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

Abstract

There are five peptidylarginine deiminase (PAD) isozymes designated 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 to PADs 1 and 3. Since 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. Since IGFBP1 is important for migration and attachment of the trophectoderm to uterine endometrium, our results suggest that PAD catalyzed citrullination may be an important post-translational mechanism for establishment of pregnancy in ewes.

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

“…Samples were deidentified and thus were exempt from University of Wyoming institutional review board (IRB) approval (protocol number 20140814BC00496). Immunohistochemistry (IHC) was performed as previously described (58,59). Briefly, slides were incubated with anti-PAD2, -PAD4, or -H3Cit2,8,17 antibodies at 1:100 in 1ϫ phosphate-buffered saline (PBS) overnight at 4°C, and negative-control slides were incubated with an equal mass of nonspecific rabbit IgG (Vector Laboratories).…”

Identification and Characterization of the Lactating Mouse Mammary Gland Citrullinome

“…Histones and cytoskeletal proteins are known substrates for PAD enzymes [4][5][6][7]. In several female reproductive tissues, PADs epigenetically regulate gene expression via citrullination of histone tail arginine residues [2,8,9]. Cytoskeletal proteins are also targets for citrullination, which is thought to alter filament stability and intermolecular interactions [10][11][12].…”

NETosis Markers in Pregnancy: Effects Differ According to Histone Subtypes

“…28 Citrullinated H3 is found in decidual cells in mice 29 and is involved in insulinlike growth factor-binding protein 1 (IGFBP1) regulation, which is important for migration and attachment of trophoectoderm to the endometrium. 30 Additionally, H3K27me3, a repressive modification, is also found in decidual cells 31 and its level increased on metalloprotease 2 and 9 (MMP2 and MMP9) promoters in third trimester placental villi compared with first trimester ones. 32 In addition, we observed a "pregnancy effect," since the pregnant group had higher levels of all proteins tested compared with the nonpregnant group but, interestingly, only histones were more expressed in complicated pregnant women.…”