Presence Of TGF-beta Research Articles

Abstract P1-07-01: Novel role of the tumor suppressor ductal epithelium associated ring chromosome 1 (DEAR1) in regulation of breast stem/progenitor cell properties

Abstract Introduction: Breast cancer is the most commonly diagnosed cancer and third-leading cause of cancer-related deaths in women in America. A quarter of lesions diagnosed annually are ductal carcinoma in situ (DCIS), one of the earliest pre-invasive forms of invasive ductal carcinoma (IDC). Without therapeutic intervention, 30-50% of DCIS cases can progress to IDC. Understanding the mechanisms regulating progression from DCIS to IDC would help identify biomarkers to stratify DCIS patients at higher risk of progression or recurrence. Cumulative literature suggests the earliest phase of dissemination from the primary tumor is driven by the epithelial-mesenchymal transition (EMT) program. DEAR1 is a tumor suppressor gene which is mutated, undergoes loss of heterozygosity in breast cancer, and is downregulated in DCIS lesions. DEAR1 regulates acinar morphogenesis and cell polarity, and is a negative regulator of TGF-beta-driven EMT by binding to and ubiquitinating SMAD3, thereby limiting the amount of SMAD3 available to activate an EMT signature. Overexpression of EMT master regulators, or exposure to TGF-beta in immortalized human mammary epithelial cells (HMECs), results in mammosphere formation and breast stem cell markers, thus linking the EMT process to acquisition of stem cell characteristics. Methods: Stable lentiviral shRNA knockdown, in vitro mammosphere assay, cytospin, immunofluorescence, immunoprecipitation, western blot analysis, ubiquitination and real-time quantitative PCR were performed. Results: DEAR1 knockdown in immortal HMECs resulted in a significant enhancement of primary mammosphere formation and growth compared to controls, suggesting that DEAR1 may regulate stem/progenitor cell properties; this effect was greater when cells were exposed to TGF-beta. To determine if DEAR1 regulates stem cell properties through regulation of SMAD3 levels, DEAR1-SMAD3 double knockdown clones were examined for mammosphere formation. Results indicated fewer mammospheres in double knockdown clones, but only in the presence of TGF-beta, suggesting that the mammosphere phenotype is partially dependent on the TGF-beta-SMAD3 pathway. The observation that loss of DEAR1 alone results in a stem/progenitor cell phenotype indicates that DEAR1 may regulate mammosphere formation independent of its role in regulating the TGF-beta pathway. HMEC mammospheres express higher levels of stem cell marker aldehyde dehydrogenase (ALDH1) and co-express luminal and basal cytokeratins, suggesting bipotential capacity. We also observed upregulation of SNAI2 and ZEB1, master EMT and stem cell regulators, in DEAR1 knockdown HMECs. DEAR1 interacted with SNAI2 by co-immunoprecipitation analysis and also promoted the polyubiquitination of SNAI2. Conclusions: Loss of the tumor suppressor/polarity regulator DEAR1 promotes stem cell properties in part through the DEAR1-TGF-beta-SMAD3 axis. We demonstrate a DEAR1-SNAI2 axis that may partially regulate stem/progenitor cell properties in HMECs through the polyubiquitination of SNAI2, a master regulator of EMT and stemness. These mechanisms governing acquisition of the stem cell properties may contribute to understanding how DCIS progress to IDC. Citation Format: Le U, Chen N, Balasenthil S, Killary AM. Novel role of the tumor suppressor ductal epithelium associated ring chromosome 1 (DEAR1) in regulation of breast stem/progenitor cell properties [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-07-01.

Specific Gamma Delta T Cells for Cellular Immunotherapy of EBV-Associated Diseases after Allo-HSCT By T-Cell Receptor Gene Modification

Epstein-Barr virus (EBV) infection may result in a spectrum of diseases in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially post-transplant lymphoproliferative disease (PTLD). Furthermore, the clinical course of EBV-associated diseases often superimposes with graft-versus-host disease (GVHD) and that increases the difficulty of treatment. Adoptive immunotherapy with EBV-specific cytotoxic T lymphocytes (EBV-CTL) is recommended as one of the first-line therapy for PTLD. Engineering of T cell receptor (TCR)-transferred T lymphocytes could be an attractive strategy to obtain sufficient T cells with an antigen specificity of choice. But TCR-modified alpha beta T cells are restricted by major histocompatibility (MHC) class I expression and low affinity. Because gamma delta T cells (GD T cells) recognize target cells independent of human leukocyte antigen (HLA), the alpha beta TCR engineering of GD T cells forms a feasible strategy to generate antigen-specific effector T cells without HLA restriction. And regulatory GD T cells (GD Tregs) (one of the novel subset of GD T cells), which express Foxp3 and primarily belong to CD27+CD25high phenotype, might have application prospect in the treatment of GVHD. In order to investigate a novel strategy with potential therapeutic benefits in EBV-associated diseases after allo-HSCT, we obtained the monoclonal EBV-CTL clone HLA-A*0201/GLCTLVAML from the PROIMMUNE company (U.K.) and detected peptide-specific CTL activity in chromium release assays, then identified the EBV-CTL associated - TCR Valpha 15 and TCR Vbeta 1 chain genes, and subsequently cloned into the eukaryotic expression vector pIRES to construct the recombinant plasmid; We transferred the EBV specific recombinant plasmid to GD T cells and GD Tregs (generated in vitro by stimulating with anti-TCR gamma delta in the presence of TGF-beta and IL-2). The TCR gene-transduced groups had special TCR Vbeta 1 expression in fluorescence detection, quantitative PCR and western Blot analysis. TCR gene-transduced GD T cells had significant cytotoxicity against the EBV+ target cell lines (including toledo cells (HLA-A2 positive) (38.17±0.86%) and daudi cells (HLA-A2 negative) (61.74±1.84%)) compared with empty vector transfected GD T cells (toledo cells (30.15±0.89%); daudi cells (53.91±4.33%)) (P<0.001, P=0.006). TCR gene-transduced GD Tregs also had significant cytotoxicity against toledo cells (56.21±5.34%) and daudi cells (68.41±2.30%) compared with empty vector transfected GD Tregs (toledo cells (48.47±0.35%); daudi cells (56.23±2.04%)) (P=0.012, P<0.001). Both TCR gene-transduced GD T cells and GD Tregs had high levels of IFN-gamma in culture supernatants. Moreover, TCR gene-transduced GD Tregs had high levels of IL-10 in culture supernatants. Interestingly, TCR-modified GD Tregs showed significant suppressive effect on autologous CD4+ responder cells in CCK8 detection and CFSE assays. In conclusion, GD T cells or GD Tregs co-transduced with TCR alpha beta genes (from EBV CTL clone) could acquire antiviral capacity. Immunomodulatory function of GD Tregs could remain after TCR gene transfection. TCR modified GD Tregs might be an alternative and attractive strategy with potential therapeutic benefits in EBV-associated diseases after allo-HSCT, especially with GVHD. DisclosuresWu:National Natural Science Foundation of China (No. 81200388), the Science and Technology Planning Project of Guangdong Province of China (No. 2012B031800403), the project of the Zhujiang Science & Technology Star of Guangzhou(No. 2013027): Research Funding. Li:National Natural Science Foundation of China (No. 81200388), the Science and Technology Planning Project of Guangdong Province of China (No. 2012B031800403), the project of the Zhujiang Science & Technology Star of Guangzhou(No. 2013027): Research Funding.

Loss of TRAF2 Impairs Alloreactive CD4+ T Cells and Maintains Tregs to Favour Islet Allograft Survival Without Exogenous Immunosuppression.

The signaling molecule, TNF-receptor associated factor 2 (TRAF2) integrates multiple TNF-family signaling pathways critical for T cell activation suggesting TRAF2 may be a potential target for promoting allograft survival.Here, the role of TRAF2 in T cell responses towards allografts is examined using T cell specific TRAF2-deficient mice (TRAF2TKO). TRAF2TKO mice permanently accepted the majority of full MHC-mismatched islet allografts and display delayed rejection of vascular heterotopic heart and skin allografts without exogenous immunosuppression. Further, TRAF2TKO failed to proliferate in an in vitro MLR - thus TRAF2 deficiency impairs the alloimmune response. Loss of TRAF2 resulted in diminished CD4+ T cell activation as shown by reduced proliferation and decreased expression of activation markers CD44 and CD25 and effector molecules granzyme B and IFN-gamma in response to alloantigen. Further, under Th1 polarizing conditions, TRAF2TKO CD4+ T cells showed abnormal expression of the Th2 cytokine IL-13. Furthermore, TRAF2TKO CD4+ T cells more readily converted to a Treg phenotype in the presence of TGF-beta; and in vivo TRAF2TKO mice harbored increased Tregs. TRAF2 deficiency delayed activation of TNF-induced JNK and NF-kB in T cells. Therefore TRAF2 coordinates extracellular signals from TNF-family ligands to maintain T cell lineage selection and effector function.

P024 Regional differences in effect of TGF-beta1 and PDGF on the early onset of intestinal fibrosis

Background: Intestinal fibrosis (IF) is one of the major complications in inflammatory bowel disease patients. IF can cause narrowing of the intestinal lumen, which may lead to stricture formation. The mechanism of IF is still unknown and adequate models are lacking. By using precision-cut intestinal slice (PCIS) from different regions of the bowel, we studied the early onset of fibrosis in mouse jejunum, ileum and colon PCIS, in the presence of transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF). Methods: Mouse jejunum, ileum and colon were excised and prepared as a segment embedded in agarose. PCIS (estimated 300-400 μm) was prepared and incubated up to 48 hr with or without the presence of TGF-beta1 and PDGF. ATP content of the PCIS was used to assess the general viability. The gene expression of different fibrosis markers including Pro-Collagen 1 A1 (COL1A1), heat shock protein 47 (HSP47), alpha-smooth muscle actin (SMA), connective tissue growth factor (CTGF), synaptophysin (SYN) and fibronectin (FN2) were determined. Results: Mouse PCIS from different segments were viable up to 48 hr. After 48 hr of incubation, HSP47 and FN2 gene expression were significantly up-regulated, compared to PCIS directly after slicing, in jejunum (3.6 and 4.9 fold, respectively) and in ileum (4.9 and 5.5 fold, respectively). When incubated with 5 ng/mL TGF-beta1, in jejunum PCIS, COL1A1, HSP47, CTGF and FN2 were significantly up-regulated compared to 48 hr control. In ileum PCIS the gene expression of HSP47 (1.9 fold) and FN2 (3.9 fold) were also significantly increased. In the presence of 50 ng/mL PDGF, only in ileum PCIS, CTGF (1.4 fold) and SYN (1.9 fold) were significantly increased compared to 48 hr control. Interestingly, in PCIS from the colon, 5 ng/ml TGF-beta1 did not affect the gene expression of the fibrosis markers. However, HSP47 (1.4 fold) and FN2 (1.7 fold) were significantly increased when colon PCIS were incubated with 50 ng/mL PDGF. Conclusions: TGF-beta1, but not PDGF, was able to induce HSP47 and FN2 in mouse jejunum and ileum PCIS. This is in contrast to the result in colon PCIS, where only PDGF was able to induce these fibrosis markers. Moreover, PDGF increased CTGF and SYN only in ileum PCIS. These results indicate differences in the effect of TGF-beta1 and PDGF on the early onset of fibrosis in different regions of the intestine.

P025 Reduced Butyricicoccus pullicaecorum levels in mucosa of UC patients correlate with aberrant CLDN1 expression

Background: Intestinal fibrosis (IF) is one of the major complications in inflammatory bowel disease patients. IF can cause narrowing of the intestinal lumen, which may lead to stricture formation. The mechanism of IF is still unknown and adequate models are lacking. By using precision-cut intestinal slice (PCIS) from different regions of the bowel, we studied the early onset of fibrosis in mouse jejunum, ileum and colon PCIS, in the presence of transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF). Methods: Mouse jejunum, ileum and colon were excised and prepared as a segment embedded in agarose. PCIS (estimated 300 400mm) was prepared and incubated up to 48 hr with or without the presence of TGF-beta1 and PDGF. ATP content of the PCIS was used to assess the general viability. The gene expression of different fibrosis markers including Pro-Collagen 1 A1 (COL1A1), heat shock protein 47 (HSP47), alpha-smooth muscle actin (SMA), connective tissue growth factor (CTGF), synaptophysin (SYN) and fibronectin (FN2) were determined. Results: Mouse PCIS from different segments were viable up to 48 hr. After 48 hr of incubation, HSP47 and FN2 gene expression were significantly up-regulated, compared to PCIS directly after slicing, in jejunum (3.6 and 4.9 fold, respectively) and in ileum (4.9 and 5.5 fold, respectively). When incubated with 5 ng/mL TGF-beta1, in jejunum PCIS, COL1A1, HSP47, CTGF and FN2 were significantly up-regulated compared to 48 hr control. In ileum PCIS the gene expression of HSP47 (1.9 fold) and FN2 (3.9 fold) were also significantly increased. In the presence of 50 ng/mL PDGF, only in ileum PCIS, CTGF (1.4 fold) and SYN (1.9 fold) were significantly increased compared to 48 hr control. Interestingly, in PCIS from the colon, 5 ng/ml TGF-beta1 did not affect the gene expression of the fibrosis markers. However, HSP47 (1.4 fold) and FN2 (1.7 fold) were significantly increased when colon PCIS were incubated with 50 ng/mL PDGF. Conclusions: TGF-beta1, but not PDGF, was able to induce HSP47 and FN2 in mouse jejunum and ileum PCIS. This is in contrast to the result in colon PCIS, where only PDGF was able to induce these fibrosis markers. Moreover, PDGF increased CTGF and SYN only in ileum PCIS. These results indicate differences in the effect of TGF-beta1 and PDGF on the early onset of fibrosis in different regions of the intestine.

Transforming growth factor-beta increases the expression of vascular smooth muscle cell Markers in human multi-lineage progenitor cells

SummaryBackgroundVascular smooth muscle cell (SMC) differentiation is an essential component of vascular repair and tissue engineering. However, currently used cell models for the study of SMC differentiation have several limitations. Multi-lineage progenitor cells (MLPCs) originate from human umbilical cord blood and are cloned from a single cell. The object of this study was to investigate whether MLPCs could differentiate into SMCs in vitro with induction by transforming growth factor β1 (TGF-β1).Material/MethodsMLPCs were treated without or with TGF-β1 (1 and 5 ng/mL) in mesenchymal stem cell media plus 1% FBS for 7 days. Total RNA was isolated from the MLPCs, and semi-quantitative real-time PCR was performed to test the following mRNA levels: early and late phase SMC-specific markers, two endothelial cell (EC)-specific markers, endothelial progenitor cell (EPC) marker CD34, TGF-β1 accessory protein CD105, and adhesion molecule CD146.ResultsTGF-β1 (1 ng/mL) significantly increased the mRNA levels of SMC-specific markers SM22α, calponin-1, SM α-actin, caldesmon, tropomyosin and MLCK as well as adhesion molecule CD146. The mRNA levels of EC-specific markers VE-cadherin and VEGFR-2, EPC marker CD34 and TGF-β1 accessory protein CD105 were decreased significantly, after MLPC were treated with TGF-β1 (1 ng/mL). TGF-β1 at 5 ng/mL showed similar effect on the expression of these genes.ConclusionsThis study demonstrates that in the presence of TGF-β1, MLPCs undergo SMC lineage differentiation indicating that MLPCs are a promising cell model for SMC lineage differentiation studies, which may contribute to advances in vascular repair and tissue engineering.

Demonstration of TGF-β and XIIIα in Endocardial Biopsies of Carcinoid Heart Disease Patients: an Immunofluorescence Study.

BackgroundSerotonin and other vasoactive substances play a critical role in the development of carcinoid heart disease, but the exact etiology of the illness is still unknown.MethodsBy using immunofluorescence microscopy, we investigated the expression of transforming growth factor-β (TGF-β) and the presence of fibrin-stabilizing factor (XIIIα) in endomyocardial biopsy specimens of patients with carcinoid heart disease. In addition, the tissue integrity of the specimens was studied by staining for laminin.ResultsBoth TGF-β and XIIIα co-localized in the endocardium beneath carcinoid plaques: while TGF-β was found within myocytes, XIIIα was detected on the surface of cells in fibrotic lesions stretching out into the tissue. Laminin staining revealed that the integrity of the endocardium was dissolved and that the tissue consisted of hypertrophic and hypotrophic myocytes.ConclusionsThe results suggest that the presence of TGF-β and XIIIα in carcinoid heart lesions indicates that endocardial damage induced by serotonin and other vasoactive substances gives rise to an overshooting wound healing process.

Evidence of Common Mechanisms Underlying the Survival Advantage of the PIG-A mutant and 13q- Hematopoietic Stem Cell Clones In Patients with Immune-Mediated Bone Marrow Failure

AbstractAbstract 4429 Background:Numerical karyotypic abnormalities of undetermined significance are occasionally detected in patients with bone marrow failure (BMF), including aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS). BMF patients with 13q deletions (13q-) are likely to respond to immunosuppressive therapy (IST) without a propensity to develop into leukemia (Ishiyama K et al, Br J Haematol; 117: 747. 2002), and they possess small populations of glycosylphosphatidyl-inositol anchored protein (GPI-AP)- deficient granulocytes and erythrocytes more frequently than patients with normal karyotypes%, 15 with AA and 8 with MDS) of 1705 patients. The proportion of 13q- clones in all metaphase cells ranged from 5% to 100% with a median of 27.5%. Fourteen of the 23 (61%) patients showed 13q- alone and 9 (39%) showed other karyotypic abnormalities in addition to 13q-. GPI-AP- cells that accounted for 0.009% to 6.7% (median 0.148%) of granulocytes were detected in all (100%) of the 14 patients with 13q- alone, while the prevalence of increased GPI-AP- cells in patients with 13q- plus other abnormalities and those with a normal karyotype was 44% and 43%, respectively. A FISH analysis of PB granulocytes in individual patients revealed apparently lower percentages of GPI-AP- granulocytes than those of 13q- granulocytes, indicating that the GPI-AP- granulocytes were derived from non 13q- HSCs. Ten patients with 13q- alone were treated with IST (ATG+cyclosporine in 4 and cyclosporine alone in 6) and all of them achieved either a partial remission or a complete remission. The SNP array analysis of 5 patients revealed common deletion of a 15 Mb (13.3 to 14.3) region containing the RB gene. Wild-type and PIG-A mutant K562 cells were incubated in the presence of TGF-beta, which can inhibit leukemic cell proliferation by down-regulating phosphorylated RB protein (pRB), to determine whether the RB gene loss in 13q- HSCs has any connection with a proliferative advantage of PIG-A mutant HSCs. PIG-A mutant K562 cells were less sensitive to TGF-beta inhibition of proliferation and down-regulation of pRB levels than wild-type K562 cells. Conclusion:The markedly high prevalence of increased GPI-AP- cells and good response to IST indicate that the presence of 13q- clones is highly associated with the immune pathophysiology of BMF. Decreased sensitivity to inhibitory signals by TGF-beta due to RB gene loss in 13q- HSCs and the loss of GPI-AP-type TGF-beta receptors in PIG-A mutant HSCs may therefore be a common mechanism that confers a survival advantage to these mutant HSCs over normal HSCs in patients with immune-mediated BMF. Disclosures:No relevant conflicts of interest to declare.

FGF‐2 abolishes the chondrogenic effect of combined BMP‐6 and TGF‐β in human adipose derived stem cells

In this study, we investigated the influence of transforming growth factor beta 3 (TGF-beta3), bone morphogenetic protein 6 (BMP-6) and basic fibroblast growth factor (FGF-2) on chondrogenesis in adipose derived stem cells (ASC). Cells were isolated from liposuction material, expanded and subjected to chondrogenic differentiation. Micromass pellets were cultured in chondrogenic medium containing 10 ng/mL TGF-beta3 which was additionally supplemented with 10 ng/mL BMP-6, 10 ng/mL FGF-2 or a combination of both. We quantitatively evaluated the cartilage specific gene expression after 14 days of culture. The end point measurements on day 35 included glycosaminoglycan (GAG) quantification, histological staining for chondrogenic markers, and transmission electron microscopy (TEM). In comparison to cultures induced with TGF-beta3/FGF-2, the presence of TGF-beta3/BMP-6 demonstrated strong induction of collagen type II, collagen type IX and aggrecan mRNA expression. This was corroborated by quantification and histological staining for GAGs and immunohistological staining for collagen II. However, when a combination of BMP-6 and FGF-2 in addition to TGF-beta3 was added, FGF-2 counteracted BMP-6, as indicated by reduced marker gene expression and weak to absent staining for GAGs. In conclusion, this study demonstrates that BMP-6 combined with TGF-beta3 is a potent inducer of chondrogenesis in human ASC. In contrast, FGF-2 does not contribute to differentiation, but rather suppresses the chondrogenic potential of BMP-6.

Effect of budesonide on TGF‐β1‐enhanced VEGF production by lung fibroblasts

VEGF (vascular endothelial growth factor) is a potent proangiogenic cytokine, and vascular change is one of the characteristic features of airway remodelling. Since the glucocorticoids have shown antifibrosis properties, we sought to investigate whether budesonide, a widely used glucocorticoid in clinical practice, could attenuate TGF-beta1 (transforming growth factor-beta1)-induced VEGF production by HFL-1 (human lung fibroblasts). HFL-1 fibroblasts were treated with various concentrations of budesonide (10(-11) M, 10(-9) M and 10(-7) M) in the absence or presence of TGF-beta1. Postculture media were collected for ELISA of VEGF at the indicated times. The cell lysates were subjected to Western blotting analysis to test TGF-beta1/Smad and MAP (mitogen-activated protein) kinase signalling activation, respectively. The results suggested that budesonide pretreatment reduced the significant increase of VEGF release induced by TGF-beta1 in HFL-1 fibroblasts in a dose-dependent manner, and suppressed the increase of phospho-Smad3 and phosphor-ERK (extracellular signal-regulated kinase) protein levels. In conclusion, budesonide may reduce TGF-beta1-induced VEGF production in the lung, probably through the Smad/ERK signalling pathway and, thus, may provide new sight into the molecular mechanism underlying glucocorticoid therapy for airway inflammatory diseases.

MEK/ERK and p38 MAPK regulate chondrogenesis of rat bone marrow mesenchymal stem cells through delicate interaction with TGF‐β1/Smads pathway

This study was carried out to reveal functions and mechanisms of MEK/ERK and p38 pathways in chondrogenesis of rat bone marrow mesenchymal stem cells (BMSCs), and to investigate further any interactions between the mitogen-activated protein kinase (MAPK) and transforming growth factor-beta1 (TGF-beta1)/Smads pathway in the process. Chondrogenic differentiation of rat BMSCs was initiated in micromass culture, in the presence of TGF-beta1, for 2 weeks. ERK1/2 and p38 kinase activities were investigated by Western Blot analysis. Specific MAPK inhibitors PD98059 and SB20350 were employed to investigate regulatory effects of MEK/ERK and p38 signals on gene expression of chondrocyte-specific markers, and TGF-beta1 downstream pathways of Smad2/3. ERK1/2 was phosphorylated in a rapid but transient manner, whereas p38 was activated in a slow and sustained way. The two MAPK subtypes played opposing roles in mediating transcription of cartilage-specific genes for Col2alpha and aggrecan. TGF-beta1-stimulated gene expression of chondrogenic regulators, Sox9, Runx2 and Ihh, was also affected by activity of PD98059 and SB203580, to different degrees. However, influences of MAPK inhibitors on gene expression were relatively minor when not treated with TGF-beta1. In addition, gene transcription of Smad2/3 was significantly upregulated by TGF-beta1, but was regulated more subtly by treatment with MAPK inhibitors. MAPK subtypes seemed to regulate chondrogenesis with a delicate balance, interacting with the TGF-beta1/Smads signalling pathway.

The Response of Bone Marrow-Derived Mesenchymal Stem Cells to Dynamic Compression Following TGF-β3 Induced Chondrogenic Differentiation

The objective of this study was to investigate the hypothesis that the application of dynamic compression following transforming growth factor-beta3 (TGF-beta3) induced differentiation will further enhance chondrogenesis of mesenchymal stem cells (MSCs). Porcine MSCs were encapsulated in agarose hydrogels and cultured in a chemically defined medium with TGF-beta3 (10 ng/mL). Dynamic compression (1 Hz, 10% strain, 1 h/day) was initiated at either day 0 or day 21 and continued until day 42 of culture; with TGF-beta3 withdrawn from some groups at day 21. Biochemical and mechanical properties of the MSC-seeded constructs were evaluated up to day 42. The application of dynamic compression from day 0 inhibited chondrogenesis of MSCs. This inhibition of chondrogenesis in response to dynamic compression was not observed if MSC-seeded constructs first underwent 21 days of chondrogenic differentiation in the presence of TGF-beta3. Spatial differences in sGAG accumulation in response to both TGF-beta3 stimulation and dynamic compression were observed within the constructs. sGAG release from the engineered construct into the surrounding culture media was also dependent on TGF-beta3 stimulation, but was not effected by dynamic compression. Continued supplementation with TGF-beta3 appeared to be a more potent chondrogenic stimulus than the application of 1 h of daily dynamic compression following cytokine initiated differentiation. In the context of cartilage tissue engineering, the results of this study suggest that MSC seeded constructs should be first allowed to undergo chondrogenesis in vitro prior to implantation in a load bearing environment.

Abstract 4102: Mesenchymal stem cells induce epithelial-to-mesenchymal transition (EMT) in metastatic SP cells in pancreatic cancer

Abstract In malignant cells, some reports demonstrated that Side population (SP) cells expressed tumor-initiating activity, chemotherapy resistance and metastatic potential. We previously reported that SP cells play a pivotal role of local invasion and in vivo liver metastasis in pancreatic cancer (Int-J-Cancer 124:2771, 2009). The augmentation of metastatic activity in SP cells appeared to relate to epithelial-to-mesenchymal transition (EMT), as SP cells were highly responsive to TGF-beta-mediated EMT and local invasion. However, the mechanisms by which SP cells undergo EMT in vivo remains unknown. Cancer associate fibroblasts (CAFs) have recently been implicated in important aspects of epithelial solid tumor biology, considered migrate to tumor sites and contribute to neoplastic progression, tumor growth, angiogenesis, and invasion/metastasis. In addition, recent reports have demonstrated that CAFs are derived from hematopoietic precursor/stem cells from bone marrow. Thus, bone marrow derived mesenchymal stem cells (MSCs) would be an attractive candidate for CAFs which enhance cancer metastasis by mediating EMT. The AIM of present study is to examine whether bone marrow-derived MSCs have ability to mediate EMT of pancreatic cancer SP cells. METHODS: Human bone marrow-derived MSCs were isolated by flowcytometry. Panc-1 cells were co-cultured with or without MSCs, and tumorigenesity was compared by implantation into NOD/SCID mice. Either unsorted panc-1 cells or Panc-1-derived SP cells were co-cultured in the presence or absence of MSCs using transwell culture dish for 7 days. Invasion activity was assessed by using matrigel invasion assay. In addition, real-time PCR experiments were performed to quantitate E-cadherin mRNA levels. RESULTS: When Panc-1 cells were mixed with MSCs and co-injected into NOD/SCID mice, tumorigenic activity was enhanced as compared to single injection of the equivalent number of cancer cells. In vitro co-culturing with MSCs changed the shape of Panc-1-derived SP cells to spindle-like appearance. The cell-to-cell contact underwent loosely, these morphological changes were consistent with EMT. E-cadherin mRNA levels were decreased after co-culturing. In addition, SP cell matrigel invasion activity was enhanced by co-culturing with MSCs. Similar results were observed in SP cells when EMT was induced by incubation in the presence of TGF-beta, a known inducer of EMT. It was surprising that when SP cells were co-incubated with MSCs, most of the cells fell into non-SP fraction by re-analysis of Hoechst33342 staining. This alteration was reversible as the cells recovered SP-phenotype by removing the MSCs. In CONCLUSION, MSC-derived soluble factors may induce EMT which is concordant with augmentation of invasion activity in metastatic SP cells. The SP-phenotype appears to be transient and reversible, which could be regulated by MSC-derived factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4102.

651 VERAPAMIL AND PENTOXIFYLLINE INHIBIT TGF-BETA-STIMULATED TIMP-3 PRODUCTION IN PEYRONIE'S DISEASE FIBROBLASTS: EVIDENCE OF CAMP- AND CALCIUM-DEPENDENT SIGNALING CROSSTALK

You have accessJournal of UrologyPenis/Testis/Urethra: Benign & Malignant Disease II1 Apr 2010651 VERAPAMIL AND PENTOXIFYLLINE INHIBIT TGF-BETA-STIMULATED TIMP-3 PRODUCTION IN PEYRONIE'S DISEASE FIBROBLASTS: EVIDENCE OF CAMP- AND CALCIUM-DEPENDENT SIGNALING CROSSTALK Marcello Del Carlo, Ada Cole, and Laurence Levine Marcello Del CarloMarcello Del Carlo More articles by this author , Ada ColeAda Cole More articles by this author , and Laurence LevineLaurence Levine More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1025AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The calcium channel blocker verapamil and the phosphodiesterase type 4 (PDE4) inhibitor pentoxifylline have been reported to be useful as a treatment alternative for patients with Peyronie's Disease (PD). The fibrotic effects of TGF-beta overexpression in PD are well documented but the role of cAMP and calcium as secondary intermediates of TGF-beta signaling is poorly understood. The purpose of this study was to determine the effects of these anti-fibrotic agents on TGF-beta mediated tissue inhibitor of matrix metalloproteinase (TIMP) production as a potential mechanism of action. METHODS Fibroblasts were isolated from fibrotic plaque tissue removed from patients undergoing surgical correction for PD. The effects of verapamil (100 uM) and pentoxifylline (500 uM) on TGF-beta (10 ng/ml) mediated TIMP-3 production was analyzed by immunoblotting conditioned media from monolayer cultures 48 hours after stimulation, and reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify TIMP-3 mRNA in cells 8 hours after stimulation. Intracellular [calcium] fluxes were analyzed by confocal microscopy (lambda-ex = 340/380 nm, lambda-em = 510 nm) using Fura-2/AM (2 uM) loaded cells in the presence and absence of either the PKA activator forskolin (10 uM) or the PKA inhibitor H89 (10 uM) to modulate intracellular [cAMP]. Luciferase reporter constructs were transfected in cells to quantify CREB binding activity. RESULTS Pre-treatment with either verapamil or pentoxifylline reduced TGF-beta mediated TIMP-3 mRNA expression and protein production, and also prevented increased cytosolic calcium fluxes observed in cells stimulated with TGF-beta. Similar results were obtained with the calcium channel blocker nifedipine and the PKA activator forskolin. Pretreatment with either the cell-permeable calcium-chelator BAPTA/AM (5 uM) or the PKA inhibitor H89 had no effect. Treatment with either verapamil or pentoxifylline alone significantly induced CREB activity, which was marginally attenuated in the presence of TGF-beta. CONCLUSIONS Decreased TGF-beta mediated TIMP-3 expression represents an attractive mechanism for the anti-fibrotic effects displayed by verapamil and pentoxifylline therapy in patients with PD. The increased activity of cAMP-sensitive calcium channels caused by TGF-beta overexpression may represent an important biochemical mechanism for the localized fibrosis observed in PD. Chicago, IL© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e255 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Marcello Del Carlo More articles by this author Ada Cole More articles by this author Laurence Levine More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Th17-specific demethylation and methylation-sensitive STAT3 binding at a DNA element in the IL-17 promoter (88.13)

Abstract CD4+ helper T cells can be instructed by non-antigen-specific signals to differentiate into functionally distinct subsets with exclusive patterns of cytokine production. Activation in the presence of TGFbeta and IL-6 results in the development of Th17 cells that produce IL-17, an inflammatory cytokine that is not expressed by Th1 or Th2 lineage cells. The promoter region of the IL-17A gene contains 12 CpG dinucleotides, leading us to test whether the IL-17 gene is regulated epigenetically by DNA methylation. In Th1 and Th2 cells, all CpG dinucleotides were heavily methylated (80-100%), with the exception of the -33 dinucleotide adjacent to the TATA element, which was 70% methylated in all cell lineages. The Th17 differentiation program was associated with a selective, 4-fold increase in demethylation (from 90% to 60%) at CpG dinucleotides -144 and +13 bp from the transcriptional start site. Interestingly, the -144 CpG is located within a consensus element (CCpGTCA) for STAT3, a transcription factor that is required for IL-17 gene induction and Th17 differentiation. We find that STAT3 from Th17 nuclear extracts binds with high affinity to this CCpGTCA element in vitro, but shows low affinity binding to the methylated CmeCpGTCA element. These results suggest that signals from the TGFbeta and/or IL-6 receptors lead to demethylation of a STAT3 element in the IL17A promoter, and epigenetically program the IL-17 locus for transactivation by STAT3 in the Th17 lineage.

TGF-beta is an essential factor for IL-9 production (36.60)

Abstract Interleukin-9 (IL-9) is a multifunctional cytokine involved in proliferation, differentiation, and effector functions of various cell types that include T cells, B cells, mast cells, eosinophiles neutrophils, and epithelial cells. In previous studies, Th2 cells were shown to produce IL-9. Recent studies suggest that IL-9 can also be produced by so-called Th9 cells. In this study, we report that TGF-beta is an essential factor for in vitro cultured human peripheral blood mononuclear cells (PBMC) into subsets that secrete IL-9. PBMC were activated in the presence of anti-CD3/CD28 antibodies and polarizing cytokines with or without the addition of TGF-beta. Using flow cytometry to detect both intracellular IL-9 expression and soluble IL-9 secretion, we found that IL-9 was only produced in the presence of TGF-beta. Addition of other cytokines, especially IL-4 further increased IL-9 production. Interestingly, a subset of CD4+IL-9+ T cells was seen to also express IL-17, but not IL-4, IL-10 or IL-12. Additional studies will address the plasticity between the Th9 and Th17 CD4 T cell subsets.

Effects of TGF-β3 and preculture period of osteogenic cells on the chondrogenic differentiation of rabbit marrow mesenchymal stem cells encapsulated in a bilayered hydrogel composite

In this work, injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) and gelatin microparticles (MPs) were used to fabricate a bilayered osteochondral construct. Rabbit marrow mesenchymal stem cells (MSCs) were encapsulated with transforming growth factor-beta3 (TGF-beta3)-loaded MPs in the chondrogenic layer and cocultured with cells of different periods of osteogenic preculture (0, 3, 6 and 12 days) in the osteogenic layer to investigate the effects of TGF-beta3 delivery and coculture on the proliferation and differentiation of cells in both layers. The results showed that, in the chondrogenic layer, TGF-beta3 significantly stimulated chondrogenic differentiation of MSCs. In addition, cells of various osteogenic preculture periods in the osteogenic layer, along with TGF-beta3, enhanced gene expression for MSC chondrogenic markers to different extents. In the osteogenic layer, cells maintained their alkaline phosphatase activity during the coculture; however, mineralization was delayed by the presence of TGF-beta3. Overall, this study demonstrated the fabrication of bilayered hydrogel composites which mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers, while illustrating that encapsulated cells of different degrees of osteogenic differentiation can significantly influence the chondrogenic differentiation of cocultured progenitor cells in both the presence and absence of chondrogenic growth factors.

Smad3 Protein Levels Are Modulated by Ras Activity and during the Cell Cycle to Dictate Transforming Growth Factor-β Responses

Transforming growth factor beta (TGF-beta) regulates many biological processes, and aberrant TGF-beta signaling is implicated in tumor development. Smad3 is a central component of the TGF-beta signaling pathway, and once activated, Smad3 forms complexes with Smad4 or other receptor-regulated Smads, which accumulate in the nucleus to transcriptionally regulate TGF-beta target genes. Because Smad3 plays a significant role in mediating the activities of TGF-beta, we examined its regulation during tumor development using a well characterized tumor model. We demonstrate that Smad3 levels are dramatically reduced in the tumorigenic cell line transformed with activated H-Ras compared with the normal parental epithelial cells. Interestingly, we also observe a cell cycle-dependent regulation of Smad3 in both cell types, with high Smad3 levels in quiescent cells and a significant drop in Smad3 protein levels in proliferating cells. Smad3 is regulated at the mRNA level and at the level of protein stability. In addition, functional analysis indicates that down-regulation of Smad3 levels is required for the tumor cells to proliferate in the presence of TGF-beta, because ectopic expression of Smad3 in the tumorigenic cell line restores the growth inhibitory response to TGF-beta. In contrast, expression of high levels of Smad3 did not interfere with the ability of these cells to undergo epithelial to mesenchymal transition upon TGF-beta stimulation. Altogether, our results suggest that the level of Smad3 protein is an important determinant of the progression of tumorigenesis. High levels of Smad3 are required for the tumor suppressor activities of TGF-beta, whereas lower levels are sufficient for the tumor promoting functions.

A novel inhibitor of Smad‐dependent transcriptional activation suppresses tissue fibrosis in mouse models of systemic sclerosis

Tissue fibrosis is a major cause of morbidity and mortality in systemic sclerosis (SSc), and an increasing number of promising molecular targets for antifibrotic therapies have been described recently. Transforming growth factor beta (TGFbeta) is well known to be the principal factor that leads to tissue fibrosis. The present study was undertaken to investigate the ability of HSc025, a novel small compound that antagonizes TGFbeta/Smad signaling through the activation of nuclear translocation of Y-box binding protein 1, to prevent tissue fibrosis in vitro or in mouse models of SSc. Human dermal fibroblasts were exposed to HSc025 at various concentrations in the presence of TGFbeta, and levels of collagen or fibronectin expression were determined. HSc025 (15 mg/kg/day for 14 days) was administered orally to tight skin mice and to mice with bleomycin-induced pulmonary fibrosis. Improvement of tissue fibrosis was evaluated by histologic or biochemical examination in each model. Pretreatment with HSc025 prevented Smad-dependent promoter activation, in a dose-dependent manner; however, HSc025 had no effect on TGFbeta-induced phosphorylation of Smad3. The inhibitory effects of HSc025 on TGFbeta-induced collagen or fibronectin expression were also confirmed in vitro. Orally administered HSc025 significantly reduced hypodermal thickness and hydroxyproline content in tight skin mice, and markedly decreased the histologic score and hydroxyproline content in the lungs of bleomycin-treated mice. These results demonstrate that HSc025 is a novel inhibitor of TGFbeta/Smad signaling, resulting in the improvement of skin and pulmonary fibrosis. Orally available HSc025 might therefore be useful in the treatment of SSc.

Dynamic Compression Stimulates Proteoglycan Synthesis by Mesenchymal Stem Cells in the Absence of Chondrogenic Cytokines

The objective of this study was to evaluate the effect of dynamic compression on mesenchymal stem cell (MSC) chondrogenesis. Dynamic compression was applied to agarose hydrogels seeded with bone marrow-derived adult equine MSCs. In the absence of the chondrogenic cytokine transforming growth factor beta (TGFbeta), dynamic compression applied for 12 h per day led to significantly greater proteoglycan synthesis than in unloaded TGFbeta-free cultures, although at a rate that was approximately 20% to 35% of unloaded TGFbeta cultures. These data suggest that the emergence of aggrecan dominated a chondrogenic response to loading as increases in proteoglycan synthesis. Cross-sectional analyses were conducted to subjectively identify potential spatial distributions of heterogeneous differentiation. In loaded samples, cell viability and metachromatic staining was low near the porous compression platen interface but increased with depth, reaching levels in the lower portion of the hydrogel that resembled unloaded TGFbeta cultures. These results suggest that the combination of high hydrostatic pressure and low dynamic strain and fluid flow had a stronger effect on chondrogenesis than did low hydrostatic pressure coupled with high dynamic strain and fluid flow. Next, the 12-h per day loading protocol was applied in the presence of TGFbeta. Biosynthesis in loaded cultures was less than in unloaded TGFbeta samples. Taken together, these data suggest that the duration of loading necessary to stimulate mechanoinduction of MSCs may not be optimal for neo-tissue accumulation in the presence of chondrogenic cytokines.