Cross-talk between 1,25-dihydroxyvitamin D-3 and Transforming Growth Factor-beta Signaling requires Binding of VDR and Smad3 Proteins to their cognate DNA Recognition Elements

1,25-Dihydroxyvitamin D-3 (vitamin D) and transforming growth factor-beta (TGF-beta) regulate diverse biological processes including cell proliferation and differentiation through modulation of the expression of target genes. Members of the Smad family of proteins function as effecters of TGF-beta signaling pathways whereas the vitamin D receptor (VDR) confers vitamin D signaling. We investigated the molecular mechanisms by which TGF-beta and vitamin D signaling pathways interact in the regulation of the human osteocalcin promoter. Synergistic activation of the osteocalcin gene promoter by TGF-beta and vitamin D was observed in transient transfection experiments. However, in contrast to a previous report by Yanagisawa, J., Yanagi, Y., Masuhiro, Y., Suzawa, M., Watanabe, M., Kashiwagi, K., Toriyabe, T., Rawabata, M., Miyazono, K., and Kato, S. (1999) Science, 283, 1317-1321, synergistic activation was not detectable when the osteocalcin vitamin D response element (VDRE) alone was linked to a heterologous promoter. Inclusion of the Smad binding elements (SBEs) with the VDRE in the heterologous promoter restored synergistic activation. Furthermore, this synergy was dependent on the spacing between VDRE and SBEs. The Smad3-Smad4 heterodimer was found to bind in gel shift assay to two distinct DNA segments of the osteocalcin promoter: -1030 to -989 (SBE3) and -418 to -349 (SBE1). Deletion of SBE1, which is proximal to the VDRE, brit not the distal SBE3 in this promoter reporter abolished TGF-beta responsiveness and eliminated synergistic co-activation with vitamin D. Thus the molecular mechanism, whereby Smad3 and VDR mediate cross-talk between the TGF-beta acid vitamin D signaling pathways, requires both a VDRE and a SBE located in close proximity to the target promoter

Enhanced transactivation activity of vitamin D receptor B1 associated with focal nuclear accumulation and cofactor binding

Biological responses to 1,25 dihydroxyvitamin D3 are mediated by the vitamin D receptor (VDR), of which there are two protein isoforms. The A/B region of the recently identified VDRB1 isoform is extended at the N-terminus by 50 amino acids producing a 73 amino acid A/B region, compared with the 23 amino acids of the conventional VDRA receptor. As A/B regions of nuclear receptors often contain ligand-independent activation function (AF-1) domains, the functional properties of this region of VDRB1 might differ from those of VDRA. The VDRB1 isoform exhibited greater (144% ± 17%) transactivation of a rat 1,25-dihydroxvitamin D-24-hydroxylase reporter construct than VDRA in transiently transfected COS-1 cells 6 hr post treatment with 10nM 1,25-dihydroxyvitamin D3. This difference was not apparent at 16 hr post treatment. Evidence for AF-1 activity in the VDRB1 N-terminal A/B region was observed in yeast and mammalian one-hybrid experiments. The VDRB1 A/B region exhibited strong AF-1 activity (8-fold ligand-independent activation), compared to the VDRA A/B region (3-fold activation). Similarly, there was stronger interaction between the A/B region of VDRB1 and the VDR ligand binding domain (LBD) in mammalian two-hybrid and GST pull-down assays (3-fold in the presence of ligand, whereas interaction between the VDRA A/B domain and the LBD was weak. Introduction of an E420Q mutation into the LBD, which inactivates C-terminal ligand-dependent transactivation (AF-2) activity by abolishing recruitment of coactivators, markedly reduced but did not abolish ligand-dependent interaction with the VDRB1 A/B domain. This suggests that, as well as cofactor mediated interaction between the VDRB1 A/B domain and the LBD, there may be a direct AF-1/AF-2 interaction. In the absence of ligand, GFP-tagged VDRB1 accumulated in discrete nuclear foci whereas VDRA exhibited a diffuse nuclear signal. VDRB1 foci were not observed with the E420Q mutation, suggesting that speckle formation may be mediated at least in part by A/B domain-LBD interaction or by cofactor recruitment in the absence of ligand. Ligand-independent interaction of p160 co-activator proteins with full-length wildtype VDRB1, but not with the E420Q mutant VDRB1 or wildtype VDRA in GST-pull-down assays, supports the latter concept. Thus, accumulation of VDRB1 in nuclear foci may relate to its differential binding to transcriptional co-activators in the absence of ligand and the physical association of cofactors in the absence of ligand may allow for a more rapid response to ligand leading to the more effective transactivation observed

Degarelix treatment is compatible with diabetes and antithrombotic therapy in patients with prostate cancer

Suguru Tokiwa,1 Hiroaki Shimmura,1 Shuhei Nomura,2–4 Ryota Watanabe,1 Minoru Kurita,1 Naoto Yoshida,1 Kaori Yamashita,1 Yoshitaka Nishikawa,5 Alexander Kouzmenko,6 Shigeaki Kato4 1Department of Urology, Jyoban Hospital, Tokiwa Foundation, Iwaki, Fukushima, Japan; 2Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, London, UK; 3Department of Global Health Policy, Graduate School of Medicine, The University of Tokyo, Tokyo, 4Research Institute of Innovative Medicine, Tokiwa Foundation, Iwaki, 5Department of Health Informatics, School of Public Health, Kyoto University, Kyoto, Japan; 6Department of Life Sciences, Alfaisal University, Riyadh, Kingdom of Saudi Arabia Introduction: Therapeutically induced androgen deficiency (AD) is a standard treatment for patients with prostate cancer, but it is often associated with various adverse effects (AEs) that may lead to discontinuation. Some AEs may depend on the patient’s health condition, while others may be due to complications of the drug delivery method. Degarelix is a gonadotropin-releasing hormone (GnRH) antagonist widely used for the treatment of androgen-dependent prostate cancer. This study aimed to ascertain the following: 1) the compatibility of degarelix treatment with diabetes and 2) any specific causal associations of degarelix injections with increased blood clotting and antithrombotic therapy requirements.Patients and methods: The medical records of 162 patients with prostate cancer who had undergone degarelix treatment were retrospectively examined. The association of a medical history of diabetes and anticoagulant co-treatment with degarelix treatment discontinuation was analyzed statistically.Results: Rapid and significant decreases in prostate-specific antigen (PSA) levels during the course of degarelix treatment were detected for patients with prostate cancer regardless of clinical state. During the 27 months of treatment, 68 subjects (48%) ceased degarelix treatment, owing to several reasons, mainly financial issues. Among these subjects, 19 had diabetes, while 35 were treated with antithrombotics. Extensive statistical analysis indicated that there were no causal associations between degarelix treatment discontinuation and preexisting diabetes or antithrombotic therapy.Conclusion: Our study suggests that preexisting diabetes and antithrombotic therapy were not significant factors for the discontinuation of degarelix treatment in patients with prostate cancer. Keywords: GnRH antagonists, prostate cancer, degarelix, discontinuation, diabetes, antithrombotic treatmen

Lentiviral gene therapy vector with UCOE stably restores function in iPSC-derived neutrophils of a CDG patient

A recent gamma-retroviral clinical Chronic Granulomatous Disease (CGD) gene therapy (GT) trial achieved proof-of-concept but was accompanied by activation of oncogenes and transgene silencing. The ubiquitous chromatin opening element (UCOE) comprises the sequences of two divergently oriented house-keeping gene promoters and is known to have anti-silencing properties. In a screen we identified two novel UCOE constructs that prevent adjacent promoter methylation in P19 cells. Experiments were continued with the shorter UCOE constructs in induced pluripotent stem cells (iPSC) derived from a p47phox-deficient CGD patient. The iPSC line was transduced with the lentiviral GT vectors expressing p47 under the constitutively active SFFV promoter with UCOE element (UCOE_SF) and without UCOE element (SF) adjacent to the SFFV promoter. The iPSC were expanded before propagation towards neutrophils. 20 days after transduction the UCOE_SF vector was protected from methylation in iPSC as previously shown in P19 cells, whereas the SF vector was heavily methylated in iPSC. The UCOE_SF vector maintained stable transgene expression in iPSC, macrophages and neutrophils, whereas the SF vector was strongly silenced. The UCOE_SF vector stably restored ROS production in neutrophils, whereas for the SF vector the count of ROS producing cells was marginal. To conclude, we have shown that the prevention of transgene silencing by UCOE is functionally and mechanistically preserved upon terminal neutrophil differentiation