Canine Mammary PAD2 Expression Figure 2. PAD2 mRNA and protein are differentially expressed across the canine estrous cycle with highest levels detected during late diestrus. (A) RNA was purified from canine mammary tissue samples, reverse transcribed and resulting cDNA was used in qPCR reactions with intron spanning primers specific for the 75 kDa isoform of PAD2 and RPS5, a characterized canine reference gene. All values are normalized to anestrus samples and bars represent the means 6 SEM. Means were separated by one-way ANOVA using Tukey’s HSD and the letters a and b represent significant differences (P,0.01). (B) Canine mammary tissue samples from anestrus, estrus/early diestrus and late diestrus were subject to SDS-PAGE and probed with an anti-PAD2 antibody which detected a 75 kDa band, the estimated molecular weight of PAD2. doi:10.1371/journal.pone.0011768.g002 PADs target histone H3 from mammary gland epithelial cells for citrullination only during diestrus and EGF can stimulate histone H3 citrullination The expression pattern and functional role of PAD catalyzed citrullination in mammary tissue has not previously been examined. To address this issue, canine mammary tissue sections from anestrus, estrus/early diestrus, and late diestrus were assessed by western blot analysis with an antibody that recognizes citrullinated proteins (anti-pan-citrulline antibody) as previously described [21]. Pan-citrulline western blots detected three strong bands corresponding to 33, 15, and 10 kDa in diestrus canine mammary tissue samples while citrullinated proteins were not detected (or weakly detected) at other stages (Figure 4B). Based on their molecular weights, we speculate that the 33 kDa band is nucelophosmin, a known PAD substrate, and that the 10 and 15 kDa bands are likely histone H3 and H4. To further validate that the lower molecular weight proteins from the diestrus sample were histones, we citrullinated bulk histones using purified PAD2 and found that similarly sized histone bands at 10 and 15 kDa were observed (Figure 4B, control). In order to establish where PAD-mediated citrullination was occurring within mammary tissue, sections from anestrus, estrus/ early diestrus and late diestrus were subject to IF and interrogated with the pan-citrulline antibody. As expected, only the late diestrus samples showed fluorescent pan-citrulline signal. Further, we also found that this signal was primarily limited to the nucleus of a subset of epithelial cells (Figure 4C). Our finding that mammary gland epithelial cell histones appear to be a target for citrullination at diestrus raises the exciting possibility that one function of PADs in mammary tissue is to regulate gene activity via histone citrullination. To further test this hypothesis, western blots of diestrus mammary tissue samples were probed with the H3 cit 2-8- 17, H3 cit 26, and H4 cit 3 site-specific anti-citrullinated histone antibodies. Purified PAD2 in vitro citrullinated CMT25 cell histones were included as a positive control. Interestingly, we found that citrullination was limited to the arginine residues at 2, 8, and 17 on the histone H3 tail suggesting that these residues are the primary targets of PAD activity in mammary epithelial cells (Figure 4D). Next we examined the ability of EGF, presumably acting via PAD2, to induce citrullination of histone H3 tails by western blot. EGF treated CMT25 cells show an increase citrullination of histone 3 tail residues 2, 8, and 17 similar to what we found in mammary tissue suggesting a role for EGF in not only PAD2 expression, but also activity (Figure 4E). Given that PAD2 activity is restricted to diestrus in vivo, the main secretory phase of the estrous cycle, our findings support the hypothesis that PAD2 may play a role in the regulation of the expression of lactation related genes in mammary epithelial cells via histone citrullination, and we are currently directly testing this hypothesis. Discussion Canine mammary glands undergo dramatic molecular and morphological reorganization during the estrous cycle to convert mammary tissue to an active secretory gland. Specific stages of the estrous cycle are defined by morphologic and molecular signatures such as generalized edema and proliferation during estrus/early diestrus, production of lactation related gene products during late diestrus and apoptosis during involution towards the metestrus to anestrus crossover. These dramatic cellular transformations are clearly orchestrated, in part, by sex steroid hormones. However, the downstream targets and molecular mechanisms catalyzed by these hormones that effect cellular reorganization are poorly understood. Given that PADs are regulated by E2 in rodents and that citrullination of target proteins by PADs has been found to cause dramatic reorganization in both the cytoplasm and nuclear architecture, these enzymes are strong candidates for mediating hormonal changes throughout the canine estrous cycle. IHC and IF studies of canine mammary tissue illustrate that the patterns and intensity of PAD2 expression change over the estrous cycle. For example, in estrus/early diestrus PAD2 expression is sparse, yet distinct, in cells within the alveolar end units. During diestrus, PAD2 expression is more widespread potentially indicating that, although expression is initiated during estrus, expression expands to additional epithelial cells throughout diestrus. Given this expression pattern, we hypothesize that PAD2 may possibly be involved in converting the mammary gland to an active secretory organ. Studies in rodent models have also suggested that PAD2 may be involved in a secretory function in the uterus [22]. PLoS ONE | www.plosone.org 5 July 2010 | Volume 5 | Issue 7 | e11768