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Endocrinology Vol. 140, No. 5 2224-2231
Copyright © 1999 by The Endocrine Society

ARTICLES

Positive and Negative Regulations of the Renal 25-Hydroxyvitamin D3 1{alpha}-Hydroxylase Gene by Parathyroid Hormone, Calcitonin, and 1{alpha},25(OH)2D3 in Intact Animals1

Akiko Murayama, Ken-ichi Takeyama, Sachiko Kitanaka, Yasuo Kodera, Yoshindo Kawaguchi, Tatsuo Hosoya and Shigeaki Kato

The Institute of Molecular and Cellular Biosciences (A.M., K-i.T., S.K., Y.K., S.K.), The University of Tokyo, Yayoi 1–1-1, Bunkyo-ku, Tokyo 113-0032, Japan; Second Department of Internal Medicine (A.M., Y.K., T.H.), Jikei University School of Medicine, Nishishinbashi 3–25-8, Minato-ku, Tokyo 105-8461; CREST (S.K.), Japan Science and Technology Corporation, Honcho 4–1-8, Kawaguchi, Saitama 332-0012

Address all correspondence and requests for reprints to: Shigeaki Kato, Ph.D., The Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1–1-1, Bunkyo-ku, Tokyo 113-0032, Japan. E-mail: uskato{at}hongo.ecc.u-tokyo.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reflecting the prime role of 1{alpha},25(OH)2D3 in calcium homeostasis, the activity of 25-hydroxyvitamin D3 1{alpha}-hydroxylase, a key enzyme for 1{alpha},25(OH)2D3 biosynthesis, is tightly regulated by 1{alpha},25(OH)2D3, PTH and calcitonin. Its significant activity is found in kidney, though the enzymatic activity is also reported in extra-renal tissues. In the present study, we found that the 1{alpha}-hydroxylase gene abundantly expresses in kidney, and at low levels in other tissues and in some cell lines. Positive and negative regulations of 1{alpha}-hydroxylase gene by PTH, calcitonin, or 1{alpha},25(OH)2D3 were observed at transcriptional levels in kidneys of animals and in a mouse proximal tubule cell line. Moreover, the protein kinase A inhibitor abrogated the PTH-mediated positive regulation. In mice lacking the vitamin D receptor, the 1{alpha}-hydroxylase gene expression was overinduced, and the inducible effect of either PTH or calcitonin, but not the repression by 1{alpha},25(OH)2D3, was evident. Thus, vitamin D receptor is essential for the negative regulation by 1{alpha},25(OH)2D3. Moreover, we demonstrate that renal 1{alpha}-hydroxylase gene expression in chronic renal failure model rats was decreased and the positive effect by PTH and calcitonin was diminished. The present study demonstrates that PTH and calcitonin positively regulate renal 1{alpha}-hydroxylase gene expression via PKA-dependent and independent pathway, respectively, and that 1{alpha},25(OH)2D3 negatively regulates it mediated by vitamin D receptor. Furthermore, in a moderate state of chronic renal failure, renal cells expressing the 1{alpha}-hydroxylase gene appear to have diminished potential in response to PTH and calcitonin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RENAL 1{alpha}-hydroxylation of 25-hydroxyvitamin D3 occurs by means of 25-hydroxyvitamin D3 1{alpha}-hydroxylase (1{alpha}-hydroxylase) and is the key metabolic step in the biosynthesis of 1{alpha},25(OH)2D3, a most biologically active form of vitamin D (1, 2, 3). The 1{alpha}-hydroxylase, a cytochrome P450 enzyme acting as a mixed-function oxidase, is present in the inner membrane of mitochondria (4, 5). The significant activity is detected only in kidney, though the activity is also reported in several extra-renal tissues (6, 7, 8, 9). Reflecting the critical role of 1{alpha},25(OH)2D3 in calcium homeostasis, the activity of 1{alpha}-hydroxylase is tightly regulated by various factors such as 1{alpha},25(OH)2D3 and calciotropic hormones (10). Calciotropic peptide hormones such as PTH and calcitonin are positive regulators for the activity of 1{alpha}-hydroxylase (11, 12). Though this positive effect of PTH on 1{alpha}-hydroxylase activity is mediated at least in part by the protein kinase A (PKA) signaling pathway (13, 14), calcitonin seems to induce the activity by a pathway different from PKA (15). Serum calcium and phosphate levels are critical for the 1{alpha}-hydroxylase activity; however, the regulation by calcium is considered to be indirect (16, 17, 18). On the other hand, as a negative regulation, the inhibitory actions of 1{alpha},25(OH)2D3 are well described (19, 20).

The activity of 1{alpha}-hydroxylase is known to be modulated in some pathologic states. Abnormal vitamin D metabolism is reported in patients with chronic renal failure (CRF) (21, 22, 23). This abnormality is considered to play a role in the development of renal osteodystrophy (24), a serious complication of CRF. Reduction of serum 1{alpha},25(OH)2D levels, irrespective of high PTH levels in these patients (25), suggests reduced 1{alpha}-hydroxylase activity or unresponse of 1{alpha}-hydroxylase activity to PTH. However, the mechanism underlying the reduced 1{alpha}-hydroxylase activity remained to be determined at the gene expression level.

Despite numerous observations on the regulation of the 1{alpha}-hydroxylase activity and accumulating clinical interest, it is unclear whether these regulations occur at transcriptional or posttranscriptional levels, and cloning of 1{alpha}-hydroxylase complementary DNA (cDNA) remained to be done. We recently cloned mouse 1{alpha}-hydroxylase cDNA, using a novel VDR-mediated expression cloning method, from the kidney of VDR knockout (VDR-KO) mice (26). Subsequently, we isolated human 1{alpha}-hydroxylase cDNA and demonstrated a significant role of 1{alpha}-hydroxylase by the observation that vitamin D-dependent rickets type I (VDDR I) is caused by inactive mutations in the 1{alpha}-hydroxylase gene (27). Other investigators also isolated human (28, 29) and rat 1{alpha}-hydroxylase cDNA (30, 31). Although they demonstrated some regulation of 1{alpha}-hydroxylase gene expression in vitamin D deficient animals, a precise study on its regulation has not been performed yet.

In this study, we tried to examine the regulation of renal 1{alpha}-hydroxylase gene by PTH, calcitonin and 1{alpha},25(OH)2D3 in vivo. Moreover, to clarify the link between 1{alpha}-hydroxylase gene expression and abnormal vitamin D metabolism in pathological states, we investigated the gene expression using model animals for chronic renal failure and VDDR II.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental protocols
Seven-week-old male Wistar rats (Tokyo Laboratory Animals Science Co., LTD; Tokyo, Japan), weighing 180–200 g were fed a standard rodent chow and free access to food and top water was provided. For production of rats with CRF, 5/6 nephrectomy was performed by a two-thirds heminephrectomy of the left kidney, followed by removal of the right kidney 7 days later, with the rats under pentobarbital anesthesia (100 mg/kg, ip) (32). Control animals underwent a sham operation, which involved exposure of the kidneys and subsequent closure of the two separate flank incisions. The sham-operated and 5/6-nephrectomized rats were maintained on the standard chow for 5 weeks after the surgery, and the rats were used for experiments. VDR-KO mice were generated as previously described (33). VDR-KO mice and littermate mice (VDR+/+) were used at 3 week of age.

The treated rats were killed 4 h after an ip injection of calcitonin (20 µg/100 g BW), 3 h after an iv injection of PTH (10 µg/100 g BW) and 5 h after an ip injection of 1{alpha},25(OH)2D3 (0.8 nmol/100 g BW). Actinomycin D (80 µg/100 g BW, ip) was given 3 h before hormone treatments. The doses and the time schedule were determined by dose-response curve and time course studies to be the maximal dose and the peak time.

The animal studies were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals.

Measurement of serum calcium, phosphorus, urea nitrogen (BUN), creatinine, PTH, calcitonin and vitamin D metabolites in rats
Blood samples were collected at time of killing the rats, and the serum was obtained immediately. Serum calcium, phosphorus, BUN, and creatinine levels were measured using an autoanalyzer. Serum PTH levels in the rats were measured by RIA using two-site immunoradiometric assay kits (34). Serum calcitonin levels in the rats were measured by RIA kits (35). These PTH or calcitonin measurements do not detect the injected rat PTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or salmon calcitonin. Serum aliquots were stored at -80 C before measurement of vitamin D metabolites using commercial RRA kits (36).

Cells and culture conditions
MCT cells, an SV40-transformed mouse proximal tubule cell line (37), were cultured on 100 mm culture-grade plastic dishes in DMEM supplemented with 2 mM glutamine, 10% FCS, and 5.5 mM glucose in a humidified atmosphere of 95% air and 5% CO2. MCT cells, originally developed by Dr. Neilson (University of Pennsylvania), is a gift from Dr. Logman-Adham (University of Utah). Experiments were started when the cells were approximately 90% confluent. Serum were removed 18–24 h before the experiments. The cells were treated for 3 h with PTH (10-7 M), calcitonin (10-7 M), forskolin (10-4 M), and 1{alpha},25(OH)2D3 (10-8 M). 1{alpha},25(OH)2D3 and H89 (5 x 10-7 M) were added 1 h before treatments with PTH, calcitonin, or forskolin. Incubation was halted by aspiration of the medium and washing the cells once with ice-cold PBS. Following isolation of polyadenylated RNA [poly(A)+-RNA], the 1{alpha}-hydroxylase messenger RNA (mRNA) was quantified by Northern blot analysis. HOS cells, a human osteogenic sarcoma cell line, obtained from American Type Culture Collection (38) were cultured on 100 mm culture-grade plastic dishes in DMEM supplemented with 2 mM glutamine, 10% FCS, and 5.5 mM glucose in a humidified atmosphere of 95% air and 5% CO2. Isolation of poly(A)+-RNA were started when the cells were approximately 90% confluent.

Tissues
Human placental and decidual tissues were independently obtained from full-term delivery and from 8-week legal abortion, as described (39, 40). Normal human kidney and brain tissues were obtained from the normal part of the renal carcinoma or malignant brain tumor, respectively. Following isolation of poly(A)+-RNA, the expression of the mRNA was examined by RT-PCR.

RNA isolation and Northern blot analysis
Total RNA was extracted from tissues of rats and mice, and from cells, using the acid guanidinium thiocyanate/phenol/chloroform (AGPC) method (41). Poly(A)+-RNA was further purified by means of oligo(dT) affinity chromatography. Poly(A)+-RNA was separated by electrophoresis on 1% agarose-1.1 M formaldehyde gels, then transferred to nitrocellulose membranes by capillary action in 20 x SSC (1 x SSC = 0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.0). Membranes were cross-linked under UV light and prehybridized at 42 C in 50% formamide, 5 x SSPE (1 x SSPE = 0.1 M sodium chloride, 10 mM NaH2PO4, and 1 mM EDTA, pH 7.0), 5 x Denhardt’s reagent (1 x Denhardt’s = 0.02% polyvinylpyrrolidone, 0.02% BSA, and 0.02% Ficoll 400), 1 mg/ml salmon sperm DNA, and 0.1% SDS for 4 h. Thereafter, the membranes were hybridized at 42–45 C for 12 h in 5 x SSPE, 50% formamide, 0.2 mg of denatured salmon sperm DNA/ml and 1 x Denhardt’s reagent and 1 x 106 cpm/ml specific cDNA probe. Rat 1{alpha}-hydroxylase cDNA fragment [333 bp : 888- 1221 (31)] and mouse 1{alpha}-hydroxylase cDNA N-terminal fragment [750 bp: 30–780 (26)] were used as probes. The cDNA probes were labeled with [32P]deoxy-CTP by the random primer method. The membranes were washed at room temperature for 15 min in 2 x SSPE, 0.03% sodium pyrophosphate and 0.1% SDS, then the most stringent wash was performed at 65 C in 0.1 x SSPE containing 1.0% SDS and 0.03% sodium pyrophosphate. After dehybridizing the membranes for 30 min in 0.1 x SSPE, 0.1% SDS at 90 C, they were hybridized with either rat or mouse ß-actin, or mouse glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probes. The membranes were exposed to x-ray film at -80 C between intensifying screens. The relative abundance of transcripts is corrected by the ß-actin transcripts and indicated as the mean ± SE for at least three samples from different rats, mice or cells.

RT-PCR
Poly(A)+-RNA was extracted from the tissues and cells as described above. Two hundred ng of mRNA was then converted to cDNA in a 20 µl reaction using Superscript II reverse transcriptase (Gibco BRL, Canadian Life Technologies) as recommended by the manufacturer (42). One µl of each RT reaction was then added to a standard 50 µl PCR mixture. After 5 min of preincubation at 95 C, amplification was performed for 30 cycles consisting of 1 min of denaturing at 95 C, 1 min of annealing at 56 C, and 1 min extension at 72 C. The sequence of human cDNA (27) primers is as follows: 5'-CCCTCAAGTACGCCTCCAGAG-3' and 5'-CATCGCCATGGTCAACAGCG-3'. PCR products were separated on 1% agarose gel. Preliminary experiments established that under this condition of PCR, the amounts of 1{alpha}-hydroxylase transcript are semiquantitative.

Statistical analysis
Data are expressed as means ± SE. The statistical significance of differences between groups was determined using Student’s t test. A P < 0.05 value was taken to indicate statistical significance.

Materials
Actinomycin D, rat PTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), salmon calcitonin and forskolin were purchased from Sigma Chemical Co. H89 was purchased from BIOMOL Research Laboratories, Inc., 1{alpha},25(OH)2D3 was kindly provided by Chugai Pharmaceutical Co., Ltd.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tissue distribution of the 1{alpha}-hydroxylase gene expression
Previous reports revealed significant activity of 1{alpha}-hydroxylase in the kidney. We examined the tissue distribution of the 1{alpha}-hydroxylase transcript, using Northern blot analysis (Fig. 1AGo). In wild-type mice (VDR+/+), a transcript of approximately 2.5 kb was abundantly detected only in the kidney. This is in good agreement with observations that the kidney is a prime site of the 1{alpha}-hydroxylase activity (43). In model mice of VDDR II (VDR-KO mice), the transcript was greatly overexpressed 27.5-fold up-regulation by loss of VDR in the kidney, as we previously reported (26). However, even with a longer exposure in Northern blotting, the expression was not detectable in extra-renal tissues of the VDR-KO mice. In normal rats, expression of the 1{alpha}-hydroxylase gene was also evident only in the kidney, and was not detected in extra-renal tissues (data not shown), as was the case in human tissues (27, 28).



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  		Figure 1. Tissue distribution of 1{alpha}-hydroxylase transcript. A, Northern blot analysis of 1{alpha}-hydroxylase transcript in various tissues of wild-type (VDR+/+) and VDR-KO (VDR-/-) mice at 7 weeks of age. Total RNA (50 µg) was electrophoresed and the 750 bp cDNA fragment corresponding to the N-terminal region of mouse 1{alpha}-hydroxylase was used as a probe. The hybridized membranes were exposed to x-ray film for 48 h. Single band was detected only in the kidney in both VDR+/+ and VDR-/- mice. Similar results were obtained in three independent sets of experiments. As an internal control, the G3PDH transcript was used to confirm the presence of intact mRNAs from the tissues. B, RT-PCR analysis of 1{alpha}-hydroxylase transcript in human tissues. Poly(A)+-RNA (200 ng) from the indicated tissue were reverse-transcribed and amplified by PCR using human 1{alpha}-hydroxylase specific primers. Amplified fragment (704 bp) corresponding to the N-terminal was detected in the kidney, decidua and HOS by agarose gel electrophoresis. Similar results were obtained using primer pairs for C-terminal and intermediate parts. The sequence of these fragments were completely identical to those of renal 1{alpha}-hydroxylase transcript. The representative data are shown at the PCR condition, where the amount of 1{alpha}-hydroxylase transcripts is semiquantitative.

 
It is reported that 1{alpha}-hydroxylase activity is detected in extra-renal tissues such as placental decidual cells, keratinocytes, cultured bone cells and an osteosarcoma cell line (6, 7, 8, 9). Accordingly, we further investigated the expression of 1{alpha}-hydroxylase gene in human decidual cells with more sensitive method a semiquantitative RT-PCR to detect the 1{alpha}-hydroxylase transcript. As shown in Fig. 1BGo, the expected 1{alpha}-hydroxylase cDNA fragments were detected from human decidual cells, at much less amounts than from kidney but was not detected from placenta. The RT-PCR products were verified as the cloned renal 1{alpha}-hydroxylase by DNA sequencing. The expression was also detected by RT-PCR in an osteosarcoma cell line, HOS cells (Fig. 1BGo), and also in rat bone tissue (data not shown). Fu et al. (29) reported the expression of 1{alpha}-hydroxylase gene in human cultured keratinocytes, however, we could detect very low levels of the expression by RT-PCR in mouse skin (data not shown), which might contain only small numbers of keratinocytes.

Taken together, although 1{alpha}-hydroxylase is expressed in several extra-renal tissues, it is detectable only in kidney by Northern blotting. Thus, 1{alpha}-hydroxylase gene is expressed most abundantly in kidney, and it is considered that kidney is a prime tissue for 1{alpha}-hydroxylation. Therefore, hereafter we examined the regulation of 1{alpha}-hydroxylase gene expression by hormones only in the kidney.

Positive and negative regulations by PTH, calcitonin and 1{alpha},25(OH)2D3 in renal 1{alpha}-hydroxylase gene expression in rats
Calciotropic hormones and 1{alpha},25(OH)2D3 regulate positively and negatively renal 1{alpha}-hydroxylase activity; however, it is unclear whether these regulations occur at a level of gene expression. PTH, calcitonin or 1{alpha},25(OH)2D3 was then administered to rats and levels of the 1{alpha}-hydroxylase transcript were measured using Northern blot analysis. In rats given either PTH or calcitonin, serum 1,25(OH)2D levels were elevated, as expected, compared with findings in control rats (Table 1Go), possibly due to an enhanced activity of 1{alpha}-hydroxylase. In these rats, a significant increase in 1{alpha}-hydroxylase transcript level was observed as shown in Fig. 2Go, lanes 3 and 5. In the sharp contrast, injection of 1{alpha},25(OH)2D3 apparently reduced the expression (lane 2).


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  		Table 1. Serum biochemical indices in control and 5/6 nephrectomized rats

 


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  		Figure 2. Positive and negative regulation of the rat 1{alpha}-hydroxylase gene expression by PTH, calcitonin and 1{alpha},25(OH)2D3. The expression of 1{alpha}-hydroxylase gene was analyzed by Northern blotting. Poly(A)+ -RNA (5 µg) from the kidneys of the rats was electrophoresed and the 333 bp cDNA fragment corresponding to the 888-1221 region of rat 1{alpha}-hydroxylase was used as a probe. Rats were treated with PTH, calcitonin (CT), or both together with or without 1{alpha},25(OH)2D3. A representative Northern blot analysis is shown in the upper panel. We calculated the relative abundance of the 1{alpha}-hydroxylase gene transcript normalized with the ß-actin transcript from more than three rats for one group.

 
When both PTH and calcitonin were given together, an additive effect occurred regarding induction of the 1{alpha}-hydroxylase gene expression (lane 7). However, 1{alpha},25(OH)2D3 abrogated any induction by either PTH or calcitonin (lanes 4 and 6). These findings indicate that positive and negative regulation of the 1{alpha}-hydroxylase activity by PTH, calcitonin and 1{alpha},25(OH)2D3 occurred at a gene expression level.

Regulations of the 1{alpha}-hydroxylase gene expression occur at transcriptional levels
To determine if the regulations of the 1{alpha}-hydroxylase gene expression occur at a transcriptional level, we studied the effects of the RNA synthesis inhibitor, actinomycin D (Act D) on regulations by the hormones. As shown in Fig. 3Go, the effect of either PTH or calcitonin on 1{alpha}-hydroxylase gene expression was abolished in the presence of Act D (lanes 4 and 6). These results indicate that the effects by PTH, calcitonin or 1{alpha},25(OH)2D3 on 1{alpha}-hydroxylase gene expression are, at least in part, transcriptional events.



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  		Figure 3. A transcriptional inhibitor blocked the induction of 1{alpha}-hydroxylase gene by PTH and calcitonin and the suppression by 1{alpha},25(OH)2D3. The expression of 1{alpha}-hydroxylase gene was analyzed by Northern blotting, using 5 µg each of poly(A)+-RNA from kidneys of the rats, as described for Fig. 2Go. PTH, calcitonin (CT) or 1{alpha},25(OH)2D3 were administered to rats pretreated with actinomycin D (Act D). N.D., Not detectable.

 
PTH induced transcriptional control of the 1{alpha}-hydroxylase gene expression mediated by a PKA signaling pathway.
We also studied regulation of 1{alpha}-hydroxylase gene expression, using the MCT cell line. The MCT cell derives from mouse proximal tubules and are considered to possess the activity of 1{alpha}-hydroxylase. At first, we confirmed both the expression of 1{alpha}-hydroxylase gene and the existence of receptors for PTH and calcitonin in MCT cells by RT-PCR (44). As observed in intact animals, PTH and calcitonin acted as positive regulators for 1{alpha}-hydroxylase gene expression, and 1{alpha},25(OH)2D3 abrogated induction by PTH and calcitonin as shown in Fig. 4AGo. Previous reports suggested that PTH actions are mediated through a PKA signaling pathway (13, 14). Taking advantage of this cell line, we examined whether the PKA signaling pathway is involved in the transcriptional control by PTH and calcitonin. For these studies, forskolin, an adenylate cyclase activator, and H89, a PKA inhibitor, were used. As shown in Fig. 4BGo, both forskolin and PTH significantly enhanced 1{alpha}-hydroxylase gene expression, whereas H89 clearly blocked the induction by both PTH and forskolin (lanes 4 and 8). However, H89 had no apparent effect on the calcitonin-induced 1{alpha}-hydroxylase gene expression (lanes 5 and 6). These observations suggest that transcriptional control of the 1{alpha}-hydroxylase gene by PTH, but not by calcitonin, is mediated through a PKA-dependent pathway.



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  		Figure 4. Regulations of 1{alpha}-hydroxylase gene expression by hormones in the proximal tubule cell line, MCT cells. The expression of 1{alpha}-hydroxylase gene was analyzed by Northern blotting, using 10 µg each of poly(A)+-RNA from MCT cell lines, as described for Fig. 1Go. The N-terminal region of mouse 1{alpha}-hydroxylase was used as a probe. A representative Northern blot analysis is shown in the upper panel. We calculated the relative abundance of the 1{alpha}-hydroxylase gene transcripts normalized with the ß-actin transcript from more than three for one group. A, MCT cells treated with PTH, calcitonin or both, together with or without 1{alpha},25(OH)2D3. B, MCT cells treated with PTH, calcitonin or forskolin, together with or without H89.

 
PTH and calcitonin induced the 1{alpha}-hydroxylase gene expression in the VDDR II model mice (VDR-KO mice)
In the VDDR II patients, the serum 1{alpha},25(OH)2D level is elevated, possibly due to the enhanced activity of the 1{alpha}-hydroxylase. To determine if the enhanced activity of 1{alpha}-hydroxylase is due to transcriptional control, we examined expression of the 1{alpha}-hydroxylase gene in VDDR II model mice (VDR-KO mice) at 3 weeks, and studied the effect of either PTH or calcitonin on the induction. As shown in Fig. 5Go, the 1{alpha}-hydroxylase gene in the VDR-KO mice was overexpressed 3-fold of VDR+/+ mice (lane 3). When either PTH or calcitonin was given to VDR-KO mice, an enhancement was observed in the 1{alpha}-hydroxylase transcript level (lanes 5 and 7). Nevertheless, 1{alpha},25(OH)2D3 had no apparent effect on the 1{alpha}-hydroxylase gene expression in the VDR-KO mice either untreated or treated with PTH and calcitonin (lanes 4, 6, and 8). From these observations, it is likely that the inducible effect of either PTH or calcitonin on renal 1{alpha}-hydroxylase gene expression is mainly not mediated by VDR, though VDR is essential for the negative regulation by 1{alpha},25(OH)2D3. The high serum levels of 1{alpha},25(OH)2D in VDDR II patients is probably due to both elevated levels of serum calciotropic hormones, and absence of the VDR-mediated repression of the 1{alpha}-hydroxylase gene expression.



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  		Figure 5. Induction of 1{alpha}-hydroxylase gene by PTH and calcitonin in VDDR II model mice. The expression of 1{alpha}-hydroxylase gene was analyzed by Northern blotting, using 2 µg of poly(A)+-RNA from the kidneys of the mice, as described in Fig. 1Go. Mice were treated with PTH or calcitonin, together with or without 1{alpha},25(OH)2D3. A representative Northern blot analysis is shown above. We calculated the relative abundance of the 1{alpha}-hydroxylase gene transcripts normalized with the ß-actin transcript from more than three mice for one group.

 
1{alpha}-hydroxylase gene expression and regulation in chronic renal failure model rats
As a model animal of CRF, a state of CRF was induced by 5/6 nephrectomy in rats and was confirmed by increased serum creatinine and BUN levels (Table 1Go). Serum PTH and calcitonin levels in these rats were significantly higher than those in sham-operated controls. In these rats with CRF, serum calcium concentrations remained normal and serum phosphorus concentrations were slightly higher than in controls. In such moderate stage of CRF, 1{alpha}-hydroxylase transcripts without any treatment were lower than those in sham-operated rats (Fig. 6Go, lane 2). We further examined the induction of 1{alpha}-hydroxylase gene expression by PTH and calcitonin. These treatments only slightly induced the 1{alpha}-hydroxylase gene expression (Fig. 6Go, lanes 3 and 4) compared with findings in normal rats (Fig. 2Go). When PTH and calcitonin were administrated to the rats with CRF, serum levels of 1{alpha},25(OH)2D remained unchanged (Table 1Go). These findings reflected the expression of renal 1{alpha}-hydroxylase gene in uremic rats. Thus, in moderate stage of CRF, renal cells expressing the 1{alpha}-hydroxylase gene appear to have a diminished potential to respond to PTH and calcitonin.



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  		Figure 6. Slight induction of the 1{alpha}-hydroxylase gene by PTH and calcitonin in chronic renal failure model animals. The expression of 1{alpha}-hydroxylase gene was analyzed by Northern blotting, using 5 µg each of poly(A)+-RNA from the kidneys of the CRF rats as described in Fig. 2Go. CRF rats were treated with PTH or calcitonin. A representative Northern blot analysis is shown above. N.D., Not detectable.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
1{alpha}-hydroxylase is the prime enzyme physiologically critical for metabolic conversion of vitamin D into a hormonal form of vitamin D, 1{alpha},25(OH)2D3 (1, 2). There are a number of reports that the 1{alpha}-hydroxylase activity is stimulated by PTH and calcitonin, and is suppressed by 1{alpha},25(OH)2D3 (10). Nevertheless, the regulatory mechanism of 1{alpha}-hydroxylase activity in the kidney has heretofore not been studied at the molecular level because cDNA of 1{alpha}-hydroxylase had not been cloned. In earlier work, we cloned cDNAs for mouse and human 1{alpha}-hydroxylase (26, 27). In our series on going, we asked if regulations of the activity occur at a transcriptional or a posttranscriptional level. Consequently, we demonstrated that positive and negative regulations by PTH, calcitonin and 1{alpha},25(OH)2D3 are transcriptional events. These results suggest the presence of regulatory elements for PTH, calcitonin and 1{alpha},25(OH)2D3 in the 1{alpha}-hydroxylase promoter. This idea is supported by our recent observation that the cloned human 1{alpha}-hydroxylase gene promoter (about 4.0 kb) confers responses to PTH, calcitonin and 1{alpha},25(OH)2D3 (44). A regulatory element for PTH was identified also in the mouse 1{alpha}-hydroxylase promoter (1.7 kb) (45).

Though both PTH and calcitonin act as positive regulators for activity and transcriptional control of the 1{alpha}-hydroxylase gene, the signaling pathways downstream from these two factors do not seem to be identical. PTH activates the PKA via cAMP production, and indeed activation of the PKA signaling pathway mimicked the PTH action in the induction of the 1{alpha}-hydroxylase activity (13). In the present study, we demonstrated that the 1{alpha}-hydroxylase gene expression is induced by activating PKA in a mouse proximal tubule cell line, MCT cells. This finding clearly indicates that the transcription is activated by the PKA signaling pathways, possibly through its promoter. This idea is further supported by the finding that a PKA inhibitor, H89 abrogated the PTH-induced 1{alpha}-hydroxylase gene expression. However, H89 had no apparent effect on calcitonin action in the induction of the 1{alpha}-hydroxylase gene expression, so that another signaling pathway must be involved in the regulation by calcitonin.

We next showed that 1{alpha},25(OH)2D3 suppresses the 1{alpha}-hydroxylase gene expression even when its expression is enhanced by PTH and calcitonin. However, this suppression was not seen in the mice deficient of VDR (VDR-KO mice), which still responded to PTH and calcitonin. These findings strongly suggested that VDR is essential for the negative regulation by 1{alpha},25(OH)2D3. As elevated levels of PTH were seen in the VDDR II patients (46), the high serum levels of 1{alpha},25(OH)2D in the VDDR II patients may be due to both enhanced levels of serum PTH and the absence of VDR-mediated repression in the 1{alpha}-hydroxylase gene expression.

Stimulation of transcription of other genes by 1{alpha},25(OH)2D3 through positive vitamin D response elements (VDREs) has been examined in great detail. However, the mechanism of transcriptional repression by 1{alpha},25(OH)2D3 is not well understood. To date, only two negative VDREs have been described in the gene promoters for the human and avian PTH and rat bone sialoprotein gene (47, 48). Demay et al. (49) reported that negative VDRE of the human PTH promoter differs from positive VDRE both in sequence composition and in affinity for VDR/RXR heterodimer. However, analysis of the two negative VDRE did not lead identification of consensus negative VDRE. The present study suggests the existence of a strong negative VDRE in the 1{alpha}-hydroxylase gene promoter. Organization of negative and positive elements in this promoter to elicit complex transcriptional regulations is of particular interest, in addition to identification of its kidney-specific enhancer element and its binding factor.

In patients with chronic renal failure (24), decreased serum 1{alpha},25(OH)2D levels, possibly as a result of reduced 1{alpha}-hydroxylase activity are observed. Decreased serum 1{alpha},25(OH)2D levels might play a role in secondary hyperparathyroidism which subsequently may cause renal osteodystrophy. Therefore, to demonstrate whether the hormonal regulation of the renal 1{alpha}-hydroxylase gene expression are affected in a state of chronic renal failure or not, we used 5/6 nephrectomized rats that have been established as model rats for chronic renal failure (50). The remained renal tissue, which was hypertrophied, was confirmed to contain comparable amount of cells by detecting the ß-actin transcript. We found in these rats that the 1{alpha}-hydroxylase transcripts are detectable, but lower than those seen in sham-operated rats. Moreover, we found in these rats that the response to PTH and calcitonin was diminished (Fig. 6Go). From these findings, it is suggested that the 1{alpha}-hydroxylase gene expression in CRF patients is reduced, irrespective of hyperparathyroidism. We do not know why cells expressing the 1{alpha}-hydroxylase gene lose the normal response to caltiotropic hormones in moderate stage of chronic renal failure. Such unresponsiveness might be somehow explained by 1{alpha}-hydroxylase gene repression by phosphate levels. However, the mechanism of gene regulation by phosphate remains unknown and further investigation may be necessary.

We have studied the transcriptional regulations of the 1{alpha}-hydroxylase gene mostly in kidney. However, we found that 1{alpha}-hydroxylase gene is also expressed in extra-renal tissues. It has been reported that the regulations of 1{alpha}-hydroxylase activity are distinct among tissues (51). For instance, in alveolar macrophages and pulmonary T cells, the 1{alpha}-hydroxylase activity appears to be stimulated by {gamma}-interferon, but not by PTH and calcitonin (52). Thus, it will be of much interest to further investigate the regulations of the 1{alpha}-hydroxylase gene in extra-renal tissues.

In conclusion, we found that renal 1{alpha}-hydroxylase gene expression is positively regulated by PTH and calcitonin, and negatively by 1{alpha},25(OH)2D3 at transcriptional levels in intact animals. The positive control by PTH is considered to be mediated through the PKA pathway, however, the signal pathway of calcitonin seem to be different. VDR is essential for the negative regulation by 1{alpha},25(OH)2D3. Identification of the regulatory elements in the 1{alpha}-hydroxylase promoter will be interesting.


   Acknowledgments
 
We would like to thank Naoko Yagishita, Takashi Sato and Nobuaki Joh for technical assistance, Junn Yanagisawa, Tatsuya Yoshizawa, Yugo Shibagaki, Itsuro Inoue and Senya Matsufuji for helpful discussions, Michiko Watanabe, Hitoshi Tai, Akinori Sugiyama, Hideyuki Harada and Chifumi Kitanaka for preparing tissue samples, Toshio Takeyama and Health Science Research Institute inc. for measurement of serum data, Chugai Pharmaceutical Co., Ltd. for vitamin D relative compounds, CSK Research Park for the maintenance of VDR-KO mice.


   Footnotes
 
1 This research was supported by a grant-in-aid for priority areas from the Ministry of Education, Science, Sports and Culture of Japan (Sigeaki Kato). Back

Received October 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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