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

•Steroid sulfatase (STS) converts inactive steroids to active ones in peripheral tissues.•STS activity is present in mouse NIH-3T3 fibroblasts and in mouse ear and tail fibroblasts.•Glucocorticoids decrease STS activity in NIH-3T3 cells, not due to substrate inhibition.•STS in fibroblasts may increa...

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Bibliographic Details
Published in:Steroids 2021-10, Vol.174, p.108890, Article 108890
Main Authors: Selcer, Kyle, Balasubramonian, Barathi, Miller, Dylan, Kerr, Jade, DiFrancesco, Mia, Ojha, Sanjana, Urbano, Rachel
Format: Article
Language:English
Subjects:
Cortisol
Estrogens
Estrone sulfate
Fibroblasts
Glucocorticoids
Mouse
NIH-3T3 cells
Regulation
Steroid sulfatase
Steryl-Sulfatase
Sulfated steroids
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Summary:•Steroid sulfatase (STS) converts inactive steroids to active ones in peripheral tissues.•STS activity is present in mouse NIH-3T3 fibroblasts and in mouse ear and tail fibroblasts.•Glucocorticoids decrease STS activity in NIH-3T3 cells, not due to substrate inhibition.•STS in fibroblasts may increase estrogens and cortisol may decrease them, affecting inflammation. 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 µM using E1S as substrate. Primary fibroblasts prepared from mouse ears and tails also had measurable STS activity, as indicated by 3H-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 µM and 1.0 µM but not at 0.1 µM. 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 µM estradiol-17β, a known substrate inhibitor of E1S fo
ISSN:0039-128X
1878-5867
1878-5867
DOI:10.1016/j.steroids.2021.108890