Canine Mammary PAD2 Expression Figure 1. PAD2 expression in canine mammary tissue. (A) PAD2 is expressed in the canine mammary gland in an estrous cycle dependant fashion with the highest expression in alveolar unit epithelium during late diestrus. Anestrus (a 2006, b 6306), estrus/early diestrus (c 2006, d 6306), and late diestrus (e 2006, f 6306) mammary tissue sections were probed with anti-PAD2 antibody or equal concentration of rabbit IgG as a control (see Supplemental Figure 1A) and counterstained with hematoxylin. Arrow in estrus/early diestrus depicts occasional alveolar end units expressing PAD2. Arrow in diestrus depicts lack of PAD2 epithelial staining in ductule. (B) PAD2 (green fluorescent signal) is first detected in estrus/early diestrus, a time of high cellular proliferation, but PAD2 is widely expressed in late diestrus during active lactation. In estrus the red fluorescent signal represents the proliferation marker Ki67 (4006), while in diestrus the red fluorescent signal is WGA (4006) which stains the apical membrane of actively lactating mammary glands and all nuclei are stained with DAPI. (C) PAD2 expression is restricted to canine mammary luminal epithelial cells. PAD2 expression is shown as green fluorescent signal during anestrus and diestrus. In 3A, the red fluorescent signal represents cytokeratin, a marker for luminal epithelial cells, while in 3B the red signal is p63, a marker of myoepithelial cells, and all nuclei are stained with DAPI (4006). doi:10.1371/journal.pone.0011768.g001 (Figure 2B). Additionally, the PAD2 antibody used in both the IHC and western blot experiments is specific for the 75 kDa isoform of PAD2 and does not appear to cross-react with other PAD family members (Figure S2). PAD2 expression is induced by EGF in the Canine Mammary Tumor (CMT) 25 primary mammary carcinoma epithelial cell line Given the observed estrous cycle dependant changes in expression of PAD2 in canine mammary tissue samples, we decided to directly test the hypothesis that PAD2 expression is regulated by specific hormones and/or growth factors in mammary epithelial cells. Thus, we examined the effect of hormones and growth factors such as E2, EGF, prolactin and P4 on PAD2 expression in canine mammary epithelial cells using two canine mammary tumor-derived cell lines: CMT12 and CMT25 cells [16,20]. Preliminary experiments found that PAD2 was only expressed in the CMT25 cell line, indicating differential regulation of PAD2 expression across these two cell lines (data not shown). Our analysis of the CMT25 cells, however, found that this line constitutively expressed PAD2 in the presence of low serum levels. Given estrous cycle dependent changes in PAD2 expression, we predicted that E2, P4, or prolactin would potentially induce PAD2 expression. Surprisingly, however, treatment of CMT25 cells with these hormones did not significantly alter PAD2 expression with the exception of cells treated with EGF, where PAD2 expression was significantly increased (P,0.01) (Figure 3A). Further exam- ination of PAD2 expression by qPCR of hormone/growth factor treated cells confirmed that PAD2 transcription was only affected by EGF stimulation (P,0.05) (Figure 3B). Presently, the specific signal transduction pathway responsible for EGF induced expression of PAD2 is under investigation. PAD2 is expressed in both the cytoplasmic and nuclear compartment of mammary epithelial cells Interestingly, initial IHC and IF studies suggested that a fraction of PAD2 localized to the nucleus. To further investigate the subcellular localization of PAD2, mammary tissue sections were evaluated by IF, and high magnification images were taken. Results show that in diestrus samples, PAD2 can be visualized as punctate foci in euchromatic (i.e. nuclear regions that are stained poorly by DAPI) regions of the nucleus suggesting that PAD2 enzymatic targets may be nuclear in nature (Figure 4A, arrows). While a role for PAD2 in nuclear function has not been previously described, reports have shown that PAD2 can target histones for citrullination in vitro [12]. PLoS ONE | www.plosone.org 4 July 2010 | Volume 5 | Issue 7 | e11768