epithelial cells. To date PAD2 expression and function in other tissues is thought to be limited to the cytoplasm. Thus, our finding suggests that PAD2 localization and function may be dependent to some degree upon the cell type in which it is expressed, and that the role and targets of PAD2 in the mammary gland may be different from those of other tissue types. Presently, studies are underway to determine the possible mechanism by which PAD2 translocates to the cell nucleus in mammary epithelial cells. Given that no previous studies have documented PAD expression in the mammary gland nor have previous reports demonstrated that PAD-mediated citrullination occurs in mam- mary tissue, we believe that this study provides significant new insight into PAD biology. For example, our finding that PAD- mediated citrullination in mammary tissue appears to be restricted to epithelial cells during a distinct stage of the estrous cycle and limited to a small subset of targets (likely histones and nucleophosmin) suggests that PAD activity is likely facilitating a specific event during diestrus. Further, the observation that histone citrullination correlates with PAD2 expression in mammary epithelium at diestrus and that a fraction of PAD2 is nuclear in these cells, raises the possibility that PAD2 may play a role regulating gene activity in the mammary epithelium. Currently, we are testing whether the observed histone citrullination activity is directly regulated by PAD2 or is possibly indirectly regulated by PAD2 via crosstalk with PAD4. Subsequent findings directly demonstrating that PADs regulate gene activity in the mammary gland epithelium would likely lead to the identification of new regulatory pathways important for mammary function. References 1. Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ (2003) PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays 25: 1106–1118. 2. Gyorgy B, Toth E, Tarcsa E, Falus A, Buzas EI (2006) Citrullination: a posttranslational modification in health and disease. Int J Biochem Cell Biol 38: 1662–1677. 3. Chavanas S, Mechin MC, Takahara H, Kawada A, Nachat R, et al. (2004) Comparative analysis of the mouse and human peptidylarginine deiminase gene clusters reveals highly conserved non-coding segments and a new human gene, PADI6. Gene 330: 19–27. 4. Vossenaar ER, Nijenhuis S, Helsen MM, van der Heijden A, Senshu T, et al. (2003) Citrullination of synovial proteins in murine models of rheumatoid arthritis. Arthritis Rheum 48: 2489–2500. 5. Musse AA, Li Z, Ackerley CA, Bienzle D, Lei H, et al. (2008) Peptidylarginine deiminase 2 (PAD2) overexpression in transgenic mice leads to myelin loss in the central nervous system. Dis Model Mech 1: 229–240. 6. Ishigami A, Ohsawa T, Hiratsuka M, Taguchi H, Kobayashi S, et al. (2005) Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer’s disease. J Neurosci Res 80: 120–128. 7. Chang X, Han J (2006) Expression of peptidylarginine deiminase type 4 (PAD4) in various tumors. Mol Carcinog 45: 183–196. 8. Takahara H, Kusubata M, Tsuchida M, Kohsaka T, Tagami S, et al. (1992) Expression of peptidylarginine deiminase in the uterine epithelial cells of mouse is dependent on estrogen. J Biol Chem 267: 520–525. 9. Senshu T, Akiyama K, Nagata S, Watanabe K, Hikichi K (1989) Peptidylargi- nine deiminase in rat pituitary: sex difference, estrous cycle-related changes, and estrogen dependence. Endocrinology 124: 2666–2670. 10. Vossenaar ER, Radstake TR, van der Heijden A, van Mansum MA, Dieteren C, et al. (2004) Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages. Ann Rheum Dis 63: 373–381. 11. Wood DD, Ackerley CA, Brand B, Zhang L, Raijmakers R, et al. (2008) Myelin localization of peptidylarginine deiminases 2 and 4: comparison of PAD2 and PAD4 activities. Lab Invest 88: 354–364. Canine Mammary PAD2 Expression Supporting Information Figure S1 Immunohistochemitry in canine mammary with PAD-4. (A) As an IHC control, anestrus, estrus/early diestrus, and late diestrus mammary tissue sections were probed with equal concentration of rabbit IgG (compared to anti-PAD2) and counterstained with hematoxylin. (B) Anestrus, estrus/early diestrus, and late diestrus mammary tissue sections were are also probed with an anti-PAD4 antibody, and opposite of PAD2 staining, showed highest PAD4 levels in anestrus with lowest staining during late diestrus. Found at: doi:10.1371/journal.pone.0011768.s001 (2.96 MB TIF) Figure S2 The PAD2 antibody is not cross reactive with other PAD family members. Mammalian expression plasmids contain- ing the cDNA for human PADs 1, 2, 3, and 4 were transiently transfected into human embryonic kidney 293 cells. After 24 hours, cells were harvested and overexpression lysates run on a western blot probed with anti-PAD2. Found at: doi:10.1371/journal.pone.0011768.s002 (0.12 MB TIF) Author Contributions Conceived and designed the experiments: BDC JJW. Performed the experiments: BDC EPM AMS JJW. Analyzed the data: BDC EPM AMS SC JJW. Contributed reagents/materials/analysis tools: SC. Wrote the paper: BDC. 12. Sarmento OF, Digilio LC, Wang Y, Perlin J, Herr JC, et al. (2004) Dynamic alterations of specific histone modifications during early murine development. J Cell Sci 117: 4449–4459. 13. Uva P, Aurisicchio L, Watters J, Loboda A, Kulkarni A, et al. (2009) Comparative expression pathway analysis of human and canine mammary tumors. BMC Genomics 10: 135. 14. Munson L, Moresco A (2007) Comparative pathology of mammary gland cancers in domestic and wild animals. Breast Dis 28: 7–21. 15. Cardiff RD (2001) Validity of mouse mammary tumour models for human breast cancer: comparative pathology. Microsc Res Tech 52: 224–230. 16. Wolfe LG, Smith BB, Toivio-Kinnucan MA, Sartin EA, Kwapien RP, et al. (1986) Biologic properties of cell lines derived from canine mammary carcinomas. J Natl Cancer Inst 77: 783–792. 17. Brinkhof B, Spee B, Rothuizen J, Penning LC (2006) Development and evaluation of canine reference genes for accurate quantification of gene expression. Anal Biochem 356: 36–43. 18. Rehm S, Stanislaus DJ, Williams AM (2007) Estrous cycle-dependent histology and review of sex steroid receptor expression in dog reproductive tissues and mammary gland and associated hormone levels. Birth Defects Res B Dev Reprod Toxicol 80: 233–245. 19. Gama A, Alves A, Gartner F, Schmitt F (2003) p63: a novel myoepithelial cell marker in canine mammary tissues. Vet Pathol 40: 412–420. 20. Ahern TE, Bird RC, Bird AE, Wolfe LG (1996) Expression of the oncogene c- erbB-2 in canine mammary cancers and tumor-derived cell lines. Am J Vet Res 57: 693–696. 21. Senshu T, Sato T, Inoue T, Akiyama K, Asaga H (1992) Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane. Anal Biochem 203: 94–100. 22. Takahara H, Tsuchida M, Kusubata M, Akutsu K, Tagami S, et al. (1989) Peptidylarginine deiminase of the mouse. Distribution, properties, and immunocytochemical localization. J Biol Chem 264: 13361–13368. 23. Donnay I, Wouters-Ballman P, Devleeschouwer N, Leclercq G, Verstegen J (1995) Changes in oestrogen, progesterone and epidermal growth factor receptor concentrations and affinities during the oestrous cycle in the normal mammary gland and uterus of dogs. Vet Res Commun 19: 101–113. PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11768