Active TGF-beta Research Articles

Antifibrotic effects of adiponectin include induction of BMP-activated and membrane bound inhibitor (Bambi) in liver cells which antagonizes TGF-beta activity

Objectives: TGF-beta induces activation of hepatic stellate cells and an epithelial mesenchymal transition of hepatocytes, and thereby promotes the progression of liver fibrosis. BMP-activated and membrane bound inhibitor (Bambi) mimics TGF-beta receptor I, but lacks an intracellular kinase domain and interaction of Bambi with TGF-beta receptor II results in a non functional TGF-beta pseudoreceptor. Adiponectin, whose systemic levels are reduced in patients with non-alcoholic fatty liver disease, protects from liver fibrosis, and it was analysed whether this adipokine induces Bambi in human liver cells. Methods: Real-time RT-PCR and immunoblot were performed using primary human hepatocytes, hepatic stellate cells and liver tissues of patients with and without liver steatosis. Human liver tissue for immunoblot analysis and cell isolation was obtained according to the guidelines of the charitable state-controlled foundation Human Tissue & Cell Research (HTCR) with the patient's informed consent. Results: Bambi protein was found reduced in whole liver lysates of human fatty liver compared to non-steatotic liver. Adiponectin induced Bambi in primary human hepatocytes (PHH) and hepatic stellate cells in vitro. Specific antagonists were used to show that the upregulation of Bambi in PHH is driven via NF-kappaB and ERK1/2 signalling pathways. Preincubation with adiponectin lowered phosphorylation of Smad 2 and Smad 3 in hepatocytes upon TGF-beta incubation. Overexpression of Bambi in HepG2 cells significantly impaired TGF-beta mediated induction of connective tissue growth factor (CTGF). Conclusion: Adiponectin impairs TGF-beta activity in hepatocytes partly by induction of Bambi protein. Low levels of Bambi in human fatty liver may contribute to enhanced TGF-beta signalling and progression of fibrosis. The antifibrogenic effects of adiponectin make this adipokine an interesting therapeutic target in non-alcoholic fatty liver disease.

Neuropilin-1 exerts co-receptor function for TGF-beta-1 on the membrane of cancer cells and enhances responses to both latent and active TGF-beta

Neuropilin (Nrp)-1 and Nrp-2 are multifunctional proteins frequently expressed by cancer cells and contribute to tumor progression by mechanisms that are not well understood. They are co-receptors for vascular endothelial growth factor and class 3 semaphorins, but recently we found that Nrp1 also binds latent and active transforming growth factor (TGF)-β1, and activates the latent form latency-associated peptide (LAP)-TGF-β1. Here, we report that Nrp1 has affinity for TGF-β receptors TβRI and TβRII, the signaling TGF-β receptors, as well as TβRIII (betaglycan), as determined in binding assays, pull down assays and confocal microscopy. Nrp1 had a higher affinity for TβRI than TβRII and could form a complex with these receptors. In breast cancer cells, Nrp1 and TβRI cointernalized in the presence of TGF-β1. Nrp1 acted as a TGF-β co-receptor by augmenting canonical Smad2/3 signaling. Importantly, Nrp-positive cancer cells, unlike negative cells, were able to activate latent TGF-β1 and respond. We examined two other membrane proteins that bind LAP-TGF-β, i.e. an RGD-binding integrin (αvβ3) and Glycoprotein A repetitions predominant (CLRRC32). RGD-binding integrins are frequently expressed by cancer cells, and glycoprotein A repetitions predominant is expressed by activated regulatory T cells that appear linked to poor tumor immunity. In vitro, these receptors did not activate LAP-TGF-β1, but subsequent addition of Nrp1 activated the cytokine. Thus, Nrp1 might collaborate with other latent TGF-β receptors in TGF-β capture and activation. We also show that Nrp2 has activities similar to Nrp1. We conclude that Nrp1 is a co-receptor for TGF-β1 and augments responses to latent and active TGF-β. Since TGF-β promotes metastasis this is highly relevant to cancer biology.

TEL-AML1 Affects the Regulation of Cytoskeleton and Causes Alteration In Cellular Adhesive and Migratory Properties In An In Vitro Model of Pre-Leukemia.

AbstractAbstract 3624 Introduction:The t(12;21) chromosome translocation generating TEL-AML1 chimeric fusion gene is a frequent initiating event in childhood leukaemia. Its impact is to generate a clone of covert, clinically silent pre-leukemic B cell progenitors. The leukemia arises only following second, post-natal hit/genetic events occurring years later. Moreover, relapse of leukemia is frequently arising from the pre-leukemic clone. Aimof our study is to investigate how TEL-AML1 expression can sustain this covert condition for many years. In a recent paper we described that the fusion gene rendered the B precursors resistant to the inhibitory activity of TGFbeta. Here we want to inquire into other factors that can explain the positive selection of the pre-leukemic clones over the normal counterpart. In particular, given the importance of the interaction with the microenvironment for survival signals for normal and leukemic stem cells, we question if the fusion gene causes changes in cellular adhesive and migratory properties. Methods:the study was performed by using two different models: i) a TEL-AML1 inducible expression system on the murine pro-B Ba/F3 cell line and ii) murine primary B lymphocytes (pre-BI cells) isolated from fetal liver, stably transduced with the pMIGR1-TEL-AML1-IRES-GFP construct. Gene expression assays were performed by using TaqMan (Applied Biosystems) and PCR Array technologies (SABioscences). Results:The expression of TEL-AML1 in Ba/F3 cell line causes over-expression of genes regulators of the cytoskeleton, specifically involved in cellular movement and in the regulation of actin dynamics. This gene expression alteration results in changes in the cellular morphology and phenotype: the cells acquire long extensions and several molecules involved in cell adhesion and migration are disregulated. Moreover, the TEL-AML1 positive cells present an increased ability to adhere to the ICAM1 substrate, but they also show a significant defect in the chemotactic response to CXCL12 in transwell migration assays in vitro, although the expression and the recycling of CXCR4 receptor are unaffected. This inability is not due to defects to migrate in general, as spontaneous motility is enhanced, but it is associated with a defect in CXCR4 signaling. In particular, CXCL12 calcium flux and ERK phosphorylation were inhibited. Those results have been confirmed in murine PreBI primary cells. Conclusions:in our murine models, TEL-AML1 affects the cytoscheleton regulation and causes alteration in cellular adhesive and migratory properties. We are now investigating how these alterations can give advantages to the pre-leukemic cells in the pathogenesis of TEL-AML1–expressing leukemia. Disclosures:No relevant conflicts of interest to declare.

Angiotensin II increases mRNA levels of all TGF‐β isoforms in quiescent and activated rat hepatic stellate cells

AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF-beta (transforming growth factor-beta). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF-beta actions. Here, we studied the effect of AII on the mRNA levels of TGF-beta isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF-beta1, TGF-beta2 and TGF-beta3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF-beta isoforms, particularly TGF-beta2and TGF-beta3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF-beta protein after AII treatment. The mRNA expression of TGF-beta isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF-beta expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.

Evaluation of the Transforming Growth Factor-β Activity in Normal and Dry Eye Human Tears by CCL-185 Cell Bioassay

To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye. Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay. The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in normal controls (4129.3 +/- 1342.9 pg/mL) (P < 0.05). Among patients with DE, TGF-beta bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-beta in DE tears and 37.6% TGF-beta in normal tears were found to be biologically active. The CCL-185 cell assay was found to be a suitable tool for assessing TGF-beta activity in human tears. Tear TGF-beta bioactivity increases in DE, particularly in Sjögren syndrome, where elevated levels of TGF-beta1 transcripts in the conjunctival epithelium have been previously detected.

Effects of porcine 25 kDa amelogenin and its proteolytic derivatives on bone sialoprotein expression

Amelogenins are hydrophobic proteins that are the major component of developing enamel. Enamel matrix derivative has been used for periodontal regeneration. Bone sialoprotein is an early phenotypic marker of osteoblast differentiation. In this study, we examined the ability of porcine amelogenins to regulate bone sialoprotein transcription. To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene by amelogenins, we conducted northern hybridization, transient transfection analyses and gel mobility shift assays using the osteoblast-like ROS 17/2.8 cells. Amelogenins (100 ng/mL) up-regulated bone sialoprotein mRNA at 3 h, with maximal mRNA expression occurring at 12 h (25 and 20 kDa) and 6 h (13 and 6 kDa). Amelogenins (100 ng/mL, 12 h) increased luciferase activities in pLUC3 (nucleotides -116 to +60), and 6 kDa amelogenin up-regulated pLUC4 (nucleotides -425 to +60) activity. The tyrosine kinase inhibitor inhibited amelogenin-induced luciferase activities, whereas the protein kinase A inhibitor abolished 25 kDa amelogenin-induced bone sialoprotein transcription. The effects of amelogenins were abrogated by 2-bp mutations in the fibroblast growth factor 2 response element (FRE). Gel-shift assays with radiolabeled FRE, homeodomain-protein binding site (HOX) and transforming growth factor-beta1 activation element (TAE) double-strand oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells at 3 h (25 and 13 kDa) and 6 h (20 and 6 kDa). These results demonstrate that porcine 25 kDa amelogenin and its proteolytic derivatives stimulate bone sialoprotein transcription by targeting FRE, HOX and TAE in the bone sialoprotein gene promoter, and that full-length amelogenin and amelogenin cleavage products are able to regulate bone sialoprotein transcription via different signaling pathways.

Transcription of the Transforming Growth Factor β Activating Integrin β8 Subunit Is Regulated by SP3, AP-1, and the p38 Pathway

Integrin alphavbeta8 is a critical regulator of transforming growth factor beta activation in vasculogenesis during development, immune regulation, and endothelial/epithelial-mesenchymal homeostasis. Recent studies have suggested roles for integrin beta8 in the pathogenesis of chronic obstructive pulmonary disease, brain arteriovenous malformations, and select cancers (Araya, J., Cambier, S., Markovics, J. A., Wolters, P., Jablons, D., Hill, A., Finkbeiner, W., Jones, K., Broaddus, V. C., Sheppard, D., Barzcak, A., Xiao, Y., Erle, D. J., and Nishimura, S. L. (2007) J. Clin. Invest. 117, 3551-3562; Su, H., Kim, H., Pawlikowska, L., Kitamura, H., Shen, F., Cambier, S., Markovics, J., Lawton, M. T., Sidney, S., Bollen, A. W., Kwok, P. Y., Reichardt, L., Young, W. L., Yang, G. Y., and Nishimura, S. L. (2010) Am. J. Pathol. 176, 1018-1027; Culhane, A. C., and Quackenbush, J. (2009) Cancer Res. 69, 7480-7485; Cambier, S., Mu, D. Z., O'Connell, D., Boylen, K., Travis, W., Liu, W. H., Broaddus, V. C., and Nishimura, S. L. (2000) Cancer Res. 60, 7084-7093). Here we report the first identification and characterization of the promoter for ITGB8. We show that a SP binding site and a cyclic AMP response element (CRE) in the ITGB8 core promoter are required for its expression and that Sp1, Sp3, and several AP-1 transcription factors form a complex that binds to these sites in a p38-dependent manner. Furthermore, we demonstrate the requirement for Sp3, ATF-2, and p38 for the transcription and protein expression of integrin beta8. Additionally, reduction of SP3 or inhibition of p38 blocks alphavbeta8-mediated transforming growth factor beta activation. These results place integrin beta8 expression and activity under the control of ubiquitous transcription factors in a stress-activated and pro-inflammatory pathway.

Inhibition of NF-κB Signaling Reduces Virus Load and Gammaherpesvirus-Induced Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disorder of unknown etiology. Several studies have demonstrated an association between pulmonary infection with a herpesvirus and IPF. Based on those observations, we have developed a mouse model in which interferon (IFN)gammaR(-/-) mice infected intranasally with murine gammaherpesvirus 68 (MHV68) develop lung fibrosis. We hypothesize that viral load was a critical factor for the development of fibrosis. Because nuclear factor (NF)-kappaB signaling is required to efficiently establish gammaherpesvirus, latency we infected IFNgammaR(-/-) mice with a MHV68 virus that expresses a mutant dominant inhibitor of the NF-kappaB signaling pathway, called IkappaBalphaM. Striking differences were observed at the onset of the chronic infection, which correlated with a decreased virus load in mice infected with MHV68-IkappaBalphaM compared with mice infected with control MHV68 (MHV68-MR). IFNgammaR(-/-) mice infected with MHV68-IkappaBalphaM lacked vasculitis and fibrosis 15 to 120 days post infection. Inhibition of NF-kappaB in MHV68-infected cells of the lungs diminished the expression of the fibrocyte recruiting chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL12, ameliorated macrophage expression of markers of alternative activation, and failed to increase expression of the integrin alphavbeta6, which is implicated in the activation of the profibrotic factor TGF-beta. Thus, the inhibition of NF-kappaB signaling in the infected lung cells of IFNgammaR(-/-) mice reduces virus persistence and ameliorates profibrotic events. Host determinants of latency might therefore represent new therapeutic targets for gammaherpesvirus-associated pulmonary fibrosis.

Smad3 Regulates Rho Signaling via NET1 in the Transforming Growth Factor-β-induced Epithelial-Mesenchymal Transition of Human Retinal Pigment Epithelial Cells

We previously demonstrated that RhoA-dependent signaling regulates transforming growth factor-beta1 (TGF-beta1)-induced cytoskeletal reorganization in the human retinal pigment epithelial cell line ARPE-19. Smad pathways have also been shown to mediate TGF-beta1 activity. Here, we examined what regulates Rho GTPase activity and tested whether Smad signaling cross-talks with Rho pathways during TGF-beta1-induced actin rearrangement. Using small interfering RNAs, we found that NET1, the guanine nucleotide exchange factor of RhoA, is critical for TGF-beta1-induced cytoskeletal reorganization, N-cadherin expression, and RhoA activation. In ARPE-19 cells lacking NET1, TGF-beta1-induced stress fibers and N-cadherin expression were not observed. Interestingly, in dominant-negative Smad3-expressing or constitutively active Smad7 cells, TGF-beta1 failed to induce NET1 mRNA and protein expression. Consistent with these results, both dominant-negative Smad3 and constitutively active Smad7 blocked the cytoplasmic localization of NET1 and inhibited interactions between NET1 and RhoA. Finally, we found that NET1 is a direct gene target of TGF-beta1 via Smad3. Taken together, our results demonstrate that Smad3 regulates RhoA activation and cytoskeletal reorganization by controlling NET1 in TGF-beta1-induced ARPE-19 cells. These data define a new role for Smad3 as a modulator of RhoA activation in the regulation of TGF-beta1-induced epithelial-mesenchymal transitions.

Silibinin attenuates cardiac hypertrophy and fibrosis through blocking EGFR‐dependent signaling

Cardiac hypertrophy is a major determinant of heart failure. The epidermal growth factor receptor (EGFR) plays an important role in cardiac hypertrophy. Since silibinin suppresses EGFR in vitro and in vivo, we hypothesized that silibinin would attenuate cardiac hypertrophy through disrupting EGFR signaling. In this study, we examined this hypothesis using neonatal cardiac myocytes and fibroblasts induced by angiotensin II (Ang II) and animal model by aortic banding (AB) mice. Our data revealed that silibinin obviously blocked cardiac hypertrophic responses induced by pressure overload. Meanwhile, silibinin markedly reduced the increased generation of EGFR. Moreover, these beneficial effects were associated with attenuation of the EGFR-dependent ERK1/2, PI3K/Akt signaling cascade. We further demonstrated silibinin decreased inflammation and fibrosis by blocking the activation of NF-kappaB and TGF-beta1/Smad signaling pathways in vitro and in vivo. Our results indicate that silibinin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through blocking EGFR activity and EGFR-dependent different intracellular signaling pathways.

Inhibition of fatty acid oxidation activates transforming growth factor-beta in cerebrospinal fluid and decreases spontaneous motor activity

We have previously reported that transforming growth factor (TGF)-beta in the cerebrospinal fluid (CSF) is involved in the mechanism underlying the regulation of spontaneous motor activity (SMA) by the central nervous system after exercise. However, it remained unclear what physiological condition triggers the activation of TGF-beta. We hypothesized that the shortage of energy derived from fatty acid (FA) oxidation observed in the early phase of exercise activated TGF-beta in the CSF. To test this hypothesis, we investigated whether mercaptoacetate (MA), an inhibitor of FA oxidation, could induce an activation of TGF-beta in the CSF and a decrease in SMA. Intraperitoneal (i.p.) administration of MA activated TGF-beta in CSF in rats and depressed SMA; 2-deoxyglucose, an inhibitor of carbohydrate oxidation, on the other hand, depressed SMA but failed to activate CSF TGF-beta. Intracisternal administration of anti-TGF-beta antibody abolished the depressive effect of MA on SMA. We also found that the depression of SMA and the activation of TGF-beta in the CSF by i.p. MA administration were eliminated by vagotomy. Our data suggest that TGF-beta in the CSF is activated by the inhibition of FA oxidation via the vagus nerve and that this subsequently induces depression of SMA.

The Sensing of Environmental Stimuli by Follicular Dendritic Cells Promotes Immunoglobulin A Generation in the Gut

In the Peyer's patches (PPs), germinal centers (GCs) are chronically induced by bacteria and are the major sites for generation of gut immunoglobulin A (IgA) immune responses. Whether follicular dendritic cells (FDCs) within the GCs directly contribute to the IgA production in PPs is unknown. We showed here that direct stimulation of FDCs by bacterial products and retinoic acid synergistically enhanced the expression of the chemokine CXCL13, the survival factor BAFF, and molecules that facilitate the secretion and activation of the cytokine TGF-beta1. A reduced production of these molecules by PP FDCs associated with deficiencies in the Toll-like receptor pathway or vitamin A resulted in decreased numbers of GC B cells and defective generation of IgA(+) B cells within PP GCs. Our data indicate that PP FDCs are conditioned by environmental stimuli to express key factors for B cell migration, survival, and preferential generation of IgA in gut.

Thy-1-Integrin αvβ5 Interactions Inhibit Lung Fibroblast Contraction-induced Latent Transforming Growth Factor-β1 Activation and Myofibroblast Differentiation

Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin alpha(v)beta(5)-dependent latent transforming growth factor (TGF)-beta1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin alpha(v)beta(5), both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin alpha(v)beta(5) interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin alpha(v)beta(5)-dependent latent TGF-beta1 activation and TGF-beta1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-alpha(v)beta(5) interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-beta1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin alpha(v)beta(5) interactions inhibit contraction-induced latent TGF-beta1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-beta1 with integrin alpha(v)beta(5). Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.

TGF-β Receptor Deletion in the Renal Collecting System Exacerbates Fibrosis

TGF-beta plays a key role in upregulating matrix production in injury-induced renal fibrosis, but how TGF-beta signaling in distinct compartments of the kidney, such as specific segments of the nephron, affects the response to injury is unknown. In this study, we determined the role of TGF-beta signaling both in development of the renal collecting system and in response to injury by selectively deleting the TGF-beta type II receptor in mice at the initiation of ureteric bud development. These mice developed normally but demonstrated a paradoxic increase in fibrosis associated with enhanced levels of active TGF-beta after unilateral ureteral obstruction. Consistent with this observation, TGF-beta type II receptor deletion in cultured collecting duct cells resulted in excessive integrin alphavbeta6-dependent TGF-beta activation that increased collagen synthesis in co-cultured renal interstitial fibroblasts. These results suggest that inhibiting TGF-beta receptor-mediated function in collecting ducts may exacerbate renal fibrosis by enhancing paracrine TGF-beta signaling between epithelial and interstitial cells.

Expression of Transforming Growth Factor-β1 Limits Renal Ischemia-Reperfusion Injury

Renal ischemia-reperfusion injury (IRI) largely contributes to kidney transplant dysfunction and acute kidney injury, but its pathogenesis is not fully understood. In this study, the role of transforming growth factor (TGF)-beta1 in renal IRI is investigated using TGF-beta1 deficient mice. Human renal tubular epithelial cells (TEC) line (HK-2) was used as an in vitro model, and cell apoptosis was determined by flow cytometric analysis. Renal IRI was induced in mice by clamping renal vein and artery for 45 min at 32 degrees C. Here, we showed that in cultures of HK-2 cells, TGF-beta1 expression was up-regulated by tumor necrosis factor (TNF)-alpha. Neutralization of TGF-beta1 activity increased both spontaneous and TNF-alpha-mediated apoptosis, and knockdown of TGF-beta1 expression increased the sensitivity of cell apoptosis to TNF-alpha. In a mouse model of renal IRI, a deficiency in TGF-beta1 expression increased the severity of renal injury, as indicated by more severe renal tubular damage, higher levels of serum creatinine or blood urea nitrogen in TGF-beta1 deficient mice as compared with those in wild-type controls. Further experiments showed that the antiapoptosis of TGF-beta1 correlated with up-regulation of Bcl-2 in kidney cells. Expression of TGF-beta1 in TECs, potentially induced by proinflammatory TNF-alpha, renders TECs resistance to cell death. In mice, TGF-beta1 deficiency results in more prone to IRI. These data imply that TGF-beta1 may act as a feedback survival factor in the resistance to kidney injury and maintenance of epithelium homeostasis.

Krüppel-like Factor 4 Promotes Differentiation by Transforming Growth Factor-β Receptor-mediated Smad and p38 MAPK Signaling in Vascular Smooth Muscle Cells

KLF4 (Krüppel-like factor 4) has been implicated in vascular smooth muscle cell (VSMC) differentiation induced by transforming growth factor beta (TGF-beta). However, the role of KLF4 and mechanism of KLF4 actions in regulating TGF-beta signaling in VSMCs remain unclear. In this study, we showed that TGF-beta1 inhibited cell cycle progression and induced differentiation in cultured rat VSMCs. This activity of TGF-beta1 was accompanied by up-regulation of KLF4, with concomitant increase in TbetaRI (TGF-beta type I receptor) expression. KLF4 was found to transduce TGF-beta1 signals via phosphorylation-mediated activation of Smad2, Smad3, and p38 MAPK. The activation of both pathways, in turn, increased the phosphorylation of KLF4, which enabled the formation of KLF4-Smad2 complex in response to TGF-beta1. Chromatin immunoprecipitation studies and oligonucleotide pull-down assays showed the direct binding of KLF4 to the KLF4-binding sites 2 and 3 of the TbetaRI promoter and the recruitment of Smad2 to the Smad-responsive region. Formation of a stable KLF4-Smad2 complex in the promoter's Smad-responsive region mediated cooperative TbetaRI promoter transcription in response to TGF-beta1. These results suggest that KLF4-dependent regulation of Smad and p38 MAPK signaling via TbetaRI requires prior phosphorylation of KLF4 through Smad and p38 MAPK pathways. This study demonstrates a novel mechanism by which TGF-beta1 regulates VSMC differentiation.

Induction of Bystander Response in Human Glioma Cells using High-Energy Electrons: A Role for TGF-β1

We examined bystander cell death produced in T98G cells by exposure to irradiated cell conditioned medium (ICCM) produced by high-energy 20 MeV electrons at a dose rate of 10 Gy min(-1) and doses up to 20 Gy. ICCM induced a bystander response in T98G glioma cells, reducing recipient cell survival by more than 25% below controls at 5 and 10 Gy. Higher doses increased survival to near control levels. ICCM was analyzed for the presence of transforming growth factor alpha (TGF-alpha) and transforming growth factor beta1 (TGF-beta1). Monoclonal antibodies for TGF-alpha (mAb TGF-alpha) and TGF-beta1 (mAb TGF-beta1) were added to the ICCM to neutralize any potential effect of the cytokines. The results indicate that TGF-alpha was not present in the ICCM and addition of mAb TGF-alpha to the ICCM had no effect on bystander cell survival. No active TGF-beta1 was present in the ICCM; however, addition of mAb TGF-beta1 completely abolished bystander death of reporter cells at all doses. These results indicate that bystander cell death can be induced in T98G glioma if a large enough radiation stress is applied and that TGF-beta1 plays a downstream role in this response.

Cyclosporin A-induced gingival overgrowth is not associated with myofibroblast transdifferentiation

Cyclosporin A (CyA) induces gingival overgrowth via its stimulatory effects on expression of transforming growth factor-beta1 (TGF-beta1) and collagen. It is not known whether CyA has a direct effect on gingival fibroblasts or induces its effect indirectly via stimulation of myofibroblast transdifferentiation. The present study was undertaken to examine the in vivo and in vitro effect of CyA on myofibroblast transdifferentiation. Rats were treated for 60 days with a daily subcutaneous injection of CyA, and the gingival overgrowth tissue was analyzed by immunohistochemistry. In vitro, fibroblasts from normal gingiva (NG) were cultured in the presence of different concentrations of CyA, and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction and western blot. Although CyA treatment stimulated TGF-beta1 expression by NG fibroblasts, it lacked to induce expression and production of isoform alpha of smooth muscle actin (alpha-SMA), the specific myofibroblast marker. The expression levels of connective tissue growth factor (CTGF), which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-beta1 activation, were unaffected by CyA. Our results demonstrate that CyA-induced gingival overgrowth is not associated with activation of myofibroblast transdifferentiation, since CyA is not capable to increase CTGF expression.

An integrative view on the role of TGF-β in the progressive tubular deletion associated with chronic kidney disease

Transforming growth factor-beta (TGF-beta) is a cytokine known to participate in several processes related to the development of chronic kidney disease (CKD), including tubular degeneration. This is thought to occur mainly through apoptosis and epithelial-to-mesenchymal transition (EMT) of tubule epithelial cells, which give rise to a reduction of the tubular compartment and a scarring-like, fibrotic healing process of the interstitial compartment. In vivo blockade of TGF-beta action has been shown to reduce CKD-associated tubular damage. However, a direct action of TGF-beta on tubule cells is controversial as the underlying mechanism. On the one hand, TGF-beta is known to induce EMT of tubular cells, although its incidence in vivo can hardly explain the extent of the damage. On the other hand, a few publications have reported that TGF-beta induces a mild degree of apoptosis in cultured tubular cells. This most likely reflects the consequence of the cell-cycle arrest rather than a direct pro-apoptotic effect of TGF-beta. The implications of these observations are analyzed in the pathological context, where normal tubular cells do not normally proliferate, but they might divide for repair purposes. Furthermore, renal fibrosis, a TGF-beta-mediated event, is integrated as a potential, indirect effect contributing to tubule deletion.

Co-regulated expression of matrix metalloproteinase-2 and transforming growth factor-β in melanoma development and progression

Previous studies have found that matrix metalloproteinase-2 (MMP-2) and transforming growth factor-beta (TGF-beta) can be considered as biomarkers and indices of disease progression in several human cancers. In this study, we investigated the plasma levels of MMP-2 and TGF-beta and their correlation in 49 primary cutaneous melanoma and 10 metastatic melanoma. Plasma MMP-2 and TGF-beta levels in patients with primary melanoma were significantly higher than those of healthy controls. These protein levels were significantly higher in patients with metastatic melanoma. A positive correlation between plasma levels of MMP-2 and TGF-beta in melanoma patients supports the hypothesis that TGF-beta triggers the release of MMP-2. The immunohistochemistry analysis shows that MMP-2 and TGF-beta were highly expressed in tumor tissues as well as in matched plasma samples. This finding suggests that these proteins are released from tumor cells. Overall, our data indicate that MMP-2 and TGF-beta may represent novel diagnostic markers and therapeutic targets in melanoma and the determination of their concentration could be a useful diagnostic and prognostic indicator. TGF-beta, leading the tissue invasion mediated by MMP-2, is a strong promoter of tumor progression. Therefore, reducing or blocking the activity of TGF-beta may represent a promising target in therapeutic strategies for limiting the growth of melanoma.