Valerie F. Curtis

Tryptophan metabolite activation of the aryl hydrocarbon receptor regulates IL-10 receptor expression on intestinal epithelia

IL-10 is a potent anti-inflammatory cytokine that inhibits the production of proinflammatory mediators. Signaling by IL-10 occurs through the IL-10 receptor (IL-10R), which is expressed in numerous cell types, including intestinal epithelial cells (IECs), where it is associated with development and maintenance of barrier function. Guided by an unbiased metabolomics screen, we identified tryptophan (Trp) metabolism as a major modifying pathway in interferon-γ (IFNγ)-dominant murine colitis. In parallel, we demonstrated that IFNγ induction of indoleamine 2,3-dioxygenase 1, an enzyme that catalyzes the conversion of Trp to kynurenine (Kyn), induces IL-10R1 expression. Based on these findings, we hypothesized that IL-10R1 expression on IEC is regulated by Trp metabolites. Analysis of the promoter region of IL-10R1 revealed a functional aryl hydrocarbon response element, which is induced by Kyn in luciferase-based IL-10R1 promoter assays. Additionally, this analysis confirmed that IL-10R1 protein levels were increased in response to Kyn in IEC in vitro. Studies using in vitro wounding assays revealed that Kyn accelerates IL-10-dependent wound closure. Finally, reduction of murine dextran sodium sulfate colitis through Kyn administration correlates with colonic IL-10R1 expression. Taken together, these results provide evidence on the importance of IL-10 signaling in intestinal epithelia and implicate AHR in the regulation of IL-10R1 expression in the colon.

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses

There is interest in understanding posttranslational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down‐regulated 8 (NEDD8) to promote protein stabilization. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia‐inducible factor (HIF; via Cullin‐2). Here, we elucidate the role of human deneddylase‐1 (DEN‐1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN‐1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4.7 nM) stabilized HIF‐1a, activated HIF promoter activity by 2.5‐fold, and induced HIF‐target genes in human epithelial cells up to 5‐fold. Knockdown of DEN‐1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0.5‐fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN‐1 is a regulator of cullin neddylation and fine‐tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease.—Curtis, V. F., Ehrentraut, S. F., Campbell, E. L., Glover, L. E., Bayless, A., Kelly, C. J., Kominsky, D. J., Colgan, S. P., Stabilization of HIF through inhibition of Cullin‐2 neddylation is protective in mucosal inflammatory responses. FASEB J. 29, 208–215 (2015). www.fasebj.org

The inflammatory tissue microenvironment in IBD.

Abstract: A current view of the inflammatory bowel diseases (IBDs) includes the luminal triggering of innate immune disease in a genetically susceptible host. Given the unique anatomy and complex environment of the intestine, local microenvironmental cues likely contribute significantly to both disease progression and resolution in IBD. Compartmentalized tissue and microbe populations within the intestine result in significant metabolic shifts within these tissue microenvironments. During active inflammatory disease, metabolic demands often exceed supply, resulting in localized areas of metabolic stress and diminished oxygen delivery (hypoxia). There is much recent interest in harnessing these microenvironmental changes to the benefit of the tissue, including targeting these pathways for therapy of IBD. Here, we review the current understanding of metabolic microenvironments within the intestine in IBD, with discussion of the advantages and disadvantages of targeting these pathways to treat patients with IBD.

Metabolic regulation of intestinal epithelial barrier during inflammation

The gastrointestinal mucosa has proven to be an interesting tissue for which to investigate disease-related metabolism. In this review, we outline some evidence that implicates metabolic signaling as important features of barrier in the healthy and disease. Studies from cultured cell systems, animal models and human patients have revealed that metabolites generated within the inflammatory microenvironment are central to barrier regulation. These studies have revealed a prominent role for hypoxia and hypoxia-inducible factor (HIF) at key steps in adenine nucleotide metabolism and within the creatine kinase pathway. Results from animal models of intestinal inflammation have demonstrated an almost uniformly beneficial influence of HIF stabilization on disease outcomes and barrier function. Studies underway to elucidate the contribution of immune responses will provide additional insight into how metabolic changes contribute to the complexity of the gastrointestinal tract and how such information might be harnessed for therapeutic benefit.

HIF-dependent regulation of AKAP12 (gravin) in the control of human vascular endothelial function

Hypoxia has been widely implicated in many pathological conditions, including those associated with inflammation and tumorigenesis. A number of recent studies have implicated hypoxia in the control of vasculogenesis and permeability, the basis for which is not fully understood. Here we examine the transcriptional regulation of angiogenesis and permeability by hypoxia in endothelial cells. Guided by a global profiling approach in cultured endothelial cells, these studies revealed the selective induction of human gravin (protein kinase A anchoring protein 12) by hypoxia. Analysis of the cloned gravin promoter identified a functional hypoxia‐responsive region including 2 binding sites for hypoxia‐inducible factor (HIF). Site‐directed mutagenesis identified the most distal HIF‐binding site as essential for the induction of gravin by hypoxia. Further studies examining gravin gain and loss of function confirmed strong dependence of gravin in control of microvascular endothelial tube formation, wherein gravin functions as a “braking” system for angiogenesis. Additional studies in confluent endothelia revealed that gravin functionally couples to control endothelial barrier function in response to protein kinase A (PKA) agonists. Taken together, these results demonstrate transcriptional coordination of gravin by HIF‐1α and amplified PKA‐dependent endothelial responses. These findings provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.—Weissmüller, T., Glover, L. E., Fennimore, B., Curtis, V. F., MacManus, C. F., Ehrentraut, S. F., Campbell, E. L., Scully, M., Grove, B. D., Colgan, S. P. HIF‐dependent regulation of AKAP12 (gravin) in the control of human vascular endothelial function. FASEB J. 28, 256–264 (2014). www.fasebj.org

Perturbation of neddylation-dependent NF-κB responses in the intestinal epithelium drives apoptosis and inhibits resolution of mucosal inflammation

Targeting of the neddylation pathway in intestinal epithelial cells inhibits NF-κB, resulting in unabated apoptosis. These studies provide new insight into neddylation pathways within the mucosal epithelium and highlight an important role for NF-κB in the resolution of ongoing inflammation.

Actions of Adenosine on Cullin Neddylation: Implications for Inflammatory Responses

There is intense interest in understanding how the purine nucleoside adenosine functions in health and during disease. In this review, we outline some of the evidence that implicates adenosine signaling as an important metabolic signature to promote inflammatory resolution. Studies derived from cultured cell systems, animal models and human patients have revealed that nucleotide metabolism is significant component of the overall inflammatory microenvironment. These studies have revealed a prominent role for the transcription factors NF-κB and hypoxia-inducible factor (HIF) and that these molecules are post-translationally regulated through similar components, namely the neddylation of cullins within the E3 ligase that are controlled through adenosine receptor signaling. Studies defining differences and similarities between these responses have taught us a number of important lessons about the complexity of the inflammatory response. A clearer definition of these pathways has provided new insight into disease pathogenesis and importantly, the potential for new therapeutic targets.