TGF-β Neutralizing Research Articles

Abstract 633: Inhibition of TGFβ as a strategy to convert the irradiated tumor into in situ individualized vaccine

Abstract Accumulating data support the concept that ionizing radiation therapy (RT) has the potential to convert the tumor into an in situ, individualized vaccine; however this potential is rarely realized by RT alone. Transforming growth factor β (TGFβ) is an immunosuppressive cytokine that is activated by RT and inhibits the antigen-presenting function of dendritic cells and the differentiation of effector CD8+ T cells. Here we tested the hypothesis that TGFβ hinders the ability of RT to promote anti-tumor immunity. Development of tumor-specific immunity was examined in two pre-clinical models of metastatic breast cancer and analyzed in patients with metastatic breast cancer treated with local radiotherapy and the TGFβ-neutralizing antibody Fresolimumab. Mice bearing established 4T1 and TSA mouse mammary carcinomas treated with pan-isoform specific TGFβ neutralizing antibody, 1D11, showed significantly improved control of the irradiated tumor and non-irradiated metastases, but no effect in the absence of RT. Notably, whole tumor transcriptional analysis demonstrated the selective upregulation of genes associated with immune-mediated rejection only in tumors of mice treated with RT+TGFβ blockade. Mice treated with RT+TGFβ blockade exhibited cross-priming of CD8+ T cells producing IFNγ in response to three tumor-specific antigens in tumor-draining lymph nodes, which was not evident for single modality treatment. Likewise, HLA-A2.1+ metastatic breast cancer patients (n=8) enrolled in NCT01401062 trial of local RT and fresolimumab were examined for CD8+ T cells specific for the tumor antigen survivin by tetramer staining. Three patients developed increased frequencies of survivin-specific CD8+ T cells in the blood during treatment, while two patients negative at baseline became positive. Analysis of the immune infiltrate in mouse tumors showed a significant increase in CD4+ and CD8+ T cells only in mice treated with the combination of RT+TGFβ blockade. Depletion of CD4+ or CD8+ T cells abrogated the therapeutic benefit of RT+TGFβ blockade. These data identify TGFβ as a master inhibitor of the ability of RT to generate an in situ tumor vaccine, which supports testing inhibition of TGFβ during radiotherapy to promote therapeutically effective anti-tumor immunity. Supported by DOD BCRP Multi-Team Award BC100481. Citation Format: Claire I. Vanpouille-Box, Julie M. Diamond, Jiri Zavadil, James Babb, Dörthe Schaue, Mary Helen Barcellos-Hoff, William H. McBride, Silvia C. Formenti, Sandra Demaria. Inhibition of TGFβ as a strategy to convert the irradiated tumor into in situ individualized vaccine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 633. doi:10.1158/1538-7445.AM2014-633

Neutralizing murine TGFβR2 promotes a differentiated tumor cell phenotype and inhibits pancreatic cancer metastasis.

Elevated levels of TGFβ are a negative prognostic indicator for patients diagnosed with pancreatic cancer; as a result, the TGFβ pathway is an attractive target for therapy. However, clinical application of pharmacologic inhibition of TGFβ remains challenging because TGFβ has tumor suppressor functions in many epithelial malignancies, including pancreatic cancer. In fact, direct neutralization of TGFβ promotes tumor progression of genetic murine models of pancreatic cancer. Here, we report that neutralizing the activity of murine TGFβ receptor 2 using a monoclonal antibody (2G8) has potent antimetastatic activity in orthotopic human tumor xenografts, syngeneic tumors, and a genetic model of pancreatic cancer. 2G8 reduced activated fibroblasts, collagen deposition, microvessel density, and vascular function. These stromal-specific changes resulted in tumor cell epithelial differentiation and a potent reduction in metastases. We conclude that TGFβ signaling within stromal cells participates directly in tumor cell phenotype and pancreatic cancer progression. Thus, strategies that inhibit TGFβ-dependent effector functions of stromal cells could be efficacious for the therapy of pancreatic tumors. Cancer Res; 74(18); 4996-5007. ©2014 AACR.

Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p<0.05; >10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.

Gli1 activation and protection against hepatic encephalopathy is suppressed by circulating transforming growth factor β1 in mice

Hepatic encephalopathy (HE) is a neurologic disorder that develops during liver failure. Few studies exist investigating systemic-central signalling during HE outside of inflammatory signalling. The transcription factor Gli1, which can be modulated by hedgehog signalling or transforming growth factor β1 (TGFβ1) signalling, has been shown to be protective in various neuropathies. We measured Gli1 expression in brain tissues from mice and evaluated how circulating TGFβ1 and canonical hedgehog signalling regulate its activation. Mice were injected with azoxymethane (AOM) to induce liver failure and HE in the presence of Gli1 vivo-morpholinos, the hedgehog inhibitor cyclopamine, Smoothened vivo-morpholinos, a Smoothened agonist, or TGFβ-neutralizing antibodies. Molecular analyses were used to assess Gli1, hedgehog signalling, and TGFβ1 signalling in the liver and brain of AOM mice and HE patients. Gli1 expression was increased in brains of AOM mice and in HE patients. Intra-cortical infusion of Gli1 vivo-morpholinos exacerbated the neurologic deficits of AOM mice. Measures to modulate hedgehog signalling had no effect on HE neurological decline. Levels of TGFβ1 increased in the liver and serum of mice following AOM administration. TGFβ neutralizing antibodies slowed neurologic decline following AOM administration without significantly affecting liver damage. TGFβ1 inhibited Gli1 expression via a SMAD3-dependent mechanism. Conversely, inhibiting TGFβ1 increased Gli1 expression. Cortical activation of Gli1 protects mice from induction of HE. TGFβ1 suppresses Gli1 in neurons via SMAD3 and promotes the neurologic decline. Strategies to activate Gli1 or inhibit TGFβ1 signalling might be developed to treat patients with HE.

Critical Role for CD39+ Tregs in Humans and Non-Human Primates in Peripheral Tolerance to SALSAAs.

Self Antigens Left Sequestered After Activation [SALSAAs] represent a class of autoantigens that are transiently made available to the immune system during immunologic activation of tissues, and subsequently are rendered inaccessible following repair and return to a resting state. Since injury/repair processes are common events within tissues, the immune response to SALSAAs must remain under strict control, to avoid a potentially deadly spiral of fibro-obliterative pathologies. Guided by reports of specific self antigens that have been found to be targeted during cardiac allograft vasculopathy & obliterative bronchiolitis, we used a trans-vivo DTH assay(tvDTH) -based, “tolerance-breaking” approach, to identify the immunodominant SALSAAs of mammalian hosts. In normal PBMC, TGFβ neutralization, or removal of CD39+ or CD25+ Tregs, revealed a remarkably strong footpad swelling response to: a) components of the “pro-inflammatory matrix”, such as collagen type V (Col V), and its homologue Col XI; b) cytoskeletal proteins like Vimentin and kα1-tubulin; and c) HSP65. In contrast, no response to ubiquitous collagens I,II & IV, or to cardiac myosin or myelin basic protein was revealed. Strong tvDTH responses to SALSAAs were obtained in normal human and NHP controls, and to a lesser extent, in rats and mice, by removal of CD39+ or CD25+ Tregs, by drugs blocking CD39 ATPase activity or adenosine receptor A2b, or by mAb neutralization of TGFβ1 or IL-35. Neutralization of IL-17, or addback of CD39+ T cells restored low responses to SALSAAs. Conclusion: Since only primed/ memory T effector cells can mediate a tvDTH response to antigen, the strong response to SALSAAs revealed in normal human & NHP subjects by removal of CD39+ Tregs implies that T cells primed to these specific self antigens were not deleted in the thymus. Peripheral tolerance therefore requires homeostasis between SALSAA-specific Th17 effector and CD39+ Treg populations. Imbalance between Th17 effector and CD39+ Treg populations, both donor and host-derived, may account for autoimmunity to SALSAAs post-transplant, associated with primary graft dysfunction and chronic rejection. It may also be the cause of native organ failure, as in Col V-driven coronary atherosclerosis and idiopathic pulmonary fibrosis. DISCLOSURE:Wilkes, D.: Other, Immune Works, Founder and CEO of company.

Platelets provoke distinct dynamics of immune responses by differentially regulating CD4+ T‐cell proliferation

Platelets regulate responses of type 1 T helper (Th1), Th17, and regulatory T (Treg) cells. However, little is known about how platelets influence the dynamics of CD4(+) T-cell responses. To investigate the dynamics of platelet-regulated CD4(+) T-cell activation and cross-talk and their underlying mechanisms. Human CD3/CD28-challenged CD4(+) T cells were cultured without or with autologous platelets. Th1, Th17, and Treg responses were monitored during 5days. Platelets simultaneously enhanced activation of Th1, Th17, and Treg cells within 48-h coculture. Thereafter, platelets remained augmentative for Treg but turned suppressive for Th1/Th17 responses. Without platelets, FoxP3 blockade inhibited Treg activation, which subsequently enhanced Th1 activation. In platelet-T-cell cocultures, however, FoxP3 blockade had no effect on Treg or Th1 activation. Neutralization of platelet-derived transforming growth factor β, but not Treg-derived interleukin-10, enhanced Th1 activation. These data suggest that Treg cells have limited impact on, while platelets are the primary regulator for Th1 suppression during the second phase of coculture. Combining carboxyfluorescein succinimidyl ester and FoxP3 staining, platelets were found to enhance Treg response by promoting cell proliferation of FoxP3(+) T cells and to induce the suppression phrase of Th1 responses by inhibiting FoxP3(-) T-cell proliferation. The latter was markedly attenuated by TGFβ neutralization. Platelets constantly promote Treg cell response but exert a biphasic regulation on Th1/Th17 activation, namely a transient enhancement followed by a secondary suppression. The distinct regulations are achieved by transforming growth factor β-mediated selective inhibition of FoxP3(-) T-cell proliferation. This represents a novel mechanism of platelet-regulated CD4(+) effector cell responses.

Functional Effects of TGF-β1 on Mesenchymal Stem Cell Mobilization in Cockroach Allergen–Induced Asthma

Mesenchymal stem cells (MSCs) have been suggested to participate in immune regulation and airway repair/remodeling. TGF-β1 is critical in the recruitment of stem/progenitor cells for tissue repair, remodeling, and cell differentiation. In this study, we sought to investigate the role of TGF-β1 in MSC migration in allergic asthma. We examined nestin expression (a marker for MSCs) and TGF-β1 signaling activation in airways in cockroach allergen extract (CRE)-induced mouse models. Compared with control mice, there were increased nestin(+) cells in airways and higher levels of active TGF-β1 in serum and p-Smad2/3 expression in lungs of CRE-treated mice. Increased activation of TGF-β1 signaling was also found in CRE-treated MSCs. We then assessed MSC migration induced by conditioned medium from CRE-challenged human epithelium in air/liquid interface culture in Transwell assays. MSC migration was stimulated by epithelial-conditioned medium, but was significantly inhibited by either TGF-β1-neutralizing Ab or TβR1 inhibitor. Intriguingly, increased migration of MSCs from blood and bone marrow to the airway was also observed after systemic injection of GFP(+) MSCs and from bone marrow of Nes-GFP mice following CRE challenge. Furthermore, TGF-β1-neutralizing Ab inhibited the CRE-induced MSC recruitment, but promoted airway inflammation. Finally, we investigated the role of MSCs in modulating CRE-induced T cell response and found that MSCs significantly inhibited CRE-induced inflammatory cytokine secretion (IL-4, IL-13, IL-17, and IFN-γ) by CD4(+) T cells. These results suggest that TGF-β1 may be a key promigratory factor in recruiting MSCs to the airways in mouse models of asthma.

Abstract 511: Emilin1 Controls Myogenic Response of Resistance Arteries by Modulation of TRPC6 Through the TGFß/ALK5/ADAM17/EGFR Pathway

Emilin1 (E1), a protein of the extracellular matrix expressed in the cardiovascular system, regulates TGFβ bioavailability through proteolysis of the proTGFβ, that releases the growth factor. E1 null mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 null phenotype results from a developmental defect or lack of a homeostatic role exerted during the adult life. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (SMCs) of adult mice, by the use of floxed E1 and CreER T2 [a tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. TAM induced Cre activity selectively in SMCs, as shown in mice with the Rosa26R mutation. When E1 fl/fl mice carrying the Smmhc-CreER T2 (Smmhc-CreER T2 E1 fl/fl ) were given TAM, E1 protein completely disappeared and blood pressure significantly increased, as compared to E1 fl/fl . While no structural changes of arteries were detected, we found that mesenteric arteries of Smmhc-CreER T2 E1 fl/fl displayed an increased myogenic response to pressure as compared to E1 fl/fl . Since the hypertensive phenotype of Smmhc-CreER T2 E1 fl/fl mice was rescued by a treatment with a TGFβ neutralizing antibody, we investigated the molecular mechanism in SMCs that, downstream to this pathway, could lead to an increased Ca 2+ -mediated contractility. In particular, also the increased myogenic response was rescued by exposure of resistance arteries to a TGFβ neutralizing antibody and, more important, by the inhibition of TGFβ receptor ALK5, obtained by in vivo vessels transfection with an adenovirus carrying a dominant negative ALK5 mutant. Moreover, we found that the increased TGFβ signaling, by phosphorylating the ADAM17/EGFR cascade, converged on overactivation of the Transient receptor potential cation channel C6 (TRPC6), rescued by in vivo vessels transfection with an adenovirus carrying a dominant negative TRPC6 mutant. Our results demonstrate that E1 exerts a homeostatic control of myogenic response by regulating the TRPC6 Ca 2+ channel, through a modulation of the TGFβ/ALK5/ADAM17/EGFR signaling pathway in SMCs.

Contextual regulation of pancreatic cancer stem cell phenotype and radioresistance by pancreatic stellate cells

Background and purposeProgression of pancreatic ductal adenocarcinoma (PDAC) is promoted by desmoplasia induced by pancreatic stellate cells (PSC). Contributory to this progression is epithelial mesenchymal transition (EMT), which shares many characteristics with the cancer stem cell (CSC) hypothesis. We investigated the role of these processes on the radioresponse and tumorigenicity of pancreatic cancer cells. Materials and methodsWe used an in vitro sphere model and in vivo xenograft model to examine the role of PSC in EMT and CSC processes. ResultsWe demonstrated that PSC enhanced the CSC phenotype and radioresistance of pancreatic cancer cells. Furthermore, the expression of several EMT and CSC markers supported enhanced processes in our models and that translated into remarkable in vivo tumorigenicity. Multi-dose TGFβ neutralizing antibody inhibited the EMT and CSC processes, sensitized cells to radiation and reduced in vivo tumorigenicity. A proteomic screen identified multiple novel factors that were regulated by PSC in pancreatic cells. ConclusionThese results are critical in highlighting the role of PSC in tumor progression and radioresistance by manipulating the EMT and CSC processes. TGFβ and the novel factors identified are important targets for better therapeutic outcome in response to PSC mediated mechanisms.

Medical therapy of stricturing Crohn’s disease: what the gut can learn from other organs - a systematic review

Crohn’s disease (CD) is a chronic remitting and relapsing disease. Fibrostenosing complications such as intestinal strictures, stenosis and ultimately obstruction are some of its most common long-term complications. Despite recent advances in the pathophysiological understanding of CD and a significant improvement of anti-inflammatory therapeutics, medical therapy for stricturing CD is still inadequate. No specific anti-fibrotic therapy exists and the incidence rate of strictures has essentially remained unchanged. Therefore, the current therapy of established fibrotic strictures comprises mainly endoscopic dilation as well as surgical approaches. However, these treatment options are associated with major complications as well as high recurrence rates. Thus, a specific anti-fibrotic therapy for CD is urgently needed. Importantly, there is now a growing body of evidence for prevention as well as effective medical treatment of fibrotic diseases of other organs such as the skin, lung, kidney and liver. In face of the similarity of molecular mechanisms of fibrogenesis across these organs, translation of therapeutic approaches from other fibrotic diseases to the intestine appears to be a promising treatment strategy. In particular transforming growth factor beta (TGF-β) neutralization, selective tyrosine kinase inhibitors, blockade of components of the renin-angiotensin system, IL-13 inhibitors and mammalian target of rapamycin (mTOR) inhibitors have emerged as potential drug candidates for anti-fibrotic therapy and may retard progression or even reverse established intestinal fibrosis. However, major challenges have to be overcome in the translation of novel anti-fibrotics into intestinal fibrosis therapy, such as the development of appropriate biomarkers that predict the development and accurately monitor therapeutic responses. Future clinical studies are a prerequisite to evaluate the optimal timing for anti-fibrotic treatment approaches, to elucidate the best routes of application, and to evaluate the potential of drug candidates to reach the ultimate goal: the prevention or reversal of established fibrosis and strictures in CD patients.

Mesenchymal Stem Cells Recruited by Active TGFβ Contribute to Osteogenic Vascular Calcification

Vascular calcification is an actively regulated process that culminates in organized extracellular matrix mineral deposition by osteoblast-like cells. The origins of the osteoblastic cells involved in this process and the underlying mechanisms remain to be defined. We previously revealed that active transforming growth factor (TGFβ) released from the injured arteries mobilizes mesenchymal stem cells (MSCs) to the blood stream and recruits the cells to the injured vessels for neointima formation. In this study, we used a low-density lipoprotein receptor (LDLR)-deficient mouse model (ldlr(-/-)), which develop progressive arterial calcification after having fed high-fat western diets (HFD), to examine whether TGFβ is involved in the mobilization of MSCs during vascular calcification. Nestin(+)/Sca1(+) cells were recruited to the diseased aorta at earlier time points, and osteocalcin(+) osteoblasts and the aortic calcification were seen at later time point in these mice. Importantly, we generated parabiotic pairs with shared blood circulation by crossing ldlr(-/-)mice fed HFD with transgenic mice, in which all the MSC-derived cells were fluorescently labeled. The labeled cells were detected not only in the peripheral blood but also in the arterial lesions in ldlr(-/-) mouse partners, and these blood circulation-originated cells gave rise to Ocn(+) osteoblastic cells at the arterial lesions. Both active TGFβ1 levels and MSCs in circulating blood were upregulated at the same time points when these cells appeared at the aortic tissue. Further, conditioned medium prepared by incubating the aortae from ldlr(-/-)mice fed HFD stimulated the migration of MSCs in the ex vivo transwell assays, and either TGFβ neutralizing antibody or the inhibitor of TGFβ Receptor I kinase (TβRI) antagonized this effect. Importantly, treatment of the mice with TβRI inhibitor blocked elevated blood MSC numbers and their recruitment to the arterial lesions. These findings suggest that TGFβ-recruited MSCs to the diseased vasculature contribute to the development of osteogenic vascular calcification.

Neural Stem/Progenitor Cells Induce Conversion of Encephalitogenic T Cells into CD4+-CD25+- FOXP3+Regulatory T cells

An immune role of neural stem/progenitor cells (NSPCs) has been proposed in many recent studies; however much still remains to be elucidated. In the current investigation, we report that NSPCs possess the ability to convert encephalitogenic T cells into CD4(+)-CD25(+)-FOXP3(+) regulatory T cells (T(reg)). Encephalitogenic and nonencephalitogenic T cells isolated from sham and Japanese encephalitis virus (JEV) infected animals were co-cultured with mouse NSPCs. Post co-culture, significant increase in the number of T(regs) was observed from encephalitogenic T cells co-cultured with NSPCs. This increased conversion was found to be dependent on direct contact between T cells and NSPCs. Neutralization of TGF-β and IFN-γ in NSPC cultures abrogated this increased conversion of encephalitogenic T cells into T(regs). Flow cytometric, quantitative RT-PCR, and immunoblot analysis of both T cells and NSPCs revealed surface and intracellular changes post co-culture. Co-stimulatory molecules (B7) and ICAM-1 were increased on NSPCs post co-culture; levels of TGFβ, IFNγ, and TGFβR1 were also increased in NSPCs. This study provides a basic insight into the interaction between CNS-infiltrating encephalitogenic T cells and NSPCs during viral encephalitis. Conversion of encephalitogenic T cells into CD4(+)-CD25(+)-FOXP3(+) T(regs) through interaction with NSPCs indicates an attempt in regulation of excessive inflammation in the CNS.

Transforming growth factor-β1 in the microenvironment of ischemia reperfusion-injured kidney enhances the chemotaxis of mesenchymal stem cells to stromal cell-derived factor-1 through upregulation of surface chemokine (C-X-C motif) receptor 4

Acute renal failure is one of the most common complications observed in hospitals. Although extensive studies have been carried out to search for therapeutic treatments, no effective cure has been established. In recent years, stem cell therapy for tissue engineering and repair has become a key area of study. Bone marrow mesenchymal stem cells (MSCs) have been demonstrated to exhibit a reparative role in ischemia reperfusion-injured kidney tissue, and the stromal cell-derived factor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis has been found to play an important role in the migration and homing of MSCs. In the present study, transforming growth factor-β1 (TGF-β1) in the homogenate of the acute ischemia reperfusion-injured renal tissue was found to markedly increase the CXCR4 surface expression of MSCs, which contributes to the migration of MSCs to SDF-1. Neutralization of TGF-β1 inhibited the migration in an antibody concentration-dependent manner, through downregulation of CXCR4 localized to the membrane. These observations suggest a potential mechanism for MSC migration to the kidney which may provide a possible therapeutic target for curing acute renal failure.

TMEPAI/PMEPA1 enhances tumorigenic activities in lung cancer cells

TMEPAI/PMEPA1 is a transmembrane protein that was originally identified as a prostatic RNA, the synthesis of which is induced by testosterone or its derivatives. We have recently identified TMEPAI as a direct target gene of transforming growth factor-β (TGF-β)/Smad signaling that participates in negative feedback control of the duration and intensity of TGF-β/Smad signaling. TMEPAI is constitutively and highly expressed in many types of cancer and is associated with poor prognosis. Here, we report that TMEPAI is highly expressed in the lung adenocarcinoma cell lines Calu3, NCI-H23, and RERF-LC-KJ. Expression of TMEPAI in these cancer cells was significantly suppressed by a TGF-β receptor kinase antagonist, SB208, and by TGF-β neutralizing antibodies. These results suggest that constitutive expression of TMEPAI in these cancer cells depends on autocrine TGF-β stimulation. Knockdown of TMEPAI in Calu3 and NCI-H23 cells enhanced levels of Smad2 phosphorylation and significantly suppressed cell proliferation in the presence of TGF-β, indicating that highly expressed TMEPAI suppresses levels of Smad phosphorylation in these cancer cells and reduces the growth inhibitory effects of TGF-β/Smad signaling. Furthermore, knockdown of TMEPAI in Calu3 and NCI-H23 cells suppressed sphere formation in vitro and tumor formation in s.c. tissues and in lungs after tail vein injection in NOD-SCID mice in vivo. Together, these experiments indicate that TMEPAI promotes tumorigenic activities in lung cancer cells.

The role of SIRT6 in the differentiation of vascular smooth muscle cells in response to cyclic strain

Vascular smooth muscle cells (VSMCs) may switch their phenotype between a quiescent contractile phenotype and a synthetic phenotype in response to cyclic strain, and this switch may contribute to hypertension, atherosclerosis, and restenosis. SIRT 6 is a member of the sirtuin family, and plays an important role in different cell processes, including differentiation. We hypothesized that cyclic strain modulates the differentiation of VSMCs via a transforming growth factor-β1 (TGF-β1)-Smad-SIRT6 pathway. VSMCs were subjected to cyclic strain using a Flexercell strain unit. It was demonstrated that the strain stimulated the secretion of TGF-β1 into the supernatant of VSMCs. After exposed to the strain, the expressions of contractile phenotype markers, including smooth muscle protein 22 alpha, alpha-actin, and calponin, and phosphorylated Smad2, phosphorylated Smad5, SIRT6 and c-fos were up-regulated in VSMCs by western blot and immunofluorescence. And the expression of intercellular-adhesion molecule-1 (ICAM-1) was also increased detected by flow cytometry. The strained-induced up-regulation of SIRT6 was blocked by a TGF-β1 neutralizing antibody. Furthermore, the effects of strain on VSMCs were abrogated by SIRT6-specific siRNA transfection via the suppression c-fos and ICAM-1. These results suggest that SIRT6 may play a critical role in the regulation of VSMC differentiation in response to the cyclic strain.

Regulation of Renal Fibrosis by Smad3 Thr388 Phosphorylation

Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease.

Modulation of the immune response in HTLV-1-infections by SM29 antigen in vitro is dependent of IL-10, TGF-β and CTLA-4

The HTLV-1 is the causal agent of HTLV-1-associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). The immune response in HTLV-1 infection is polarized to the Th1-type, and previous data from our group had demonstrated that the use of Schistosoma antigens to cell cultures in HTLV-1 infection resulted in down-modulation of Th1/inflammatory response. The aim of this study was to evaluate whether the modulation of inflammatory response by S. mansoni antigens depends upon the presence of IL-10, TGF-β and CTLA-4. The antibodies anti-IL-10, anti-TGF-β and anti-CTLA-4 with or without the S. mansoni antigen Sm29 were added to the PBMC cultures of HTLV-1- infected individuals and the levels of cytokines in the supernatants were measure using ELISA sandwich method. Compared to the levels of cytokine in non stimulated cultures (1.073 pg/ml, median values), the levels of IFN-gproduction (552 pg/ml, 1.002 pg/ml and 548 pg/ml, respectively; p<0.05). On the other hand, the levels of IL-10 were increased by the presence of Sm29 (it passed from 26 pg/ml to102 pg/ml; p=0.02), however when anti-TGF-β and anti-CTLA-4 were added to the cultures there was a reduction in the levels of IL-10 (19 pg/ml and 23 pg/ml, respectively; p<0.05). The regulation of IFN-y production by Sm29 antigen in HTLV-1-infected individuals is dependent on IL-10, TGF-β and CTLA-4. Also the levels of IL-10 were affected by the neutralization of TGF-β and CTLA-4.

Sphingosylphosphorylcholine induces α-smooth muscle actin expression in human lung fibroblasts and fibroblast-mediated gel contraction via S1P2 receptor and Rho/Rho-kinase pathway

Chronic airway diseases like COPD and asthma are usually accompanied with airway fibrosis. Myofibroblasts, which are characterized by expression of smooth muscle actin (α-SMA), play an important role in a variety of developmental and pathological processes, including fibrosis and wound healing. Sphingosylphosphorylcholine (SPC), a sphingolipid metabolite, has been implicated in many physiological and pathological conditions. The current study tested the hypothesis that SPC may modulate tissue remodeling by affecting the expression of α-SMA in human fetal lung fibroblast (HFL-1) and fibroblast mediated gel contraction. The results show that SPC stimulates α-SMA expression in HFL-1 and augments HFL-1 mediated collagen gel contraction in a time- and concentration-dependent manner. The α-SMA protein expression and fibroblast gel contraction induced by SPC was not blocked by TGF-β1 neutralizing antibody. However, it was significantly blocked by S1P2 receptor antagonist JTE-013, the Rho-specific inhibitor C3 exoenzyme, and a Rho-kinase inhibitor Y-27632. These findings suggest that SPC stimulates α-SMA protein expression and HFL-1 mediated collagen gel contraction via S1P2 receptor and Rho/Rho kinase pathway, and by which mechanism, SPC may be involved in lung tissue remodeling.

Osteosarcoma cells promote the production of pro-tumor cytokines in mesenchymal stem cells by inhibiting their osteogenic differentiation through the TGF-β/Smad2/3 pathway

Mesenchymal stem cells (MSCs) are among the most important components of the osteosarcoma microenvironment and are reported to promote tumor progression. However, the means by which osteosarcoma cells modulate MSC behavior remains unclear. The aim of this study was to determine the effects of osteosarcoma cells on both the production of pro-tumor cytokines by mesenchymal stem cells (MSCs) and the osteogenic differentiation of MSCs. High level of transforming growth factor-β (TGF-β) was detected in three osteosarcoma cell lines. Conditioned media (CM) from the osteosarcoma cell lines Saos-2 and U2-OS were used to stimulate the cultured MSCs. We found that osteosarcoma cells promoted the production of IL-6 and VEGF in MSCs by inhibiting their osteogenic differentiation. Furthermore, TGF-β in tumor CM was proved to be an important factor. The TGF-β neutralizing antibody antagonized the effects induced by osteosarcoma CM. The inhibition of Smad2/3 by siRNA significantly decreased the production of IL-6 and VEGF in MSCs and induced their osteogenic differentiation. We also found that Smad2/3 enhanced the expression of β-catenin in MSCs by decreasing the level of Dickkopf-1 (DKK1). Although the inhibition of β-catenin did not affect the production of IL-6 or VEGF, or the gene expression of the early osteogenic markers Runx2 and ALP, it did enhance the gene expression of osteocalcin. Taken together, our data indicate that osteosarcoma cells secrete TGF-β to maintain the stemness of MSCs and promote the production of pro-tumor cytokines by these cells.

Brucella abortus Induces Collagen Deposition and MMP-9 Down-Modulation in Hepatic Stellate Cells via TGF-β1 Production

In patients with active brucellosis, the liver is frequently affected by histopathologic lesions, such as granulomas, inflammatory infiltrations, and parenchymal necrosis. Herein, we examine some potential mechanisms of liver damage in brucellosis. We demonstrate that Brucella abortus infection inhibits matrix metalloproteinase-9 (MMP-9) secretion and induces collagen deposition and tissue inhibitor of matrix metalloproteinase-1 secretion induced by hepatic stellate cells (LX-2). These phenomena depend on transforming growth factor-β1 induction. In contrast, supernatants from B. abortus-infected hepatocytes and monocytes induce MMP-9 secretion and inhibit collagen deposition in hepatic stellate cells. Yet, if LX-2 cells are infected with B. abortus, the capacity of supernatants from B. abortus-infected hepatocytes and monocytes to induce MMP-9 secretion and inhibit collagen deposition is abrogated. These results indicate that depending on the balance between interacting cells and cytokines of the surrounding milieu, the response of LX-2 cells could be turned into an inflammatory or fibrogenic phenotype. Livers from mice infected with B. abortus displayed a fibrogenic phenotype with patches of collagen deposition and transforming growth factor-β1 induction. This study provides potential mechanisms of liver immune response induced by B. abortus-infected hepatic stellate cells. In addition, these results demonstrate that the cross talk of these cells with hepatocytes and macrophages implements a series of interactions that may contribute to explaining some of mechanisms of liver damage observed in human brucellosis.