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J Am Soc Nephrol 10: 963–970, 1999 ␣ Cloning of Porcine 25-Hydroxyvitamin D3 1 -Hydroxylase and Its Regulation by cAMP in LLC-PK1 Cells

TADASHI YOSHIDA, NORIKO YOSHIDA, AKIRA NAKAMURA, TOSHIAKI MONKAWA, MATSUHIKO HAYASHI, and TAKAO SARUTA Department of Internal , School of Medicine, Keio University, Tokyo, Japan.

␣ Abstract. The 25-hydroxyvitamin D3 1 -hydroxylase, also re- and expression of CYP27B1 mRNA was measured by a quan- ferred to as CYP27B1, is a mitochondrial P450 titative reverse transcription-PCR. At the completion of 3-, 6-, that catalyzes the biosynthesis of 1␣, 25-dihydroxyvi- 12-, and 24-h incubations, 500 ␮mol/L 8-bromo-cAMP had ␣ tamin D3 (1 ,25(OH)2D3) from 25-hydroxyvitamin D3 in renal significantly increased CYP27B1 mRNA expression (260 to proximal tubular cells. Recently, human, , and 340%). The adenylate cyclase activator forskolin at 50 ␮mol/L CYP27B1 cDNA have been cloned, however the regula- also had a stimulatory effect at 6 h (190%). Moreover, the tion has not been fully elucidated. In the present study, porcine kinase A inhibitor H-89 reduced the cAMP effect. On ␣ CYP27B1 cDNA was cloned, and the effects of cAMP and the other hand, 1 ,25(OH)2D3 had no effect on CYP27B1 vitamin D3 on the regulation of CYP27B1 mRNA expression mRNA expression at 10 and 100 nmol/L, whereas expression in LLC-PK1 cells were examined. PCR cloning revealed that of 25-hydroxyvitamin D3 24-hydroxylase (CYP24) mRNA ␣ porcine CYP27B1 cDNA consisted of 2316 bp, encoding a was markedly increased by 1 ,25(OH)2D3. These findings protein of 504 amino acids. The deduced sequence suggest that LLC-PK1 cells express CYP27B1 mRNA, and that showed over 80% identity to the human, mouse, and rat en- cAMP is an upregulating factor of the CYP27B1 gene in vitro. zyme. LLC-PK1 cells were incubated with humoral factors,

␣ The physiologically active form of vitamin D3,1 , 25-dihy- kidney (10). Recently, molecular defects in the CYP27B1 gene ␣ droxyvitamin D3 (1 ,25(OH)2D3), plays an important role in have been identified in one American, four Japanese, and four calcium , bone growth, and cell differentiation. The French-Canadian patients (8,11,12). ␣ biosynthesis of 1 ,25(OH)2D3 from 25-hydroxyvitamin D3 The enzymatic activity of CYP27B1 is regulated by a num- ␣ (25(OH)D3) is catalyzed by 25-hydroxyvitamin D3 1 -hydrox- ber of physiologic factors, such as parathyroid (PTH), ␣ ylase, a mitochondrial cytochrome P450 enzyme in renal prox- 1 ,25(OH)2D3, calcitonin, insulin-like growth factor-I, cal- ␣ imal tubular cells (1–3). The level of 1 ,25(OH)2D3 is primar- cium, and phosphorus (13–19). Numerous reports have dem- ily controlled by this enzyme rather than the other onstrated that PTH is the most potent upregulator of CYP27B1 involved in vitamin D3 metabolism. In 1997, we and other activity (13,14). PTH exerts its effects through binding to investigators succeeded in molecular cloning of rat, mouse, and PTH/PTH-related peptide (PTHrP) receptor and then activating ␣ human 25-hydroxyvitamin D3 1 -hydroxylase cDNA (4–8), the protein kinase A (PKA) and/or protein kinase C pathway which were later assigned the name CYP27B1 by the Cyto- (20). It has been demonstrated previously that the effect of chrome P450 Gene Superfamily Nomenclature Committee. PTH on the CYP27B1 activity is due to of the PKA ␣ dependency rickets type I is an autosomal recessive pathway (14). On the other hand, 1 ,25(OH)2D3 is a down- ␣ disorder characterized by early onset of hypocalcemia, second- regulator of its own synthesis (15). In general, 1 ,25(OH)2D3 ary hyperparathyroidism, and severe rachitic lesions (9). It is alters transcriptional activity by binding to vitamin D receptor known to be caused by impaired CYP27B1 activity in the in target cells. However, Brenza et al. (21), who cloned the 5Ј flanking region of mouse cyp27b1 gene, reported that ␣ 1 ,25(OH)2D3 did not suppress basal promoter activity of its gene by assay. The precise regulation of the Received September 3, 1998. Accepted November 20, 1998. CYP27B1 gene has not yet been clarified. The nucleotide sequence data reported in this article will appear in the In the present study, to investigate the humoral regulation of DDBJ/EMBL/GenBank nucleotide sequence databases with the accession no. AB018743. the CYP27B1 gene in vitro, we first cloned porcine CYP27B1 Correspondence to Dr. Matsuhiko Hayashi, Department of Internal Medicine, cDNA in LLC-PK1 cells. Although LLC-PK1 cells retained School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo many functional characteristics of renal proximal tubular cells, 160-8582, Japan. Phone and Fax: 81-3-3359-2745; E-mail: matuhiko@ they were not responsive to PTH because they lack the PTH/ mc.med.keio.ac.jp PTHrP receptor (22,23). We therefore examined the effects of 1046-6673/1005-0963$03.00/0 ␣ Journal of the American Society of Nephrology cAMP, a second messenger of PTH, and 1 ,25(OH)2D3 on Copyright © 1999 by the American Society of Nephrology regulation of CYP27B1 mRNA expression in these cells. 964 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 963–970, 1999

Materials and Methods Reverse Transcription-PCR

Total RNA was isolated from LLC-PK1 cells (American Type Culture Collection, Rockville, MD) by acid guanidinium thiocyanate- phenol- extraction (24). Approximately 1 ␮g of total RNA was reverse-transcribed by random hexamer (GeneAmp RNA PCR Core kit; Applied Biosystems, Foster City, CA). PCR was performed by using several pairs of primers (Table 1 and Figure 1). The resulting PCR products of predicted size were extracted and subcloned. Figure 1. Porcine CYP27B1 cDNA fragments amplified by PCR. Arrows indicate the direction of the primers. Isolation of the cDNA Ends Ј ␮ To obtain the 3 -cDNA ends, 1 g of total RNA from LLC-PK1 cells was reverse-transcribed with adapter primer using a rapid am- 70 to 80% confluence, LLC-PK cells were maintained for 12 h in plification of 3Ј-cDNA ends (3Ј-RACE) system (Life Technologies 1 serum-free Dulbecco’s modified Eagle’s medium supplemented with BRL, Grand Island, NY) according to the protocol. PCR was carried 0.1% bovine serum albumin. Then humoral factors were added to the out with 1A-H-52 primer (Table 1 and Figure 1) and universal medium and incubation was continued for 3, 6, 12, and 24 h. The amplification primer. humoral factors used were 5, 50, and 500 ␮mol/L 8-bromo-cAMP, 50 Rapid amplification of 5Ј-cDNA ends (5Ј-RACE) was performed ␮mol/L forskolin, 10 ␮mol/L H-89, 10 and 100 nmol/L 1␣,25(OH) using a 5Ј-RACE system (Life Technologies BRL). First-strand 2 D , 100 nmol/L 25(OH)D , and 100 nmol/L 24,25(OH) D . After cDNA was synthesized using 1A-H-51 primer and 1 ␮g of total RNA 3 3 2 3 Ј treatment with the humoral factors, total RNA was extracted. from LLC-PK1 cells. The 5 end of cDNA was amplified with 1A-H-53 primer and 5Ј-RACE abridged anchor primer followed by nested PCR with 1A-H-54 primer and abridged universal amplifica- Quantitative Reverse Transcription-PCR tion primer. Expression of CYP27B1 mRNA was measured by the quantitative reverse transcription (RT)-PCR/Southern blot technique described DNA Sequencing previously (25). Briefly, first-strand cDNA was synthesized from 1 The PCR products were ligated into pT7-Blue T vector (Novagen, ␮g of total RNA using random hexamers. After an initial denaturation Madison, WI) and subcloned. Nucleotide sequences were determined for 3 min at 93°C, 26 cycles of PCR were performed for CYP27B1 with an ABI Dyedeoxy Terminator Cycle Sequencing kit (Applied with 1A-H-61 and 1A-H-62 primers, and 18 cycles for ␤-actin with Biosystems) and analyzed on DNA sequencing systems (model 310; ␤-actin-04 and ␤-actin-05 primers (Table 1). The preliminary exper- Applied Biosystems). All sequences were confirmed by reading both iments showed linear relationship between PCR products and PCR DNA strands of more than four independent clones. cycles around these cycles. Each cycle was programmed as follows: 1-min denaturation at 93°C, 2-min annealing at 67°C or 64°C (for Cell Culture CYP27B1 and ␤-actin, respectively), and 2-min extension at 72°C.

LLC-PK1 cells were cultured in Dulbecco’s modified Eagle’s me- No products were detected when RT was performed in the absence of dium supplemented with 10% fetal bovine serum. When cells were at reverse transcriptase.

Table 1. Oligonucleotide primers used in this experiment

Primer Name Sequence Sense or Antisense

1A-H-41 5Ј-TTCTGCAAGGGGGGGCTGTC-3Ј Sense 1A-H-42 5Ј-CAGCTGTGATCTCTGAGTGG-3Ј Antisense 1A-H-43 5Ј-GTGGGCTCGGTGTTTGTGTC-3Ј Sense 1A-H-44 5Ј-AGCTGGACGAAAAGAATTTGGCTC-3Ј Antisense 1A-H-51 5Ј-AGCCAGCTGGGCATCGCCATGGT-3Ј Antisense 1A-H-52 5Ј-TATGCCACTTCAAGGGACCC-3Ј Sense 1A-H-53 5Ј-TGGACCACGTCATGCAGGG-3Ј Antisense 1A-H-54 5Ј-ACTGCGGAGCCTCTGCCATTCTTCACC-3Ј Antisense 1A-H-61 5Ј-TGCCGCGACTGGGACCAGATG-3Ј Sense 1A-H-62 5Ј-AGGAGAGTGTGTTGGACACCG-3Ј Antisense 1A-H-63 5Ј-AGCCAGGGAAAGCCTGAGGAG-3Ј Sense ␤-actin-04 5Ј-TGGAGAAGAGCTACGAGCTG-3Ј Sense ␤-actin-05 5Ј-ACTTCATGATGGAGTTGAAGG-3Ј Antisense ␤-actin-06 5Ј-AGGTCATCACCATCGGCAACG-3Ј Sense 24-H-03 5Ј-ATGAGAAGAGATTTGGGCTCC-3Ј Sense 24-H-04 5Ј-GTCACAGACGCATACAATTCC-3Ј Antisense 24-H-05 5Ј-ACCTCAACACCAAGGTCTGG-3Ј Sense ␣ J Am Soc Nephrol 10: 963–970, 1999 Porcine 1 -Hydroxylase in LLC-PK1 Cells 965

Figure 3. (A) Amino acid sequence comparison between porcine, Figure 2. Nucleotide sequence of porcine CYP27B1 cDNA and its human, mouse, and rat CYP27B1. Identical residues are indicated by deduced amino acid sequence. The consensus sequence for the - asterisks. The sequence of the heme-binding domain and amino acids binding domain is underlined. The conserved amino acid character- for at Lys-367, Lys-371, and Arg-375 are istics of the ferredoxin binding site are boxed. boxed. (B) Partial amino acid sequence comparison between porcine and human CYP24. Identical residues are indicated by asterisks. Analysis of the PCR Products The PCR products specific for CYP27B1 and ␤-actin were com- sequences were relatively conserved in these species to obtain bined and subjected to electrophoresis on a 3% agarose gel. For partial sequences of porcine CYP27B1 cDNA. RT-PCR of Southern blot analysis, gels were blotted onto Hybond Nϩ nylon total RNA from LLC-PK1 cells yielded an 889-bp and a 621-bp membranes (Amersham Corp., Arlington Heights, IL), and the mem- sequence (RT-PCR-1 and RT-PCR-2, respectively) (Figure 1), 32 branes were hybridized with P-labeled CYP27B1-specific 1A-H-63 and they showed significant with human, rat, and probe. After washing, membranes were exposed to imaging plates and mouse CYP27B1 cDNA. We then performed 3Ј-RACE and 5Ј- analyzed (BAS2000; Fuji Film Institution, Tokyo, Japan). To quantify RACE to clone the full length of its cDNA. As shown in Figure the mRNA expression for CYP27B1, the membranes were rehybrid- ized with 32P-labeled ␤-actin-06 probe. We calculated the ratio of 2, the nucleotide sequence of porcine CYP27B1 cDNA was CYP27B1 to ␤-actin from each sample. 2316 bp in length and contained an open-reading frame of 1512 bp, starting at base 36 and ending at base 1547. The predicted Statistical Analyses protein was composed of 504 amino acids, and showed 88, 81, Data are presented as means Ϯ SEM. Statistical analyses were and 80% sequence identities to the human, rat, and mouse performed by one-way ANOVA with a post hoc Fisher protected least enzyme, respectively (Figure 3A). It was particularly notewor- significant difference test. P values Ͻ 0.05 were considered signifi- thy that the ferredoxin binding site at Lys-367, Lys-371, and cant. Arg-375, and the heme-binding domain recognized from resi- dues 446 to 464 were almost completely conserved among Results these species. On the contrary, the predicted protein had only Cloning of Porcine CYP27B1 cDNA in LLC-PK1 Cells 43% amino acid identity to vitamin D3 25-hydroxylase, and We and other investigators have recently succeeded in the had less than 35% identity to other including 25- cloning of rat, mouse, and human CYP27B1 cDNA (4–8,11), hydroxyvitamin D3 24-hydroxylase (CYP24) (26). We there- and these clones showed high homology with one another. We fore concluded that this is the porcine homologue of performed RT-PCR with oligonucleotide primers in which the CYP27B1. 966 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 963–970, 1999

Figure 5. Effect of cAMP removal on CYP27B1 mRNA expression. ␮ LLC-PK1 cells were treated with 500 mol/L 8-bromo-cAMP for 6 h, and then 8-bromo-cAMP was removed or not. (A) Representative RT-PCR/Southern blots. Upper and lower panels represent mRNA expression for CYP27B1 and ␤-actin, respectively. (B) The ratios of CYP27B1 to ␤-actin were calculated. Cells were treated with 8-bro- mo-cAMP for 6 h, then medium was changed to the medium without 8-bromo-cAMP (F) or unchanged (E). An arrow indicates the time the medium was changed. An arbitrary value of 100 was assigned to the sample before treatment (control). Values represent means Ϯ SEM Figure 4. Time course of the effects of cAMP and vitamin D3 on of four independent experiments. *P Ͻ 0.05 compared with control. CYP27B1 mRNA expression. LLC-PK1 cells were treated with 500 ␮ ␣ mol/L 8-bromo-cAMP or 10 nmol/L 1 , 25-dihydroxyvitamin D3 ␣ (1 ,25(OH)2D3) for 0, 3, 6, 12, and 24 h. Total RNA was extracted and analyzed by quantitative reverse transcription (RT)-PCR/South- P Ͻ 0.05) (Figure 4, A and C). The maximal induction of ern blot. Representative RT-PCR/Southern blots with 8-bromo-cAMP CYP27B1 mRNA level by 8-bromo-cAMP was seen at 6 h. To ␣ (A) and 1 ,25(OH)2D3 (B) treatment are shown. Upper and lower confirm the effect of cAMP on CYP27B1 gene expression, we ␤ panels represent mRNA expression for CYP27B1 and -actin, respec- examined whether the effect of 8-bromo-cAMP was reversible. ␤ tively. (C) The ratios of CYP27B1 to -actin were calculated. Cells LLC-PK cells were incubated with 500 ␮mol/L 8-bromo- were treated with 8-bromo-cAMP (E), 1␣,25(OH) D (‚), or vehicle 1 2 3 cAMP for 6 h, and then the medium was changed into that (F). An arbitrary value of 100 was assigned to the sample before treatment (control). Values represent means Ϯ SEM of four indepen- without 8-bromo-cAMP. Removal of 8-bromo-cAMP from the dent experiments. *P Ͻ 0.05 compared with control. medium caused a rapid decrease in CYP27B1 mRNA expres- sion within 3 h (Figure 5). The dose–response relationship for the effect of 8-bromo-cAMP on CYP27B1 mRNA was then Effect of cAMP on CYP27B1 mRNA Expression examined. Although 500 ␮mol/L 8-bromo-cAMP significantly ␮ LLC-PK1 cells were incubated with humoral factors, and increased CYP27B1 mRNA levels, neither 50 nor 5 mol/L expression of CYP27B1 mRNA was measured by the quanti- 8-bromo-cAMP had a significant effect at 6 h (137 Ϯ 7 and tative RT-PCR/Southern blot procedure. We first examined the 109 Ϯ 10%, respectively) (Figure 6A). Because 8-bromo- time course of the effect of 8-bromo-cAMP on CYP27B1 cAMP at 50 and 5 ␮mol/L did not reach its effective concen- mRNA expression. When cells were incubated with 500 tration (27,28), it would have no significant effect. The other ␮mol/L 8-bromo-cAMP, CYP27B1 mRNA expression was agent that increases intracellular cAMP levels, forskolin at 50 significantly increased at 3-, 6-, 12-, and 24-h incubations ␮mol/L, increased CYP27B1 mRNA expression (188 Ϯ 19%, (294 Ϯ 42, 342 Ϯ 47, 314 Ϯ 53, and 261 Ϯ 76%, respectively, P Ͻ 0.05) after6hofincubation (Figure 6B). The action of ␣ J Am Soc Nephrol 10: 963–970, 1999 Porcine 1 -Hydroxylase in LLC-PK1 Cells 967

␮ Figure 6. Effect of cAMP on CYP27B1 mRNA expression. LLC-PK1 cells were treated with 5, 50, and 500 mol/L 8-bromo-cAMP (A), 50 ␮mol/L forskolin (B), and the combination of 10 ␮mol/L H-89 and 50 or 500 ␮mol/L 8-bromo-cAMP (C), for 6 h, then total RNA was extracted. (Top) Upper and lower panels represent mRNA expression for CYP27B1 and ␤-actin, respectively, by quantitative RT-PCR/ Southern blot. (Bottom) The ratios of CYP27B1 to ␤-actin were calculated. An arbitrary value of 100 was assigned to the control. Values represent means Ϯ SEM of four independent experiments. *P Ͻ 0.05 compared with control; ¶P Ͻ 0.05 compared with 500 ␮mol/L 8-bromo-cAMP-treated cells.

cAMP generally occurs through the activation of PKA. We stimulatory effect on CYP24 mRNA expression, but 100

therefore examined the effect of the PKA inhibitor H-89 on nmol/L 24,25(OH)2D3 failed to stimulate expression at all cAMP-induced CYP27B1 mRNA expression. Combined treat- (Figure 7). ment with 10 ␮mol/L H-89 and 500 ␮mol/L 8-bromo-cAMP Ϯ significantly reduced CYP27B1 mRNA levels (202 10%) Combined Effect of cAMP and Vitamin D3 on compared to treatment with 500 ␮mol/L 8-bromo-cAMP only CYP27B1 mRNA Expression (Figure 6C). The effect of coincubation with 8-bromo-cAMP and ␣ 1 ,25(OH)2D3 was examined. Although CYP27B1 mRNA ␮ Effect of Vitamin D3 on CYP27B1 mRNA Expression expression was significantly increased by 500 mol/L 8-bro- ␮ We next examined the effects of vitamin D3 metabolites on mo-cAMP treatment, combined treatment with 500 mol/L ␣ CYP27B1 mRNA expression in LLC-PK1 cells. As shown in 8-bromo-cAMP and 10 nmol/L 1 ,25(OH)2D3 did not reduce ␣ Figure 4B, 10 nmol/L 1 ,25(OH)2D3, the active form of vita- its expression (Figure 8). In physiologic conditions in which ␣ min D3, had no effect on CYP27B1 mRNA expression after 3-, elevated levels of 1 ,25(OH)2D3 have been generated, the 6-, 12-, and 24-h incubations. Treatment with other vitamin D3 cAMP levels would tend to be low due to the suppression of metabolites, 100 nmol/L 25(OH)D3 and 100 nmol/L PTH and due to the elevation of serum calcium. Hence, the 24,25(OH)2D3, did not alter the CYP27B1 mRNA levels, nor effect of a combination of lower cAMP concentrations (5 and ␣ ␮ ␣ did 10 and 100 nmol/L 1 ,25(OH)2D3 (Figure 7). We also 50 mol/L 8-bromo-cAMP) and 10 nmol/L 1 ,25(OH)2D3 measured the level of CYP24 mRNA in LLC-PK1 cells. Partial was also examined. As shown in Figure 8, any combined sequence of porcine CYP24 cDNA was cloned, and deduced treatment had no difference with cAMP treatment alone. amino acid sequence was shown in Figure 3B. The quantitative RT-PCR was performed using porcine CYP24-specific 24- Discussion ␣ H-03 and 24-H-04 primers, followed by Southern blotting with PTH and 1 ,25(OH)2D3 are the two major physiologic fac- 24-H-05 oligonucleotide probe (Table 1). Treatment with 10 tors involved in the regulation of 1␣-hydroxylase and 24- ␣ and 100 nmol/L 1 ,25(OH)2D3 markedly stimulated CYP24 hydroxylase activities of 25(OH)D3 in the kidney mRNA expression, and 100 nmol/L 25(OH)D3 had a weak (13–15,17,29). The mechanism of regulation of CYP27B1 has 968 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 963–970, 1999

Figure 8. Combined effect of cAMP and vitamin D3 on CYP27B1 mRNA expression. LLC-PK1 cells were treated with the combination ␣ ␮ of 100 nmol/L 1 ,25(OH)2D3 and 5, 50, or 500 mol/L 8-bromo- cAMP, or 8-bromo-cAMP alone for 6 h. Total RNA was extracted and analyzed by quantitative RT-PCR/Southern blot. (A) A representative RT-PCR/Southern blot. Upper and lower panels represent mRNA expression for CYP27B1 and ␤-actin, respectively. (B) The ratios of CYP27B1 to ␤-actin were calculated. An arbitrary value of 100 was assigned to the control. Values represent means Ϯ SEM of four independent experiments. *P Ͻ 0.05 compared with control.

Figure 7. Effect of vitamin D3 on CYP27B1 mRNA expression. LLC-PK cells were treated with 10 and 100 nmol/L 1␣,25(OH) D , 1 2 3 analysis (4). Moreover, porcine cDNA has extremely high 100 nmol/L 25(OH)D3, and 100 nmol/L 24,25(OH)2D3, for 6 h, then total RNA was extracted. (A) Upper, middle, and lower panels rep- homology to human CYP27B1 cDNA, and their ferredoxin resent mRNA expression for CYP27B1, ␤-actin, and CYP24, respec- binding site and heme-binding domain matched perfectly. tively, by quantitative RT-PCR/Southern blot. (B) The ratios of Thus, we confirmed that this clone is the porcine homologue of CYP27B1 to ␤-actin were calculated. An arbitrary value of 100 was CYP27B1 cDNA. assigned to the control. Values represent means Ϯ SEM of four PTH has been shown to stimulate CYP27B1 enzyme activity independent experiments. in cultured avian and mammalian kidney cells (30,31). PTH increases intracellular cAMP levels by stimulating adenylate cyclase activity. The effect of PTH on CYP27B1 enzyme been studied only in terms of its enzyme activity, whereas the activity is mediated mainly by the cAMP signaling pathway molecular mechanism of regulation of CYP24 gene has been (14). In this study, we have shown that the elevation of intra- clarified (17,29). Recent cloning of rat (4,5), mouse (6), and cellular cAMP has a stimulatory effect on CYP27B1 mRNA human (7,8,11) CYP27B1 cDNA has made it possible to expression. Both 8-bromo-cAMP and forskolin increased the investigate the molecular mechanism of regulation of the CYP27B1 mRNA level in LLC-PK1 cells. This effect of 8-bro- CYP27B1 gene. In this study, we have cloned porcine mo-cAMP was reversible and was blocked by the PKA inhib- CYP27B1 cDNA, and demonstrated that the CYP27B1 mRNA itor. Our observation is in agreement with the enzymatic data level was significantly increased by cAMP, but unaffected by described previously (14), and shows that the elevation of ␣ 1 ,25(OH)2D3 in LLC-PK1 cells. CYP27B1 enzyme activity by cAMP was due to elevation of LLC-PK1 cells, originally isolated from an unknown site in its mRNA level. In a promoter analysis of the mouse cyp27b1 the renal cortex of the Hampshire pig, retain many functional gene, PTH markedly increased its promoter activity in AOK- characteristics of renal proximal tubular cells (23). In a previ- B50 cells, a modified LLC-PK1 cell line that expresses stably ous study, Condamine et al. demonstrated that LLC-PK1 cells, transfected PTH/PTHrP receptors (21,32). They also observed but not opossum kidney cells, are able to produce that stimulation of promoter activity by PTH could be mim- ␣ 1 ,25(OH)2D3 (18). They found that the production of icked by forskolin. Accordingly, cAMP-induced expression of ␣ 1 ,25(OH)2D3 was stimulated by phosphate deprivation or CYP27B1 mRNA would be mostly a result of transcriptional insulin-like growth factor-I treatment of these cells. These activation rather than the reduction of degradation in this gene. ␣ findings are in agreement with the results of several studies on Although 1 ,25(OH)2D3 is known to be a downregulator of regulation of the enzymatic activity of CYP27B1 in vivo (19). the enzyme activity of CYP27B1 (15), our data failed to ␣ On the basis of their report, we attempted to clone porcine demonstrate that 1 ,25(OH)2D3 had an inhibitory effect on the CYP27B1 cDNA in LLC-PK1 cells. The porcine CYP27B1 expression of CYP27B1 mRNA in LLC-PK1 cells. The addi- ␣ cDNA was approximately 2.3 kb long, and consistent with the tion of 1 ,25(OH)2D3 to 8-bromo-cAMP-treated cells did not ␣ size of the transcript from pig kidney tissue by Northern blot reduce its mRNA level either. By contrast, 1 ,25(OH)2D3 ␣ J Am Soc Nephrol 10: 963–970, 1999 Porcine 1 -Hydroxylase in LLC-PK1 Cells 969

markedly increased CYP24 mRNA expression in LLC-PK1 al-dominanten Erbgang: Die heredita¨re PseudoMangelrachitis. cells. This is in agreement with the previous data that Helv Paediatr Acta 16: 452–468, 1961 ␣ 10. Fraser D, Kooh SW, Kind P, Tanaka Y, DeLuca HF: Pathogen- 1 ,25(OH)2D3 induces CYP24 mRNA levels in vivo (17). Our findings concerning CYP24 mRNA expression mean that the esis of hereditary vitamin D-dependent rickets: An inborn error transactivation system by 1␣,25(OH) D was well conserved of metabolism involving defective conversion of 25-hydroxyvi- 2 3 tamin D to 1␣, 25-dihydroxyvitamin D. N Engl J Med 289: in these cells, whereas no transrepression system by ␣ 817–822, 1973 1 ,25(OH)2D3 was demonstrated. In the transfection analysis 11. Kitanaka S, Takeyama K, Murayama A, Sato T, Okumura K, ␣ by Brenza et al. (21), 1 ,25(OH)2D3 also failed to suppress Nogami M, Hasegawa Y, Niimi H, Yanagisawa J, Tanaka T, basal promoter activity of mouse cyp27b1 gene in AOK-B50 Kato S: Inactivating mutations in the 25-hydroxyvitamin D3 cells. Reduction of CYP27B1 enzyme activity by 1␣-hydroxylase gene in patients with pseudovitamin D-defi- ␣ 1 ,25(OH)2D3 itself may be a different mechanism from the ciency rickets. N Engl J Med 338: 653–661, 1998 direct suppression of CYP27B1 mRNA expression. Another 12. Yoshida T, Monkawa T, Tenenhouse HS, Goodyer P, Shinki T, possibility is that loss of transrepression in the CYP27B1 gene Suda T, Wakino S, Hayashi M, Saruta T: Two novel 1␣-hydrox- ␣ ylase mutations in French-Canadians with vitamin D dependency in response to 1 ,25(OH)2D3 was specific to LLC-PK1 cells. Several are negatively controlled at the transcription rickets type I. Kidney Int 54: 1437–1443, 1998 level by 1␣,25(OH) D , and this transrepression apparently 13. Garabe´dian M, Holick MF, DeLuca HF, Boyle IT: Control of 2 3 25-hydroxycholecalciferol metabolism by parathyroid glands. occurs by mechanisms different from the well known transac- Proc Natl Acad Sci USA 69: 1673–1676, 1972 tivation mechanism (33). An unknown required for ␣ 14. Horiuchi N, Suda T, Takahashi H, Shimazawa E, Ogata E: In negative regulation by 1 ,25(OH)2D3 may be deficient in these vivo evidence for the intermediary role of 3Ј,5Ј-cyclic AMP in cells. Additional studies will be needed to clarify this issue. parathyroid hormone-induced stimulation of 1alpha, 25-dihy- droxyvitamin D3 synthesis in . Endocrinology 101: 969–974, 1977 Acknowledgments 15. Henry HL: Regulation of the of 25-hydroxyvita-

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