Peptidylarginine deiminase enzymes and citrullinated proteins in female reproductive physiology and associated diseases†.

Citrullination, the post-translational modification of arginine residues, is catalyzed by the four catalytically active peptidylarginine deiminase (PAD or PADI) isozymes and alters charge to affect target protein structure and function. PADs were initially characterized in rodent uteri and, since then, have been described in other female tissues including ovaries, breast, and the lactotrope and gonadotrope cells of the anterior pituitary gland. In these tissues and cells, estrogen robustly stimulates PAD expression resulting in changes in levels over the course of the female reproductive cycle. The best-characterized targets for PADs are arginine residues in histone tails, which, when citrullinated, alter chromatin structure and gene expression. Methodological advances have allowed for the identification of tissue-specific citrullinomes, which reveal that PADs citrullinate a wide range of enzymes and structural proteins to alter cell function. In contrast to their important physiological roles, PADs and citrullinated proteins are also involved in several female-specific diseases including autoimmune disorders and reproductive cancers. Herein, we review current knowledge regarding PAD expression and function and highlight the role of protein citrullination in both normal female reproductive tissues and associated diseases.

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 µM 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, α-tubulin, and ß-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 µg/mL prolactin for 10 min induced a 2-fold increase in histone H2A citrullination and a 4.5-fold increase in α-tubulin citrullination. We next investigated if prolactin-induced citrullination regulates the expression of lactation genes ß-casein (Csn2) and butyrophilin (Btn1a1). Prolactin treatment for 12 h increased ß-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.

Histone Citrullination Represses MicroRNA Expression, Resulting in Increased Oncogene mRNAs in Somatolactotrope Cells.

Peptidylarginine deiminase (PAD) enzymes convert histone arginine residues into citrulline to modulate chromatin organization and gene expression. Although PADs are expressed in anterior pituitary gland cells, their functional role and expression in pituitary adenomas are unknown. To begin to address these issues, we first examined normal human pituitaries and pituitary adenomas and found that PAD2, PAD4, and citrullinated histones are highest in prolactinomas and somatoprolactinomas. In the somatoprolactinoma-derived GH3 cell line, PADs citrullinate histone H3, which is attenuated by a pan-PAD inhibitor. RNA sequencing and chromatin immunoprecipitation (ChIP) studies show that the expression of microRNAs (miRNAs) let-7c-2, 23b, and 29c is suppressed by histone citrullination. Our studies demonstrate that these miRNAs directly target the mRNA of the oncogenes encoding HMGA, insulin-like growth factor 1 (IGF-1), and N-MYC, which are highly implicated in human prolactinoma/somatoprolactinoma pathogenesis. Our results are the first to define a direct role for PAD-catalyzed histone citrullination in miRNA expression, which may underlie the etiology of prolactinoma and somatoprolactinoma tumors through regulation of oncogene expression.

Citrullination regulates the expression of insulin-like growth factor-binding protein 1 (IGFBP1) in ovine uterine luminal epithelial cells.

There are five peptidylarginine deiminase (PAD) isozymes designated as PADs 1, 2, 3, 4 and 6, and many are expressed in female reproductive tissues. These enzymes post-translationally convert positively charged arginine amino acids into neutral citrulline residues. Targets for PAD-catalyzed citrullination include arginine residues on histone tails, which results in chromatin decondensation and changes in gene expression. Some of the first studies examining PADs found that they are localized to rodent uterine epithelial cells. Despite these findings, the function of PAD-catalyzed citrullination in uterine epithelial cells is still unknown. To address this, we first examined PAD expression in uterine cross-sections from pregnant ewes on gestation day 25 (d25). Immunohistochemistry revealed that the levels of PADs 2 and 4 are robust in luminal and glandular epithelia compared with those of PADs 1 and 3. As PADs 2 and 4 have well-characterized roles in histone citrullination, we next hypothesized that PADs citrullinate histones in these uterine cells. Examination of caruncle lysates from pregnant ewes on gestation d25 and an ovine luminal epithelial (OLE) cell line shows that histone H3 arginine residues 2, 8, 17 and 26 are citrullinated, but histone H4 arginine 3 is not. Using a pan-PAD inhibitor, we next attenuated histone citrullination in OLE cells, which resulted in a significant decrease in the expression of insulin-like growth factor-binding protein 1 (IGFBP1) mRNA. As IGFBP1 is important for the migration and attachment of the trophectoderm to uterine endometrium, our results suggest that PAD-catalyzed citrullination may be an important post-translational mechanism for the establishment of pregnancy in ewes.

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 µg/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.