TY - JOUR
T1 - Characterization of steroid sulfatase in the MC3T3-E1 mouse pre-osteoblastic cell line
AU - Selcer, K. W.
AU - DiFrancesca, H. M.
N1 - Funding Information:
The authors thank Dr. Pui-Kai Li for providing the EMATE. Laura Vollmer provided excellent technical assistance. This work was funded by a Commonwealth Universal Research Enhancement Grant from the Pennsylvania Department of Health to K.W. Selcer.
PY - 2012/5
Y1 - 2012/5
N2 - Regulation of bone density is partly dependent upon steroid hormones, with estrogens playing an important role. Inactive conjugated estrogens may serve as precursors to active estrogens, especially in post-menopausal women, via steroid sulfatase, which converts conjugated estrogens into unconjugated estrogens. The purpose of this study was to characterize steroid sulfatase in the MC3T3-E1 mouse pre-osteoblastic cell line. Enzyme conversion assays were performed on whole MC3T3-E1 cells in culture and on microsomes prepared by differential centrifugation. 3H-E 1S and 3H-DHEAS were used as tracers, and radioinert E 1S and DHEAS were used as substrate. Whole cells and microsomes exhibited steroid sulfatase activity, which was blocked by the specific inhibitor estrone-3-O-sulfamate (EMATE). The K m of steroid sulfatase in microsomes averaged 83 μM when using E 1S as substrate and 64 μM when using DHEAS. Western blotting of MC3T3-E1 microsomes for steroid sulfatase was performed, after SDS-PAGE, using an antibody generated against a peptide based on a conserved region of steroid sulfatase. Western blotting revealed three bands of cross-reactivity, ranging from 50 to 79 kDa. Reverse transcriptase polymerase chain reaction (RT-PCR), using specific primers, resulted in a single cDNA band of the expected size (100 bp) and sequence, indicating the presence of steroid sulfatase mRNA. Growth assays revealed that the MC3T3-E1 cells were stimulated by estradiol-17β, and also by estrone sulfate and DHEAS, revealing that the cells can use steroid sulfatase to produce active estrogens. Furthermore, growth of these cells in the presence of estradiol, estrone and estrone sulfate was inhibited by the estrogen receptor blocker ICI 182,780, indicating that stimulation of cell growth is mediated by the estrogen receptor. In our studies, four lines of evidence (enzyme activity, immunoassay, RT-PCR and growth assays) demonstrated the presence of steroid sulfatase in mouse MC3T3-E1 bone cells. The existence of steroid sulfatase in these pre-osteoblastic cells, along with the ability of sulfated steroids to promote their growth, suggest the possibility that this enzyme is involved in regulation of bone density in mice.
AB - Regulation of bone density is partly dependent upon steroid hormones, with estrogens playing an important role. Inactive conjugated estrogens may serve as precursors to active estrogens, especially in post-menopausal women, via steroid sulfatase, which converts conjugated estrogens into unconjugated estrogens. The purpose of this study was to characterize steroid sulfatase in the MC3T3-E1 mouse pre-osteoblastic cell line. Enzyme conversion assays were performed on whole MC3T3-E1 cells in culture and on microsomes prepared by differential centrifugation. 3H-E 1S and 3H-DHEAS were used as tracers, and radioinert E 1S and DHEAS were used as substrate. Whole cells and microsomes exhibited steroid sulfatase activity, which was blocked by the specific inhibitor estrone-3-O-sulfamate (EMATE). The K m of steroid sulfatase in microsomes averaged 83 μM when using E 1S as substrate and 64 μM when using DHEAS. Western blotting of MC3T3-E1 microsomes for steroid sulfatase was performed, after SDS-PAGE, using an antibody generated against a peptide based on a conserved region of steroid sulfatase. Western blotting revealed three bands of cross-reactivity, ranging from 50 to 79 kDa. Reverse transcriptase polymerase chain reaction (RT-PCR), using specific primers, resulted in a single cDNA band of the expected size (100 bp) and sequence, indicating the presence of steroid sulfatase mRNA. Growth assays revealed that the MC3T3-E1 cells were stimulated by estradiol-17β, and also by estrone sulfate and DHEAS, revealing that the cells can use steroid sulfatase to produce active estrogens. Furthermore, growth of these cells in the presence of estradiol, estrone and estrone sulfate was inhibited by the estrogen receptor blocker ICI 182,780, indicating that stimulation of cell growth is mediated by the estrogen receptor. In our studies, four lines of evidence (enzyme activity, immunoassay, RT-PCR and growth assays) demonstrated the presence of steroid sulfatase in mouse MC3T3-E1 bone cells. The existence of steroid sulfatase in these pre-osteoblastic cells, along with the ability of sulfated steroids to promote their growth, suggest the possibility that this enzyme is involved in regulation of bone density in mice.
KW - Estrogens
KW - Mouse bone
KW - Osteoblast cells
KW - Sulfatase
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U2 - 10.1016/j.steroids.2012.02.024
DO - 10.1016/j.steroids.2012.02.024
M3 - Article
C2 - 22426324
AN - SCOPUS:84859807719
SN - 0039-128X
VL - 77
SP - 696
EP - 702
JO - Steroids
JF - Steroids
IS - 6
ER -