Potential Role for Peptidylarginine Deiminase 2 (PAD2) in Citrullination of Canine Mammary Epithelial Cell Histones Brian D. Cherrington1, Eric Morency1, Angela M. Struble2, Scott A. Coonrod1, Joseph J. Wakshlag 2 * 1 J.A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America, 2 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America Abstract Peptidylarginine Deiminases (PADs) convert arginine residues on substrate proteins to citrulline. Previous reports have documented that PAD2 expression and activity varies across the estrous cycle in the rodent uterus and pituitary gland, however, the expression and function of PAD2 in mammary tissue has not been previously reported. To gain more insight into potential reproductive roles for PAD2, in this study we evaluated PAD2 expression and localization throughout the estrous cycle in canine mammary tissue and then identified possible PAD2 enzymatic targets. Immunohistochemical and immunofluorescence analysis found PAD2 expression is low in anestrus, limited to a distinct, yet sparse, subset of epithelial cells within ductal alveoli during estrus/early diestrus, and encompasses the entire epithelium of the mammary duct in late diestrus. At the subcellular level, PAD2 is expressed in the cytoplasm, and to a lesser extent, the nucleus of these epithelial cells. Surprisingly, stimulation of canine mammary tumor cells (CMT25) shows that EGF, but not estrogen or progesterone, upregulates PAD2 transcription and translation suggesting EGF regulation of PAD2 and possibly citrullination in vivo. To identify potential PAD2 targets, anti-pan citrulline western blots were performed and results showed that citrullination activity is limited to diestrus with histones appearing to represent major enzymatic targets. Use of site-specific anti- citrullinated histone antibodies found that the N-terminus of histone H3, but not H4, appears to be the primary target of PAD activity in mammary epithelium. This observation supports the hypothesis that PAD2 may play a regulatory role in the expression of lactation related genes via histone citrullination during diestrus. Citation: Cherrington BD, Morency E, Struble AM, Coonrod SA, Wakshlag JJ (2010) Potential Role for Peptidylarginine Deiminase 2 (PAD2) in Citrullination of Canine Mammary Epithelial Cell Histones. PLoS ONE 5(7): e11768. doi:10.1371/journal.pone.0011768 Editor: Neil A. Hotchin, University of Birmingham, United Kingdom Received February 22, 2010; Accepted July 1, 2010; Published July 26, 2010 Copyright: 2010 Cherrington et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Department of Defense (DOD) Era of Hope Scholar Award W81XWH-07-1-0372, Scott A. Coonrod. 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. * E-mail: Jw37@cornell.edu Introduction The peptidylarginine deiminases (PADs) are a family of calcium- dependent enzymes that post-translationally convert arginine residues on substrate proteins to the non-standard amino acid citrulline. PAD catalyzed citrullination, with concomitant loss of the positive imine group, converts the strongly basic arginine residue to a neutral amino acid. Loss of basic charge caused by citrullination is thought to disrupt charge distribution within the substrate protein and alter its ability to interact with other molecules [1,2]. The PAD family consists of five members (1–4 and 6) located within a gene cluster encompassing ,300 kb at human chromosome 1p36.13. PADs 1 and 3 and PADs 4 and 6, respectively, are closely aligned [1,3] while PAD2, the apparent ancestral homolog, is set apart from the other PADs on chromosome 1 and is oriented in the opposite direction. Additionally, PAD2 is the most widely expressed and largest of the PAD genes with a long, distinctive 39 untranslated region (UTR). The PAD enzymes and citrullinated proteins are associated with multiple human diseases including rheumatoid arthritis, multiple sclerosis, Alzheimer’s disease, and, more recently, with cancer [4–7]. PAD expression in mammary tissue has not been documented. However, previous reports have shown that PAD2 is expressed in other reproductive tissues in a hormone dependent manner. For example, both PAD2 and citrullination levels were found to be higher in the female rodent pituitary gland than in males and PAD2 was also found to be expressed in the luminal and glandular epithelia of the uterine endometrium with expression levels changing in an estrous cycle-dependent manner [8,9]. Further, ovariectomized mice treated with estrogen (E2) displayed both increased PAD2 mRNA levels and increased citrullination in uterine samples compared to vehicle treated controls suggesting E2-mediated regulation. Potential PAD2 targets in reproductive tissues have not been previously identified. However, two in vivo substrates for PAD2 have been described in other tissues: myelin basic protein (MBP) in neurons and vimentin in skeletal muscle and macrophages. In macrophages, the presence of high calcium levels cause PAD2 to citrullinate vimentin resulting in the breakdown of the vimentin intermediate filament network potentially to play a role in apoptotic events [10]. The brain expresses PAD2 where it citrullinates MBP a major component of the myelin sheath that covers the axons of nerves. MBP normally contains non- citrullinated arginine residues allowing compact myelin sheaths to form; citrullinated MBP is not capable of forming tight sheaths which is hypothesized to lead to neurodegeneration and possibly PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11768