HDAC1 and HDAC2 Restrain the Intestinal Inflammatory Response by Regulating Intestinal Epithelial Cell Differentiation

et al. (2013) HDAC1 and HDAC2 Restrain the Intestinal Inflammatory Response by Regulating Intestinal Epithelial Cell Differentiation. PLoS ONE 8(9): e73785. doi:10.1371/journal.pone.0073785 HDAC1 and HDAC2 Restrain the Intestinal Inflammatory Response by Regulating Intestinal Epithelial Cell Differentiation Naomie Turgeon 0 Mylne Blais 0 Julie-Moore Gagn 0 Vronique Tardif 0 Franois Boudreau 0 Nathalie 0 Perreault 0 Claude Asselin 0 Emiko Mizoguchi, Massachusetts General Hospital, United States of America 0 Departement d'anatomie et Biologie Cellulaire, Faculte de Medecine et des Sciences de la Sante, Pavillon de recherche appliquee sur le cancer, Universite de Sherbrooke , Sherbrooke, Quebec , Canada Acetylation and deacetylation of histones and other proteins depends on histone acetyltransferases and histone deacetylases (HDACs) activities, leading to either positive or negative gene expression. HDAC inhibitors have uncovered a role for HDACs in proliferation, apoptosis and inflammation. However, little is known of the roles of specific HDACs in intestinal epithelial cells (IEC). We investigated the consequences of ablating both HDAC1 and HDAC2 in murine IECs. Floxed Hdac1 and Hdac2 homozygous mice were crossed with villin-Cre mice. Mice deficient in both IEC HDAC1 and HDAC2 weighed less and survived more than a year. Colon and small intestinal sections were stained with hematoxylin and eosin, or with Alcian blue and Periodic Acid Schiff for goblet cell identification. Tissue sections from mice injected with BrdU for 2 h, 14 h and 48 h were stained with anti-BrdU. To determine intestinal permeability, 4-kDa FITC-labeled dextran was given by gavage for 3 h. Microarray analysis was performed on total colon RNAs. Inflammatory and IEC-specific gene expression was assessed by Western blot or semi-quantitative RT-PCR and qPCR with respectively total colon protein and total colon RNAs. HDAC1 and HDAC2deficient mice displayed: 1) increased migration and proliferation, with elevated cyclin D1 expression and phosphorylated S6 ribosomal protein, a downstream mTOR target; 2) tissue architecture defects with cell differentiation alterations, correlating with reduction of secretory Paneth and goblet cells in jejunum and goblet cells in colon, increased expression of enterocytic markers such as sucrase-isomaltase in the colon, increased expression of cleaved Notch1 and augmented intestinal permeability; 3) loss of tissue homeostasis, as evidenced by modifications of claudin 3 expression, caspase-3 cleavage and Stat3 phosphorylation; 4) chronic inflammation, as determined by inflammatory molecular expression signatures and altered inflammatory gene expression. Thus, epithelial HDAC1 and HDAC2 restrain the intestinal inflammatory response, by regulating intestinal epithelial cell proliferation and differentiation. - Funding: This work has been supported by a grant from the Crohn's and Colitis Foundation of Canada (www.ccfc.ca). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Continuous intestinal epithelial cell renewal is sustained by crypt stem cells generating multiple IEC lineages [1]. Differentiation and maintenance of intestinal stem cells is regulated by different pathways, including the Notch pathway which controls secretory cell and enterocyte determination [2]. While absorptive enterocytes, mucin-producing goblet cells and enteroendocrine cells reside in small intestinal villi, antimicrobial peptide-secreting Paneth cells remain in the crypts. The colonic epithelium contains colonocytes as well as goblet and enteroendocrine cells, without Paneth cells. All gut epithelium lineages contribute to mucosal barrier function. This barrier is both physical, with the presence of tight junctions [3], and chemical, through production of mucins and the mucus layer by goblet cells [4], and of antimicrobial proteins by Paneth cells as well as other IECs, including enterocytes and goblet cells [5]. In addition to this barrier function, epithelial cells translate signals coming from intestinal luminal contents, including the microbiota, to different immune cells, in order to maintain intestinal homeostasis [6]. For example, while the mucous layer limits bacterial colonization at IEC surfaces, Paneth cells, enterocytes and colonocytes relay microbiotaderived signals in order to induce antimicrobial peptide production [7]. Thus, IECs, bacteria and immune cells communicate to insure intestinal homeostasis. However, disruption of various mechanisms preserving this equilibrium may lead to inappropriate inflammatory responses observed in inflammatory bowel diseases [8,9]. Whereas many pathways involved in the regulation of murine intestinal differentiation, proliferation and homeostasis have been discovered, the extent of epigenetic dependent transcriptional mechanisms such as acetylation and the role of various acetylation regulators, including histone deacetylases (HDAC), remain to be fully determined. Lysine-targeted acetylation and deacetylation of histones and non-histone proteins are regulated respectively by histone acetyltransferases (HAT) and HDAC [10]. Histone acetylation decreases histone interactions with DNA, resulting in relaxed chromatin, and creates docking sites for bromodomain containing proteins, which ultimately affect chromatin structure [11]. Protein acetylation levels are regulated by HDACs, which remove acetyl groups from histones to stimulate chromatin condensation, and from non-histone proteins, resulting in either gene repression or gene activation. Indeed, transcriptomic experiments suggest that HDACs display repressive as well as activating transcriptional activities, depending on the promoter and chromatin context [11]. HDACs are divided in four classes. Of these, ubiquitously expressed and highly homologous nuclear class I HDAC1 and HDAC2 form homo- or heterodimers, and are recruited to chromatin as part of large Sin3, CoREST and NuRD multiprotein complexes, among others [12,13]. These complexes contain additional chromatinmodifying activities, such as the LSD1 H3K4 demethylase in CoREST complexes, and the MI-2 chromatin remodelling enzyme in NuRD complexes. HDAC1 and HDAC2 display both overlapping and nonredundant functions [14]. Indeed, while HDAC1 deficiency leads to pre-natal death and proliferative defects in mice, HDAC2 knockout results in perinatal lethality and cardiac arrhythmias [15]. HDAC1, and to a lesser extent HDAC2, is a negative regulator of cell proliferation [14]. HDAC inhibitors and down-regulation of specific HDACs, including HDAC1 and HDAC2, inhibit colon cancer cell proliferation [16] and modulate both inflammation and immunity [17]. Acetylated targets include, in addition to histones, transcription factors which may be acetylated by HATs and deacetylated by HDACs. For examp (...truncated)