Please cite this article in press as: Kulkarni, N.N., et al., Phenylbutyrate induces cathelicidin expression via the vitamin D receptor: Linkage to inflammatory and growth factor cytokines pathways. Mol. Immunol. (2014), http://dx.doi.org/10.1016/j.molimm.2014.10.007 ARTICLE IN PRESS G Model MIMM-4499; No. of Pages 10 Molecular Immunology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Molecular Immunology j ourna l ho me pa ge: www.elsevier.com/locate/molimm Phenylbutyrate induces cathelicidin expression via the vitamin D receptor: Linkage to inflammatory and growth factor cytokines pathways Nikhil N. Kulkarni a , Zhiqian Yi a , Carolin Huehnken a , Birgitta Agerberth b , Gudmundur H. Gudmundsson a,∗ a Biomedical Center and Department of Life and Environmental Sciences, University of Iceland, Reykjavík, Iceland b Department of Laboratory Medicine, Division of Clinical Microbiology Karolinska University Hospital, Karolinska Institutet, Huddinge, Stockholm, Sweden a r t i c l e i n f o Article history: Received 13 August 2014 Received in revised form 29 September 2014 Accepted 11 October 2014 Available online xxx Keywords: Antimicrobial peptides CAMP gene LL-37, hBD-1, Pseudomonas aeruginosa a b s t r a c t Antimicrobial peptides (AMPs) constitute an indispensable arm of innate immunity against infectious microbes in humans. Induction of endogenous AMPs may become an alternative therapy against infec- tions. Our previous studies have demonstrated phenylbutyrate (PBA) as a novel inducer of the AMPs cathelicidin (encoded by the CAMP gene) and human beta-defensin-1 in the human bronchial epithe- lial cell line VA10. In this work, we have continued by studying molecular mechanisms of PBA mediated induction of LL-37 expression and associated pathways in the human bronchial epithelial cell line VA10. In this study we demonstrate vitamin D receptor (VDR) as a key transcription factor required for PBA medi- ated up-regulation of the CAMP gene expression. PBA also increases mRNA expression of the vitamin D3 regulated genes CYP24A1 and CD14. The siRNA knockdown of VDR reduced PBA mediated increase in CAMP, CYP24A1 and CD14 expression. Furthermore, we demonstrate that PBA enhances Toll-Like Recep- tor 5 ligand flagellin regulated mRNA expression of the inflammatory cytokine TNF and chemokine CXCL8. PBA also up-regulates the expression of the genes encoding the growth factor cytokines trans- forming growth factor (TGF) , TGF1 and TGF2. Our results indicate that TGF type I receptor and epidermal growth factor receptor are involved in PBA mediated CAMP regulation. Finally, we show that co-treatment with PBA and vitamin D3 reduces Pseudomonas aeruginosa growth in vitro. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Cationic antimicrobial peptides (AMPs) constitute a vital arm of the innate immune system (Hilchie et al., 2013; Zasloff, 2002). Despite their ancient lineage, AMPs have eluded emergence of resistant bacteria (Zasloff, 2002). Cathelicidins and defensins com- prise two main families of AMPs in mammals (Zasloff, 2002). AMPs are highly active against enteric pathogens and have been shown to govern the composition of the intestinal microbiota (Salzman et al., 2010). In humans, LL-37 peptide is the only known member of the cathelicidin family encoded by the cathelicidin antimicrobial peptide (CAMP) gene. The storage form of cathelicidin pro-LL-37 (hCAP18) is processed extracellular to the mature form LL-37 by specific proteases (Morizane et al., 2010; Sørensen et al., 2001). This ∗ Corresponding author. Tel.: +3545255276; fax: +354 525 4069. E-mail address: ghrafn@hi.is (G.H. Gudmundsson). mature form has been demonstrated to have direct anti-microbial and immune-modulatory effects (Cederlund et al., 2011). It has also been indicated to play an important role in wound healing, den- dritic cell differentiation, angiogenesis and mast cell degranulation (Kahlenberg and Kaplan, 2013). Pathogen colonization and invasion is associated with decreased CAMP expression and increased infection in respiratory diseases such as cystic fibrosis (Bergsson et al., 2009). The physiological rel- evance of cathelicidin is underlined by the fact that the cathelicidin deficient mice (Cnlp -/-) are susceptible to infections (Nizet et al., 2001). Although pathogens, such as Shigella, can subvert the ini- tial defenses by suppression of AMP expression (Islam et al., 2001), we have shown that regulation and reversal of this suppression can be achieved by treatment with butyrate in a rabbit model of shigellosis (Raqib et al., 2006). The steroid hormone 1, 25- dihydroxyvitamin D 3 (1,25D3) is a direct inducer of the CAMP gene, acting via the vitamin D receptor (VDR) (Gombart et al., 2005; Wang et al., 2004). Calcidiol (25D3), the inactive storage form of vita- min D3 is converted to the hormonally active form 1,25D3 by the http://dx.doi.org/10.1016/j.molimm.2014.10.007 0161-5890/© 2014 Elsevier Ltd. All rights reserved.