P-221 Creatine Kinase, a Novel HIF-2a Target, Regulates Apical Junction Complex Assembly in Intestinal Epithelial Cells

• Published in: Inflammatory bowel diseases Vol. 18; no. suppl_1; p. S101
• Main Authors: Glover, Louise, Bowers, Brittelle, Saeedi, Bejan, Ehrentraut, Stefan, Kominsky, Douglas, Campbell, Eric, Kelly, Caleb, Colgan, Sean
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford University Press 01.12.2012
• Subjects: Enzymes; Homeostasis; Hypoxia; Inflammatory bowel disease; Kinases
• ISSN: 1078-0998; 1536-4844
• DOI: 10.1097/00054725-201212001-00254

Intestinal epithelial cells (IEC) lining the GI tract exhibit a uniquely adaptive oxygen profile subject to frequent, prodigious fluctuations. Adaptive responses to hypoxia are orchestrated through coordinated transcriptional changes driven largely by hypoxia-inducible factor (HIF). Recent studies have highlighted a protective role for HIF-1 in epithelial barrier function and homeostasis. We therefore sought to define the relative contribution of HIF-1 and HIF-2 transactivation to epithelial hypoxic responses. A ChIP-on-chip screen highlighted creatine kinase (CK) isozymes as a novel gene family regulated by HIF-2a. Cytosolic CK enzymes temporally regulate cellular ATP regeneration and energy homeostasis through the transfer of high-energy phosphate from phosphocreatine (PCr) to ADP, thus acting as a metabolic sensor specifically at subcellular regions of high and fluctuating energy demand.Methods Cell Culture: Human intestinal epithelial Caco-2 and T84 cells were cultured under normoxic (21% O2) or hypoxic conditions (1% O2) ChIP-on-chip: ChIP was performed with a polyclonal HIF-2a antibody. ChIP-enriched and input DNA were Cy-labeled, hybridized to a promoter microarray, and log2 ratios (input-Cy3/HIF-ChIP-Cy5) analyzed to identify sequences specific for HIF-2a binding. ChIPqPCR was used to quantify HIF-2 occupancy of candidate loci. Transfections and Promoter-luciferase assay: 1.4kb of 5′ flanking promoter region of human CKM and CKB were cloned into pGL3 basic backbone, and co-transfected with oxygenstable HIF-1a or HIF-2a expression plasmids into Caco-2 using Lipofectamine-LTX. Luciferase activity was normalized to co-transfected Renilla reporter. Calcium Switch and TER measurements: T84 plated on transwell inserts were incubated for 16h in low Ca2+ medium (LCM) or 5 mins in HBSS with 2 mM EDTA before switching to normal Ca2+ conditions [1.8 mM]. Transepithelial resistance (TER) was measured over time using a voltameter, and expressed in ohm.cm2. TNBS Colitis and creatine supplementation: Female C57/BL6 mice were fed 2% Cr-supplemented or normal chow for 3 weeks. TNBS was induced by intrarectal administration of 2.5% TNBS in 40% EtOH; controls received EtOH alone.ResultsImmunolocalization studies indicated that CK enzymes CKM and CKB localize to apical junctions in both model polarized epithelia and in human colonic mucosa, and are transcriptionally induced in hypoxia. Promoter-reporter and ChIP-qPCR analyses revealed that HIF-2, but not HIF-1, binds and transactivates CKM and CKB promoters at proximal HRE sites. To investigate whether CK contributes to epithelial junction regulation, we performed calcium switch on T84 monolayers. Pharmacological inhibition of CK with 10uM DNFB significantly attenuated apical junctional re-assembly, as indicated by TER measurement and analysis of junctional protein localization, implicating a prominent role for the Cr-PCr shuttle. Dietary creatine supplementation in a murine TNBS model of acute colitis ameliorated clinical symptoms, including mortality, weight and crypt loss. Quantitative RT-PCR analyses further revealed reduced expression of CK enzymes in colonic mucosa of IBD patients, suggestive of an impaired CK/PCr energy circuit in chronic intestinal inflammation.Conclusion(s)Taken together, these results support a prominent role for HIFregulated creatine kinase in apical junction homeostasis, and implicate a compelling link between cellular energy status and junction integrity. Moreover, modulation of mucosal creatine levels through creatine supplementation may represent a novel therapeutic strategy for IBD.