However, more experiments are necessary to determine if the BFAinsensitive pathway described here is indeed independent of the Golgi. Our results are consistent with a previous report where two distinct targeting pathways were proposed for TIP3;1 and BP-80 using a transient assay in tobacco protoplasts [17], and for a BFAinsensitive pathway for TIP3;1 trafficking in the same system [21]. Multiple pathways for membrane proteins to the vacuole were also identified in transient assays using tobacco leaf epidermis. In that system, the calcineurin binding protein CBL6 was trafficked in a COPII-independent manner, while aTIP/TIP3;1 and Vam3/ SYP22 were not [23]. It is unclear if the targeting of TIP3;1 via the BFA-insensitive pathway is also COPII-dependent in Arabidopsis. While BFA treatments may be used to differentiate these two pathways as well, BFA affects all Golgi-dependent pathways and it also has major effects on endomembrane morphology and endocytosis [53,61]. The lack of effects of C834 on the localization of at least twelve endomembrane markers suggests that C834 is a unique new tool that may be used for targeted inhibition of the BFA-insensitive pathway for tonoplast proteins in Arabidopsis roots. C834 may inhibit an important component of the endomembrane system involved in the BFA-insensitive pathway for membrane proteins. Identification of the C834 target(s) may shed light into the mechanisms of this pathway and its interactions with other trafficking pathways. However, the possibility exists that, in contrast to that of hypocotyls and protoplasts, the trafficking of the TIP3;1-YFP and GFP-TIP2;1 is Golgi-dependent in Arabidopsis roots, and in this case C834 would target a highly specific but unknown mechanism for TIP3;1 and TIP2;1 traffic to the vacuole. If this were the case, this would add one more layer of complexity to the regulation of the endomembrane system. We cannot fully exclude the possibility that C834 acts by enhancing a retrograde traffic of tonoplast proteins from the vacuole back to the ER instead of acting as an inhibitor of anterograde traffic. However, the loss of TIP3;1-YFP and GFPTIP2;1 from the vacuolar membrane during C834 treatments are not consistent with this possibility, as proteins that reach the ER by these means would be expected to eventually enter the anterograde pathway and reach the vacuole to some extent. More mechanistic and detailed analysis of the C834 inhibition could be used to differentiate between these possibilities, and our lab is moving in this direction.

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.

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. A number of recent reports are identifying new biological processes that might be influenced by endogenous receptor ligands. For example,
descriptions of mice harboring a null allele at the Ahr locus indicate that receptor signaling plays an important role in normal cardiovascular development and function [3,4].

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