Faculty Bibliography

Antioxidants cause stabilization and nuclear translocation of NF-E2-related factor 2 (Nrf2), where it binds to the antioxidant response element (ARE) and induces up-regulation of defensive genes that protect cells against oxidative and electrophilic stress. Bach1, the negative regulator of Nrf2, competes with Nrf2 for binding to the ARE in the human NQO1 promoter. In this study, we demonstrate that Bach1 exits the nucleus within 1-2 h upon antioxidant treatment. Genistein, an inhibitor of tyrosine kinases, blocked nuclear export of Bach1. Site-directed mutagenesis and immunoprecipitation assays identified tyrosine 486 that was phosphorylated in response to the antioxidant and was essential for nuclear export of Bach1. Chromatin immunoprecipitation assays revealed a competitive interplay between Bach1 and Nrf2 at 1-2 and 4 h for binding to the human NQO1 ARE. Luciferase and real time PCR assays showed a significant decrease in antioxidant induction of reporter activity and mRNA levels in cells transfected with mutant Bach1 compared with wild type. This decrease was due to the absence of nuclear export of the mutant protein. Bach1 levels inside the nucleus returned to normal at 4 h after antioxidant treatment in the absence but not in the presence of protein synthesis inhibitor cycloheximide. In addition, antioxidant treatment increased the transcription of Bach1 as shown by pulse chase and real time PCR experiments. Taken together, these results indicate that increased synthesis of Bach1 restored its nuclear levels to normal at 4 h. In conclusion, antioxidant-induced tyrosine 486 phosphorylation leads to nuclear exit of Bach1, thus allowing Nrf2 access to the ARE.

Endoplasmic reticulum (ER) stress has been linked to the onset and progression of many diseases. SIL1 is an adenine nucleotide exchange factor of the essential ER lumen chaperone HSPA5/BiP that senses ER stress and is involved in protein folding. Mutations in the Sil1 gene have been associated with Marinesco-Sjogren syndrome, hallmarks of which include ataxia and cerebellar atrophy. We have previously shown that loss of SIL1 function in mouse results in ER stress, ubiquitylated protein inclusions, and degeneration of specific Purkinje cells in the cerebellum. Here, we report that overexpression of HYOU1/ORP150, an exchange factor that works in parallel to SIL1, prevents ER stress and rescues neurodegeneration in Sil1(-/-) mice, whereas decreasing expression of HYOU1 exacerbates these phenotypes. In addition, loss of DNAJC3/p58(IPK), a co-chaperone that promotes ATP hydrolysis by BiP, ameliorates ER stress and neurodegeneration in Sil1(-/-) mice. These findings suggest that alterations in the nucleotide exchange cycle of BiP cause ER stress and neurodegeneration in Sil1-deficient mice. Our results present the first evidence of important genetic modifiers of Marinesco-Sjogren syndrome, and provide additional pathways for therapeutic intervention for this, and other ER stress-induced, diseases

Diabetic wounds are a significant public health burden, with slow or nonhealing diabetic foot ulcers representing the leading cause of non-traumatic lower limb amputation in developed countries. These wounds heal poorly as a result of compromised blood vessel formation in response to ischemia. We have recently shown that this impairment in neovascularization results from a high glucose-induced defect in transactivation of hypoxia-inducible factor-1alpha (HIF-1alpha), the transcription factor regulating vascular endothelial growth factor (VEGF) expression. HIF-1 dysfunction is the end result of reactive oxygen species-induced modification of its coactivator p300 by the glycolytic metabolite methylglyoxal. Use of the iron chelator-antioxidant deferoxamine (DFO) reversed these effects and normalized healing of humanized diabetic wounds in mice. Here, we present additional data demonstrating that HIF-1alpha activity, not stability, is impaired in the high glucose environment. We demonstrate that high glucose-induced impairments in HIF-1alpha transactivation persist even in the setting of constitutive HIF-1alpha protein overexpression. Further, we show that high glucose-induced hydroxylation of the C-terminal transactivation domain of HIF-1alpha (the primary pathway regulating HIF-1alpha/p300 binding) does not alter HIF-1alpha activity. We extend our study of DFO's therapeutic efficacy in the treatment of impaired wound healing by demonstrating improvements in tissue viability in diabetic mice with DFO-induced increases in VEGF expression and vascular proliferation. Since DFO has been in clinical use for decades, the potential of this drug to treat a variety of ischemic conditions in humans can be evaluated relatively quickly.

Cancer cells exist in harsh microenvironments that are governed by various factors, including hypoxia and nutrient deprivation. These microenvironmental stressors activate signaling pathways that affect cancer cell survival. While others have previously measured microenvironmental stressors in tumors, it remains difficult to detect the real-time activation of these downstream signaling pathways in primary tumors. In this study, we developed transgenic mice expressing an X-box binding protein 1 (XBP1)-luciferase construct that served as a reporter for endoplasmic reticulum (ER) stress and as a downstream response for the tumor microenvironment. Primary mammary tumors arising in these mice exhibited luciferase activity in vivo. Multiple tumors arising in the same mouse had distinct XBP1-luciferase signatures, reflecting either higher or lower levels of ER stress. Furthermore, variations in ER stress reflected metabolic and hypoxic differences between tumors. Finally, XBP1-luciferase activity correlated with tumor growth rates. Visualizing distinct signaling pathways in primary tumors reveals unique tumor microenvironments with distinct metabolic signatures that can predict for tumor growth.

Development involves the establishment of boundaries between fields specified to differentiate into distinct tissues. The Drosophila larval eye-antennal imaginal disc must be subdivided into regions that differentiate into the adult eye, antenna and head cuticle. We have found that the transcriptional co-factor Chip is required for cells at the ventral eye-antennal disc border to take on a head cuticle fate; clones of Chip mutant cells in this region instead form outgrowths that differentiate into ectopic eye tissue. Chip acts independently of the transcription factor Homothorax, which was previously shown to promote head cuticle development in the same region. Chip and its vertebrate CLIM homologues have been shown to form complexes with LIM-homeodomain transcription factors, and the domain of Chip that mediates these interactions is required for its ability to suppress the eye fate. We show that two LIM-homeodomain proteins, Arrowhead and Lim1, are expressed in the region of the eye-antennal disc affected in Chip mutants, and that both require Chip for their ability to suppress photoreceptor differentiation when misexpressed in the eye field. Loss-of-function studies support the model that Arrowhead and Lim1 act redundantly, using Chip as a co-factor, to prevent retinal differentiation in regions of the eye disc destined to become ventral head tissue

Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to 1) delineate cell membrane protein signals and associate them with a specific nucleus; 2) compute a coupled representation of the multiplexed DNA content with membrane proteins; 3) rank computed features associated with such a multidimensional representation; 4) visualize selected features for comparative evaluation through heatmaps; and 5) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables

Formate transport across the inner membrane is a critical step in anaerobic bacterial respiration. Members of the formate/nitrite transport protein family function to shuttle substrate across the cytoplasmic membrane. In bacterial pathogens, the nitrite transport protein is involved in protecting bacteria from peroxynitrite released by host macrophages. We have determined the 2.13-A structure of the formate channel FocA from Vibrio cholerae, which reveals a pentamer in which each monomer possesses its own substrate translocation pore. Unexpectedly, the fold of the FocA monomer resembles that found in water and glycerol channels. The selectivity filter in FocA consists of a cytoplasmic slit and a central constriction ring. A 2.5-A high-formate structure shows two formate ions bound to the cytoplasmic slit via both hydrogen bonding and van der Waals interactions, providing a structural basis for the substrate selectivity of the channel

Several lines of evidence point to the central role of WNT signaling in the initiation of intestinal tumorigenesis, most often due to loss of APC, a negative regulator of the WNT-betaCATENIN/TCF pathway. Modeling human colon cancers in mice through loss of Apc has shown that inappropriate activation of Wnt signaling is sufficient to induce adenoma formation. More recent analyses have also demonstrated a key role for HEDGEHOG-GLI (HH-GLI) signaling in human colon cancers. However, how the WNT and HH pathways interact during intestinal development, homeostasis and cancer is not clear. Marker analyses suggest predominant paracrine signaling from rare Shh producing cells in the crypt's bottom to adjacent Gli1(+) mesenchymal cells in normal adult mice. Using conditional KO models, we show that inhibition of the function of the critical Hh mediator Smoothened (Smo) rescues the lethality and intestinal phenotypes of loss of Apc. The results uncover an essential role of the Hh pathway in tumors induced by hyperactive Wnt signaling, suggest the action of the Hh pathway in parallel or downstream of Wnt signaling, and validate this model for its use in preclinical work testing Hh pathway antagonists.