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. 2010 Jul 26;5(7):e11768.
doi: 10.1371/journal.pone.0011768.

Potential role for peptidylarginine deiminase 2 (PAD2) in citrullination of canine mammary epithelial cell histones

Brian D Cherrington  1 , Eric Morency, Angela M Struble, Scott A Coonrod, Joseph J Wakshlag
Affiliations

Potential role for peptidylarginine deiminase 2 (PAD2) in citrullination of canine mammary epithelial cell histones

Brian D Cherrington et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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 200×, b 630×), estrus/early diestrus (c 200×, d 630×), and late diestrus (e 200×, f 630×) 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 (400×), while in diestrus the red fluorescent signal is WGA (400×) 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 (400×).
Figure 2
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 ± 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.
Figure 3
Figure 3. Treatment of the canine mammary tumor (CMT25) cell line with EGF results in an increase in PAD2 expression levels.
(A) Western blot illustrating that only EGF treatment increases PAD2 protein levels with β-actin shown as a loading control. Densitometry measurements quantified treatment groups compared to vehicle treated control in three independent experiments and bars represent the means ± SEM. * indicates a statistically significant difference from other treatments (P<0.01) (B) Quantitative PCR analysis revealed elevated PAD2 transcript levels in CMT25 cells treated with EGF compared to vehicle treated control and * indicates a statistically difference from the other treatments (P<0.05).
Figure 4
Figure 4. PAD2 association with histone citrullination.
(A) PAD2 is detected in the nucleus and cytoplasm of mammary epithelial. PAD2 expression is shown in green and nuclear DAPI stain is in blue with white arrows denoting punctuate PAD2 staining in euchromatic nuclear regions (1000×). (B) PADs _target histones from mammary gland epithelial cells for citrullination and this activity is restricted to late diestrus. Canine mammary tissue samples from anestrus, estrus/early diestrus and late diestrus were examined by SDS-PAGE and probed with an anti-pan-citrulline antibody. (C) Late diestrus mammary tissue sections were subject to a modified IF protocol using the anti-pan-citrulline antibody. The panel shows pan-citrulline staining in green and nuclear DAPI stain in blue (1000×). (D) Late diestrus canine mammary tissue samples and in vitro citrullinated CMT25 histones (as a positive control) were examined by SDS-PAGE and probed with the histone tail citrullination specific antibodies H3cit 2-8-17, H3cit 26, or H4cit 3. (E) Western blot analysis of CMT25 cells treated with EGF for 24 hours show increased histone tail citrullination using a H3 cit 2-8-17 specific antibody. Equal loading was confirmed using a mouse histone 3 antibody.
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