TY - JOUR
T1 - 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
AU - Chandler, J. S.
AU - Chandler, S. K.
AU - Pike, J. W.
AU - Haussler, M. R.
PY - 1984
Y1 - 1984
N2 - 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 (K(d)) 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, 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 μM) at 37°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 ≥ 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 functionally but escapes detection by standard binding techniques.
AB - 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 (K(d)) 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, 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 μM) at 37°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 ≥ 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 functionally but escapes detection by standard binding techniques.
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M3 - Article
C2 - 6321462
AN - SCOPUS:0021338173
SN - 0021-9258
VL - 259
SP - 2214
EP - 2222
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -