with IL-6 levels was significantly decreased than that of the allergy group (p<0.05). Moreover, the mRNA expression levels of CD25, forkhead box P3 and TGF-β were significantly higher in the spleen of L. acidophilus-treated group, while the mRNA expression levels of IL-17A, RORγt and IL-10 were significantly lower than that in the allergy group (p<0.05). In conclusion, the suppression of major allergic symptoms by oral administration of L.acidophilus was probably due to improve the regulatory T (Treg)/Th17 balance and inhibit the IL-6 production.

Melatonin reduces X-ray radiation-induced lung injury in mice by modulating oxidative stress and cytokine expression

Purpose: The modification of radiation-induced lung injuries by melatonin was studied by measuring changes in oxidative stress, cytokine expression and histopathology in the lung tissue of mice following irradiation.Materials and methods: The thoraces of C57BL/6 mice were exposed to a single X-ray radiation dose of 12 Gy with or without 200 mg/kg of melatonin pretreatment. The level and localization of transforming growth factor (TGF)-β1 protein were measured using an enzyme-linked immunosorbent assay (ELISA) method and immunohistochemical staining, respectively. Real-time quantitative polymerase chain reaction (PCR) was established to evaluate the relative mRNA expression levels of TGF-β1, tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6.Results: Malondialdehyde (MDA) levels increased after irradiation and then significantly reduced (1.9-fold) under melatonin treatment. Changes in superoxide dismutase (SOD) and catalase activities, as well as glutathione (GSH) levels, after irradiation were significantly reduced by melatonin, including a notable 5.4-fold difference in catalase activity. We observed increased expression of TGF-β1 and TNF-α after irradiation and a significant reduction in the elevation of their expression by melatonin treatment. Furthermore, irradiation-induced histopathologic alterations were obviously abated in the melatonin-pretreated mice.Conclusions: The present results suggest that melatonin reduces radiation-induced lung injury via a significant reduction of oxidative stress and of the production of cytokines, such as TGF-β1 and TNF-α, the production of which increased following lung irradiation.

Mouse Skeletal Muscle Fiber-Type-Specific Macroautophagy and Muscle Wasting Are Regulated by a Fyn/STAT3/Vps34 Signaling Pathway

Skeletal muscle atrophy induced by aging (sarcopenia), inactivity, and prolonged fasting states (starvation) is predominantly restricted to glycolytic type II muscle fibers and typical spares oxidative type I fibers. However, the mechanisms accounting for muscle fiber-type specificity of atrophy have remained enigmatic. In the current study, although the Fyn tyrosine kinase activated the mTORC1 signaling complex, it also induced marked atrophy of glycolytic fibers with relatively less effect on oxidative muscle fibers. This was due to inhibition of macroautophagy via an mTORC1-independent but STAT3-dependent reduction in Vps34 protein levels and decreased Vps34/p150/Beclin1/Atg14 complex 1. Physiologically, in the fed state endogenous Fyn kinase activity was increased in glycolytic but not oxidative skeletal muscle. In parallel, Y705-STAT3 phosphorylation increased with decreased Vps34 protein levels. Moreover, fed/starved regulation of Y705-STAT3 phosphorylation and Vps34 protein levels was prevented in skeletal muscle of Fyn null mice. These data demonstrate a Fyn/STAT3/Vps34 pathway that is responsible for fiber-type-specific regulation of macroautophagy and skeletal muscle atrophy.

Renal Protection of In Vivo Administration of Tempol in Streptozotocin-Induced Diabetic Rats

The present study was carried out to investigate the protective effects of tempol on renal function and the underlying mechanism in streptozotocin-induced diabetic rats. The diabetic rats were randomly divided into the model group (without tempol) and tempol group (1 mM tempol in drinking water for 6 weeks). Nondiabetic rats were served as the Control group. The mRNA expression of canonical transient receptor potential 6 (TRPC6), transforming growth factor (TGF)-β1, and type IV collagen (Col IV) were examined. The malondialdehyde (MDA) level, activities of superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in renal tissues were measured to assess redox status in kidneys. We found that tempol significantly reduced 24-h urine output and urine albuminuria excretion in the diabetic rats. Compared with the model group, the concentration of MDA was significantly lower in the tempol group. In addition, diabetes decreased activities of SOD and GSH-Px and these responses were prevented by tempol treatment. Moreover, in diabetic rats, the mRNA expression levels of TGF-β1 and Col IV were upregulated. TRPC6 mRNA expression level was down-regulated in diabetic kidneys. However, all of these diabetic effects were significantly suppressed by tempol treatment. These results suggest that chronic treatment of diabetic rats with tempol can protect kidneys, possibly by reducing expression of TGF-β1, Col IV, and upregulating TRPC6 expression level.

Association of Transforming Growth Factor-β1 Gene Polymorphisms With Genetic Susceptibility to Acute Myocardial Infarction

IntroductionTransforming growth factor beta 1 (TGF-β1) gene plays an important role in acute myocardial infarction (AMI); however, little is known about the relation of variations within the gene and risk of cardiovascular diseases. In this study, the authors evaluated the influence of TGF-β1 polymorphisms on the onset and progression of AMI in Iranian patients comparing with healthy individuals. MethodsGenomic DNA and peripheral blood mononuclear cells of 900 enrolled patients with AMI and 900 control subjects were extracted. The −509 C/T, 868T/C, 913G/C and 11929C/T TGF-β1 polymorphisms were detected. The messenger RNA (mRNA) expression and serum levels of TGF-β1 were analyzed by real-time reverse-transcriptase polymerase chain reaction and ELISA, respectively. ResultsThe frequency of “T” allele in −509 C/T, “C” allele in 868T/C, “C” allele in 913G/C and “T” allele in 11929C/T polymorphisms were significantly higher in the patients than control subjects (P < 0.001). There were significant differences in circulating levels of TGF-β1 in the patients than in control subjects (P < 0.001). These concentrations are associated with its gene polymorphism. The mRNA expression levels of TGF-β1 were significantly higher in the patient serums compared with controls (P < 0.001). ConclusionsOur results confirmed the association between the TGF-β1 polymorphisms and risk of AMI, which suggest that genetic polymorphisms in TGF-β1 might be helpful for determining susceptibility to AMI in Iranian patients. There are also significant relationship between serum TGF-β1 and occurrence of AMI. In addition, susceptibility to AMI might be related to TGF-β1 gene expression, which affects its serum levels.

Lysophosphatidylcholine enhances the suppressive function of human naturally occurring regulatory T cells through TGF-β production

Naturally occurring CD4 +CD25 + regulatory T cells (nTregs) play a pivotal role in the maintenance of self-tolerance and immune homeostasis. To gain insight into the mechanism of action of nTregs in pathological and physiological immune responses, it is important to analyze bioactive molecules that modulate the maintenance and function of nTregs. From a library of bioactive lipids, we obtained lysophosphatidylcholine (LPC) as a molecule that enhanced the Foxp3 expression and suppressive function of human nTregs significantly in comparison with those of DMSO-treated nTregs (control). The expression levels of TGF-β1 mRNA and protein in LPC-treated nTregs were significantly higher than those in control nTregs. After treatment with anti-TGF-β1 antibody, the increases in Foxp3 expression and the suppressive properties of LPC-treated nTregs returned to the levels observed in control nTregs. These findings indicate that LPC enhances Foxp3 expression and the suppressive function of nTregs through TGF-β1 produced by nTregs themselves. Experimental knockdown of G2A and GPR4 showed that this LPC-induced TGF-β1 expression in nTregs was due to G2A signaling, and did not involve GPR4. Moreover, JNK was a major contributor to LPC-induced TGF-β1 expression in nTregs, although LPC activated MAPKs including ERK1/2, p38 MAPK, and JNK via G2A. LPC is a bioactive lysolipid highly abundant in the circulation. Therefore, LPC may contribute to the maintenance and function of human nTregs in vivo.

Reduction of transforming growth factor-β1 expression in leukemia and its possible role in leukemia development

The expression of transforming growth factor-β1 (TGF-β1) in leukemic cells and sera from patients with leukemia and its possible role in leukemia development were studied. TGF-β1 levels in culture supernatants from leukemic cells were significantly lower than those from normal bone marrow mononuclear cells. Serum TGF-β1 levels in leukemic patients were significantly lower compared with healthy controls, but returned to normal in patients achieving complete remission, and decreased when patients relapsed. TGF-β1 mRNA expression levels were significantly higher in normal bone marrow mononuclear cells but lower in leukemic cells compared with normal CD34 + cells. After transfection of the TGF-β1 gene to HL-60 cells, cell apoptosis was detected. Moreover, by flow cytometry analysis, cells arrested in G1 phase were 62% for TGF-β1 transfected cells and 44% for controls. Transfection of exogenous TGF-β1 gene inhibited HL60 cells xenograft growth in nude mice, and prolonged survival of tumor-bearing mice compared with the controls. Decreased endogenous TGF-β1 expression in leukemia cells may be involved in leukemia development, Transfection of exogenous TGF-B1 gene to HL60 can inhibit the proliferation of the cells and induce cell apoptosis by down regulating bcl-2, hTERT (human telomerase reverse transcriptase) and c-myc expression.

Abstract 5266: TGF-β1 modulates the homeostasis between MMPs and MMPs inhibitors through p38 MAPK and ERK½ in highly invasive human breast cancer cells

Abstract Matrix metalloproteinases (MMPs) and their specific inhibitors, known as tissue inhibitors of MMPs (TIMPs) and the membrane-associated MMP inhibitor (RECK), are involved in several steps of the metastatic process. Previous results from our laboratory showed a positive correlation between high mRNA expression levels of MMPs and their inhibitors upon breast cancer progression, both in cellular models and in tumor tissue samples. However, the molecular mechanisms underlying this coordinate regulation of MMPs, TIMPs and RECK expression remain unknown. Since transforming growth factor beta 1 (TGF-β1) has been related to mechanisms which regulate the expression of some MMPs and MMPs inhibitors in different models, we investigated whether this multifunctional cytokine could have a role as a common regulator of MMPs, TIMPs and RECK in the human breast cancer model. The mRNA expression levels of TGF-β isoforms and their receptors were examined, by qRT-PCR, in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. We demonstrated that TGF-β1, TGF-β2 and TβRII are significantly overexpressed in more aggressive mammary cell lines. Furthermore, using the Spearman correlation test, we showed a significantly positive correlation between the mRNA expression levels of TGF-β1 and MMP-2, MMP-14, TIMP-1, TIMP-3 and RECK. In addition, upon treatment with TGF-β1, the MDA-MB-231 breast cancer cell line presented significantly increased mRNA expression levels of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 was also able to induce the protein expression levels of MMP-2, MMP-9, MMP-14 and TIMP-2. On the other hand, RECK protein expression was significantly downregulated in TGF-β1-treated-MDA-MB-231 cells. In addition to the classical TGF-β-induced signal transduction by Smads, it is well known that this cytokine also signals in a Smad-independent manner, by induction of other pathways. Here, we analyzed the involvement of p38 MAPK and ERK½, two well established Smad-independent pathways, in the proposed mechanism of coordinate regulation of MMPs, TIMPs and RECK by TGF-β1 in the breast cancer model. Firstly, we confirmed that TGF-β1 is able to induce phosphorylation of p38MAPK and ERK½ proteins in the MDA-MB-231 cell line. Inhibition of ERK½ pathway led to increased levels of the MMPs inhibitors (TIMP-2 and RECK) mRNA in MDA-MB-231 cells treated (or not) with TGF-β1. In contrast, cells treated with a pharmacological p38MAPK inhibitor showed blockage of TGF-β1-induced mRNA expression of MMPs (MMP-2, MMP-9 and MMP-14) and MMPs inhibitors (TIMP-2 and RECK). Altogether, our results suggest that the balance between p38MAPK and ERK½ activities mediates the role of TGF-β1 as an important homeostasis regulator between MMPs and MMPs inhibitors in the MDA-MB231 breast cancer cell line. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5266. doi:10.1158/1538-7445.AM2011-5266

The study of effects of pirfenidone on the pulmonary fibrosis induced by paraquat in mice

To study the curative effects of pirfenidone (PF) on pulmonary fibrosis induced by paraquat (PQ) in mice and to provide the theoretical basis for clinical treatment. Ninety adult healthy male ICR mice were randomly divided into six groups: control group, PQ group, 2 mg/kg Dexamethasone group, 25 mg/kg PF group, 50 mg/kg PF group and 100 mg/kg PF group, there were 15 mice in each group. The corresponding volume of normal saline was given to the each mouse in control group according to the weight, after 2 h 0.1% CMC was given to the each mouse of control group one time by intragastric administration, then the CMC was administrated at regular time until sacrifice. All mice for other 5 groups were exposed to 100 mg/kg PQ by intragastric administration. At 2 h after exposure to PQ, 0.02 ml/10 g dexamethasone and 25, 50, 100 mg/kg PF were given to mice for dexamethasone group and for 3 PF groups by intragastric administration each day for 49 days, respectively. The lung coefficient was calculated and pathological changes of lung tissue were observed by HE staining for each mouse. The hydroxyproline (HYP) level in lung tissue was measured for each mouse. The mRNA level of and the protein level of TGF-β(1) in lung tissue for each mouse were determined, and the protein level of TGF-β(1) in the bronchus-alveolus lavage fluid (BALF) of each mouse was detected. The survival rates on the 3rd day in PQ group, 3 PF groups and dexamethasone group were 53.33%, 46.67%, 73.33%, 86.67% and 80%, respectively. The survival rates on the 3rd day in dexamethasone group, 50 mg/kg and 100 mg/kg PF groups were significantly higher than those of PQ group and 25 mg/kg PF group (P < 0.05). The lung coefficients of 3 PF groups were significantly lower than that of the PQ group (P < 0.05). The lung tissue HYP levels of dexamethasone group and 3 PF groups were 50.95 ± 11.65, 44.52 ± 9.48, 43.27 ± 6.01 and 40.82 ± 5.90 mg/g respectively, which were significantly lower than that (74.27 ± 3.68) of PQ group (P < 0.01). The TGF-β(1) protein levels of BALF in dexamethasone group, 50 and 100 mg/kg PF groups were 22.03 ± 7.27, 27.75 ± 5.84 and 21.31 ± 6.82 ng/ml respectively, which were significantly lower than that (52.52 ± 15.51) ng/ml of PQ group (P < 0.01) The expression level of TGF-β(1) mRNA in 100 mg/kg PF group decreased significantly, as compared with PQ group (P < 0.01). PF could reduce the collagen deposition and pulmonary fibrosis induced by PQ in mice lungs.

Systemic Immune Tuning in Renal Cell Carcinoma

Renal cell carcinoma (RCC) can inhibit protective immunity by induction of immunosuppressive cells that produce inhibitory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β. If this immunosuppression influences response to kinase inhibitors such as sorafenib is not known. Therefore, we asked for the prognostic influence of cells with immunosuppressive properties in peripheral blood (pB) in a cohort of metastatic clear cell renal cell carcinoma (mRCC) patients uniformly receiving sorafenib treatment. IL-10 and TGF-β mRNA levels, regulatory T-cell (Treg) counts, and frequencies of IL-10/TGF-β producing mononuclear cell subsets were determined in pB from 46 patients with mRCC before receiving sorafenib treatment. Relationship between established clinical and laboratory prognostic factors and outcome were examined by univariate and multivariate Cox regression analysis. IL-10 and TGF-β1 mRNA levels, and frequencies of CD4(+)CD25high/CD3(+) and CD4(+)CD25highFoxP3(+)/CD3(+)Treg cells were significantly higher in mRCC patients compared with healthy individuals. Monocytes were suggested as main producers of IL-10 and TGF-β. In a multivariate analysis low ECOG score and-surprisingly-high TGF-β1 mRNA levels were independently associated with favorable progression-free survival (P=0.005 and P=0.003, respectively) and overall survival (P=0.001 and P=0.039, respectively). In conclusion, mRCC is associated with an immunosuppressive phenotype in peripheral blood. The positive prognostic influence of high TGF-β1 mRNA expression levels may reflect immune promoting functions of TGF-β in combined activity with inflammatory cytokines.