1,25-Dihydroxyvitamin D3 induces 25-hydroxyvitamin D3-24-hydroxylase in a cultured monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity receptor for the hormone

• Published in: The Journal of biological chemistry Vol. 259; no. 4; pp. 2214 - 2222
• Main Authors: Chandler, J S, Chandler, S K, Pike, J W, Haussler, M R
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.02.1984; American Society for Biochemistry and Molecular Biology
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Calcifediol - metabolism; Calcitriol - metabolism; Calcitriol - pharmacology; Cell Line; Cell Nucleus - enzymology; Cytochrome P-450 Enzyme System; Cytosol - enzymology; Fundamental and applied biological sciences. Psychology; Haplorhini; Kidney - enzymology; Other biological molecules; Receptors, Calcitriol; Receptors, Steroid - metabolism; Steroid Hydroxylases - metabolism; Swine; Terpenes, steroids. Hormones; Vitamin D3 24-Hydroxylase; Cell culture; Steroid hormone; Induction; Monkey; Biosynthesis; Cholecalciferol(dihydroxy-1,25); Kidney; Vertebrata; Mammalia; Cell line; Primates; Cholecalciferol(dihydroxy-24,25); Hormonal receptor
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)43340-0

A consequence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) action in kidney is the enhanced production of 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). We have studied this apparent induction phenomenon in two established mammalian cell lines of renal origin. A porcine kidney cell line, LLC-PK1, was found to possess typical receptors for 1,25-(OH)2D3 which sediment at 3.3 S and bind to immobilized DNA. Saturation analysis of LLC-PK1 cell cytosol revealed an equilibrium binding constant (Kd) for 1,25-(OH)2D3 of 7.8 X 10(-11) M and a concentration of 5400 binding sites/cell. In the presence of serum, intact LLC-PK1 cells also internalize and bind 1,25-(OH)2D3. In contrast, a monkey kidney cell line, LLC-MK2, was found to contain a negligible concentration of the 1,25-(OH)2D3 receptor by all criteria examined. However, both renal cell lines respond to 1,25-(OH)2D3 with a 2- to 20-fold increase in basal levels of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity. Incubation of viable cell suspensions with 25-hydroxy[26,27-3H]vitamin D3 (0.5 microM) at 37 degrees C for 30 min followed by subsequent analysis of lipid extracts via high performance liquid chromatography was carried out to assess 24,25-(OH)2[3H]D3 formation. Enzyme induction was found to be specific for 1,25-(OH)2D3 in both cell lines with half-maximal stimulation of 24-hydroxylase activity observed at 0.2 and greater than or equal to 1.0 nM 1,25-(OH)2D3 in LLC-PK1 and LLC-MK2, respectively. The response in LLC-PK1 was more rapid (1-4 h) than in LLC-MK2 (4-8 h) following 1,25-(OH)2D3 treatment of cultures in situ. In both cell lines, actinomycin D abolished the 1,25-(OH)2D3-dependent increase in 24-hydroxylase activity. Our results suggest that the high affinity 1,25-(OH)2D3 receptor may not be required for 1,25-(OH)2D3-dependent induction of renal 24-hydroxylase activity. Alternatively, LLC-MK2 cells could contain an atypical form of the 1,25-(OH)2D3 receptor protein which retains functionality but escapes detection by standard binding techniques.