Steroid sulfatase in the mouse NIH-3T3 fibroblast cell line: Characterization, and downregulation by glucocorticoids

被引:7
作者
Selcer, Kyle [1 ]
Balasubramonian, Barathi [1 ]
Miller, Dylan [1 ]
Kerr, Jade [1 ]
DiFrancesco, Mia [1 ]
Ojha, Sanjana [1 ]
Urbano, Rachel [1 ]
机构
[1] Duquesne Univ, Dept Biol Sci, Mellon Hall Room 201, Pittsburgh, PA 15282 USA
关键词
Fibroblasts; Cortisol; Glucocorticoids; Estrogens; Estrone sulfate; NIH-3T3; cells; Regulation; Steroid sulfatase; Sulfated steroids; Mouse; ESTRONE SULFATASE; CANCER CELLS; EXPRESSION; INHIBITION; INDUCTION;
D O I
10.1016/j.steroids.2021.108890
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Steroid hormones often circulate in the blood as inactive sulfated forms, such as estrone sulfate and dehydroepiandrosterone sulfate. The enzyme steroid sulfatase (STS) converts these steroids into active forms, mainly estrogens, in peripheral tissues. We have previously characterized STS activity in human and mouse breast and bone tissues, and we have shown that STS can provide estrogens to these tissues from circulating sulfated precursors. This study was designed to characterize STS activity in a mouse fibroblast cell line (NIH-3T3). Using a radioactive estrone sulfate (E1S) conversion assay, we detected STS activity in cultured NIH-3T3 cells. This activity was blocked by the STS inhibitors EMATE and STX-64, indicating authentic STS activity. We also found that microsomes prepared from NIH-3T3 cells had relatively high STS activity and that cytosols had low activity, consistent with the known distribution of this enzyme to the endoplasmic reticulum. Michaelis-Menten analysis of the NIH-3T3 microsomes indicated a Km of 10.9 mu M using E1S as substrate. Primary fibroblasts prepared from mouse ears and tails also had measurable STS activity, as indicated by H-3-E1S conversion assay, further supporting the conclusion that fibroblasts possess STS. Furthermore, Western blotting confirmed the presence of immunoreactive STS in NIH-3T3 microsomes. With regard to regulation, treatments of cultured NIH-3T3 cells revealed that cortisol and the synthetic glucocorticoids dexamethasone and prednisolone decreased STS activity, as we have found for cell lines from other tissues. The effect of cortisol was seen at both 10 mu M and 1.0 mu M but not at 0.1 mu M. Western blotting also indicated a decrease in STS immunoreactivity in cortisol-treated microsomes. The reduction in STS activity by dexamethasone in whole cells was reversed by the glucocorticoid receptor antagonist RU-486, indicating that glucocorticoid downregulation of STS activity is receptor mediated. An inhibition assay on NIH-3T3 microsomes revealed that STS activity was inhibited significantly by 10 mu M estradiol 17 beta, a known substrate inhibitor of E1S for STS, but not by 10 mu M cortisol. This is consistent with the idea that cortisol inhibits STS in NIH-3T3 cells through a regulatory mechanism rather than by substrate inhibition. Our results could have important implications regarding local estrogen production by STS in fibroblasts, which are the most common connective tissue cells in the body, and on possible regulation of local estrogen levels by cortisol.
引用
收藏
页数:9
相关论文
共 50 条
  • [21] A proteomic approach for dissecting H-Ras signaling networks in NIH/3T3 mouse embryonic fibroblast cells
    Park, JW
    Kim, S
    Bahk, YY
    PROTEOMICS, 2006, 6 (08) : 2433 - 2443
  • [22] HSP110 expression is induced by cadmium exposure but is dispensable for cell survival of mouse NIH3T3 fibroblasts
    Ridley, Wakako
    Nishitai, Gen
    Matsuoka, Masato
    ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY, 2010, 29 (03) : 260 - 265
  • [23] 槲皮素抗NIH-3T3细胞凋亡的作用
    宫璀璀
    郑乃刚
    张成德
    曹阳
    孟明利
    吴景兰
    河南医学研究, 2007, (02) : 108 - 110
  • [24] Behaviors of NIH-3T3 Fibroblasts on Graphene/Carbon Nanotubes: Proliferation, Focal Adhesion, and Gene Transfection Studies
    Ryoo, Soo-Ryoon
    Kim, Young-Kwan
    Kim, Mi-Hee
    Min, Dal-Hee
    ACS NANO, 2010, 4 (11) : 6587 - 6598
  • [25] Protection of Fibroblasts (NIH-3T3) against Oxidative Damage by Cyanidin-3-rhamnoglucoside Isolated from Fig Fruits (Ficus carica L.)
    Solomon, Anat
    Golubowicz, Sara
    Yablowicz, Zeev
    Bergman, Margalit
    Grossman, Shlomo
    Altman, Arie
    Kerem, Zohar
    Flaishman, Moshe A.
    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 2010, 58 (11) : 6660 - 6665
  • [26] EFFECT OF CALCIUM-ANTAGONISTS ON RNA-SYNTHESIS OF NIH-3T3 CELLS
    ANDRAWIS, NS
    ABERNETHY, DR
    AMERICAN JOURNAL OF THE MEDICAL SCIENCES, 1993, 306 (03) : 137 - 140
  • [27] Construction of TSC2 knockout cell line using CRISPR/Cas9 system and demonstration of its effects on NIH-3T3 cells
    Xu Wang
    Yang Zhao
    Zhan Wang
    Zhangcheng Liao
    Yushi Zhang
    Cell Biochemistry and Biophysics, 2022, 80 : 681 - 687
  • [28] Effects of Ca2+-ionophore A23187 and calmodulin antagonists on regulatory mechanisms of glycolysis and cell viability of NIH-3T3 fibroblasts
    Ashkenazy-Shahar, M
    Beitner, R
    MOLECULAR GENETICS AND METABOLISM, 1999, 67 (04) : 334 - 342
  • [29] Cloning and Stable Expression of cDNA Coding For Platelet Endothelial Cell Adhesion Molecule -1 (PECAM-1, CD31) in NIH-3T3 Cell Line
    Salehi-Lalemarzi, Hamed
    Shanehbandi, Dariush
    Shafaghat, Farzaneh
    Abbasi-Kenarsari, Hajar
    Baradaran, Behzad
    Movassaghpour, Ali Akbar
    Kazemi, Tohid
    ADVANCED PHARMACEUTICAL BULLETIN, 2015, 5 (02) : 247 - 253
  • [30] Tissue inhibitor of metalloproteinase-1 promotes NIH3T3 fibroblast proliferation by activating p-Akt and cell cycle progression
    Lu, Yang
    Liu, Shuxin
    Zhang, Shujia
    Cai, Guangyan
    Jiang, Hongwei
    Su, Huabin
    Li, Xiaofan
    Hong, Quan
    Zhang, Xueguang
    Chen, Xiangmei
    MOLECULES AND CELLS, 2011, 31 (03) : 225 - 230