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.

Epidemiological data indicate that low serum vitamin D concentrations are associated with an increased risk of a variety of human tumours. Cutaneous mast cell tumours (MCT) occur more frequently in dogs than in any other species. Canine MCT express the vitamin D receptor, and vitamin D derivatives have in vitro and in vivo anti-tumour activity. We sought to examine the association between vitamin D serum level and MCT in Labrador retrievers, a dog breed predisposed to MCT development. To examine this association, serum 25-hydroxyvitamin D3 (25(OH)D3) concentrations were examined in eighty-seven Labrador retrievers, including thirty-three with MCT and fifty-four unaffected controls. The relationship between cases and controls and 25(OH)D3 level, age and body condition score were evaluated using univariate and multivariate analyses. Potential differences in vitamin D oral intake, calculated on the basis of a dietary questionnaire, were also evaluated between groups. Mean 25(OH)D3 concentration (104 (SD 30) nmol/l) in dogs with MCT was significantly lower than that of unaffected dogs (120 (SD 35) nmol/l; P = 0.027). The mean calculated vitamin D intake per kg body weight in Labrador retrievers with MCT was not statistically different from that of unaffected Labrador retrievers (0.38 (SD 0.25) and 0.31 (SD 0.22) μg/kg body weight, respectively; P = 0.13). These findings suggest that low levels of 25(OH)D3 might be a risk factor for MCT in Labrador retrievers. Prospective cohort studies are warranted.

BackgroundVitamin D plays a pivotal role in cardiac function, and there is increasing evidence that vitamin D deficiency is associated with the development of congestive heart failure (CHF) in people.HypothesisSerum vitamin D concentration is lower in dogs with CHF compared with unaffected controls and serum vitamin D concentration is associated with clinical outcome in dogs with CHF.AnimalsEighty‐two client‐owned dogs.MethodsIn this cross‐sectional study, we examined the association between circulating 25‐hydroxyvitamin D [25(OH)D], a measure of vitamin D status, and CHF in dogs. In the prospective cohort study, we examined whether 25(OH)D serum concentration was associated with clinical outcome in dogs with CHF.ResultsMean 25(OH)D concentration (100 ± 44 nmol/L) in 31 dogs with CHF was significantly lower than that of 51 unaffected dogs (123 ± 42 nmol/L; P = .023). The mean calculated vitamin D intake per kg of metabolic body weight in dogs with CHF was no different from that of unaffected dogs (1.37 ± 0.90 μg/kg metabolic body weight versus 0.98 ± 0.59 μg/kg body weight, respectively, P = .097). There was a significant association of serum 25(OH)D concentration on time to clinical manifestation of CHF or sudden death (P = .02).Conclusion and Clinical RelevanceThese findings suggest that low concentrations of 25(OH)D may be a risk factor for CHF in dogs. Low serum 25(OH)D concentration was associated with poor outcome in dogs with CHF. Strategies to improve vitamin D status in some dogs with CHF may prove beneficial without causing toxicity.