Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming, USA.
Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Reproduction. 2021 Jul 8;162(2):117-127. doi: 10.1530/REP-21-0132.
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.
肽基精氨酸脱亚氨酶（PAD）酶最初在子宫中被描述，但此后很少有研究检查它们在该组织中的功能。PAD 酶将靶蛋白中的精氨酸残基转化为瓜氨酸，并在绵羊的肉阜上皮和绵羊子宫腔上皮（OLE）衍生细胞系中高度表达。孕酮（P4）不仅维持子宫上皮，还调节编码构成组织培养物的蛋白质的子宫内膜基因的表达，这些蛋白质在早期妊娠中至关重要。鉴于此，我们测试了 P4 是否刺激 PAD 催化的组蛋白瓜氨酸化，以表观遗传调控 OLE 细胞中组织培养物基因胰岛素样生长因子结合蛋白 1（IGFBP1）的表达。100 nM P4 显著增加 IGFBP1 mRNA 的表达；然而，用 100 nM 孕激素受体拮抗剂 RU486 或 2 µM 泛 PAD 抑制剂预处理 OLE 细胞会减弱这种增加。P4 处理 OLE 细胞还刺激组蛋白 H3 精氨酸残基 2、8 和 17 的瓜氨酸化，导致绵羊 IGFBP1 基因启动子的富集。由于 PAD2 核易位和催化活性需要钙，我们接下来研究了 P4 是否触发 OLE 细胞中的钙内流。用 10 nM 尼卡地平预处理 OLE 细胞，尼卡地平是一种 L 型钙通道阻滞剂，然后用 P4 刺激。使用 fura2-AM 成像，我们发现 P4 在 OLE 细胞中通过 L 型钙通道引发快速的钙内流。此外，这种内流对于 PAD2 核易位和组蛋白 H3 精氨酸残基 2、8 和 17 的瓜氨酸化是必需的。我们的工作表明，P4 通过 L 型钙通道引发快速的钙内流，从而启动 PAD 催化的组蛋白瓜氨酸化，并增加 IGFBP1 的表达。
