Genome-wide analysis reveals PADI4 cooperates with Elk-1 to activate c-Fos expression in breast cancer cells.

Peptidylarginine deiminase IV (PADI4) catalyzes the conversion of positively charged arginine and methylarginine residues to neutrally charged citrulline, and this activity has been linked to the repression of a limited number of target genes. To broaden our knowledge of the regulatory potential of PADI4, we utilized chromatin immunoprecipitation coupled with promoter tiling array (ChIP-chip) analysis to more comprehensively investigate the range of PADI4 target genes across the genome in MCF-7 breast cancer cells. Results showed that PADI4 is enriched in gene promoter regions near transcription start sites (TSSs); and, surprisingly, this pattern of binding is primarily associated with actively transcribed genes. Computational analysis found potential binding sites for Elk-1, a member of the ETS oncogene family, to be highly enriched around PADI4 binding sites; and coimmunoprecipitation analysis then confirmed that Elk-1 physically associates with PADI4. To better understand how PADI4 may facilitate gene transactivation, we then show that PADI4 interacts with Elk-1 at the c-Fos promoter and that, following Epidermal Growth Factor (EGF) stimulation, PADI4 catalytic activity facilitates Elk-1 phosphorylation, histone H4 acetylation, and c-Fos transcriptional activation. These results define a novel role for PADI4 as a transcription factor co-activator

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 mug/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation

Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.

BACKGROUND: We have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects. METHODS: RNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes. RESULTS: We show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 x 106) in luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45alpha, and Ki67. CONCLUSION: Together, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy

Potential Role for Peptidylarginine Deiminase 2 (PAD2) in Citrullination of Canine Mammary Epithelial Cell Histones

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

GnRH Stimulates Peptidylarginine Deiminase Catalyzed Histone Citrullination in Gonadotrope Cells

Peptidylarginine deiminase (PAD) enzymes convert histone tail arginine residues to citrulline resulting in chromatin decondensation. Our previous work found that PAD isoforms are expressed in female reproductive tissues in an estrous cycle-dependent fashion, but their role in the anterior pituitary gland is unknown. Thus, we investigated PAD expression and function in gonadotrope cells. The gonadotrope-derived LbetaT2 cell line strongly expresses PAD2 at the protein level compared with other PAD isoforms. Consistent with this, PAD2 protein expression is highest during the estrous phase of the estrous cycle and colocalizes with the LH beta-subunit in the mouse pituitary. Using the GnRH agonist buserelin (GnRHa), studies in LbetaT2 and mouse primary gonadotrope cells revealed that 30 minutes of stimulation caused distinct puncta of PAD2 to localize in the nucleus. Once in the nucleus, GnRHa stimulated PAD2 citrullinates histone H3 tail arginine residues at sites 2, 8, and 17 within 30 minutes; however, this effect and PAD2 nuclear localization was blunted by incubation of the cells with the pan-PAD inhibitor, biphenyl-benzimidazole-Cl-amidine. Given that PAD2 citrullinates histones in gonadotropes, we next analyzed the functional consequence of PAD2 inhibition on gene expression. Our results show that GnRHa stimulates an increase in LHbeta and FSHbeta mRNA and that this response is significantly reduced in the presence of the PAD inhibitor biphenyl-benzimidazole-Cl-amidine. Overall, our data suggest that GnRHa stimulates PAD2-catalyzed histone citrullination in gonadotropes to epigenetically regulate gonadotropin gene expression

Progesterone stimulates histone citrullination to increase IGFBP1 expression in uterine cells

Peptidylarginine deiminases (PAD) enzymes were initially characterized in uteri, but since then little research has examined their function in this tissue. PADs post-translationally convert arginine residues in target proteins to citrulline and are highly expressed in ovine caruncle epithelia and ovine uterine luminal epithelial (OLE)-derived cell line. Progesterone (P4) not only maintains the uterine epithelia but also regulates the expression of endometrial genes that code for proteins that comprise the histotroph and are critical during early pregnancy. Given this, we tested whether P4 stimulates PAD-catalyzed histone citrullination to epigenetically regulate expression of the histotroph gene insulin-like growth factor binding protein 1 (IGFBP1) in OLE cells. 100 nM P4 significantly increases IGFBP1 mRNA expression; however, this increase is attenuated by pre-treating OLE cells with 100 nM progesterone receptor antagonist RU486 or 2 microM of a pan-PAD inhibitor. P4 treatment of OLE cells also stimulates citrullination of histone H3 arginine residues 2, 8, and 17 leading to enrichment of the ovine IGFBP1 gene promoter. Since PAD2 nuclear translocation and catalytic activity require calcium, we next investigated whether P4 triggers calcium influx in OLE cells. OLE cells were pre-treated with 10 nM nicardipine, an L-type calcium channel blocker, followed by stimulation with P4. Using fura2-AM imaging, we found that P4 initiates a rapid calcium influx through L-type calcium channels in OLE cells. Furthermore, this influx is necessary for PAD2 nuclear translocation and resulting citrullination of histone H3 arginine residues 2, 8, and 17. Our work suggests that P4 stimulates rapid calcium influx through L-type calcium channels initiating PAD-catalyzed histone citrullination and an increase in IGFBP1 expression

Identification and Characterization of the Lactating Mouse Mammary Gland Citrullinome

Citrullination is a post-translational modification (PTM) in which positively charged peptidyl-arginine is converted into neutral peptidyl-citrulline by peptidylarginine deiminase (PAD or PADI) enzymes. The full protein citrullinome in many tissues is unknown. Herein, we used mass spectrometry and identified 107 citrullinated proteins in the lactation day 9 (L9) mouse mammary gland including histone H2A, alpha-tubulin, and beta-casein. Given the importance of prolactin to lactation, we next tested if it stimulates PAD-catalyzed citrullination using mouse mammary epithelial CID-9 cells. Stimulation of CID-9 cells with 5 microg/mL prolactin for 10 min induced a 2-fold increase in histone H2A citrullination and a 4.5-fold increase in alpha-tubulin citrullination. We next investigated if prolactin-induced citrullination regulates the expression of lactation genes beta-casein (Csn2) and butyrophilin (Btn1a1). Prolactin treatment for 12 h increased beta-casein and butyrophilin mRNA expression; however, this increase was significantly inhibited by the pan-PAD inhibitor, BB-Cl-amidine (BB-ClA). We also examined the effect of tubulin citrullination on the overall polymerization rate of microtubules. Our results show that citrullinated tubulin had a higher maximum overall polymerization rate. Our work suggests that protein citrullination is an important PTM that regulates gene expression and microtubule dynamics in mammary epithelial cells

Potential Role of Peptidylarginine Deiminase Enzymes and Protein Citrullination in Cancer Pathogenesis

The peptidylarginine deiminases (PADs) are a family of posttranslational modification enzymes that catalyze the conversion of positively charged protein-bound arginine and methylarginine residues to the uncharged, nonstandard amino acid citrulline. This enzymatic activity is referred to as citrullination or, alternatively, deimination. Citrullination can significantly affect biochemical pathways by altering the structure and function of target proteins. Five mammalian PAD family members (PADs 1–4 and 6) have been described and show tissue-specific distribution. Recent reviews on PADs have focused on their role in autoimmune diseases. Here, we will discuss the potential role of PADs in tumor progression and tumor-associated inflammation. In the context of cancer, increasing clinical evidence suggests that PAD4 (and possibly PAD2) has important roles in tumor progression. The link between PADs and cancer is strengthened by recent findings showing that treatment of cell lines and mice with PAD inhibitors significantly suppresses tumor growth and, interestingly, inflammatory symptoms. At the molecular level, transcription factors, coregulators, and histones are functional targets for citrullination by PADs, and citrullination of these targets can affect gene expression in multiple tumor cell lines. Next generation isozyme-specific PAD inhibitors may have therapeutic potential to regulate both the inflammatory tumor microenvironment and tumor cell growth