ORIGINAL ARTICLE Intestinal renineangiotensin system is stimulated after deletion of Lkb1 Boris Y Shorning, 1 Thierry Jarde ´, 1 Afshan McCarthy, 2 Alan Ashworth, 2 Wendy W J de Leng, 3 George Johan A Offerhaus, 3,4 Nicoletta Resta, 5 Trevor Dale, 1 Alan R Clarke 1 ABSTRACT Background and aims LKB1 is a serine-threonine kinase, mutation of which can lead to the development of multiple benign intestinal hamartomas (PeutzeJeghers syndrome). In this study, the authors investigate the mechanisms underlying this phenotype by exploring the transcriptional changes associated with Lkb1 deletion in intestinal epithelium. Methods The authors used mice with Lkb1 deleted in the intestinal epithelium using a Cyp1a1-specific inducible Cre recombinase and used Affymetrix (Santa Clara, California, USA) microarray analysis to examine the transcriptional changes occurring immediately after Lkb1 loss. The authors also generated cryptevillus organoid culture to analyse Lkb1 role in intestinal responses to exogenous stimuli. Results Affymetrix analysis identified the most significant change to be in Ren1 expression, a gene encoding a protease involved in angiotensinogen processing. Lkb1 deletion also enhanced ACE expression and subsequently angiotensin II (AngII) production in the mouse intestine. Intestinal apoptosis induced by Lkb1 deficiency was suppressed by ACE inhibitor captopril. Lkb1-deficient intestinal epithelium showed dynamic changes in AngII receptor type 1, suggesting a possible compensatory response to elevated AngII levels. A similar reduction in epithelial AngII receptor type 1 was also observed in human PeutzeJeghers syndrome tumours contrasting with high expression of the receptor in the tumour stroma. Mechanistically, the authors showed two pieces of data that position Lkb1 in renin expression regulation, and they implied the importance of Lkb1 in linking cell responses with nutrient levels. First, the authors showed that Lkb1 deletion in isolated epithelial organoid culture resulted in renin upregulation only when the organoids were challenged with external cues such as AngII; second, that renin upregulation was dependent upon the MEK/ERK pathway in a circadian fashion and corresponded to active feeding time when nutrient levels were high. Conclusions Taken together, these data reveal a novel role for Lkb1 in regulation of the gastrointestinal renineangiotensin system. INTRODUCTION LKB1 gene inactivation is associated with PeutzeJeghers syndrome (PJS). 1 Patients with PJS develop multiple gastrointestinal hamartomatous polyps, and they are prone to malignancies in the small intestine, the stomach, the colon and non- gastrointestinal tissues. 2 Lkb1 controls a wide spectrum of cellular processes by phosphorylating a range of kinases implicated in nutrient sensing, metabolism and cell polarity. 34 We have previously shown that conditional deletion of Lkb1 in the murine intestinal epithelium leads to significant changes in cell differentiation. 5 To further address mechanisms underlying these changes, we have now used Affymetrix (Santa Clara, California, USA) microarray analysis to examine the transcriptional changes occurring immediately after Lkb1 deletion from the intestinal epithelium. In this study, we report a marked increase in Ren1 expression immediately after loss of Lkb1. Therefore, we focused our study on Lkb1 involvement in the local intestinal renine angiotensin system (RAS) regulation. Local RAS systems differ from circulating RAS, while < Additional materials are published online only. To view these files please visit the journal online (http://gut.bmj. com/content/61/2.toc). 1 Cardiff School of Biosciences, Cardiff University, Cardiff, UK 2 The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK 3 Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands 4 Department of Pathology, Academic Medical Center Amsterdam, Amsterdam, The Netherlands 5 Dipartimento di Biomedicina dell’Eta ` Evolutiva, Universita ` degli Studi di Bari, Bari, Italy Correspondence to Dr Boris Y Shorning, Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK; shorningb@cardiff.ac.uk Revised 4 May 2011 Accepted 27 May 2011 Published Online First 3 August 2011 Significance of this study What is already known about this subject? < Mutations in Lkb1 tumour suppressor are associated with PeutzeJeghers syndrome char- acterised by gastrointestinal hamartomatous polyps. Conditional deletion of Lkb1 in the murine intestinal epithelium leads to significant changes in cell differentiation. Local intestinal renineangiotensin system controls apoptosis, cell growth and differentiation as well as ion absorption and secretion, cholesterol absorption and glucose transport. What are the new findings? < Lkb1 deletion drastically increased renin messenger RNA levels, enhanced levels of ACE and increased angiotensin II (AngII) production in the mouse intestine. This increase is likely because of Lkb1 serving as a checkpoint protein in MEK/ERK-directed physiological events. Lkb1-deficient small intestinal epithelium showed decreased AngII receptor type 1 expression. Human PJS tumours showed switch towards stromal AngII receptor type 1 localisation. How might it impact on clinical practice in the foreseeable future? < The study suggests possible involvement of local renineangiotensin system in intestinal tumouri- genesis. 202 Gut 2012;61:202e213. doi:10.1136/gutjnl-2011-300046 GI neoplasia group.bmj.com on April 15, 2012 - Published by gut.bmj.com Downloaded from