The histone deacetylase Hdac1 regulates inflammatory signalling in intestinal epithelial cells

Gonneaud et al. Journal of Inflammation The histone deacetylase Hdac1 regulates inflammatory signalling in intestinal epithelial cells Alexis Gonneaud 0 Julie Moore Gagn 0 Naomie Turgeon Claude Asselin 0 Equal contributors 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 J1E 4K8 , Canada Background: It has recently been found that both nuclear epithelial-expressed histone deacetylases Hdac1 and Hdac2 are important to insure intestinal homeostasis and control the mucosal inflammatory response in vivo. In addition, HDAC inhibitors modulate epithelial cell inflammatory responses in cancer cells. However, little is known of the specific role of different HDAC, notably Hdac1, in the regulation of inflammatory gene expression in intestinal epithelial cells (IEC). Methods: We investigated the role of Hdac1 in non-transformed IEC-6 rat cells infected with lentiviral vectors expressing specific Hdac1 shRNAs, to suppress Hdac1 expression. Proliferation was assessed by cell counting. Deacetylase activity was measured with a colorimetric HDAC assay. Cells were treated with IL-1 and/or the JQ1 bromodomain acetyl-binding inhibitor. Nuclear protein levels of Hdac1, Hdac2, phosphorylated or unphosphorylated NF-B p65 or C/EBP, and NF-B p50 and actin were determined by Western blot. Chemokine and acute phase protein expression was assessed by semi-quantitative RT-PCR analysis. Secreted cytokine and chemokine levels were assessed with a protein array. Chromatin immunoprecipitation experiments were done to assess RNA polymerase II recruitment. Results: Reduced Hdac1 protein levels led to Hdac2 protein increases and decreased cell proliferation. Hdac1 depletion prolonged nuclear IL-1-induced phosphorylation of NF-B p65 protein on Ser536 as opposed to total p65, and of C/EBP on Ser105. In addition, semi-quantitative RT-PCR analysis revealed three patterns of expression caused by Hdac1 depletion, namely increased basal and IL-1-stimulated levels (Hp, Kng1), increased IL-1-stimulated levels (Cxcl2) and decreased basal levels with normal IL-1 induction levels (Ccl2, Ccl5, Cxcl1, C3). Secreted cytokine and chemokine measurements confirmed that Hdac1 played roles both as an IL-1 signalling repressor and activator. Hdac1 depletion did not alter the JQ1 dependent inhibition of basal and IL-1-induced inflammatory gene expression. Hdac1 depletion led to decreased basal levels of RNA polymerase II enrichment on the Ccl2 promoter, as opposed to the Gapdh promoter, correlating with decreased Ccl2 basal mRNA expression. Conclusions: Hdac1 is a major nuclear HDAC controlling IL-1-dependent inflammatory response in IEC, notably by regulating gene-specific transcriptional responses. Hdac1 may be important in restricting basal and inflammatory-induced gene levels to defined ranges of expression. Hdac1; Inflammation; Intestinal epithelial cell; Chemokine expression - Background The intestinal epithelium plays important roles as a physical and biochemical sensor of the luminal environment [1]. Indeed, intestinal epithelial cells (IEC) regulate gut homeostasis by sensing luminal bacterial products or by responding to inflammatory signals emanating from the mucosal immune system [2,3]. In turn, these inflammatory signals regulate an IEC-specific inflammatory response, characterized by the expression of various cytokines, chemokines and acute phase proteins [4]. In recent years, epigenetic modifications, which include DNA methylation, histone methylation and acetylation among others, have been shown to act as receivers and transmitters of environmental changes, leading to variations in gene expression. Indeed, it has been suggested that many inflammatory diseases, including inflammatory bowel diseases, are affected by epigenetic modifications [5,6]. One important modification, namely lysine acetylation, is regulated by histone acetyltransferases and by histone deacetylases (HDAC), which respectively add or remove the acetyl group on histones as well as non-histone proteins [7]. Histone acetylation plays a dual role, either by reducing histone-DNA interactions, thereby creating an open chromatin configuration, or by acting as an anchor recognized by bromodomaincontaining chromatin modifiers [8]. Some of these recruited proteins, such as Brd family members, play important roles in the regulation of transcriptional elongation as well as inflammation. HDAC-dependent regulation of protein acetylation levels leads to cell- and gene-specific transcriptional repression or activation [8]. Of the eighteen HDAC, class I Hdac1 gains access to chromatin as a homodimer or heterodimer with Hdac2, in Sin3, CoREST and NuRD multiprotein complexes [9,10]. Hdac1 is considered as a positive regulator of cell proliferation as Hdac1 depletion in mice results in growth deficiencies, correlating with increased expression of the p21 cyclin-dependent inhibitor [11,12]. In contrast to single gene deletion, tissue-specific dual deletion of Hdac1 and Hdac2 leads to homeostatic phenotypes, such as epidermal differentiation defects when deleted in epidermal cells [13], and intestinal homeostatic perturbations when deleted in intestinal epithelial cells [14]. Treating colon cancer cell lines with HDAC inhibitors or reducing Hdac1 expression suppresses colon cancer cell proliferation [15], and alters inflammatory signalling [16]. Hdac1 is also considered as a negative regulator of transcription factors involved in inflammatory responses. For example, Hdac1 deacetylates the p65 NF-B subunit, leading to reduced transcriptional activity during inflammatory responses [17-19]. Both phosphorylation and acetylation modifications interact to insure full NF-B transcriptional activity [18,20,21]. Likewise, C/EBP acetylation leads to positive or negative interactions with co-regulators, including HDAC [22]. HDAC inhibitors are being considered as pharmacological agents to modulate inflammatory responses. However, many studies have revealed opposite effects of HDAC inhibitors as suppressor or stimulator of inflammatory responses and gene expression [23]. These differences may result from the use of different cell lines or mouse models, suggesting cell-specific effects, or of HDAC inhibitors with different selectivity to the various HDAC isoforms, suggesting target-specific effects. As more specific HDAC inhibitors are being generated, it is thus of importance to assess the role of specific HDAC in distinct cell types critical to the regulation of inflammation, including IEC. Little is known of the role of specific HDAC in the control of IEC inflammatory responses. In order to understand the role of Hdac1 in the IEC inflammatory response, we have used the non-transformed intestinal epithelial cell line IEC-6 to avoid the increased sensitivity to HDAC inhibition found in cancer cells. We had previously observed (...truncated)