Gestational changes in PRMT1 expression of murine placentas
Introduction
The placenta is essential for the fetal development, forming an interface connection and separating the maternal and the fetal circulations during pregnancy [1]. It is well known that the fetal growth is dependent on nutrients, oxygen, metabolites, several pregnancy-associated hormones, and growth factors [2]. Thus, proper formation and functional maintenance of placenta are of importance for transporting these materials into fetal circulation [3]. The placenta is composed of various cell types, including trophoblast cells (TCs), vascular endothelial cells and blood cells, in which trophoblasts are the most important for the structure and function of the placenta [4].
In mammals, TCs that form the outer layer of blastocyst develop into a large part of the placenta and play roles either in altering maternal physiology and blood flow to promote fetal growth or in nutrient uptake [3,5]. Furthermore, differentiation and proliferation of TCs are accurately controlled through environmental factors and cellular molecules, such as oxygen tension within the maternal–fetal interface and hormones and growth factors [6]. Meanwhile, it has been reported that cellular processes of TCs, such as the cell growth, differentiation, apoptosis, migration, and invasion are modulated by transcriptional, epigenetic and metabolic regulations during placental development [7]. In addition, they are generated by protein post-translational modifications (PTMs), including phosphorylation, acetylation, glycosylation, ubiquitination, and lysine methylation during placental development [[8], [9], [10], [11]].
The protein arginine methylation is also one of the PTMs and is identified in arginine residues of histones, transcription factors, RNA binding proteins, and signal transduction factors, which are involved in various biological phenomena related to maintaining cell differentiation, proliferation, growth regulation and apoptosis [[12], [13], [14]]. The reaction of arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) that form monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Currently, nine PRMTs have been identified in mammalian cells. Type I PRMTs (PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8) catalyze the formation of MMA and subsequently ADMA. PRMT5 and PRMT9, as a type II PRMTs, produce SDMA via MMA, while PRMT7, a Type III PRMT, only catalyze the formation of MMA [15,16].
PRMT1, the most predominant of these enzymes, is responsible for over 85% of cellular arginine methylation activity [17] and is ubiquitously expressed in the developing embryo [18,19]. PRMT1 has two major splice variants which differ in their N-terminus and in subcellular localizations. The variant 1 is mainly localized to the nucleus and the nuclear export signal -containing variant 2 is predominantly localized in the cytoplasm and may contribute to the methylation of multiple proteins [20]. However, the physiological functions of PRMT1 have not been understood in mammals, because homozygous mutant mice exhibit embryonic lethality at embryonic (E) day 6.5 [21]. Recently, we reported that the central nervous system (CNS)-specific PRMT1 knockout mice show hypomyelination and developmental defects [22]. In addition, vascular endothelial cell PRMT1-deficient mice revealed angiodysplasia that resulted in death by E15 [23]. These studies indicate that PRMT1 has crucial roles in developmental stages. Interestingly, it is also known that the expression level of PRMT1 in the fetus is higher than in prenatal mice [21]. Thus, although it is considered that PRMT1 plays a dynamic role in the development of placenta via the regulation of arginine methylation activity during pregnancy, there have been no reports concerning the developmental expression of PRMT1 in placenta.
In the present study, we found that the placental PRMT1 was highly expressed in mid-gestation stages. Immunohistochemistry and immunofluorescence analyses using both a bacterial β-galactosidase reporter gene (LacZ)-reporter-tagged heterozygous PRMT1 (prmt1+/LacZ) and wild-type mice showed that PRMT1 was localized in TCs or the parietal trophoblast giant cells (P-TGCs) of the ectoplacental cone (EPC), the chorion and the decidua (De) at E9, and the labyrinth (La)-restricted expression at E13. Moreover, we also found that the levels of ADMA in placental proteins were comparable to the developmental expression pattern of PRMT1. Our findings provide fundamental information regarding the PRMT1-mediated arginine methylation during placental development.
Section snippets
Antibodies
The antibodies used in this study included anti-PRMT1 antibody (Millipore, 07–404), anti-PRMT1 antibody (Abcam, ab92299), anti-GAPDH antibody (Cell Signaling Technology, 5174), anti-rabbit IgG HRP-linked antibody (GE Healthcare, NA934), and anti-rabbit IgG biotinylated antibody (Vector Laboratories, BA-1000).
Animals
All mice were housed under a 12 h light–12 h dark cycle, and they had free access to commercial chow and filtered water. All animal experiments were carried out humanely after approval from
Changes in placental PRMT1 expression during the gestational period
We characterized the expression profiles of PRMT1 mRNAs and proteins in mouse placentas at E9, E11, E13, E16, and E19. As shown in Fig. 1A, PRMT1 mRNAs were highly expressed in mid-gestation stages (E9, E11 and E13), compared to late stages of pregnancy (E16 and E19). Next, we evaluated the expression of PRMT1 proteins by Western blotting and revealed that the levels of PRMT1 variant 1 (v1) were also increased from E9 to E13, and decreased starting at E16. By contrast, variant 2 (v2) levels
Discussion
Protein arginine methylation is found on various intracellular proteins, and is catalyzed by a family of nine PRMTs in mammals [[14], [15], [16]]. The expression of placental PRMTs mRNAs showed ontogenic changes during pregnancy, in which there had different patterns among PRMTs (Fig. 1A and Supplementary figure 2), suggesting they play roles in a variety of cellular processes. PRMT1 is known to methylate histones and various non-histone proteins that are involved in the regulation of cellular
Funding
This work was supported by Grant-in-Aid for Scientific Research (A) (to A.F., Grant No. 25252062) and Grant-in-Aid for Scientific Research (C) (to A.I., Grant No. 15K10687, and to K.K., Grant No. 26350957 and 17K01942) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Conflicts of interest
The authors declare that they have no conflicts interest.
Acknowledgments
We thank Ms. Xie Yuying, Mr. Chulwon Kwon, Mr. Weizhe Lu, Dr. Misuzu Hashimoto, Dr. Tomohiro Ishimaru, and Dr. Kazuya Murata for technical assistance, support and helpful advice throughout this project. The Tpbpa-Cre transgenic mouse strain was kindly provided by the Canadian Mouse Mutant Repository. We also thank the members of Fukamizu laboratory for the helpful discussions.
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