Regulation of Steroid Sulfatase During Differentiation of Mouse MC3T3-E1 Pre-osteoblastic Cells.

Abstract

Regulation of bone density is partly dependent upon steroid hormones, with estrogens playing a particularly important role. Inactive conjugated estrogens may serve as precursors to active estrogens because of their high levels in blood. The enzyme steroid sulfatase is required for the conversion of sulfoconjugated estrogens into unconjugated estrogens. Osteoblasts proceed through a well-defined process of proliferation, matrix maturation, and extracellular mineralization. Because estrogens have been shown to be important in normal osteoblast function, steroid sulfatase may be necessary during the differentiation of these cells. Using three lines of evidence (enzyme activity, immunoassay, and RT-PCR), our lab has previously shown that undifferentiated mouse preosteoblastic MC3T3-E1 cells contain significant levels of steroid sulfatase. In the present study, we sought to determine if steroid sulfatase is present throughout differentiation of these cells. We developed a 21-day protocol suitable to assess the expression and activity of steroid sulfatase in MC3T3-E1 cells during the differentiation process. Steroid sulfatase expression was assessed by RT-PCR, and steroid sulfatase activity was determined using a whole-cell, tritiated-estrone-sulfate conversion assay. Cells were grown in whole medium alone (OS-), whole medium containing a commonly used osteogenic supplement to enhance differentiation (OS+) or whole medium containing OS+ and an estrogen receptor blocker (ICI 182780). Differentiation was assessed by alkaline phosphatase enzyme activity and by osteocalcin mRNA expression. Osteogenic supplement significantly retarded growth, a result consistent with enhanced differentiation. Indeed, alkaline phosphatase activity peaked much earlier in the OS+ cells than in OS- cells. Interestingly, alkaline phosphatase peaked even earlier, and reached higher levels, in the OS+/ICI 182780 treated cells. Steroid sulfatase activity showed a peak on Days 7 and 14 in OS- cells, a smaller peak on Day 14 in OS+ cells and no peak in the OS+/ICI 182780 treated cells. Steroid sulfatase mRNA was present, and apparently constant, throughout the differentiation process under all test conditions. These data indicate that steroid sulfatase activity varies during differentiation, being higher in the early stages of differentiation. Furthermore, our data indicate that estrogen-receptor-mediated processes may not be required for mouse osteoblasts to undergo differentiation. The exact role of increased steroid sulfatase activity during differentiation remains to be elucidated.