| In addition to its classic role as a regulator of calcium homeostasis, 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D], inhibits growth and induces differentiation of a variety of cell types, including prostate cells. The enzyme responsible for the synthesis of 1alpha,25(OH)2D from 25-hydroxyvitamin D (25(OH)D), 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase), was identified in human prostate cancer cell lines and in primary cultures of human prostate cells using high performance liquid chromatography (HPLC), radioreceptor binding assay (RRA), and reverse transcriptase polymerase chain reaction (RT-PCR). 1alpha-OHase activity in four prostate cell lines (LNCaP, DU-145, PC-3, PZ-HPV-7) was measured. Primary cultures prepared from men with prostate cancer, with benign prostatic hyperplasia (BPH), and normal organ donors were also assayed for 1alpha-OHase activity. 1alpha-OHase activity in prostate cancer cells was significantly lower than in normal prostate cells. Transfection of cells lacking 1alpha-OHase activity (LNCaP) with 1alpha-OHase cDNA restored 1alpha-OHase enzyme activity. Restoration of 1alpha-OHase activity potentiated the inhibitory effect of the prohormone, 25(OH)D3, on cell growth. Known regulators of the renal 1alpha-OHase, parathyroid hormone and calcium, did not alter prostate 1alpha-OHase activity. 1alpha,25(OH)2D3 and insulin-like binding protein 3 (IGFBP3) decreased prostate 1alpha-OHase activity. Epidermal growth factor (EGF) increased prostate 1alpha-OHase activity. To elucidate the molecular mechanism by which 1alpha,25(OH)2D3-induced cellular differentiation and decreased proliferation in the prostate, real time RT-PCR measured expression of potential downstream targets of 1alpha,25(OH) 2D3. 1alpha,25(OH)2D3 up-regulated 25(OH)D-24R-Hydroxylase (24-OHase) expression in a dose and time-dependent fashion. p27(Kip1), a cyclin-dependent kinase inhibitor, IGFBP3, and transforming growth factor beta increased one to eight fold over controls. The vitamin D 25-hydroxylase [25-OHase(Cyp 27)] was identified by RT-PCR. Vitamin D3 and 25(OH)D3 induced 24-OHase in a dose-dependent manner, implying vitamin D3 was converted to 25(OH)D3 and then to 1alpha,25(OH)2D3. The antiproliferative effect of 1alpha,25(OH)2D and the presence of 25-OHase, 1alpha-OHase, and 24-OHase in the prostate suggest that vitamin D may act in an autocrine fashion to regulate prostate cell growth. |
