1. Fig. 1 HPLC and TLC analysis of[ ³H]25OHD₃ metabolism in HKC-8 cells. A, Straight phase HPLC separation of radiolabeled vitamin D metabolites from cells incubated with 10 nmol/liter[ ³H]25OHD₃ for 3 h at 37 C. B, Reverse phase HPLC separation of straight phase HPLC fractions comigrating with authentic 1,25-(OH)₂D₃ (see A). In both A and B, results are illustrated as fractional counts per min (shaded boxes) plotted against an elution profile for vitamin D metabolites. C, TLC analysis of radiolabeled vitamin D metabolites from HKC-8 cells incubated with 3.75 nmol/liter[ ³H]25OHD₃ for 4 h at 37 C.
Constitutive Expression of 25-Hydroxyvitamin D3-1α-Hydroxylase in a Transformed Human Proximal Tubule Cell Line: Evidence for Direct Regulation of Vitamin D Metabolism by Calcium*
Endocrinology. 1999;140(5): doi: /endo
Endocrinology | Copyright © 1999 by The Endocrine Society

2. Fig. 2 Characterization of 1α-hydroxylase activity in HKC-8 cells
Fig. 2 Characterization of 1α-hydroxylase activity in HKC-8 cells. Dose-responsive conversion of[ ³H]25OHD₃ to[ ³H]1,25-(OH)₂D₃ in the presence or absence of 10 μmol/liter DPPD or 10 μmol/liter ketoconazole. Incubations were carried out at 37 C for 3 h.
Constitutive Expression of 25-Hydroxyvitamin D3-1α-Hydroxylase in a Transformed Human Proximal Tubule Cell Line: Evidence for Direct Regulation of Vitamin D Metabolism by Calcium*
Endocrinology. 1999;140(5): doi: /endo
Endocrinology | Copyright © 1999 by The Endocrine Society

3. Fig. 3 Regulation of 1α-hydroxylase and 24-hydroxylase activity in HKC-8 cells. Cells were incubated for 24 h with 10 nmol/liter 1,25-(OH)₂D₃ (D3), 10 μmol/liter forskolin (F), or 100 ng/ml PTH. A, 1α-Hydroxylase activity. B, 24-Hydroxylase activity. Data are shown as the percent conversion of[ ³H]25OHD₃ to[ ³H]1,25-(OH)₂D₃ (A) and[ ³H]24,25-(OH)₂D₃ (B) relative to that in control (con), untreated cells. Results represent the mean± sem of triplicate readings from 5–13 separate experiments (***, P < 0.001; *, P < 0.05).
Constitutive Expression of 25-Hydroxyvitamin D3-1α-Hydroxylase in a Transformed Human Proximal Tubule Cell Line: Evidence for Direct Regulation of Vitamin D Metabolism by Calcium*
Endocrinology. 1999;140(5): doi: /endo
Endocrinology | Copyright © 1999 by The Endocrine Society

4. Fig. 7 Expression of CaR mRNA in HKC-8 cells
Fig. 7 Expression of CaR mRNA in HKC-8 cells. RNA (20μ g) was analyzed in Northern blots using ³²P-labeled cDNA probes for the CaR and 18S ribosomal RNA as an internal control. RNA was prepared from control (C) cells, cells treated for 24 h with 10 nmol/liter 1,25-(OH)₂D₃ (D3) or 10μ mol/liter forskolin (F), or cells incubated for various times (24, 10, 6, and 4 h) in medium containing 0–2 mmol/liter calcium. A predominant band of approximately 2.5 kb and a minor band of approximately 3.8 kb were detected.
Constitutive Expression of 25-Hydroxyvitamin D3-1α-Hydroxylase in a Transformed Human Proximal Tubule Cell Line: Evidence for Direct Regulation of Vitamin D Metabolism by Calcium*
Endocrinology. 1999;140(5): doi: /endo
Endocrinology | Copyright © 1999 by The Endocrine Society

5. Fig. 6 Expression and regulation of 1α-hydroxylase protein in HKC-8 cells were assessed by Western blot analysis using a polyclonal antibody specific for 1α-hydroxylase. Proteins (3μ g/lane) were prepared from cells cultured in medium containing various concentrations of calcium: 1) 1 mmol/liter Ca (control cells), 2) 2 mmol/liter Ca for 10 h, and 3) 0.5 mmol/liter Ca (for 4 h). Proteins were also prepared from cells that had been treated (24 h) with 5) 10 nmol/liter 1,25-(OH)₂D₃, 6) 100 nmol/liter 1,25-(OH)₂D₃ 7) 10 μmol/liter forskolin, and 8) 100 ng/ml PTH (4 is control, untreated cells). A single band of approximately 56 kDa was detected in all treatments.
Constitutive Expression of 25-Hydroxyvitamin D3-1α-Hydroxylase in a Transformed Human Proximal Tubule Cell Line: Evidence for Direct Regulation of Vitamin D Metabolism by Calcium*
Endocrinology. 1999;140(5): doi: /endo
Endocrinology | Copyright © 1999 by The Endocrine Society

6. Fig. 4 Expression of 1α-hydroxylase mRNA and VDR and CaR protein in HKC-8 cells. A, RT-PCR analysis of RNA from HKC-8 cells using specific primers for 1α-hydroxlase enzyme detected a single band of 542 bp. Other samples included RNA from human proximal tubule cells (PT) and human keratinocytes (K). −, Negative control; M, markers. B, Nuclear proteins (10 μg) were analyzed by Western blotting using a monoclonal antibody specific for VDR. A single band of 57 kDa was detected. U937 promonocytic cells were included as a positive control. C, HKC-8 cell extracts (10 μg) were analyzed by Western blotting using a monoclonal antibody specific for CaR. Each lane represents a different sample of HKC-8 protein. A band of approximately 120 kDa was detected in each sample.
Constitutive Expression of 25-Hydroxyvitamin D3-1α-Hydroxylase in a Transformed Human Proximal Tubule Cell Line: Evidence for Direct Regulation of Vitamin D Metabolism by Calcium*
Endocrinology. 1999;140(5): doi: /endo
Endocrinology | Copyright © 1999 by The Endocrine Society

7. Fig. 5 Direct effects of calcium on vitamin D metabolism in HKC-8 cells. Cells were incubated for various times (4–24 h) in medium containing 2 mmol/liter calcium (A and C) or 0.5 mmol/liter calcium (B and D). [³H]25OHD₃ was included for the final 4 h. Data are shown as the percent conversion of[ ³H]25OHD₃ to[ ³H]1,25-(OH)₂D₃ (A and B) and[ ³H]24,25-(OH)₂D₃ (C and D) relative to that in control (con), untreated cells in standard calcium (1 mmol/liter) medium. Results represent the mean ± sem of triplicate readings from at least three separate experiments (***, P < 0.001; **, P < 0.01; *, P < 0.05).
Constitutive Expression of 25-Hydroxyvitamin D3-1α-Hydroxylase in a Transformed Human Proximal Tubule Cell Line: Evidence for Direct Regulation of Vitamin D Metabolism by Calcium*
Endocrinology. 1999;140(5): doi: /endo
Endocrinology | Copyright © 1999 by The Endocrine Society