For the markers that could be tested in roots (TIP1;1-YFP, TIP2;2-YFP and TIP2;3-YFP), we found that C834 induced a sharp decrease in protein accumulation of TIP2;2 and TIP2;3, maybe due to increased protein degradation at the ER. In contrast and similarly to the overexpression line, TIP1;1-YFP showed no change in protein accumulation or localization after C834 treatment (not shown). Even though we could not definitely determine that TIP2;2 and TIP2;3 were localized to the ER under C834 treatment, the fact that the fluorescence levels of this marker were drastically reduced indicates an effect on its targeting or stability. Therefore, the possibility exists that TIP2;2 and TIP2;3 may also be trafficked via BFA-insensitive mechanisms. Since neither of the TIP proteins contain a C-terminal di-leucine motif that was recently identified as a tonoplast targeting signal in the inositol transporter INT1 [65], targeting signals for this protein family remain unknown.
ER-Golgi traffic is BFA sensitive in hypocotyls
BFA has been used in eukaryotic cells as an inhibitor of ERGolgi traffic because its inhibition on the activity of Golgi-localized guanine-nucleotide exchange factors (GEF) for the ADP ribosylation factor (ARF) small GTPase. The active form of ARF is essential for COPI coat formation and retrograde trafficking from the Golgi to the ER [19]. Thus, BFA treatment in yeast, animal cells and most plant cells result in redistribution of Golgi membranes in to the ER. BFA also affects plasma membrane recycling in plants due to the existence of plant specific ARF-GEFs involved in recycling of plasma membrane proteins [66].
While most eukaryotic ARF-GEFs are BFA sensitive, Arabidopsis encodes two BFA resistant ARF-GEFs, GNOM-like 1 (GNL1) which is involved in ER-Golgi traffic [32,54] and BIG3 [67]. BFA does not induce Golgi redistribution to the ER in Arabidopsis roots, because GNL1 is active in those cells and instead, Golgi stacks (as labeled by gamma COP, and GNL1-YFP) cluster around BFA compartments labeled with the TGN markers VHAa1 and FM4-64, while the TGN is presumably functional [68]. However, in leaf cells, typical effects of BFA on Golgi has been demonstrated indicating that a BFA-sensitive ARF-GEF is important for ER-Golgi traffic [53]. While a previous report using a N-a-2,6-sialyltransferase(ST)-GFP fusion did not show ERGolgi traffic inhibition in hypocotyls [53], we found that some Golgi stacks do reabsorb in the ER during BFA treatment when NAG1-EGFP was used. The differences between the BFA treatments of NAG1-EGFP and ST-GFP in Arabidopsis hypocotyls may be due to compartmentalization of these markers within the Golgi stacks. ST is targeted to the trans side of the Golgi both in mammalian cells [69] and in Arabidopsis when it is expressed ectopically [70]. NAG1 is targeted to the medial Golgi in mammalian cells [71] and to an early Golgi compartment in tobacco cells, where it is required for an early N-glycosylation reaction [72]. This suggests that cis and medial Golgi stacks may be reabsorbed to the ER in Arabidopsis hypocotyls, but trans cisternae are not. Overall, our results indicated that a BFA-sensitive ARFGEF is expressed in hypocotyls and regulates retrograde traffic at the Golgi compartment, and BFA can be used in Arabidopsis hypocotyls for assessing Golgi-dependent mechanisms.with mutants with altered sensitivity to C834 will sort between these possibilities. Taken as a whole, this unique inhibitor could be a valuable tool to characterize the complexity of PIN2 vacuolar trafficking and its interactions with the BFA-insensitive pathway and the light response.
Abstract
The experimental compound SU5416 went as far as Phase III clinical trials as an anticancer agent, putatively because of its activity as a VEGFR-2 inhibitor, but showed poor results. Here, we show that SU5416 is also an aryl hydrocarbon receptor (AHR) agonist with unique properties. Like TCDD, SU5416 favors induction of indoleamine 2,3 dioxygenase (IDO) in immunologically relevant populations such as dendritic cells in an AHR-dependent manner, leading to generation of regulatory T-cells in vitro. These characteristics lead us to suggest that SU5416 may be an ideal clinical agent for treatment of autoimmune diseases and prevention of transplant rejection, two areas where regulatory ligands of the AHR have shown promise. At the same time, AHR agonism might represent a poor characteristic for an anticancer drug, as regulatory T-cells can inhibit clearance of cancer cells, and activation of the AHR can lead to upregulation of xenobiotic metabolizing enzymes that might influence the half-lives of co-administered chemotherapeutic agents. Not only does SU5416 activate the human AHR with a potency approaching 2,3,7,8-tetrachlorodibenzo-p-dioxin, but it also activates polymorphic murine receptor isoforms (encoded by the Ahrd and Ahrb1 alleles) with similar potency, a finding that has rarely been described and may have implications in identifying true endogenous ligands of this receptor.
Citation: Mezrich JD, Nguyen LP, Kennedy G, Nukaya M, Fechner JH, et al. (2012) SU5416, a VEGF Receptor Inhibitor and Ligand of the AHR, Represents a New Alternative for Immunomodulation. PLoS ONE 7(9): e44547. doi:10.1371/journal.pone.0044547 ~ Editor: Jose Carlos Alves-Filho, University of Sao Paulo, Brazil Received March 6, 2012; Accepted August 8, 2012; Published September 6, 2012 Copyright: ?2012 Mezrich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by Grant 1UL1RR025011 from the Clinical and Translational Science Award program of the National Center for Research Resources, National Institutes of Health (JDM), National Institutes of Environmental Health Services Grant R37ES005703 (CAB), National Cancer Institute Grant P30CA014520 (CAB). JDM is a John Merrill Grant Scholar of the American Society of Nephrology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: CAB has served as a scientific consultant to Dow Chemical Co. on issues related to dioxin toxicity. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. There are no patents, products in development or marketed products.
Introduction
Agonists of the aryl hydrocarbon receptor (AHR) have been of interest to the pharmaceutical industry for many years. This interest originally stemmed from the observation that the AHR is a ligand-activated transcription factor that regulates the adaptive metabolism of xenobiotics [1] and because receptor binding is a known step in the carcinogenic and toxic action of environmental pollutants like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [2]. Thus, agonism of the AHR has commonly been considered a signature for drugs that upregulate phase-I and phase-II metabolic systems and also for chemicals with pharmacological similarity to a known human carcinogen. As a result, AHR agonism has largely been considered a hazard signature for environmental chemicals and drugs in the pharmaceutical pipeline. Recent insights related to the normal physiological role of the AHR are changing our view of receptor agonism to one where agonism might be considered to hold therapeutic value.
