1,25-Dihydroxyvitamin D3 upregulates natriuretic peptide receptor-C expression in mouse osteoblasts

Noriyuki Yanaka, Hiroyuki Akatsuka, Eri Kawai, and Kenji Omori

Discovery Research Laboratory, Tanabe Seiyaku, Osaka 532-8505, Japan

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], a key regulator of mineral metabolism, regulates expression of several genes related to bone formation. The present study examined the 1,25(OH)2D3-mediated regulation of natriuretic peptide receptor-C (NPR-C) expression in osteoblasts. 1,25(OH)2D3 treatment significantly increased NPR-C-dependent atrial natriuretic peptide-binding activity and synthesis of the NPR-C protein in mouse osteoblastic cells in a cell-specific manner. Western blot analysis also demonstrated that 1,25(OH)2D3 upregulated expression of NPR-C protein in slow kinetics. Next, Northern blot analysis revealed a significant increase in the steady-state NPR-C mRNA level by 1,25(OH)2D3. Sequence analysis of the 9 kb of the 5'-flanking region of the mouse NPR-C gene revealed an absence of consensus vitamin D-response elements, and promoter analysis using osteoblastic cells stably transfected with mouse NPR-C promoter-reporter constructs showed a slight increase of promoter activity with 1,25(OH)2D3 treatment. In addition, a nuclear run-on assay exhibited that the transcriptional rate of the NPR-C gene was unchanged by 1,25(OH)2D3, whereas that of the osteopontin gene was increased. Evaluation of NPR-C mRNA half-life demonstrated that 1,25(OH)2D3 significantly increased the NPR-C mRNA stability in osteoblastic cells. 1,25(OH)2D3 attenuated intracellular cGMP production in osteoblastic cells stimulated by C-type natriuretic peptide (CNP) without a significant change of the natriuretic peptide receptor-B mRNA level, suggesting enhancement of the clearance of exogenously added CNP via NPR-C. Furthermore, NPR-C and osteopontin mRNAs in mouse calvariae were significantly increased by administration of 1,25(OH)2D3, and immunohistological analysis demonstrated that NPR-C is actually and strongly expressed in mouse periosteal fibroblasts. These findings suggest that 1,25(OH)2D3 can play a critical role for determination of the natriuretic peptide availability in bones by regulation of NPR-C expression through stabilizing its mRNA.

natriuretic peptide

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE STEROID HORMONE 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is now recognized as a key regulator of both bone formation and resorption, with a principal role in calcium homeostasis and skeletal metabolism, and influences the expression of genes related to the establishment and maintenance of the bone cell phenotype. Although the overall function of 1,25(OH)2D3 in bones is to promote resorption, it is the osteoblasts, and not the osteoclasts, that contain a nuclear receptor (vitamin D receptor, VDR) that belongs to the steroid/retinoid/thyroid hormone receptor superfamily and acts via binding to distinct vitamin D response elements (VDRE). As a consequence, the expression of several genes in osteoblasts is regulated by 1,25(OH)2D3. For instance, the expression of the type I collagen gene and the bone sialoprotein gene are downregulated by the hormone, whereas those of the osteopontin and osteocalcin, noncollagenous proteins of the bone extracellular matrix that regulates bone formation, are significantly upregulated in humans and rats (18, 25, 26). To date, the role of 1,25(OH)2D3 in the regulation of osteocalcin gene transcription has been studied extensively. However, little is known regarding the effect of 1,25(OH)2D3 on gene expression through nongenomic mechanisms.

Natriuretic peptides (NPs) are known to play important roles in cardiovascular homeostasis. Three isoforms, termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP; see Refs. 7 and 31), constitute an NP family. ANP and BNP have been considered to be responsible for systemic blood pressure control and body fluid homeostasis (3, 11). These biological functions of NPs were mediated with production of intracellular cGMP through the guanylyl cyclase (GC)-coupled receptors, termed GC-A and GC-B. Recent works have demonstrated that NPs were involved in the suppression of cell proliferation and in phenotypic development of chondrocytes and osteoblasts (12, 27, 30). In addition, CNP secreted from cultured osteoblastic cells has been shown to increase expression of alkaline phosphatase and osteocalcin and to induce formation of mineralized nodules in an autocrine manner (12). Natriuretic peptide receptor-C (NPR-C) has a very short putative intracytoplasmic extension with no GC activity (8, 10, 28). One of the possible roles of NPR-C is believed to determine the biological availabilities of NPs by elimination from circulation, therefore suggesting that the NPR-C expression is tightly associated with the maintenance of the cardiovascular system and with the regulation of bone formation. Recent work has shown that NPR-C decreased in amount during phenotypic differentiation of osteoblastic cells and chondrocytes (9, 14), suggesting that the NPR-C protein is a marker molecule representing the developmental stages of these cells. On the other hand, NPR-C has been reported to control adenylyl cyclase activity via Gi protein, phospholipid hydrolysis, thymidine kinase activity, and mitogen-activated protein kinase activity in a variety of cells (1, 2, 4, 13, 29) without any cGMP response, suggesting it has a physiological significance other than the clearance of ligands.

Here, we have demonstrated that 1,25(OH)2D3 treatment significantly increased NPR-C expression at protein and mRNA levels in mouse osteoblastic cells in a cell-specific manner. We recently reported highly specific antibody that recognizes the NPR-C cytoplasmic domain (9), and the availability of this antibody enabled us to investigate the NPR-C expression at total protein level and by immunohistochemical analysis. We focused on the significant effect of 1,25(OH)2D3 on NPR-C mRNA expression and investigated the mechanisms underlying its upregulation. The steady-state mRNA level can be affected by the rate of gene transcription and by that of mRNA degradation. Our recent studies have shown the structure of the 5'-flanking regulatory regions of the mouse and human NPR-C genes and functional features using serial 5'-deletions of the promoter regions (34, 35). Further investigations were carried out to examine the cis-acting sequences in the regulatory region of the mouse NPR-C gene and its mRNA stability using a transcription inhibitor, actinomycin D. Moreover, our findings in this study were that the NPR-C mRNA was significantly increased in mouse calvariae by 1,25(OH)2D3 administration and that NPR-C was actually expressed in mouse periosteal fibroblasts, suggesting that 1,25(OH)2D3 can modulate NPR-C expression with its physiological roles in vivo.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Restriction endonucleases and DNA-modifying enzymes were obtained from Takara Shuzo (Kyoto, Japan). [alpha -32P]dCTP, [alpha -32P]UTP, rat 125I-ANP, Hybond-N plus nylon filter, and cGMP EIA system were from Amersham. 1,25(OH)2D3 was obtained from Biomol. CNP was a product of Peptide Institute (Osaka, Japan). The ANP analog des[Gln18,Ser19,Gly20,Leu21,Gly22] ANP-(4---23)-NH2 [C-ANF-(4---23)] was purchased from Sigma. alpha -Modified Eagle's medium (alpha -MEM), Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), and G418 were obtained from GIBCO Biotech. The vectors pGVP and pGVB containing a firefly luciferase gene were obtained from Toyo Inki (Japan). The plasmid pMAM-neo was a product of Clontech.

Cell cultures. MC3T3-E1 cells, COS cells, and BALB/3T3 clone A31 were obtained from Dainippon Pharmaceutical (Osaka, Japan). C3H10T1/2 clone 8 (10T1/2) was from RIKEN Cell Bank (Tsukuba, Japan). A population of osteoblastic cells was isolated from calvariae of newborn ICR mice and Wistar rats as previously reported (12). MC3T3-E1 cells, 10T1/2 cells, and osteoblastic cells from calvariae were cultured in alpha -MEM supplemented with 10% FCS. COS cells and BALB/3T3 cells were cultured in DMEM supplemented with 10% FCS. These cells were passaged in a controlled atmosphere of 5% CO2-95% air at 37°C.

Binding assay of NPR-C. Cells in 24-well dishes were washed two times with 1 ml of ice-cold phosphate-buffered saline (PBS). Binding of 1 nM rat 125I-ANP was allowed to proceeded for 30 min at 4°C to equilibrium in the presence or absence of 1 µM of unlabeled C-ANF-(4---23). After 30 min, cells were washed two times with PBS and solubilized with 500 µl of 0.5 N NaOH. Aliquots were assayed for radioactivity in a Cobra gamma -counter (Packard).

Western blot and immunohistological analyses. The antiserum against the NPR-C protein used was obtained according to our previously published procedures (9). Confluent cells (1 × 107) were washed two times with ice-cold PBS, harvested in the homogenizing buffer [10 mM Tris · HCl (pH 7.5), 5 mM EDTA, 5 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol], sonicated for 20 s, and centrifuged at 30,000 g for 30 min. The pellet was rehomogenized in the homogenizing buffer. The solubilized membrane proteins (~30 µg/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to an Immobilon P filter (Millipore). The blots were blocked for 18 h at 4°C by soaking in 5% nonfat dried milk in PBS and were incubated for 18 h at 4°C with anti-NPR-C antiserum (diluted 1:1,000). Signals were detected using horseradish peroxidase-conjugated anti-rabbit IgG and the enhanced chemiluminescence systems (Amersham). Neonatal ICR mice were used in the immunohistological experiment. Tissues were rapidly fixed with paraformaldehyde, and 4-µm-thick sections were prepared on a cryostat. Immunostaining was performed with a primary rabbit anti-NPR-C antibody (diluted 1:1,000) with or without preabsorption by specific antigen and by the avidin-biotin-peroxidase complex (ABC) kit (Vector Laboratories, Burlingame, CA), as described in the manufacturer's directions.

Promoter analysis by stable transfection and reporter assay. Nine kilobases of the 5'-flanking region of the mouse NPR-C gene were subcloned into reporter plasmid pGVB to yield pmNPRCluc1 (35). For stable transfection of MC3T3-E1 cells, the cells were electroporated at 200 V and 960 µF using 1 µg of pmNPRCluc1 or 1 µg of control plasmid pGVP with 1 µg of pMAM-neo. After replating, the cells were treated with 500 µg/ml of G418 for 14 days. G418-resistant colonies were identified, and 10 independent colonies were repropagated until they reached a higher density. After 18 h of incubation with or without 1 × 10-8 M 1,25(OH)2D3 treatment, the cells were washed two times with PBS, collected in a microcentrifuge, and disrupted by a freeze-thaw cycle in 300 µl of cell lysis solution (Promega). The supernatants obtained by centrifugation for 5 min were pooled to measure firefly luciferase activity. Luciferase activity was measured using TD20e (Turner) with 10 µl of cell extracts.

In vivo treatment with 1,25(OH)2D3. Five-week-old ICR mice were injected intraperitoneally with 1 or 100 µg of 1,25(OH)2D3 or with vehicle and killed 18 h later. Total RNAs were isolated from calvariae that were dissected out and cleaned of soft tissues.

RNA analysis by Northern blotting hybridization. Total RNAs were isolated using ISOGEN (Nippon gene, Toyama, Japan), and poly(A)+ RNAs purified by an oligo(dT)-cellulose column chromatography using an mRNA separator kit (Clontech) were fractionated in a 1% agarose gel containing 0.66 M formaldehyde and 0.02 M MOPS (pH 7.0). Fractionated RNAs were transferred onto a nylon filter by capillary blotting and then cross-linked by ultraviolet irradiation. 32P-labeled cDNA fragments encoding mouse NPR-C (35), mouse osteopontin (5), rat beta -actin, mouse GC-B, rat beta -actin, or human glyceraldehyde phosphate dehydrogenase were used for Northern blotting hybridization as probes. Hybridization was performed as described previously (35). Values were obtained from densitometric scanning of hybridized signals using a Micro Computer Imaging Device (MCID; Imaging Research).

Nuclear run-on transcription assay. After incubation with or without 1 × 10-8 M 1,25(OH)2D3 treatment for 16 h, MC3T3-E1 cells were rinsed with ice-cold PBS two times. Cultured cells were suspended in 400 µl of buffer A [10 mM Tris · HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, and 0.5 % Nonidet P-40] and chilled on ice for 15 min. After centrifugation at 3,000 g for 5 min, nuclear fractions were resuspended in 400 µl of buffer A. The nuclei were precipitated by centrifugation at 3,000 g for 5 min and suspended in 100 µl of buffer B [50 mM Tris · HCl (pH 8.3), 0.1 mM EDTA, 5 mM MgCl2, and 40% glycerol]. To label the nascent RNA transcripts, the nuclei were incubated in 200 µl of 2× reaction buffer (10 mM Tris · HCl, 5 mM MgCl2, and 300 mM KCl) supplemented with 1 mM ATP, GTP, and CTP (Pharmacia Biotech), 250 µCi of [alpha -32P]UTP (3,000 Ci/mmol), and 5 mM dithiothreitol for 30 min at 30°C with shaking. The nuclear extracts were digested for 5 min with RNase-free DNase I (GIBCO Biotech), and the reaction was terminated by the addition of SDS buffer [83.3 mM EDTA, 330 mM Tris · HCl (pH 7.5), and 3.33% SDS] followed by treatment with proteinase K. 32P-labeled RNA transcripts were purified by precipitation on Millipore type HA (0.45 µm) filters with ice-cold 5% trichroloacetate, 30 mM sodium pyrophosphate, and ethanol precipitations. Equal counts (~4 × 106 counts/min) of radiolabeled RNA transcripts were produced from the control and 1,25(OH)2D3-treated MC3T3-E1 cells. The radiolabeled RNA transcripts were hybridized for two nights at 42°C with mouse NPR-C and mouse osteopontin cDNA probes that had been denatured and immobilized on a nylon filter. Filters were washed at room temperature in 2× saline sodium citrate (SSC) with 0.1% SDS, followed by 0.5× SSC with 0.1% SDS at 60°C for 15 min two times and subjected to autoradiography. The plasmid pUC19 was used to determine nonspecific background hybridization. Values were obtained from densitometric scanning of hybridized signals using MCID.

Measurement of intracellular cGMP accumulation. After 18 h incubation with or without 1 × 10-8 M 1,25(OH)2D3 treatment, MC3T3-E1 cells were washed two times with alpha -MEM supplemented with 10% FCS and then incubated with the indicated concentration of CNP and 0.5 mM isobutyl methylxanthine (Sigma) in alpha -MEM supplemented with 10% FCS for 30 min at 37°C. After being washed two times with PBS, cells were solubilized with 500 µl of ice-cold 50% ethanol and extracted by evaporation. The concentrations of cGMP were determined after acetylation using a cGMP EIA system (Amersham).

Statistical analysis. All values are expressed as means ± SD. Statistical significance was determined by the unpaired Student's t-test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

1,25(OH)2D3-induced NPR-C expression in a cell-specific manner. 1,25(OH)2D3 treatment increased ANP-binding activity in MC3T3-E1 cells (Fig. 1). Binding analysis with rat 125I-ANP revealed that C-ANF-(4---23), a specific NPR-C agonist, effectively competed for increased ANP-binding sites with a high affinity (data not shown). To directly estimate the NPR-C protein level, we performed Western blot analysis using the antibody against the rat NPR-C cytoplasmic domain (Fig. 2A). At the reducing condition, the solubilized NPR-C protein was detected as a single band of ~63 kDa. Strong signals corresponding to NPR-C were seen in the lysates from 1,25(OH)2D3-treated MC3T3-E1 cells compared with those from untreated cells, indicating that augmentation of the NPR-C-dependent ANP-binding activity by 1,25(OH)2D3 was accompanied by upregulation of de novo synthesis of the NPR-C protein. The relative amounts of NPR-C protein (487% increase) did not appear to correlate with the percentage increase in ANP-binding activity, suggesting that Western blot analysis could detect not only NPR-C protein on the cellular surface but also a larger amount of intracellular NPR-C protein. Next, we wanted to test whether 1,25(OH)2D3 could modulate the ANP-binding activity in a variety of cultured cells that express NPR-C protein abundantly. As shown in Fig. 1, a significant increase in NPR-C-dependent ANP-binding activity was also observed in 10T1/2 cells and in mouse cultured osteoblastic cells with 1,25(OH)2D3 treatment. However, NPR-C expression was not upregulated by 1,25(OH)2D3 in COS cells (Fig. 1A), HeLa cells, and rat aortic smooth muscle cells (data not shown). As shown in Fig. 2B, it is noteworthy that the kinetics of the increase in the NPR-C protein level were slower than that of the rapid increase of 25(OH)2D3 24-hydroxylase by 1,25(OH)2D3 treatment as previously reported (24).


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Fig. 1.   Effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on atrial natriuretic peptide (ANP)-binding activity. After treatment with the indicated concentration of 1,25(OH)2D3 for 18 h, ANP-binding activity was determined as described in MATERIALS AND METHODS. Natriuretic peptide receptor-C (NPR-C)-dependent binding activity was determined by competitive binding assay in the presence of 1 µM of unlabeled des[Gln18,Ser19,Gly20,Leu21,Gly,22] ANP-(4---23)-NH2 [C-ANF-(4---23)]. Error bars represent SD. ** P < 0.01 compared with basal.


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Fig. 2.   Effect of 1,25(OH)2D3 on the NPR-C protein level by Western blot analysis. A: after treatment with 1 × 10-8 M of 1,25(OH)2D3 for 18 h, cells (1 × 107) were washed two times with ice-cold PBS and harvested in homogenizing buffer. After centrifugation, the resulting pellets were homogenated and subjected to 10% SDS-PAGE. Transferred blots were detected using anti-NPR-C antiserum (diluted 1:1,000), horseradish peroxidase-conjugated anti-rabbit IgG, and the enhanced chemiluminescence systems as described in MATERIALS AND METHODS. B: at the day of confluence, MC3T3-E1 cells were exposed to 1 × 10-8 M of 1,25(OH)2D3 for the indicated time. Western blot analysis was performed as described in MATERIALS AND METHODS.

Northern blot analysis revealed that 1,25(OH)2D3 treatment significantly increased the steady-state level of the NPR-C mRNA in MC3T3-E1 cells (Fig. 3). The existence of two discrete NPR-C mRNA species was shown in Northern blotting analysis. We have previously isolated two different sized cDNAs corresponding to mouse NPR-C mRNA (data not shown), revealing that alternative polyadenylations in the 3'-untranslated region lead to the presence of two different species of mouse NPR-C mRNA. In the same experiment, we observed the significant increase in the osteopontin mRNA level with 1,25(OH)2D3 treatment. Previous works have shown that an AP-1 sequence was located in the 5'-flanking region of the mouse and chicken osteopontin genes and that the osteopontin mRNA level was upregulated in phorbol myristate acetate-treated MC3T3-E1 cells (5). Although our previous work has revealed an AP-1 sequence in the 5'-flanking region of the mouse NPR-C gene, no significant change of NPR-C mRNA level was observed with phorbol myristate acetate treatment for 18 h.


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Fig. 3.   RNA analysis by Northern blotting hybridization. After treatment of MC3T3-E1 cells with the indicated concentration of 1,25(OH)2D3 or with 50 ng/ml of phorbol myristate acetate (PMA) for 18 h, poly(A)+ RNAs were purified as described in MATERIALS AND METHODS. Poly(A)+ RNA (5 µg) was subjected to Northern blot analysis. Hybridization was performed using 32P-labeled mouse NPR-C, mouse osteopontin, or human glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA as probes, as described in MATERIALS AND METHODS.

Mechanism of 1,25(OH)2D3-induced increase in the NPR-C mRNA. The 1,25(OH)2D3-induced increase in steady-state NPR-C mRNA levels could be the result of transcriptional effects, an effect on NPR-C mRNA stability, or a combination of both. Our previous work revealed that no consensus VDRE was identified in 2 kb of the 5'-flanking region of the mouse NPR-C gene. We analyzed a further upstream region of the 5'-flanking region. (The nucleotide sequence has been submitted to the GenBank/EMBL Data Bank with accession number AB007853.) However, sequence analysis has shown that the 9-kb region upstream from an ATG codon contained no candidate VDRE, based on a homology search using the VDRE sequences of rat osteocalcin (21), rat calbindin-D9k (6), rat 25(OH)2D3 24-hydroxylase (24), and mouse osteopontin (23) genes. Additionally, we investigated the effect of 1,25(OH)2D3 on the mouse NPR-C promoter activity in stably transfected MC3T3-E1 cells using a reporter construct, carrying the 9 kb of the 5'-flanking region (pmNPRCP1; Fig. 4). 1,25(OH)2D3 treatment slightly increased transcriptional activity only in stable transformants carrying pmNPRCP1. Furthermore, we performed a nuclear run-on assay to determine whether 1,25(OH)2D3 increased the transcription rate of the NPR-C gene. Nuclei from the control or 1,25(OH)2D3-treated cells were isolated, and nascent transcripts were hybridized to filter-bound plasmid probes. Although 1,25(OH)2D3 significantly increased the transcriptional rate of the mouse osteopontin gene, it had no effect on that of the NPR-C gene (Fig. 5).


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Fig. 4.   Stable expression analysis of the mouse NPR-C promoter-luciferase (LUC) reporter chimeric gene in MC3T3-E1 cells. Transcriptional activity was measured by DNA transfection experiments in MC3T3-E1 cells. A: mouse NPR-C gene was inserted upstream of the firefly luciferase gene, and the nucleotide boundaries were confirmed by nucleotide sequencing. B: in each experiment, cells were treated with or without 1 × 10-8 M of 1,25(OH)2D3 for 18 h. pGVP is a control plasmid carrying the luc gene downstream of SV40 promoter. Values are relative degree of induction compared with the activity obtained without 1,25(OH)2D3. Results presented here are averages from 10 isolated independent clones. Error bars represent SD.


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Fig. 5.   Transcription rate of the NPR-C gene in 1,25(OH)2D3-treated MC3T3-E1 cells. A: nuclei were isolated from confluent MC3T3-E1 cells untreated or treated with 1 × 10-8 M of 1,25(OH)2D3 for 12 h. Labeled RNAs were hybridized to nylon membrane containing 1 µg of linearized mouse NPR-C cDNA, mouse osteopontin cDNA, and pUC19 as a background control. Hybridization was carried out as described in MATERIALS AND METHODS. B: values shown are obtained from densitometric scanning of signals (n = 3), as described in MATERIALS AND METHODS. Error bars represent SD. * P < 0.05 compared with basal.

Next, we assessed whether 1,25(OH)2D3 treatment affects the half-life of the NPR-C mRNA. After incubation in the presence or absence of 1,25(OH)2D3 for 16 h, 1,25(OH)2D3 was then removed, and parallel dishes were incubated in the presence of the transcription inhibitor actinomycin D. We estimated the NPR-C mRNA half-life in 1,25(OH)2D3-treated or -untreated MC3T3-E1 cells. The half-life of the NPR-C mRNA was significantly lengthened for 4.68 h by 1,25(OH)2D3 treatment in comparison with that in untreated cells (0.98 h). In the same experiment, we observed that 1,25(OH)2D3 had no effect on the half-life of the osteopontin mRNA (data not shown). Further experimentation was performed to test the effect of 1,25(OH)2D3 on the stability of the NPR-C mRNA. We first exposed MC3T3-E1 cells to 1,25(OH)2D3 for 16 h to increase NPR-C mRNA. 1,25(OH)2D3 was then completely removed, and parallel dishes were incubated in the presence or absence of actinomycin D, plus or minus 1,25(OH)2D3 for 6 h. We observed, in the presence of actinomycin D, that the NPR-C mRNA level without 1,25(OH)2D3 treatment was quite lower (63.5% decrease) than with its treatment (Fig. 6, B, right). These experiments were repeated independently to confirm the results described above. These results strongly suggested that 1,25(OH)2D3 could have an effect on the stability of the NPR-C transcripts.


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Fig. 6.   Effect of 1,25(OH)2D3 on NPR-C mRNA half-life in MC3T3-E1 cells. A: confluent MC3T3-E1 cells were pretreated with or without 1 × 10-8 M of 1,25(OH)2D3 for 16 h. Medium was removed and incubated in the presence of 5 µg/ml of actinomycin D for the indicated time. Poly(A)+ RNA was purified as described in MATERIALS AND METHODS. Poly(A)+ RNA (10 µg) was subjected to Northern blot analysis. Hybridization was performed using 32P-labeled mouse NPR-C cDNA fragments as a probe. Values shown are obtained from densitometric scanning of hybridized signals, as described in MATERIALS AND METHODS. B: confluent MC3T3-E1 cells were pretreated with 1 × 10-8 M of 1,25(OH)2D3 for 16 h to increase the NPR-C mRNA. After the medium was removed, the cells were incubated in the presence or absence of 5 µg/ml of actinomycin D (AcD), with or without 1 × 10-8 M of 1,25(OH)2D3 for 6 h. Poly(A)+ RNA (10 µg) was purified and subjected to Northern blot analysis. Hybridization was carried out as described in MATERIALS AND METHODS.

Effect of 1,25(OH)2D3 on cellular responsiveness to CNP. To evaluate the functional significance of 1,25(OH)2D3-mediated NPR-C upregulation, we examined the biological responsiveness to NPs. After pretreatment of MC3T3-E1 cells with or without 1 × 10-8 M of 1,25(OH)2D3, CNP (0.1 nM - 1 µM) was then added to the medium. At the end of incubation, the intracellular cGMP concentration was measured. The CNP-induced cGMP accumulation was shown to be reduced by 1,25(OH)2D3 pretreatment (Fig. 7B). cGMP production in MC3T3-E1 cells by CNP was shown to be mediated through GC-B. Northern blot analysis has shown that GC-B mRNA level was not significantly changed by 1,25(OH)2D3 treatment for 16 h (Fig. 7A). These results suggested that the attenuation of cGMP accumulation in 1,25(OH)2D3-pretreated cells is caused by the increased metabolic clearance of CNP in the medium through the NPR-C upregulation.


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Fig. 7.   Effect of 1,25(OH)2D3 on guanylate cyclase (GC)-B mRNA level and cGMP production by CNP in MC3T3-E1 cells. A: after pretreatment with or without 1 × 10-8 M of 1,25(OH)2D3 for 16 h, poly(A)+ RNAs were isolated as described in MATERIALS AND METHODS. Poly(A)+ (5 µg) RNA was subjected to Northern blot analysis. Hybridization was performed using 32P-labeled mouse GC-B or human GAPDH cDNA as probes, as described in MATERIALS AND METHODS. B: after pretreatment with or without 1 × 10-8 M of 1,25(OH)2D3 for 16 h, MC3T3-E1 cells were incubated with the indicated concentration of C-type natriuretic peptide (CNP) for 30 min. Intracellular cGMP concentration was determined as described in MATERIALS AND METHODS. Error bars represent SD. * P < 0.05 and ** P < 0.01 compared with basal.

Induction of NPR-C mRNA expression with 1,25(OH)2D3 treatment in vivo. Recent work has demonstrated that expression of the osteocalcin gene is significantly downregulated in mouse calvariae with intraperitoneal injection of 1,25(OH)2D3 as well as in 1,25(OH)2D3-treated MC3T3-E1 cells (36). Further experiments were performed to test the effect of 1,25(OH)2D3 on NPR-C expression in vivo. 1,25(OH)2D3 was injected intraperitoneally to mice, and, after 18 h, poly(A)+ RNAs from calvariae were isolated and subjected to Northern blot analysis. The steady-state NPR-C mRNA level in calvariae was markedly increased by 1,25(OH)2D3 administration (Fig. 8). This induction was also observed with 100 ng injection of 1,25(OH)2D3 (data not shown), whereas further investigations are needed to ensure the hypercalcemia did not occur in the animals with a 1,25(OH)2D3 treatment. In these experiments, a significant increase in osteopontin gene expression was also seen in calvariae from 1,25(OH)2D3-injected mice. As shown in Fig. 9A, immunohistochemical analysis by the ABC method using anti-NPR-C antiserum demonstrated positive staining in periosteal fibroblasts in 1,25(OH)2D3-treated mice. Preabsorption of anti-NPR-C antiserum by a specific antigen resulted in complete loss of the staining (Fig. 9B).


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Fig. 8.   RNA analysis by Northern blotting hybridization. Five-week-old ICR mice were injected intraperitoneally with 1 or 100 µg of 1,25(OH)2D3 or with vehicle and killed 18 h later. Poly(A)+ RNAs were isolated from mouse calvariae as described in MATERIALS AND METHODS. Poly(A)+ RNA (5 µg) was subjected to Northern blot analysis. Hybridization was performed using 32P-labeled mouse NPR-C, mouse osteopontin, or rat beta -actin cDNA fragments as probes, as described in MATERIALS AND METHODS.


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Fig. 9.   NPR-C expression at the histological level. Immunostaining was performed with a primary rabbit anti-NPR-C antiserum without (A) or with (B) preabsorption by specific antigen and control staining with hematoxylin (C), as described in MATERIALS AND METHODS. Br and M indicate brain and bone matrix, respectively. Arrows indicate positive-stained periosteal cells. Original magnification is ×40.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have investigated the regulation of the mouse NPR-C gene by 1,25(OH)2D3 and demonstrated that the expression is significantly upregulated by this hormone. 1,25(OH)2D3 is known to regulate expression of several genes related to osteoblast differentiation (28). The role of 1,25(OH)2D3 in the transcriptional regulation of the osteocalcin and osteopontin genes, which are noncollagenous proteins of the bone extracellular matrix, has been extensively studied. This regulation occurs at the transcriptional level, that is, wherein the hormone-receptor complex binds to a VDRE located in the 5'-flanking region of these genes. We have previously demonstrated the structure of the 5'-flanking regulatory region of the mouse and human NPR-C genes and functional features using serial 5'-deletions of the promoter region (34, 35). In this study, we have cloned a further upstream sequence of the 5'-flanking region of the mouse NPR-C gene. However, sequence analysis and promoter analysis using stable transfections in MC3T3-E1 cells indicated that a hormone response element for 1,25(OH)2D3 was not located in the mouse NPR-C promoter region. Although most of the VDREs identified to date are located within 1 kb of the 5'-flanking regions of the 1,25(OH)2D3-inducible genes, these observations do not exclude the possibility that VDRE is located further upstream or downstream in the NPR-C gene locus, as has been demonstrated for the regulation of the HoxA cluster by retinoic acid (16). However, nuclear run-on assays have revealed that the transcriptional rate of the NPR-C gene was not significantly affected by 1,25(OH)2D3 treatment. Furthermore, we observed the augmentation of NPR-C mRNA stability by 1,25(OH)2D3 according to experiments using a transcription inhibitor, actinomycin D. Finally, we concluded that the 1,25(OH)2D3-mediated effect is mainly dependent on stabilization of its mRNA. Although the role of 1,25(OH)2D3 in the gene transcription has been well studied, little is known regarding the effect of this hormone via the posttranscriptional pathway. Recent works have demonstrated that 1,25(OH)2D3 could increase aromatase cytochrome P-450 mRNA in the presence of actinomycin D in cultured human osteoblasts (32) and could stabilize the VDR mRNA in human MG-63 osteosarcoma cells (22). On the other hand, retinoic acid together with thyroid hormone is the main known regulator of metabolism, differentiation, and development in vertebrates (20). Gene regulation by retinoic acid follows binding to specific nuclear receptors and formation of heterodimer with members of the RXR proteins. However, several genes have been described as regulated posttranscriptionally by retinoic acid. Retinoic acid has been shown to prolong the half-life of the proteolipid protein mRNA in C6 glioma cells (19) and that of calbindin-D28k mRNA in the human medulloblastoma cell line D283 (33) without enhancing the gene transcription.

Our other finding is that 1,25(OH)2D3 mediates a significant increase of NPR-C expression in a cell-specific and a species-specific manner. 1,25(OH)2D3 was shown to be able to upregulate NPR-C-dependent ANP-binding activity in MC3T3-E1, 10T1/2, and cultured osteoblastic cells from mouse calvariae. The steady-state level of NPR-C mRNA in mouse calvariae was also markedly increased with in vivo 1,25(OH)2D3 treatment. Although MC3T3-E1 is a fibroblastic cell line from calvariae of C57BL/6 mice, 10T1/2 cells established from an early embryo have the pluripotent activity to differentiate into osteoblastic cells by bone morphogenetic protein-2 (15). Our immunohistological analysis has demonstrated that NPR-C is actually and strongly expressed in periosteal fibroblasts in 1,25(OH)2D3-treated mice, suggesting that 1,25(OH)2D3 might upregulate NPR-C expression in fibroblasts, which could differentiate into osteoblasts. Although we could not find positive staining in control mouse calvariae, it is not fully understood whether the upregulation of NPR-C mRNA in mouse calvariae with in vivo 1,25(OH)2D3 treatment could be a result of induced expression in undifferentiated fibroblasts, in osteoblasts, or a combination of both. Moreover, a significant upregulation of NPR-C expression was not observed in cultured osteoblastic cells from rat calvariae. Although recent studies demonstrated the species-specific 1,25(OH)2D3 responsiveness for regulation of osteocalcin gene transcription (17, 36), little is known regarding a species-specific effect of this hormone via the posttranscriptional pathway.

Recent work has demonstrated that an increase in intracellular cGMP by CNP leads to expression of alkaline phosphatase and osteocalcin in osteoblastic cells and formation of mineralized nodules (12). Current study suggests that one of the functional significances of upregulation of NPR-C by 1,25(OH)2D3 is to significantly attenuate biological responsiveness to NPs. To date, NPR-C has been reported to directly control adenylyl cyclase activity via Gi protein, phospholipid hydrolysis, thymidine kinase activity, and mitogen-activated protein kinase activity without any cGMP response. 1,25(OH)2D3 might modulate these biological activities of NPR-C in osteoblastic cells.

    ACKNOWLEDGEMENTS

We thank Dr. H. Hagiwara for kind instructions for preparing and culturing osteoblastic cells, K. Fujishige for technical support, and Drs. M. Sugiura and T. Ishizuka for continuous kind help.

    FOOTNOTES

The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number AB007853.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: K. Omori, Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., 16-89 Kashima-3-chome, Yodogawa-ku, Osaka 532-0085, Japan.

Received 15 May 1998; accepted in final form 6 August 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(6):E965-E973
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