The effects of 1,25-dihydroxyvitamin D3 on rat primary astrocytes
Curletto, Bryon Joseph
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Vitamin D, more specifically 1,25-dihydroxyvitamin D3, has received increasing attention over the past several years for its widespread effects on the genome. This study hypothesized that 1,25-dihydroxyvitamin D3 decreases proliferation and induces glia acitic fibrillary protein (GFAP), a marker for differentiation, mRNA in primary rat cortical astrocytes. To test the hypothesis, the presence of the vitamin D receptor in primary rat cortical astrocyte cells was confirmed using RT-PCR, western blot analysis, and immunofluorescent labeling. The presence of 1α-hydroxylase, the enzyme responsible for converting circulating 25-hydroxyvitamin D3 to biologically active 1,25-dihydroxyvitamin D3, was also investigated. Messenger RNA levels for 1α-hydroxylase fell below the detection range for RT-PCR, and western blotting was negative for 1α-hydroxylase; however, immunofluorescent labeling was positive for the enzyme. The induction of 24-hydroxylase mRNA, which codes for an enzyme that is up-regulated in response to 1,25-dihydroxyvitamin D3 in numerous cell types, was measured. Treating astrocyte cells with different levels of 1,25-dihydroxyvitamin D3 for 8 hours resulted in a dose-dependent downstream effect, up-regulating 24-hydroxylase mRNA by 40-890 fold (p<.05). To test the specific hypothesis regarding proliferation and differentiation, cells were treated with 1x10-6M 1,25-dihydroxyvitamin D3 for 24 hours. A significant decrease in 3H-thymidine incorporation into astrocyte cells was observed (p<.05) suggesting that 1,25-dihydroxyvitamin D3 regulates cell proliferation. Additionally, GFAP mRNA was significantly decreased when cells were treated with 1x10-10M 1,25-dihydroxyvitamin D3 (p<.05) for 8 hours, but no dose dependent response was observed. Results suggest that the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3, significantly reduced cell proliferation at concentrations of 1x10-6M and down-regulated GFAP mRNA at 1x10-10M in primary rat cortical astrocyte cells in vitro. Understanding the effects of 1,25-dihydroxyvitamin D3 on cells of the central nervous system is an important step towards understanding the hormone’s effects under both normal and pathological conditions in the brain.
Thesis (M.A.)--Boston University