Pivotal Role of the Nuclear Receptor PPAR γ in Colon Epithelial cells

When symbiosis between the residential intestinal flora and the epithelial cells of the gastrointestinal tract is broken one endpoint may be chronic inflammation, e.g. Inflammatory Bowel Disease (IBD). The disruption of the epithelial lining elicits activation of NF-κB and secretion of potent inflammatory mediators. In the case of IBD, elevated levels of NF-κB have been observed and interestingly several nuclear receptors have been reported to attenuate NF-κB activity. One such receptor is PPARγ. My thesis presents evidence of a possible molecular mechanism whereby commensal bacteria not only regulate the expression of PPARγ, but also its ability to export NF-κB from the nucleus, independently of IκB, suggesting a novel pathway for nuclear export of NF-κB. In paper I we show that TLR4 signalling can increase expression levels of PPARγ. Moreover we also show that germ free mice do not express PPARγ indicating that bacteria can regulate its expression. Interestingly, patients with ulcerative colitis, one of the two main forms of IBD, display a defect in protein levels of PPARγ in colonic epithelial cells compared to Crohn ́s disease patients and healthy controls. In the second paper, we assess the possible anti-inflammatory effects of Bacteroides thetaiotaomicron, a common anaerobic commensal bacteria of the gastrointestinal tract, upon co-infection in CaCo-2 cells with the pathogenic Salmonella enterica. Immunohistochemistry in combination with biochemical and functional experiments revealed that PPARγ, upon exposure to B. thetaiotaomicron, possess the ability to shuttle NFκB from the nucleus to the cytosol. In the third manuscript we extend the observations presented in paper one and assess whether there is a relationship between high levels of NF-κB and low PPARγ levels in the colonic epithelium of IBD patients. No strict correlation was observed between low intestinal levels of PPARγ and elevated NF-κB levels. Individual variations of the absolute NF-κB levels among IBD patients were however observed In addition, we show that the apoptosis inducing ligand, TRAIL, is expressed at lower levels in inflamed colon mucosa, thereby linking it to chronic inflammation. A DNA-binding protein may switch between an activator and a repressor, depending on co-factor assembly. This prompted us to assess whether such a factor could be identified for PPARγ since opposing effects of its function in colonic tumor cells had been reported. One possible factor is the transcriptional co-factor β-catenin. In the final manuscript we examine the possible interplay between PPARγ and β-catenin. Biochemical data show that β-catenin interacts with PPARγ which results in elevated protein levels of PPARγ. Functional data show that β-catenin and the known chemical agonist of Wnt signalling Lithium chloride, can activate a PPARγ dependent reporter gene in a dose dependent manner. Hence, under conditions where abnormal levels of β-catenin is observed, e.g. in colon cancer, it is tempting to speculate that the altered β-catenin levels may change the net function of PPARγ in favour of cell growth. In summary, the data presented in this thesis, provide some aspects as to how homeostasis may be tightly regulated by a very limited set of factors and that these factors may be intimately connected to execute multiple functions under stringent control. The fine tuning of these functions can be guided by epigenetic factors, such as microbes and nutrients. The strategic tissue distribution of PPARγ, and its pleiotropic functions serve as one example of an inflammatory gatekeeper that integrates gut homeostasis and metabolic control. List of Publications This thesis is based upon the following publications that will be referred to in the text by their Roman numerals: I Laurent Dubuquoy, Emmelie Å Jansson, Jean-Frédéric Colombel, Johan Auwerx, Sven Pettersson, Pierre Desreumaux (2003) Impaired expression of PPARγ in Ulcerative Colitis. The first two authors contributed equally Gastroenterology. 124:1265-1276 II Denise Kelly, Jamie I. Campell, Timothy P. King, George Grant, Emmelie A. Jansson, Alistair G. Coutts, Sven Pettersson & Shaun Conway (2004) Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclear-cytoplasmic shuttling of PPARγ and RelA. Nature Immunology. 5(1): 104-12 III Emmelie Å Jansson, Ann-Kristin Spiik, Lars-Göran Axelsson, Fransesca Bresso, Robert Löfberg and Sven Pettersson (2004) Levels of NF-κB in inflamed colonic mucosa from Inflammatory Bowel Disease patients are elevated in parallel with low levels of PPARγ and TRAIL. The first two authors contributed equally. Manuscript IV Emmelie Å Jansson, Alexandra Are, Denise Kelly, Velmurugesan Arulampalam and Sven Pettersson. (2004) A functional link between Wntsignalling/β-catenin and the Peroxisome Proliferator Activated Receptorγ. Submitted