Active TGF-beta Research Articles

The Thrombospondin1-TGF-β Pathway and Glaucoma.

Glaucoma is characterized by abnormal remodeling of the extracellular matrix (ECM) in the trabecular meshwork and in the connective tissue beams of the lamina cribrosa (LC) at the optic nerve head (ONH), which is associated with axonal damage. Mechanical strain can stimulate ECM remodeling and increased expression of matricellular proteins. Thrombospondins 1 and 2 are induced by cyclic mechanical strain in the eye in both the trabecular meshwork and in the LC region of the ONH. TGF-betas 1 and 2 are increased in glaucoma and play a role in the pathologic remodeling of the ECM in the eye in glaucoma. In this study, we address the role of thrombospondin1 as a regulator of latent TGF-beta activation and discuss the potential therapeutic use of antagonists of the thrombospondin1-TGF-beta pathway for treatment of glaucoma.

TGF‐beta Regulates Alternative Splicing of CD44 by Inducing Smad3 Binding to CD44 pre‐mRNA

During late stage of breast cancer, TGF‐beta signaling promotes tumor progression by stimulating EMT, cell invasion and disseminations to distant sites. Previous studies have showed that TGF‐beta supports tumor progression through Smad‐dependent transcription regulation and Smad‐independent translational regulation. A few reports also suggested a role of alternative splicing, which represents another important mechanism of gene regulation, in aggressive cellular behavior induced by TGF‐beta during cancer progression. However, it is still unknown how TGF‐beta regulates alternative splicing. We found that TGF‐beta regulate alternative splicing of CD44 by inducing binding of Smad3 on CD44 pre‐mRNA surrounding regions encoding variant exons to inhibit recruitment of the splicing machinery to variant exons with suboptimal splice sites, thereby promotes a switch in CD44 expression from epithelial variant isoforms to the standard isoform. This isoform switch is essential for breast cancer progression. We show that the interaction of Smad3 with CD44 pre‐mRNA requires both input from TGF‐beta signaling and activation of oncogenic signaling, which occurs selectively in the tumor cells. Thus, Smad3 enables the tumor promoting function of TGF‐beta by serving as a regulator in alternative splicing under oncogenic stimulation in tumor cells.

Enhanced SDF-1 and rantes attract the locally injected bone marrow stromal cells to remedy dental biomechanically induced temporomandibular joint osteoarthritic lesions

Enhanced SDF-1 and rantes attract the locally injected bone marrow stromal cells to remedy dental biomechanically induced temporomandibular joint osteoarthritic lesions

Integrin beta 8 (ITGB8) regulates embryo implantation potentially via controlling the activity of TGF-B1 in mice.

Integrins (ITGs) are mediators of cell-cell and cell-matrix interactions, which are also associated with embryo implantation processes by controlling the interaction of blastocyst with endometrium. During early pregnancy, ITGbeta8 (ITGB8) has been shown to interact with latent transforming growth factor (TGF) beta 1 (TGFB1) at the fetomaternal interface. However, the precise role of ITGB8 in the uterus and its association with embryo implantation has not been elucidated. Therefore, we attempted to ascertain the role of ITGB8 during the window of embryo implantation process by inhibiting its function or protein expression. Uterine plasma membrane-anchored ITGB8 was augmented at peri-implantation and postimplantation stages. A similar pattern of mRNA expression was also found during the embryo implantation period. An immunolocalization study revealed the presence of ITGB8 on luminal epithelial cells along with mild expression on the stromal cells throughout the implantation period studied; however, an intense fluorescence was noted only during the peri- and postimplantation stages. Bioneutralization and mRNA silencing of the uterine Itgb8 at preimplantation stage reduced the rate/frequency of embryo implantation and subsequent pregnancy, suggesting its indispensable role during the embryo implantation period. ITGB8 can also regulate the liberation of active TGFB1 from its latent complex, which, in turn, acts on SMAD2/3 phosphorylation (activation) in the uterus during embryo implantation. This indicates involvement of ITGB8 in the embryo implantation process through regulation of activation of TGFB1.

Constitutive activation of transforming growth factor Beta receptor 1 in the mouse uterus impairs uterine morphology and function.

Despite increasing evidence pointing to the essential involvement of the transforming growth factor beta (TGFB) superfamily in reproduction, a definitive role of TGFB signaling in the uterus remains to be unveiled. In this study, we generated a gain-of-function mouse model harboring a constitutively active (CA) TGFB receptor 1 (TGFBR1), the expression of which was conditionally induced by the progesterone receptor (Pgr)-Cre recombinase. Overactivation of TGFB signaling was verified by enhanced phosphorylation of SMAD2 and increased expression of TGFB target genes in the uterus. TGFBR1 Pgr-Cre CA mice were sterile. Histological, cellular, and molecular analyses demonstrated that constitutive activation of TGFBR1 in the mouse uterus promoted formation of hypermuscled uteri. Accompanying this phenotype was the upregulation of a battery of smooth muscle genes in the uterus. Furthermore, TGFB ligands activated SMAD2/3 and stimulated the expression of a smooth muscle maker gene, alpha smooth muscle actin (ACTA2), in human uterine smooth muscle cells. Immunofluorescence microscopy identified a marked reduction of uterine glands in TGFBR1 Pgr-Cre CA mice within the endometrial compartment that contained myofibroblast-like cells. Thus, constitutive activation of TGFBR1 in the mouse uterus caused defects in uterine morphology and function, as evidenced by abnormal myometrial structure, dramatically reduced uterine glands, and impaired uterine decidualization. These results underscore the importance of a precisely controlled TGFB signaling system in establishing a uterine microenvironment conducive to normal development and function.

Autoimmune diabetes is suppressed by treatment with recombinant human tissue Kallikrein-1.

The kallikrein-kinin system (KKS) comprises a cascade of proteolytic enzymes and biogenic peptides that regulate several physiological processes. Over-expression of tissue kallikrein-1 and modulation of the KKS shows beneficial effects on insulin sensitivity and other parameters relevant to type 2 diabetes mellitus. However, much less is known about the role of kallikreins, in particular tissue kallikrein-1, in type 1 diabetes mellitus (T1D). We report that chronic administration of recombinant human tissue kallikrein-1 protein (DM199) to non-obese diabetic mice delayed the onset of T1D, attenuated the degree of insulitis, and improved pancreatic beta cell mass in a dose- and treatment frequency-dependent manner. Suppression of the autoimmune reaction against pancreatic beta cells was evidenced by a reduction in the relative numbers of infiltrating cytotoxic lymphocytes and an increase in the relative numbers of regulatory T cells in the pancreas and pancreatic lymph nodes. These effects may be due in part to a DM199 treatment-dependent increase in active TGF-beta1. Treatment with DM199 also resulted in elevated C-peptide levels, elevated glucagon like peptide-1 levels and a reduction in dipeptidyl peptidase-4 activity. Overall, the data suggest that DM199 may have a beneficial effect on T1D by attenuating the autoimmune reaction and improving beta cell health.

MP19-06 ACTIVATION OF TGF-BETA, WNT AND CYTOSKELETAL REMODELING PATHWAYS REVEALED BY MICRORNA PROFILING IN OUTLET OBSTRUCTION-INDUCED BLADDER DYSFUNCTION

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 2014MP19-06 ACTIVATION OF TGF-BETA, WNT AND CYTOSKELETAL REMODELING PATHWAYS REVEALED BY MICRORNA PROFILING IN OUTLET OBSTRUCTION-INDUCED BLADDER DYSFUNCTION Ali Hashemi Gheinani, Fiona C. Burkhard, Hubert Rehrauer, Catharine Aquino Fournier, and Katia Monastyrskaya Ali Hashemi GheinaniAli Hashemi Gheinani More articles by this author , Fiona C. BurkhardFiona C. Burkhard More articles by this author , Hubert RehrauerHubert Rehrauer More articles by this author , Catharine Aquino FournierCatharine Aquino Fournier More articles by this author , and Katia MonastyrskayaKatia Monastyrskaya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.707AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder outlet obstruction (BOO) results in profound structural and functional changes in the detrusor. An initial hypertrophy develops into an advanced stage of bladder decompensation (fibrosis). Here we determined the miRNA profiles characteristic of defined states of BOO in human patients in order to identify the signalling pathways influencing the switch between hypertrophy and decompensation. METHODS Controls, BOO with and without detrusor overactivity (DO) and acontractile bladders were defined by urodynamics in human subjects. Bladder dome biopsies were collected and RNA isolated. MicroRNA sequencing (miRNA-seq) was used for miRNA profiling of BOO stages (4 groups, n = 5 per group). The Ingenuity Pathways Analysis (IPA) and GeneGo MetaCore software was applied to the data sets of predicted targets for the significantly altered miRNAs. RESULTS Over 500 miRNAs were expressed in patients’ biopsies and controls, and 10-15% were significantly altered at different BOO stages. MiRNA profiling showed activation of TGF-beta and WNT-dependent induction of epithelial-mesenchymal transition, cytoskeletal and extracellular matrix remodeling pathways. Some of the significantly up-regulated bladder miRNAs are known to control hypertrophy in the heart: miR-212/132 cluster (up 3 times in BOO) activates calcineurin/NFAT signaling; miR-182 (up 1.7 times) is pro-fibrotic; miR-34c (up 2.3 times) is induced by hypoxia. Pathway analysis has identified TGF, WNT and cytoskeletal remodeling as the top activated pathway (p-value 1.877e-26; FDR 1.338e-23). There was very little overlap between the miRNA profiles of BOO stages and miRNA alterations in BPS identified previously. CONCLUSIONS MiRNAs play a key role in lower urinary tract dysfunction through their ability to regulate fundamental pathways such as TGF-beta, WNT, cytoskeletal remodeling and cell adhesion. The results define miRNA expression signatures that characterize different states of LUT dysfunction during BOO. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e191 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Ali Hashemi Gheinani More articles by this author Fiona C. Burkhard More articles by this author Hubert Rehrauer More articles by this author Catharine Aquino Fournier More articles by this author Katia Monastyrskaya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A5.18 BMP2 requires TGF-BETA to induce osteophytes during experimental osteoarthritis

Background and ObjectivesOsteophytes are a major hallmark of osteoarthritis (OA). Both TGF-beta and BMP2 can induce osteophytes in murine knee joints. We demonstrated that TGF-beta could induce chondrogenesis in mesenchymal...

A5.3 Elevated levels of BMP2 compensate for loss of TGF-BETA on proteoglycan level in articular cartilage during experimental osteoarthritis

Purpose We demonstrated earlier that in aging articular cartilage TGF-beta signalling via Smad2/3 is drastically reduced and loss of Smad2/3signalling predisposed cartilage for OA. We showed that TGF-beta inhibition reduced...

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Chronic CCl4 intoxication causes liver and bone damage similar to the human pathology of hepatic osteodystrophy: a mouse model to analyse the liver–bone axis

Patients with chronic liver diseases frequently exhibit decreased bone mineral densities (BMD), which is defined as hepatic osteodystrophy (HOD). HOD is a multifactorial disease whose regulatory mechanisms are barely understood. Thus, an early diagnosis and therapy is hardly possible. Therefore, the aim of our study consisted in characterizing a mouse model reflecting the human pathomechanism. Serum samples were collected from patients with chronic liver diseases and 12-week old C57Bl6/N mice after 6-week treatment with carbon tetrachloride (CCl4). Repetitive injections of CCl4 induced liver damage in mice, resembling liver fibrosis in patients, as assessed by serum analysis and histological staining. Although CCl4 did not affect primary osteoblast cultures, μCT analysis revealed significantly decreased BMD, bone volume, trabecular number and thickness in CCl4-treated mice. In both HOD patients and CCl4-treated mice, an altered vitamin D metabolism with decreased CYP27A1, CYP2R1, vitamin D-binding protein GC and increased 7-dehydrocholesterol reductase hepatic gene expression, results in decreased 25-OH vitamin D serum levels. Moreover, both groups exhibit excessively high active transforming growth factor-beta (TGF-β) serum levels, inhibiting osteoblast function in vitro. Summarizing, our mouse model presents possible mediators of HOD, e.g. altered vitamin D metabolism and increased active TGF-β. Liver damage and significant changes in bone structure and mineralization are already visible by μCT analysis after 6weeks of CCl4 treatment. This fast response and easy transferability makes it an ideal model to investigate specific gene functions in HOD.

Immunostaining Protocol: P-Smad2 (Xenograft and Mice)

Metastasis depends on a gene program expressed by the tumor microenvironment upon TGF-beta stimulation. CRC (Colorectal cancer) cell lines did not induce robust stromal TGF-beta responses when injected into nude mice as shown by lack of p-SMAD2 accumulation in tumor-associated stromal cells. To enforce high TGF-beta signaling in xenografts, we engineered CRC cell lines to secrete active TGF-beta. Subcutaneous tumors obtained from HT29-M6TGF-β, KM12L4aTGF-β cells and SW48TGF-β cells contained abundant p-SMAD2+ stromal cells.

Latent Transforming Growth Factor-beta1 Functionalised Electrospun Scaffolds Promote Human Cartilage Differentiation: Towards an Engineered Cartilage Construct

BackgroundTo overcome the potential drawbacks of a short half-life and dose-related adverse effects of using active transforming growth factor-beta 1 for cartilage engineering, a cell-mediated latent growth factor activation strategy was developed incorporating latent transforming growth factor-β1 (LTGF) into an electrospun poly(L-lactide) scaffold.MethodsThe electrospun scaffold was surface modified with NH3 plasma and biofunctionalised with LTGF to produce both random and orientated biofunctionalised electrospun scaffolds. Scaffold surface chemical analysis and growth factor bioavailability assays were performed. In vitro biocompatibility and human nasal chondrocyte gene expression with these biofunctionalised electrospun scaffold templates were assessed. In vivo chondrogenic activity and chondrocyte gene expression were evaluated in athymic rats.ResultsChemical analysis demonstrated that LTGF anchored to the scaffolds was available for enzymatic, chemical and cell activation. The biofunctionalised scaffolds were non-toxic. Gene expression suggested chondrocyte re-differentiation after 14 days in culture. By 6 weeks, the implanted biofunctionalised scaffolds had induced highly passaged chondrocytes to re-express Col2A1 and produce type II collagen.ConclusionsWe have demonstrated a proof of concept for cell-mediated activation of anchored growth factors using a novel biofunctionalised scaffold in cartilage engineering. This presents a platform for development of protein delivery systems and for tissue engineering.

Transforming Growth Factor-Beta 1 (TGF-B1) Liberation from Its Latent Complex During Embryo Implantation and Its Regulation by Estradiol in Mouse1

Transforming growth factor-beta (TGF-B) plays an important role in embryo implantation; however, TGF-B requires liberation from its inactive latent forms (i.e., large latent TGF-B complex [LLC] and small latent TGF-B complex [SLC]) to its biologically active (i.e., monomer or dimer) forms in order to act on its receptors (TGF-BRs), which in turn activate SMAD2/3. Activation of TGF-B1 from its latent complexes in the uterus is not yet deciphered. We investigated uterine latent TGF-B1 complex and its biologically active form during implantation, decidualization, and delayed implantation. Our study, utilizing nonreducing SDS-PAGE followed by Western blotting and immunoblotting with TGF-B1, LTBP1, and latency-associated peptide, showed the presence of LLC and SLC in the uterine extracellular matrix and plasma membranous protein fraction during stages of the implantation period. A biologically active form of TGF-B1 (~17-kDa monomer) was highly elevated in the uterine plasma membranous compartment at the peri-implantation stage (implantation and nonimplantation sites). Administration of hydroxychloroquine (an inhibitor of pro-TGF-B processing) at the preimplantation stage was able to block the liberation of biologically active TGF-B1 from its latent complex at the postimplantation stage; as a consequence, the number of implantation sites was reduced at Day 5 (1000 h), as was the number of fetuses at Day 13. The inhibition of TGF-B1 showed reduced levels of phosphorylated SMAD3. Further, the delayed-implantation mouse model showed progesterone and estradiol coordination to release the active TGF-B1 form from its latent complex in the receptive endometrium. This study demonstrates the importance of liberation of biologically active TGF-B1 during the implantation period and its regulation by estradiol.

M.I.2 The matrix as a modifier for muscular dystrophy

Duchenne muscular dystrophy and the limb girdle muscular dystrophies, like other Mendelian disorders, are modified by genetic and environmental factors. We set out to identify modifiers of muscular dystrophy using a mouse model of LGMD 2C. Mice deleted for gamma sarcoglycan (Sgcg), a dystrophin associated protein, were used for mapping genetic modifiers because humans with LGMD 2C often display a variable phenotype despite having the same identical mutation. We used an intercross between Sgcg mice in the DBA2J background, which confers a severe phenotype, and Sgcg mice in the 129T2/SvEmsJ, which confers a mild background. A genomewide scan identified a locus on murine chromosome 7 that modified two different aspects of muscular dystrophy pathology. The chromosome 7 locus regulated fibrosis and also Evans blue dye uptake into skeletal muscle, a marker of sarcolemmal leakage. An insertion/deletion polymorphism was identified in the Ltbp4 gene, and this polymorphism correlated with severity of muscular dystrophy. In collaboration with the United Dystrophinopathy project, we queried whether human LTBP4 genotype correlated with outcome in DMD patients. We identified that LTBP4 acted as a modifier of DMD. LTBP4 is the latent TGFbeta binding protein and a member of the fibrillin superfamily. The members of this family reside within the extracellular matrix where they participate in binding and sequestering TGFbeta proteins. Release of TGFbeta proteins from the matrix permits ligand binding to cell surface TGFbeta receptors and activation of intracellular TGFbeta signaling. Together, these data, from mouse models and human patients, support that hyper-TGFbeta signaling is pathogenic in DMD and LGMDs, and suggest that reduction of TGFbeta signaling may be therapeutic.

50: Evaluating influences of fructans on cytokine and immunoglobulin production in a human clinical trial

A double-blind, placebo-controlled, randomized crossover clinical trial involving 30 healthy adults was conducted to examine the impact of fructans on both the gut microbiota and immune system. Along with their regular diet, trial participants consumed either an oligofructose-enriched inulin supplement as the test diet or food grade maltodextrin as the control diet for a 28 day period, with a minimum 2 week washout period before cross-over. The trial supplements were coded as “A” or “B”, and will remain coded until the completion of all analyses. Serum immunoglobulin concentrations were quantified by Luminex multiplexing technology. IgM was significantly higher after 28 days of supplement A consumption (821.3 ng/mL ± 138.3) than after supplement B consumption (485.8 ng/mL ± 66.5) ( P = 0.0118). IgA was also significantly higher ( P = 0.0092) after supplement A consumption (1967.3 ng/mL ± 374.4) than after supplement B consumption (911.7 ng/mL ± 138.2). Active transforming growth factor-beta (TGF- β ) concentration was measured in plasma and serum cytokine concentrations were quantified by Enzyme-linked immunosorbent assay (ELISA). Levels of active TGF- β were 5.4 pg/mL ± 2.1 after consumption of supplement B, in comparison to 3.4 pg/mL ± 1.5 after supplement A consumption, although this difference was not statistically significant. IL-10 concentrations were significantly lower ( P = 0.0172) after supplement B consumption (41.2 pg/mL ± 29.0) than after supplement A consumption (79.8 pg/mL ± 42.1). Granulocyte colony-stimulating factor (G-CSF) levels were also significantly lower ( P = 0.0265) after supplement B consumption (5.4 pg/mL ± 2.5) than levels after supplement A consumption (14.4 pg/mL ± 3.6). No differences were observed between subjects consuming supplement A and B in Interleukin-8 (IL-8) and IL-1Ra concentrations. Analysis of cytokine profiles will provide insight into the extent of the potential effects of dietary fructans on the immune system mediated through the gut microbiota.

Xiamenmycin Attenuates Hypertrophic Scars by Suppressing Local Inflammation and the Effects of Mechanical Stress

Hypertrophic scarring is a common disease affecting millions of people around the world, but there are currently no satisfactory drugs to treat the disease. Exaggerated inflammation and mechanical stress have been shown to be two main mechanisms of excessive fibrotic diseases. Here we found that a benzopyran natural product, xiamenmycin, could significantly attenuate hypertrophic scar formation in a mechanical stretch-induced mouse model. The compound suppressed local inflammation by reducing CD4+ lymphocyte and monocyte/macrophage retention in fibrotic foci and blocked fibroblast adhesion with monocytes. Both in vivo and in vitro studies found that the compound inhibited the mechanical stress-induced profibrotic effects by suppressing proliferation, activation, fibroblast contraction, and inactivating FAK, p38, and Rho guanosine triphosphatase signaling. Taken together, the compound could simultaneously suppress both the inflammatory and mechanical stress responses, which are the two pivotal pathological processes in hypertrophic scar formation, thus suggesting that xiamenmycin can serve as a potential agent for treating hypertrophic scar formation and other excessive fibrotic diseases.

Abstract LB-285: Pro-tumor N2 neutrophils contribute to the pre-metastatic niche.

Abstract Metastasis is the primary cause of cancer-related mortalities and the tissue microenvironment that supports tumor cell attachment and growth at metastatic sites has become an active area of investigation. Here we present evidence that pro-tumor N2 neutrophils are one of the bone marrow-derived cell types that establish this pre-metastatic niche. In the murine Lewis Lung adenocarcinoma (LLC) in vivo metastasis model, tumor cells metastasize to the lungs with increasing primary tumor growth over time. In these studies, immunocompetent mice injected subcutaneously with syngeneic LLC cells are sacrificed sequentially 10-32 days later, before and after established lung metastases. Neutrophil chemoattractants KC/CXCL1 and MIP-2/CXCL2 are significantly elevated in pre-metastatic tissue compared to normal lungs of healthy contemporary controls. MCP-1/CCL2, a marker of pro-tumor N2 neutrophils, increases steadily over time in pre-metastatic lung tissue of study mice and is significantly elevated in pre-metastatic tissue and in metastases compared to normal lung tissue. Isolated lung-infiltrating neutrophils also express N2 markers. In contrast, TNF-alpha, a marker for anti-tumor N1 cells, remains low or undetectable in lung tissue throughout metastatic tumor development. Neither active nor total TGF-beta, the putative control switch for a pro-tumor phenotype, is elevated in pre-metastatic tissue. Notably, levels of all cytokines are low or undetectable in normal lungs from naïve mice and in other organs of tumor-bearing mice (non-metastatic sites). Ly6G+ neutrophils were detected at a higher density in the lungs of pre-metastatic and metastatic mice compared to normal lungs, although the density of myeloperoxidase (MPO)-stained cells was similar in all groups. These data are consistent with recent reports that MPO activity is low or absent from tumor-associated neutrophils, although high in MDSC and naïve neutrophils. Neutrophil distribution in metastatic lung tissue is most concentrated around the tumor foci and tumor vasculature. Finally, studies of lung tissue from mice bearing LLC variants with different metastatic capacities are also consistent with our premise that neutrophils help establish the pre-metastatic niche. Understanding the factors that regulate the development and growth of malignant cells at metastatic sites is critical for improved clinical strategies in the prevention, diagnosis and treatment of metastases. Citation Format: Jered Cope Meyers, H. Keith Pittman, Hunter Story, Dare M. Imes, George A. Howard, Kathryn M. Verbanac. Pro-tumor N2 neutrophils contribute to the pre-metastatic niche. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-285. doi:10.1158/1538-7445.AM2013-LB-285

Abstract 3692: Vitamin D as a chemoprophylactic agent for liver cancer.

Abstract Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death worldwide. One third of patients with cirrhosis will develop HCC, thus comprising a well-characterized population at high-risk for the development of this deadly, yet potentially preventable cancer. Vitamin D (VD) has been implicated in the prevention of multiple cancers- some with inactivation of TGF-beta signaling. Recently, inactivation of TGF-beta signaling has been associated with HCC. The identification of subgroups of patients responsive to treatment is vital for preventing this disease effectively. In an effort to understand the role of Vit D in HCC and to identify clear responders, we analyzed the role of Vit D in the context of TGF-beta inactivation. Methods: The effect of calcitriol on cell proliferation was measured in HCC cell lines as well as in Hep-G2 cells that were knocked down to the adaptor protein β2-spectrin (Spnb2). To evaluate in vivo effects of VD on animals predisposed to developing liver cancer, Spnb2+/− mice were fed with special diets containing 1000 IU VD/kg/D or 10,000 IU VD/kg/D. After 6 weeks, the animals were euthanized and hepatocyte proliferation and the content of fat in the liver were analyzed through Ki67 staining and oil red staining, respectively. The expression of cyclin D1 was evaluated by Western blotting (WB). Lastly, liver samples from patients with HCC who had received VD supplementation in the diet were also evaluated by immunohistochemistry. Results: We observed a dramatic response to treatment with calcitriol as indicated by a significant increase in the expression of CYP24A1. Additionally, treatment with VD and calcitriol suppressed the growth of HCC cell lines. Absence of Spnb2 caused more prominent suppression of the growth of Hep-G2. From our animal studies we found that steatosis and hepatocyte proliferation were higher in Spnb2+/− mice fed with a low dose of VD, and that the high dose VD significantly reduced both parameters. The increased proliferation in Spnb2+/− mice fed with a low dose of VD was associated with higher levels of cyclin D1 expression, which was restored the level almost to that seen in the wild type mice with the higher dose of VD. Lastly, VD supplementation to patients with HCC was associated with higher expression of β2-spectrin (p<.0001) and TGFBRII (p=.013) compared with that of patients who did not receive the supplement. Conclusions: VD suppresses the proliferation of several HCC cell lines, and in agreement with this, high doses of VD in the diet suppress hepatocyte proliferation and steatosis in the HCC prone Spnb2+/− mice. Our data also indicate that VD administration appears to restore the expression of key cell cycle proteins even in the setting of TGF-β signaling disruption. Taken together, these preliminary results suggest that VD treatment strategies could potentially be pivotal in preventing obesity-induced HCC. Citation Format: Lior H. Katz, Vivek Shukla, Sara Peleg, Kirti shetty, Jian Chen, Steven Curley, Powel H. Brown, Lopa Mishra. Vitamin D as a chemoprophylactic agent for liver cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3692. doi:10.1158/1538-7445.AM2013-3692

Apc deficiency alters pulmonary epithelial cell fate and inhibits Nkx2.1 via triggering TGF-beta signaling

Wnt signaling is critical for cell fate specification and cell differentiation in many organs, but its function in pulmonary neuroendocrine cell (PNEC) differentiation has not been fully addressed. In this study, we examined the role of canonical Wnt signaling by targeting the gene for Adenomatous Polyposis Coli (Apc), which controls Wnt signaling activity via mediating phosphorylation of beta-catenin (Ctnnb). Targeting the Apc gene in lung epithelial progenitors by Nkx2.1-cre stabilized Ctnnb and activated canonical Wnt signaling. Apc deficiency altered lung epithelial cell fate by inhibiting Clara and ciliated cell differentiation and activating Uchl1, a marker of neuroendocrine cells. Similar to PNEC in normal lung, Uchl1positive cells were innervated. In mice with targeted inactivation of Ctnnb by Nkx2.1-cre, PNEC differentiation was not interrupted. These indicate that, after lung primordium formation, Wnt signaling is not essential for PNEC differentiation; however, its over-activation promotes PNEC features. Interestingly, Nkx2.1 was extinguished in Apc deficient epithelial progenitors before activation of Uchl1. Examination of Nkx2.1 null lungs suggested that early deletion of Nkx2.1 inhibits PNEC differentiation, while late repression does not. Nkx2.1 was specifically inhibited in Apc deficient lungs but not in Ctnnb gain-of-function lungs indicating a functional difference between Apc deletion and Ctnnb stabilization, both of which activate Wnt signaling. Further analysis revealed that Apc deficiency led to increased TGF-beta signaling, which inhibited Nkx2.1 in cultured lung endodermal explants. In contrast, TGF-beta activity was not increased in Ctnnb gain-of-function lungs. Therefore, our studies revealed an important mechanism involving Apc and TGF-beta signaling in regulating the key transcriptional factor, Nkx2.1, for lung epithelial progenitor cell fate determination.