1,25-Dihydroxyvitamin D3 receptors and functions in cultured pig kidney cells (LLC PK1). Regulation of 24,25-dihydroxyvitamin D3 production.

Although 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) regulates the renal metabolism of 25-hydroxyvitamin D3 (25-OH-D3), the mechanism is not well understood. The established pig kidney cell line, LLC PK1, was used to study this feedback regulation. These cells possess a receptor for 1,25-(OH)2D3 with a sedimentation coefficient of 3.2 S. Scatchard analysis of 1,25-(OH)2D3 binding to cell cytosol yielded an apparent Kd of 0.12 nM and an Nmax of 26 fmol/mg of cytosol protein. LLC PK1 cells respond to 1,25-(OH)2D3 by changes in the metabolism of 25-OH-D3. When incubated with 25-OH-[3H]D3, homogenates of untreated cells did not produce detectable 1,25-(OH)2[3H]D3 or 24,25-(OH)2[3H]D3. However, after treatment of cell monolayers with 1,25-(OH)2D3 for 8 h, homogenates converted substantial 25-OH-[3H]D2 substrate to 24,25-(OH)2[3H]D3. The appearance of this 24-hydroxylase activity in response to 1,25-(OH)2D3 was time- and dose-dependent. Half-maximal levels of enzyme activity were achieved with 0.13 nM 1,25-(OH)2D3, a concentration almost identical to the Kd of the 1,25-(OH)2D3 receptor. The stimulation of 24-hydroxylase activity was shown to be an induction event; treatment of monolayers with 13 nM 1,25-(OH)2D3 for a 4-h pulse was sufficient to induce maximal activity assayed at h. The presence of the transcriptional inhibitor, actinomycin D, during the 4-h pulse abolished the induction of 24-hydroxylase activity. These results demonstrate for the first time the presence of both 1,25-(OH)2D3 receptors and stimulation of 24-hydroxylase activity within the same established mammalian kidney cell line.