Exogenous TGF-beta Research Articles

Abstract B072: SMAD4 loss allows progression from benign to malignant disease in intraductal papillary mucinous neoplasms

Abstract As increasing numbers of MRIs and other scans are ordered for unrelated diseases, more and more people are being diagnosed with cystic neoplasms of the pancreas. While many or most of these will likely remain benign, intraductal papillary mucinous neoplasms (IPMNs) have a risk of progression to cancer and IPMN is now recognized as another precursor to pancreatic ductal adenocarcinoma. We are investigating the mechanisms by which IPMNs progress from benign to malignant disease in order to identify markers of progression and targets to block that progression. We found that loss of SMAD4 in conjunction with Krasmutation leads to IPMN that quickly becomes invasive in a mouse model. Examining human IPMN tissues, we found that only one in 46 benign IPMN tumors lacked detectable SMAD4 protein. Among 19 cases of invasive IPMNs, the sites of active invasion were SMAD4 negative in 7 cases, nuclear negative/cytoplasm positive in 3 cases (suggesting no active canonical signaling), and a mix of positive and negative cells in 3 cases, implicating complete or partial loss of SMAD-mediated signaling in 68% of actively invading cells. In order to understand how SMAD signaling blocks progression to invasion, we are examining cell lines from a benign mouse model of IPMN. While these lines do not have endogenous TGF beta signaling, they are capable of responding to exogenous TGF beta, suggesting that TGF beta-related ligands may be supplied by the tumor microenvironment rather than the tumor cells themselves. We are currently determining how different TGF beta family members contribute to regulation of growth and invasion in these cells. In summary, loss of the tumor suppressor SMAD4 allows an otherwise benign disease to progress to invasive cancer. Citation Format: Anna L. Means, Hannah Yoo, Quoc-Huy Trinh, Roshini Thiagarajan, Jinghuan Zi, Frank L. Revetta, Marcus C. Tan, Kathleen E. DelGiorno, Mary K. Washington. SMAD4 loss allows progression from benign to malignant disease in intraductal papillary mucinous neoplasms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B072.

THU0329 SAFETY, TARGET ENGAGEMENT, AND INITIAL EFFICACY OF AVID200, A FIRST-IN-CLASS POTENT AND ISOFORM-SELECTIVE INHIBITOR OF TGF-BETA 1 AND 3, IN PATIENTS WITH DIFFUSE CUTANEOUS SYSTEMIC SCLEROSIS (DCSSC): A PHASE 1 DOSE ESCALATION STUDY

Background:AVID200 is a novel, potent TGF-beta receptor ectodomain-based trap designed to selectively neutralize TGF-beta 1 and 3. These two isoforms have been implicated as central mediators of the pathogenesis of systemic sclerosis (SSc). AVID200 avoids inhibition of TGF-beta 2, the isoform that supports normal cardiac function and hematopoiesis.Objectives:This first-in-human study (AVID200-01;NCT03831438) is a Phase 1, open label, multicenter, cohort dose-escalation trial designed to evaluate safety, tolerability, pharmacokinetic (PK) profile, pharmacodynamic (PD) effects, target engagement, and preliminary efficacy in patients with diffuse cutaneous SSc (dcSSc).Methods:Eligible patients must have dcSSc of <5 years (y) duration and a modified Rodnan Skin Score (MRSS) ≥15. AVID200 at dose levels of 1, 3, 9, or 15 mg/kg IV is administered every 2 weeks (Q2W) for 6 weeks (3 doses). Patients tolerating dosing and who have not experienced disease worsening during the initial Treatment Period may receive up to 6 additional doses Q2W (Extension Period). The ability of AVID200 to selectively sequester its target is assessed in plasma by TGF-beta quantification per ELISA and a cell-based functional readout. Expression of biomarkers of TGF-beta inhibition and genes correlating with MRSS are assessed.Results:The first 2 dose cohorts have completed treatment: male/female 3 each, median age 61y (range 45-70), median MRSS at baseline 31 (range 23-39). Recruitment into cohort 3 is complete. AVID200 was well tolerated with no dose limiting toxicities or serious adverse events (SAEs). AEs, all considered possibly related, included single cases of Grade 1 dizziness and CPK elevation, and Grade 2 anemia. All patients demonstrated a decline in MRSS at 6 weeks by 3, 4, and 9 points in Cohort 1, and 2, 8, and 9 points in Cohort 2. Four of 6 patients demonstrated continued decrease in MRSS 12 weeks after the last dose, with all patients showing a decline in MRSS relative to baseline at this timepoint by 7, 6, and 7 points in Cohort 1 and 4, 8, and 13 points in Cohort 2. AVID200 in plasma engaged endogenous activated TGF-beta and potently neutralized signaling from exogenous TGF-beta 1 and 3, but not TGF-beta 2, across the treatment period. PD effects in skin biopsies, including expression of markers of SSc activity, TGF-beta activity, and myofibroblast-associated genes were assessed. Five of 6 patients showed decreased expression of PD biomarker genes, THBS1 and MS4A4A, comparing end of treatment biopsies to baseline, and all patients showed a decline in SERPINE1 expression, a marker gene for TGF-beta activity. Clustering of RNA-seq expression data showed close coregulation of COMP, THBS1, SERPINE1, LOXL, COL10A1, COL11A1, COL12A1, CTGF, and CDH11, suggesting that blocking TGF-beta inhibits this group of profibrotic genes. Single-cell sequencing data show that expression of these genes is upregulated by subsets of SSc fibroblasts.Conclusion:AVID200 at doses of 1 and 3 mg/kg was well-tolerated in this first study in dcSSc patients. Evidence of anti-fibrotic effects as indicated by rapid, persistent and clinically meaningful declines in MRSS was observed in all patients, as well as AVID200 target engagement and modulation. Recruitment into additional dose and extension cohorts is ongoing. Together, these clinical data support selective TGF-beta 1 and 3 inhibition by AVID200 as a promising therapeutic approach for dcSSc.Disclosure of Interests:Robert Lafyatis Grant/research support from: Forbius, Consultant of: Certa Therapeutics, Forbius, FBM Therapeutics, Robert Spiera Grant/research support from: Roche-Genetech, GSK, Boehringer Ingelheim, Chemocentryx, Corbus, Forbius, Sanofi, Inflarx, Consultant of: Roche-Genetech, GSK, CSL Behring, Sanofi, Janssen, Chemocentryx, Forbius, Mistubishi Tanabe, Robyn Domsic Consultant of: Forbius, Anna Papazoglou: None declared, Colin Ligon Grant/research support from: Forbius, Christina Mae Zinger Morse: None declared, Jean-François Denis Employee of: Forbius, Margaret Davis Employee of: Forbius, Tina Gruosso Employee of: Forbius, Gilles Tremblay Employee of: Forbius, Maureen O’Connor McCourt Employee of: Forbius, Sandra Sinclair Employee of: Forbius, Jonathan Delara Employee of: Forbius, Krista Alvarado Employee of: Forbius, Debra Wood Consultant of: Forbius, Symphogen, Paul Nadler Consultant of: Forbius, Symphogen, Karyopharm, Elizabeth Volkmann Grant/research support from: Forbius, Corbus Pharmaceuticals, Consultant of: Boehringer Ingelheim, Forbius, Speakers bureau: Boehringer Ingelheim

SO082LATENT TRANSFORMING GROWTH FACTOR BETA BINDING PROTEIN (LTBP4) CAN REGULATE RENAL FIBROSIS AND ALTER INFLAMMATION AND MITOCHONDRIAL DYSFUNCTION IN CHRONIC KIDNEY DISEASE (CKD)

Abstract Background and Aims: Transforming growth factor beta (TGF-beta) has been well known as a key factor for fibrosis and inflammation. LTBPs regulate TGF-beta signalling in complicated ways. Disruption and loss of LTBP4 expression is associated with abnormal accumulation of extracellular matrix (ECM) and altered TGF-beta activation. However, the role of LTBP4 in chronic kidney disease remains largely unknown. We aim to explore the mechanisms of LTBP4-related regulation in kidney disease. Method: To investigate the impact of LTBP4 on tubulointeresitial fibrosis (TIF), we generated LTBP4-overexpression human renal proximal tubule cells (HK-2), treated with exogenous TGF-beta and established a fibroblasts-HK-2 co-culture system using rat fibroblasts (NRK-49F) and HK-2 cells. Moreover, to create TIF model, we performed unilateral ureteral ligation (UUO) in Ltbp4S-/- mice and wild-type (WT) mice to check ECM deposition and phenotypic alterations. In addition, we performed RNA-Sequencing analysis to understand transcriptomic changes associate with LTBP4 overexpression. Results: Up-regulation of Ltbp4 in fibrotic kidney was noted in TIF model with UUO and in renal tissue with diabetic nephropathy. LTBP4-overexpression reduced epithelial-mesenchymal transition (EMT) with showing increased epithelial-cadherin, reduced vimentin and collagen I in the co-culture system. Moreover, higher expression of fibronectin, collagen I and alpha-smooth muscle actin (alpha-SMA) were noted in fibrotic kidneys in Ltbp4S-/- mice compared with changes in WT mice, suggesting inflammation condition could be altered by the absence of Ltbp4S. The inflammatory change in renal tissue in UUO model was studied with F4/80 stain. Increased macrophage infiltration and increased monocyte chemoattractant protein-1 (MCP-1) expression were detected in Ltbp4S-/- mice compared with that in WT mice. In addition, LTBP4-overexpression reduced mitochondrial biogenesis. Conclusion: Ltbp4 acted as a regulator of fibrosis and inflammation in fibrotic kidneys. TGF-beta- induced EMT were clearly enhanced in ltbp4 deficient environments, accompanied with reduced macrophage infiltration, which could be the major mechanism related to LTBP4/TGF-beta pathways. Furthermore, altered ATP production and usage as well as mitochondria respiration could be regulated by LTBP4 in renal failure.

TGF-beta and glutathione promote tissue repair in cigarette smoke induced injury.

We studied the effect of cigarette smoke extract (CSE) on a three-dimensional (3-D) co-culture model with epithelial cells and mesenchymal cells to clarify how epithelial cells protect lung tissue from cigarette smoke in-vivo. Two types of gels were prepared. The one was the co-culture of human fetal lung fibroblasts (HFL-I) embedded in type-I collagen gel, with alveolar epithelial cells (A549) cultured covering the top of the gel. The other was HFL-I cells alone. After 48 hours from CSE exposure, gel contraction, levels of fibronectin, transforming growth factor (TGF)-beta and GSH were assessed. CSE inhibited fibroblast-mediated gel contraction and this inhibition was lessened in co-culture associated with higher GSH concentration and TGF-beta1 level as compared to the level in HFL-I cells alone. CSE lowered fibronectin level to a lesser extent in co-culture as compared to the level in HFL-I cells alone. Exogenous TGF-beta1 restored the inhibition of gel contraction by CSE independent of GSH level. Cigarette smoke may interfere with 3-D co-culture gel contraction by diminishing GSH, fibronectin and TGF-beta1 action in the epithelial-mesenchymal interaction.

Abstract 5066: Reprogramming the TGF-beta signaling in cancer-associated fibroblasts inhibits ovarian cancer progression

Abstract Ovarian cancer is the most lethal gynecologic malignancy in the US. While major research efforts have been dedicated to identify disease related genes and mutations in cancer cells, the role of the stromal tumor microenvironment in disease progression is relatively unexplored. We previously reported that while ovarian cancer cells have minimal response to TGF-beta, TGF-beta induced secretory proteins from cancer associated fibroblasts (CAFs) promote ovarian cancer motility and invasion potential. The present study seeks to further evaluate the involvement of TGF-beta signaling in CAFs in the progression of ovarian cancer and the therapeutic potential of targeting such signaling in cancer treatment. By comparing the gene expression profiles of laser-microdissected ovarian tumor stromal tissues from high-grade serous ovarian cancer (HGSOC) patients with short and long survival, a differentially expressed gene signature was identified. Using the IPA platform, signaling pathways and upstream regulators activated in CAFs from patients with short survival were identified. TGF-beta signaling was found to be significantly activated (p = 2.47E-03), while the ligand TGF-beta 1 was identified as the most significant upstream regulator (Z-score = 5.189, p = 8.85E-29), suggested the involvement of CAF TGF-beta signaling in poor patient survival. Further immunohistochemistry and survival analyses revealed the clinical relevance of multiple TGF-beta regulated stromal genes, including VCAN, MFAP5 and COMP in poor disease prognosis. Subsequently, cell culture studies were performed to evaluate the effects of TGF-beta in ovarian cancer progression. While exogenous TGF-beta did not affect proliferation, motility and invasion potential of ovarian cancer cells directly, TGF-beta promoted ovarian cancer cells motility and invasion potential in a co-culture system of cancer cells and CAFs. Similarly, treatment of exogenous TGF-beta-induced CAF secretory proteins promoted ovarian cancer progression, suggested the presence of CAFs is required for TGF-beta mediated ovarian cancer progression and targeting such TGF-beta signaling in CAFs could be a novel modality of ovarian cancer treatment. To reprogram CAFs by targeting its TGF-beta signaling, multiple approaches were tested in co-culture systems. Using either siRNAs or small molecule inhibitors, TGF-beta signaling in CAFs was effectively suppressed and inhibited ovarian cancer progression was observed. Based on our current findings, animal studies will be performed to further evaluate the efficacy of targeting the CAF TGF-beta signaling pathway in ovarian cancer treatment. This study presents the paradigm which activation of TGF-beta signaling in ovarian CAFs is significantly associated to the poor clinical outcomes. Targeting such pathway activation in CAFs could effectively suppress tumor progression and thereby presented a novel approach in cancer treatment. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. Reprogramming the TGF-beta signaling in cancer-associated fibroblasts inhibits ovarian cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5066. doi:10.1158/1538-7445.AM2015-5066

Экспрессия факторов семейства TGFβи фактора роста фибробластов FGF2 в эмбриональных стволовых клетках мыши и человека, поддерживаемых в разных системах культивирования

Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases ofpluripotency, we examined the expression of TGFbeta family factors (ActivinA, Nodal, Leftyl, TGFbeta1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFbeta1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFbeta1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3BMP/Smad1/5/8 endogenous branches of TGFbeta signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFbeta family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smadl/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primary states of pluripotency demonstrate diverse involvement of this factor in the regulation of the pluripotent cell self-renewal.

Expression of TGFβ family factors and FGF2 in mouse and human embryonic stem cells maintained in different culture systems

Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases ofpluripotency, we examined the expression of TGFbeta family factors (ActivinA, Nodal, Leftyl, TGFbeta1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFbeta1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFbeta1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3BMP/Smad1/5/8 endogenous branches of TGFbeta signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFbeta family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smadl/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primary states of pluripotency demonstrate diverse involvement of this factor in the regulation of the pluripotent cell self-renewal.

TGF-beta1 increases cell rigidity by enhancing expression of smooth muscle actin: Keloid-derived fibroblasts as a model for cellular mechanics

Mechanical transduction contributes to appropriate cell functions. Clinically, keloid, an uncontrolled fibrous overgrowth and scarring, preferentially affects skin areas subject to higher mechanical tension than others. Keloid-derived fibroblasts have exaggerated TGF-beta1-mediated responses, including smooth muscle actin (SMA) expression, cellular contraction, and tissue remodeling, to mechanical strain compared to normal fibroblasts. This study asked if SMA contributes to cellular intrinsic rigidity using keloid -derived fibroblasts as a model. Using atomic force microscopy and confocal microscopy, we measured cellular rigidity and the expression of SMA in keloid fibroblasts treated with exogenous TGF-beta1. There was an increase of SMA expression in keloid tissue as well as keloid-derived fibroblasts. The cell rigidity increased by TGF-beta1 in keloid fibroblasts occurred concomitantly with increases in SMA expression. TGF-beta1 receptor 1 kinase inhibitors reduced TGF-beta1-induced cellular rigidity and SMA expression. Knocking down SMA with interference RNA resulted in a reduction of TGF-beta1-enhanced rigidity, suggesting that TGF-beta1 increases cell rigidity via SMA expression. We conclude that TGF-beta1 increases cell rigidity through TGF-beta1 receptor-SMA axis. This study reports that SMA, at least in part, contributes to cell rigidity in fibroblasts. SMA might be an appealing pharmaceutical target in keloids.

The Antioxidant n-Acetylcysteine Blocks Direct Regulatory T Cell Mediated Suppression of CD4+ Effector T Cells In Vitro

AbstractAbstract 2765 Background:CD4+CD25+FOXP3+ regulatory T cells (Tregs) employ a range of suppressive strategies including factors that are cytotoxic to target CD4+CD25−FOXP3− effector T cells (Teff) such as perforin and granzyme B and those that suppress proliferation, differentiation and/or cytokine production including TGF-beta1, IL-10 and CTLA-4. The relative contribution of each of these mechanisms to Treg function is unclear but from the available data their importance appears context specific. Because T cells are very sensitive to the redox state of the microenvironment, and display a pattern of impaired activation under conditions of oxidative stress, we investigated the potential contribution of an oxidant dependent suppressive pathway on direct Treg mediated suppression of Teff in vitro using the antioxidant n-acetylcysteine (NAC) to block reactive oxidants. Methods:Tregs and Teff were derived from the spleens of 2–4 month old C57BL/6 mice maintained in pathogen free conditions. T cell subsets were isolated using magnetic bead based techniques. Purity was assessed by flow cytometry. For suppression assays, Tregs were co-cultured with CFSE-labeled Teff at the indicated ratios and stimulated with anti-CD3/CD28 coated beads for 3 days. Proliferation was measured by flow cytometric evaluation of CFSE dilutional staining. Suppression was calculated by comparing proliferation of Teff cultured alone to those co-cultured with Tregs (% suppression= 1 − Tregs:Teff/Teff alone). In other experiments, CFSE-labeled Teff were cultured alone in the indicated conditions with TGF-beta +/− NAC and proliferation was assessed by CFSE staining. Intracellular reactive oxygen species (ROS) were measured by flow cytometry using the redox sensitive cell permeable indicator dye 5-(and-6)-chloromethyl-2‘,7‘-dichlorodihydrofluorescein diacetate, acetyl ester (DCFDA). Results:The presence of NAC prevented Treg suppression of Teff in a dose dependent fashion at Treg:Teff ratios of 1:2, 1:1 and 2:1 (see figure). Suppression was significantly decreased at 0.1mM NAC (p<0.05 at 1:1; p<0.01 at 2:1) and essentially absent at 1mM NAC. Proliferation of Teff was markedly higher in the setting of 1mM NAC compared to conditions without NAC, even during co-culture with Tregs at a 2:1 ratio of Tregs:Teff. Treg suppression of Teff proliferation was dependent on TGF-beta as neutralizing antibodies reversed the effect (p<0.001). The presence of NAC was sufficient to overcome the suppressive effects of exogenous TGF-beta on CD3/CD28 stimulated Teff proliferation. Treatment of CD3/CD28 stimulated Teff with TGFbeta resulted in a significant dose dependent increase in the levels of intracellular ROS (p<0.0001 at 10ng/mL TGF-beta) that inversely correlated with the degree of proliferation. Conclusion:NAC blocks Treg mediated TGF-beta dependent suppression in vitro. This suggests that TGF-beta may function to suppress proliferation of Teff via a ROS dependent mechanism and raises the possibility that targeted delivery of antioxidants may have clinical utility for modulating the effects of Tregs in vivo. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Negative Regulation of Human Granulosa Tumor Cell Survival by TGF-Beta.

Disruption to the regulatory pathways that govern granulosa cell proliferation, differentiation, and death during ovarian development and folliculogenesis can result in a variety of disorders, including granulosa cell tumor (GCT) formation. Candidate growth regulatory molecules produced within the ovary include several TGF-beta superfamily members, but the specific stimuli and intracellular signalling pathways which determine human granulosa cell proliferation and survival are still poorly understood. We have used a human ovarian cancer cell line of GCT origin, the KGN cells, to study TGF-beta superfamily actions. We have recently reported that this GCT line exhibited very little expression of the TGF-beta accessory receptor betaglycan, a TGF-beta and inhibin co-receptor which determines cellular sensitivity to these ligands. We hypothesized that GCTs would be refractory to the growth-inhibitory effects of TGF-beta and/or inhibin and that overexpression of betaglycan would restore responsiveness to TGF-beta mediated growth inhibition. To test this hypothesis, KGN cells were stably transfected with a wildtype betaglycan expression construct or the empty vector as a control. Overexpression of betaglycan in the KGN cells restored sensitivity to TGF-beta and inhibin in luciferase reporter assays but had little effect on cell proliferation, cell cycle progression, or death in either GCT cell line as determined by flow cytometry. Furthermore, treatment with either exogenous TGF-beta2 or inhibin A, INHA knockdown, or neutralisation of endogenous TGF-betas also did not significantly affect cell survival and proliferation. As the GCT cells are tumour cell lines which exhibit constitutively active NF-kappaB, which is a pro-survival factor, we pre-treated the cells with the chemical NF-kappaB inhibitor, BAY11-7082. Under these conditions, overexpression of betaglycan resulted in a decrease in proliferation (15%; p<0.05) and an increase in apoptosis (300% p<0.05). The addition of TGF-beta1 and TGF-beta2 dose-dependently decreased cell viability, and this effect was enhanced by the presence of betaglycan. However, knockdown of INHA expression did not affect cell viability even in the presence of BAY11-7082. Following treatment with exogenous TGF-beta2, INHA gene knockdown, or a combination of the two, total cell lysates were extracted from betaglycan- and vector-transfected GCT cells. The relative levels of activation of specific pathways were determined by western blot analyses using activation-specific antibodies against SMAD2, SMAD3, and SMAD1/5. This analysis revealed that overexpression of betaglycan in the GCT cells increased both the basal and TGF-beta2-induced levels of activated SMAD3, with a smaller effect observed on activated SMAD2. Collectively, these data establish the TGF-beta-SMAD2/3-betaglycan signalling pathway as an important regulator of granulosa cell viability and suggest that deficiency of this pathway and/or inhibition by NF-kappaB contributes to dysregulated granulosa cell growth and cancer. Supported by the NHMRC of Australia (RegKeys 494802; 441101; 388904) and Victorian Government Infrastructure funds. (platform)

Regression of mouse-derived renal cancer by adoptive transfer of tumor-reactive RNAi-induced TGF-beta- insensitive CD8 + T cells

Transforming growth factor beta (TGF-beta) is a potent immunosuppressant. The present study was conducted to develop a treatment strategy through adoptive transfer of tumor-reactive RNAi-induced TGF-beta-insensitive CD8 + T cells. BALB/c mice were primed with irradiated Renca cells. CD8 + T cells were isolated from the spleen of primed animals, expanded ex vivo and were rendered TGF-beta-insensitive by infecting with a retrovirus containing shRNA to mouse TGF-beta type II receptor gene (MSCV-shRNA-T). Control CD8+ T cells consist of those infected with retroviruses containing shRNA to non specific gene (MSCV-shRNA-N) and naive CD8 + T cells. The effect of all groups of CD8 + T cells on Renca cells were analyzed by semi-quantitative RT-PCR, Western-blot, in vitro and in vivo assay. MSCVshRNA-T group of CD8 + T cells were resistant to the antiproliferative effect of exogenous TGF-beta, while control groups were not. Results of Western blot showed the Smad pathway was disrupted in MSCV-shRNA-T group, which confirmed the blockade of the signal transduction pathway. In vitro cytotoxic assay revealed that these tumor-reactive, TGF-beta-insensitive CD8 + T cells killed Renca cells specifically and strongly. Adoptive transfer of these MSCV-shRNA-T CD8 + T cells to BALB/c tumorbearing mice showed strong tumor-specific cytotoxic T lymphocyte responses and antitumor immunity against Renca renal cancer. Based on these results, we predict that adoptive transfer of tumor-reactive RNAi-induced TGF-beta-insensitive CD8 + T cells may be effective to renal cancer therapy. Keywords: Transforming growth factor beta, adoptive transfer, RNA interference, renal cancer, renca cells, immunotherapy African Journal of Biotechnology Vol. 9(33), pp. 5274-5283, 16 August, 2010

TGF-β Signaling Specifies Axons during Brain Development

In the mammalian brain, the specification of a single axon and multiple dendrites occurs early in the differentiation of most neuron types. Numerous intracellular signaling events for axon specification have been described in detail. However, the identity of the extracellular factor(s) that initiate neuronal polarity in vivo is unknown. Here, we report that transforming growth factor beta (TGF-beta) initiates signaling pathways both in vivo and in vitro to fate naive neurites into axons. Neocortical neurons lacking the type II TGF-beta receptor (TbetaR2) fail to initiate axons during development. Exogenous TGF-beta is sufficient to direct the rapid growth and differentiation of an axon, and genetic enhancement of receptor activity promotes the formation of multiple axons. Finally, we show that the bulk of these TGF-beta-dependent events are mediated by site-specific phosphorylation of Par6. These results define an extrinsic cue for neuronal polarity in vivo that patterns neural circuits in the developing brain.

Transforming growth factor β2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis

Vitamin A-deficient (VAD) quail embryos lack the vitamin A-active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGFbeta2 is involved in RA-regulated cardiovascular development. In VAD embryos TGFbeta2 mRNA and protein expression are greatly elevated. The expression of TGFbeta receptor II is also elevated in VAD embryos but is normalized by treatment with TGFbeta2-specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFbeta2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFbeta2 protein to normal quail embryos mimics the excessive TGFbeta2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGFbeta2 has a major role in the retinoic acid-regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGFbeta2 signals during vertebrate heart formation.

Effects of Arginine Vasopressin on Differentiation of Cardiac Fibroblasts Into Myofibroblasts

Differentiation of cardiac fibroblasts (CFs) into myofibroblasts is a critical event in the initiation of myocardial fibrosis (MF). Previous studies have shown that arginine vasopressin (AVP) facilitates MF. However, the effects of AVP on CFs-myofibroblasts transformation, and its possible mechanisms are still unknown. CFs obtained from neonatal Sprague-Dawley rats were stimulated with AVP in the absence or presence of AVP V1a receptor specific antagonist [d(CH2)5Tyr(Me)]AVP. CFs-myofibroblast transformation was detected by expression of alpha-smooth muscle actin (alpha-SMA) and collagen synthesis. Western bolt and immunofluorescent staining were used to detect expression of alpha-SMA, [H]Proline incorporation was used to detect collagen synthesis. AVP-induced transforming growth factor-beta1 (TGF-beta1) secretion was detected by enzyme-linked immunosorbent assay. CFs was also stimulated with exogenous TGF-beta1 to find out the required dose to induce CFs-myofibroblast transformation. 10 mol/L AVP increased alpha-SMA expression and collagen synthesis significantly, and this effect was blocked by [d(CH2)5Tyr(Me)]AVP at the concentration of 10 mol/L. Meanwhile, AVP significantly increased TGF-beta1 secretion of CFs in a dose-dependent manner, and this effect was also blocked by 10 mol/L [d(CH2)5Tyr(Me)]AVP. However, the maximum production of biologic active TGF-beta1 induced by AVP is far less than the dose of exogenous TGF-beta1 needed to induce CFs-myofibroblast transformation. AVP can induce CFs-myofibroblast transformation via its V1a receptor, AVP-induced increase of TGF-beta1 synthesis, which also is mediated by V1a receptor, may play a minor role in the transformation. Inducing differentiation of CFs into myofibroblasts may be a mechanism of AVP contributing to MF.

Tranilast inhibits cell proliferation and migration and promotes apoptosis in murine breast cancer

The malignant transformation of breast epithelium involves a number of cellular pathways, including those dependent on signaling from TGF beta. Tranilast [N-(3, 4-dimethoxycinnamonyl)-anthranilic acid] is a drug that is used in Japan to control allergic disorders in patients, and its mechanism of action involves TGF beta. In view of the multiple roles of TGF beta in tumor progression, we hypothesized in this study that tranilast impacts cell proliferation, apoptosis, and migration. Using the mouse breast cancer cell line 4T1, our studies showed that tranilast increases AKT1 phosphorylation and decreases ERK1/2 phosphorylation. Alterations in the cell cycle mediators' cyclin D1, p27, cyclin A, pRB, cyclin B, and Cdc2 were observed after exposure to tranilast, favoring cell arrest beyond the G1/S phase. Tranilast reduced tumor cell proliferation even when it was amplified by exogenous TGF beta. TGF beta-neutralizing antibody did not cause a significant decrease in cell proliferation. Tranilast treatment upregulates p53, induces PARP cleavage in vitro, consistent with a promotion of tumor cell apoptosis. TGF beta-neutralizing antibody downregulates endoglin and matrix metalloproteinases (MMP)-9 levels in vitro indicating that the tranilast effect is mediated through TGF beta modulation. Tranilast treatment results in the inhibition of cell migration and invasion. Western blot analysis of tumor lysates from tranilast-treated mice shows decreased levels of TGF beta1, endoglin, and significantly higher levels of p53 and cleaved PARP. Cleaved caspase 3 expression is significantly elevated in tranilast-treated mouse breast tumors. To conclude, tranilast induces cellular and molecular changes in murine breast cancer that can be exploited in preclinical therapeutic trials.

Transforming growth factor β1 enhances tumor promotion in mouse skin carcinogenesis

Transforming growth factor beta1 (TGFbeta1) expression is elevated by tumor promoters in the mouse skin, but its role in tumor promotion has not been well defined. To investigate this, we have compared TGFbeta1+/+ and +/- mice in a two-stage skin chemical carcinogenesis protocol. Surprisingly, TGFbeta1+/- mice had fewer number and incidence of benign papillomas, reduced epidermal and tumor cell proliferation and reduced epidermal TGFbeta1 and nuclear p-Smad2 localization in response to the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) compared with TGFbeta1+/+ mice. Maximal TPA activation of protein kinase C (PKCalpha) as measured by activity assays and activation of target genes and induction of cornified envelopes correlated with TGFbeta1 gene dosage in keratinocytes and addition of exogenous TGFbeta1 restored the cornification defect in TGFbeta1+/- keratinocytes. Similarly, inhibition of ALK5-suppressed TPA-mediated PKCalpha activation suggesting that physiological levels of TGFbeta1 are required for maximal activation of PKC-dependent mitogenic responses. Paradoxically, the TPA-induced inflammatory response was greater in TGFbeta1+/- skin, but TGFbeta1+/+ papillomas had more tumor infiltrating myeloperoxidase-positive cells and pro-inflammatory gene expression was elevated in v-ras(Ha)-transduced TGFbeta1+/+ but not TGFbeta1+/- keratinocytes. Thus, ras activation switches TGFbeta1 to a pro-inflammatory cytokine. Despite this differential proliferative and inflammatory response to TPA and enhanced papilloma formation in the TGFbeta1+/+ mice, the frequency of malignant conversion was reduced compared with TGFbeta1+/- mice. Therefore, TGFbeta1 promotes benign tumors by modifying tumor promoter-induced cell proliferation and inflammation but retains a suppressive function for malignant conversion.

Attenuation of the Transforming Growth Factor β-Signaling Pathway in Chronic Venous Ulcers

Transforming growth factor beta (TGFbeta) is important in inflammation, angiogenesis, reepithelialization and connective tissue regeneration during wound healing. We analyzed components of TGFbeta signaling pathway in biopsies from 10 patients with nonhealing venous ulcers (VUs). Using comparative genomics of transcriptional profiles of VUs and TGFbeta-treated keratinocytes, we found deregulation of TGFbeta target genes in VUs. Using quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis, we found suppression of TGFbeta RI, TGFbeta RII and TGFbeta RIII, and complete absence of phosphorylated Smad2 (pSmad2) in VU epidermis. In contrast, pSmad2 was induced in the cells of the migrating epithelial tongue of acute wounds. TGFbeta-inducible transcription factors (GADD45beta , ATF3 and ZFP36L1) were suppressed in VUs. Likewise, genes suppressed by TGFbeta (FABP5, CSTA and S100A8) were induced in nonhealing VUs. An inhibitor of Smad signaling, Smad7 was also downregulated in VUs. We conclude that TGFbeta signaling is functionally blocked in VUs by downregulation of TGFbeta receptors and attenuation of Smad signaling resulting in deregulation of TGFbeta target genes and consequent hyperproliferation. These data suggest that application of exogenous TGFbeta may not be a beneficial treatment for VUs.

Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

IntroductionVasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-β) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-β signaling in fibroblasts.MethodsThe transgenic mouse strain Tβ RIIΔk-fib is characterized by balanced ligand-dependent upregulation of TGF-β signaling. Aortic and cardiac tissues were examined with histologic, biochemical, and isolated organ bath studies. Vascular and perivascular architecture was examined by hematoxylin and eosin (H&E) and special stains including immunostaining for TGF-β1 and phospho-Smad2/3 (pSmad2/3). Confirmatory aortic smooth muscle cell proliferation, phenotype, and functional assays, including signaling responses to exogenous TGF-β and endothelin-1, were performed. Aortic ring contractile responses to direct and receptor-mediated stimulation were assessed.ResultsAortic ring contractility and relaxation were diminished compared with wild-type controls, and this was associated with aortic adventitial fibrosis confirmed histologically and with Sircol assay. TGF-β1 and pSmad 2/3 expression was increased in the adventitia and smooth muscle layer of the aorta. Aortic smooth muscle cells from transgenic animals showed significant upregulation of TGF-β- responsive genes important for cytoskeletal function, such as transgelin and smoothelin, which were then resistant to further stimulation with exogenous TGF-β1. These cells promoted significantly more contraction of free floating type I collagen lattices when compared with the wild-type, but were again resistant to exogenous TGF-β1 stimulation. Aortic ring responses to receptor-mediated contraction were reduced in the transgenic animals. Specifically, bosentan reduced endothelin-mediated contraction in wild-type animals, but had no effect in transgenic animals, and endothelin axis gene expression was altered in transgenic animals. Transgenic mice developed cardiac fibrosis.ConclusionsThe histologic, biochemical, and functional phenotype of this transgenic mouse model of scleroderma offers insight into the altered biomechanical properties previously reported for large elastic arteries in human SSc and suggests a role for perturbed TGF-β and endothelin activity in this process.

Effect of transforming growth factor beta 1 (TGF-beta 1) on nitric oxide production and lipid peroxidation in oral mucosal wound healing

Wound healing is a highly orchestrated process including complex and coordinated interactions involving peptide growth factors of which transforming growth factor-beta (TGF-beta) is one of the most important modulators. Exogenous TGF-beta treatment has been shown to accelerate wound healing in normal and impaired animal models. Nitric oxide (NO) also plays a key role in wound healing. The objective of this study is to examine the effects of exogenous TGF-beta 1 treatment on NO and lipid peroxidation levels in the process of oral wound healing on different days. In this study, we used 5-month-old New Zealand albino male rabbits. After a standard surgical incision in the diestema region, the rabbits were divided into controls and TGF-beta 1 implanted groups. NO levels and malondialdeyhde (MDA) levels which are indicators of lipid peroxidation were determined by spectrophotometry. In the TGF-beta 1 implanted groups, both NO and MDA levels significantly increased only on the third day after wounding when compared to control groups. We found decreased MDA levels parallel to NO levels on the fifth day after wounding. These findings suggest that TGF-beta 1 affects mucosal wound healing by altering NO production on different days of wounding. TGF-beta 1 may regulate NO production by its dual effect in as both an activator and inhibitor an in oral mucosal healing.

Upregulation of hepatoma-derived growth factor is involved in murine hepatic fibrogenesis

Hepatoma-derived growth factor (HDGF) expression is correlated with progression of hepatocellular carcinoma. Since liver fibrosis frequently occurs before hepatoma development, this study investigated the expression profile of HDGF and its relationship with transforming growth factor-beta (TGF-beta) signaling in experimental models of hepatofibrogenesis. Liver fibrosis was induced in mice receiving bile duct ligation (BDL) or carbon tetrachloride (CCl(4)) administration. The expression levels of HDGF and other fibrosis-related markers were measured using quantitative RT-PCR, Western blotting, and enzyme-linked immunosorbent assays. Hepatic HDGF overexpression was achieved by adenovirus gene delivery. Rat hepatocytes were used to study the interplay between HDGF and TGF-beta1. In both liver fibrosis models, HDGF de novo synthesis significantly increased during the progression of fibrosis. The HDGF upregulation was observed mainly in hepatocytes and correlated with the expression of TGF-beta1 and collagen COL1A1 and COL1A2 proteins. Hepatic HDGF overexpression itself deteriorated hepatocellular structure and integrity, and aggravated the extents of BDL- and CCl(4)-induced liver fibrosis with concomitant upregulation of TGF-beta1 and COL1A1. Exogenous TGF-beta1 stimulated HDGF expression only in cultured primary hepatocytes grown on collagen matrix, whereas exogenous HDGF also increased TGF-beta1 production in hepatocytes in a collagen-dependent manner. Moreover, HDGF enhanced Smad2 phosphorylation dose-dependently and the TGF-beta1-driven luciferase activities. HDGF plays a pro-fibrogenic role during liver fibrosis in mice through activation of TGF-beta pathway. The mutual regulation between TGF-beta1 and HDGF may facilitate a vicious cycle to promote the progression of hepatic fibrogenesis.