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Endocrinology Vol. 144, No. 11 4876-4885
Copyright © 2003 by The Endocrine Society

Parathyroid Hormone-Related Protein(1–34) Regulates Phex Expression in Osteoblasts through the Protein Kinase A Pathway

Miguel Ángel Vargas, Mathieu St-Louis, Luc Desgroseillers, Jean-Louis Charli and Guy Boileau

Département de Biochimie (M.Á.V., M.S.L., L.D., G.B.), Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7; and Departamento de Genética del Desarrollo y Fisiología Molecular (M.A.V., J.-L.C.), Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, México

Address all correspondence and requests for reprints to: Guy Boileau, Département de Biochimie, Faculté de Médecine, Université de Montréal, C.P. 6128, Succ. Centre-Ville Montréal, Québec, Canada H3C 3J7. E-mail: guy.boileau{at}umontreal.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phex (a phosphate-regulating gene with homologies to endopeptidases on the X chromosome) is expressed predominantly in bone in which it has been implicated in the mineralization process. Multiple factors and hormones, including PTHrP, regulate formation, development, and/or homeostasis of bone. The purpose of the present study was to determine whether PTHrP(1–34) regulates Phex expression and identify the signaling pathway used. Phex mRNA and protein levels were analyzed by RT-PCR and immunoblotting, respectively. In UMR-106 cells, PTHrP(1–34) caused a time- and concentration-dependent decrease in Phex expression. Forskolin, an adenylate cyclase activator, had the same effect. Dibutiryl cAMP also decreased Phex expression, and its effect was blocked by H89, a protein kinase A (PKA) inhibitor. In contrast, 12-O-tetradecanoyl phorbol-13-acetate, a protein kinase C (PKC) activator, increased Phex expression in a time- and dose-dependent manner. This effect was reversed by bisindolylmaleimide I, a PKC inhibitor. Bovine PTH(3–34), which activates PKC but not PKA, had no effect. On the contrary, human PTH(1–31), which activates PKA but not PKC, decreased Phex expression. H89 but not bisindolylmaleimide I blocked the effect of PTHrP(1–34). PTHrP(1–34) also decreased Phex expression in cultures of fetal rat calvaria cells at d 7 of culture but not at later stages. These data demonstrate that PTHrP(1–34), through PKA, down-regulates Phex expression in osteoblasts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PHEX (FORMERLY PEX; a phosphate-regulating gene with homologies to endopeptidases on the X chromosome) was identified by positional cloning as the gene responsible for X-linked hypophosphatemia (XLH) in humans (1). XLH is a mendelian disorder of phosphate homeostasis characterized by growth retardation, renal defects in vitamin D metabolism, hypophosphatemia resulting from decreased phosphate reabsorption in the renal proximal tubules, and defective mineralization of teeth and bones causing rachitic and osteomalacic bone disease (2). Much of our actual understanding of the pathophysiology of XLH has been obtained using a murine model for human XLH, the Hyp mouse, which presents a large deletion in the 3' region of the Phex gene (PHEX and Phex refer to the human and mouse genes, respectively) (3) and phenotypic features similar to those of XLH patients (4). The defective renal phosphate transport in Hyp mice has been attributed to decreased renal proximal tubule expression of the type I and II sodium-dependent phosphate cotransporter genes and proteins (5, 6). Parabiosis experiments and cross-kidney transplantation between Hyp and normal mice (7, 8) suggested the presence of a circulatory phosphaturic hormone, designated phosphatonin (9), responsible for renal phosphate wasting. In accordance with this hypothesis, two groups have reported the presence of a renal phosphate transport inhibitory activity in conditioned medium of cultured osteoblasts isolated from Hyp mice (10, 11). Although the exact nature of phosphatonin remains to be determined, several candidates have been proposed (for a review see Ref. 12) including FGF 23 (13), matrix extracellular phosphoglycoprotein (14), and Frizzled-related protein-4 (15).

Human and mouse PHEX/Phex cDNAs have been cloned and sequenced (3, 16, 17, 18, 19, 20). Amino acid sequence comparisons have demonstrated homologies between PHEX/Phex protein and members of the M13 zinc metalloendopeptidase family. The peptidases of this family are type II integral membrane glycoproteins with a relatively short N-terminal intracellular region, a single-transmembrane domain, and a large extracellular domain, which contains the active site of the enzyme (21). Peptidases of the M13 family have been shown to regulate the activity of biologically active peptides either by degradation of active peptides [neprilysin (NEP), neutral endopeptidase 24.11] (22) or in processing inactive peptide precursors into active forms endothelin-converting enzymes 1 and 2 (23).

Several studies support a role of PHEX/Phex in mineralization. PHEX/Phex mRNA and protein are localized predominantly in bone and teeth, specifically in osteoblasts, osteocytes, and odontoblasts but not in preosteoblasts (3, 16, 19, 24, 25, 26). In developing bones in vivo or during osteoblast differentiation in vitro, Phex mRNA and protein expression is temporally associated with the mineralization process (19, 24, 25, 26, 27). Finally, studies in vivo and in vitro have shown that the loss of Phex function in osteoblasts causes an intrinsic defect in the mineralization process (28, 29, 30, 31). However, the mechanism by which mutations in the PHEX/Phex gene cause renal phosphate wasting and impaired bone mineralization in XLH patients and Hyp mice is not known. Based on the sequence homology of PHEX/Phex to members of the M13 family, it has been postulated that PHEX/Phex inactivates phosphatonin.

Although it is well documented that PHEX/Phex is involved in regulating phosphate homeostasis and bone mineralization, little information is available concerning the regulation of PHEX/Phex expression. Recently, it has been reported that Phex expression is down-regulated in vitro by vitamin D3 in primary osteoblasts derived from calvaria and MC3T3 cells and up-regulated by glucocorticoids in UMR-106 cells (27, 32). In vivo, Phex mRNA levels are up-regulated by glucocorticoids in calvaria and IGF-I and GH in calvaria and lungs (32, 33, 34). The present study was undertaken to determine whether PTHrP(1–34) can regulate Phex expression and identify elements of the signaling pathway used by this hormone in UMR-106 (35) cells and fetal rat calvaria cells. PTHrP(1–34) is a hormone synthesized by osteoblasts, acting auto- or paracrinally and involved in the development of bones (36, 37). Using semiquantitative RT-PCR and immunoblotting to determine Phex mRNA and protein levels, respectively, we show that PTHrP(1–34), through the protein kinase A (PKA) pathway, down-regulates Phex expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Bisindolylmaleimide I, 12-O-tetradecanoyl phorbol-13-acetate (TPA), 4{alpha}-phorbol 12,13-didecanoate (4{alpha}-PDD), forskolin, N6,2'-O-dibutiryladenosine-3', 5'-cyclic monophosphate [(Bu)2cAMP], N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), and 1,9-dideoxyforskolin were obtained from Sigma (St. Louis, MO). PTHrP(1–34) and PTHrP(107–111), rat PTH, bovine PTH(3–34) (bPTH), and human PTH(1–31) were purchased from Peninsula Laboratories (Belmont, CA) or Bachem (Torrance, CA).

Culture of UMR-106 cells and treatments
Stock cultures of UMR-106 cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM (Life Technologies, Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum (FBS), glucose (4.5 g/liter), penicillin (50 U/ml), streptomycin (50 µg/ml), fungizone (125 ng/ml), glutamine (2 mM), and sodium pyruvate (1 mM) in a water-saturated atmosphere of 95% O2 and 5% CO2 at 37 C. Cells were passaged every 3–4 d when reaching confluence and were not used beyond passage 17.

To study the regulation of Phex expression, 2 x 105 cells were plated in 60-mm tissue culture dishes in 2 ml DMEM supplemented as indicated above; the medium was changed every 2 d. At d 4 of culture, when cells reached confluence, medium was replaced with DMEM (phenol red free) supplemented as indicated above except that FBS was substituted for 10% stripped FBS. After 24 h, the medium was replaced by a fresh one, and cells were incubated with agents at indicated doses and times. Finally, cells were washed twice with PBS and kept at -80 C or immediately processed for RNA or protein extraction.

TPA (10-2 M), 4{alpha}-PDD (10-2 M), bisindolylmaleimide I (10-3 M), forskolin (10-2 M), 1,9-dideoxyforskolin (10-2 M), and H89 (10-2 M) were dissolved in dimethylsulfoxide; (Bu)2cAMP (10-1 M), hormones, and peptides (10-4 M) in water. Stock solutions of drugs were diluted with culture medium to appropriate concentration just before addition to cultures. Dimethylsulfoxide had no effect on Phex mRNA levels. Inhibitors were added 1 h before agents.

Culture of fetal rat calvaria cells and treatments
This protocol was approved by Le Comité de déontologie en expérimentation animale de l’Université de Montréal. Cells were enzymatically isolated from the calvaria of 21-d Wistar rat fetuses as described by Bonnelye et al. (38). Isolated cells were plated on 12-well plates at a density of 104 cells/well. After 24-h incubation, the medium was changed and supplemented with 50 µg/ml ascorbic acid, 10 mM sodium ß-glycerophosphate, and 10-8 M dexamethasone. Medium was changed every 2 or 3 d, and the cells were maintained in a water-saturated atmosphere of 95% O2 and 5% CO2 at 37 C. Cells were treated with PTHrP(1–34) (10-7 M), forskolin (10-6 M), or TPA (10-8 M), or vehicle alone at d 7, 12, 16, 19, and 22. Medium was changed before drug treatments, and cells were then incubated with drugs for 16 h. Cells were then washed twice with PBS and processed for RNA extraction.

RNA extraction
Total RNA was isolated using the RNeasy kit (QIAGEN, Mississauga, Ontario, Canada) according to the manufacturer’s instructions. RNA was digested with ribonuclease-free deoxyribonuclease (QIAGEN) to remove possible contaminating genomic DNA. RNA concentration was determined by absorbance at 260 nm; RNA yields were similar among treatments and reproducible (80–100 µg total RNA per dish). Only RNA samples with a 260/280 nm absorbance ratio greater than 1.6 were used in RT-PCR.

Phex and NEP mRNA semiquantification by RT-PCR
Changes in Phex or NEP mRNA levels were determined by semiquantitative RT-PCR. Total RNA (0.26 µg) was reverse transcribed and cDNA amplified by PCR using the One-Step RT-PCR kit (QIAGEN) as recommended by the manufacturer, in presence of a recombinant ribonuclease inhibitor (Applied Biosystems, Foster City, CA). The housekeeping genes ß-actin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were used as internal controls. For cDNA amplification we used the following sets of specific primers (custom synthesized by Life Technologies): 5'-CCATGTACGTAGCCATCCAG-3' and 5'-GCACGATTTCCCTCTCAGCTGT-3' sense/antisense strands, amplifying bases 2186–2422 of the rat ß-actin cDNA (accession no. V01217); 5'-AAACCCATCACCATCTTCCAGG-3' and 5'-ACCACCCTGTTGCTGTAGCC-3' sense/antisense strands, amplifying bases 238–998 of the rat G3PDH cDNA (accession no. M17701); 5'-GGATTGGCTATCCTGATGAC-3' and 5'-CCTAGTGTGTTAATTCCA-3' sense/antisense strands, amplifying bases 1514–2015 of the rat NEP cDNA (accession no. M15944); 5'-CCTGAAGGGGTTTGGTCAGAGAGA-3' and 5'-GTCCCCTGGATTACCCTTGAGAA-3' sense/antisense strands, amplifying bases 1007–1668 of the rat Phex cDNA (accession no. AJ001637).

Because initial experiments showed that the linear amplification range for each product (ß-actin, G3PDH, NEP, and Phex) occurred in different cycle ranges, cDNAs were amplified at the same time in different tubes. After initial denaturation at 95 C for 15 min, 20 or 27 cycles were carried out to amplify ß-actin/G3PDH or Phex/NEP cDNAs, respectively. Each cycle consisted of denaturation, annealing, and extension steps of 1 min at 94 C, 1 min at 58 C, and 1 min at 72 C, respectively, followed by a final extension step at 72 C for 10 min. RT-PCR was performed in a Peltier thermal cycler (MJ Research Inc., Boston, MA). The PCR products were separated by electrophoresis on 1% agarose gels and stained with ethidium bromide to visualize and quantify the bands by computerized densitometric scanning using the ChemicDoc system and the Quantity One software (Bio-Rad Laboratories, Hercules, CA). As expected, PCR amplification yielded products with sizes consistent with 237-, 761-, 502-, and 662-bp products for ß-actin, G3PDH, NEP, and Phex, respectively. Phex or NEP RT-PCR product densitometric values were normalized against those corresponding to ß-actin or G3PDH products. The intensity of ß-actin or G3PDH PCR products per microgram total RNA did not change significantly between the treatments. Two negative controls and samples without RT reaction or without RNA were usually included; in these conditions no products were visualized, eliminating genomic DNA or laboratory contaminations.

Phex protein extraction and immunoblotting
After treatments, UMR-106 cells were washed twice with PBS and resuspended by scraping in 0.5 ml PBS containing 1% n-octylglucoside. The cell suspension was incubated at 4 C for 4 h on a rotating wheel and finally centrifuged at 13,000 rpm for 30 min at 4 C. The supernatant was concentrated 15 times using Microcon centrifugal devices (Millipore, Bedford, MA). Quantitation was made using the DC protein assay kit (Bio-Rad, Mississauga, Ontario, Canada). For protein analysis, 50 µg protein were resolved by SDS-PAGE on a 7.5% gel, transferred to a nitrocellulose membrane, and immunodetected with the 13B12 anti-PHEX monoclonal antibody (25) (BioMep, Montréal, Québec, Canada). The protein/antibody complex was revealed by chemiluminescence using the Western lightning kit (Perkin-Elmer Life Sciences, Boston, MA).

cAMP accumulation assays
UMR-106 cells were grown in DMEM in 100-mm petri dishes. Before treatments, the cells were starved in serum-free medium and labeled overnight (16 h) with 1 µCi/ml [3H]adenine (Perkin-Elmer Life Science; 24.2 Ci/mmol). Radioactive medium was then replaced with fresh DMEM, and the cells were mechanically detached and thoroughly washed (three times) with PBS (4 C). Viability was assessed using trypan blue. Then 5 x 105 cells were resuspended in 300 µl assay mixture containing PBS and the different drugs at the indicated concentrations and incubated for 20 min at 37 C. The assay was terminated by the addition of 600 µl of an ice-cold solution containing 5% trichloroacetic acid, 5 mM ATP, and 5 mM cAMP. [3H]ATP and [3H]cAMP were separated by sequential chromatography on Dowex exchange resin and aluminum oxide (39). Results were expressed as the ratio of [3H]cAMP:[3H]ATP+[3H]cAMP.

Protein kinase C (PKC) activation
Cultures of UMR-106 cells were starved in serum-free medium for 20 h and incubated in the absence or presence of 10-7 M PTHrP(1–34) or 10-8 M TPA for 30 min in the same medium containing 0.1% BSA. Subcellular fractions were prepared as previously described with minor modifications (40). Cells were washed with cold PBS and scraped from the plates on ice in 750 µl buffer A [50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor, 500 µM 4-(2-aminoethyl)benzene sulfonyl fluoride, 1 mM NaF, and 1 mM sodium ortho vanadate]. Cells were allowed to swell on ice for 1 h and homogenized by passage several times through an 18-gauge needle. Homogenates were centrifuged at 600 x g for 20 min at 4 C to pellet the crude nuclear fraction. The supernatant was further centrifuged at 100,000 x g for 30 min at 4 C. Membranes were collected in the pellet and solubilized in buffer A containing 1% Triton X-100. The cytosol was recovered in the supernatant. The crude nuclear fraction was resuspended in buffer A containing 0.1% Triton X-100, layered over 30% sucrose (wt/vol in buffer A), and centrifuged at 5000 x g for 30 min at 4 C. The pellet containing the nuclear fraction was resuspended in buffer A containing 1% Triton X-100 and sonicated on ice for 15 sec.

Quantitation of proteins in the cytosolic, membrane, and nuclear fractions was made using the DC protein assay kit (Bio-Rad Laboratories). For protein analysis, 50 µg of proteins were resolved by SDS-PAGE on a 7.5% gel, transferred to a nitrocellulose membrane, and immunodetected with monoclonal antibodies specific to PKC-{alpha} and PKC-{delta} (BD Bioscience Pharmalgen, San Diego, CA). The protein/antibody complex was revealed by chemiluminescence using the Western lightning kit (Perkin-Elmer Life Sciences).

Statistical analyses
Data generally represent the mean ± SEM values from two independent cultures (each performed in triplicate), except where indicated. One-way ANOVA followed by Duncan multiple-range test was used to determine statistical significance between individual means. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTHrP(1–34) down-regulates Phex expression in UMR-106 cells
We tested the hypothesis that PTHrP(1–34), a hormone synthesized by osteoblasts and involved in the development of bones, could regulate Phex expression. For these studies, the densitometric values of Phex RT-PCR products were normalized by comparison with the values of two internal controls: the housekeeping genes ß-actin and G3PDH. PTHrP(1–34) effects on Phex mRNA levels were similar with both internal controls (Fig. 1Go, A and B). This supports the ability of our RT-PCR protocol to determine semiquantitative changes in Phex mRNA levels. Data described below are those referring to ß-actin. Incubation of cells for 16 h with PTHrP(1–34) at concentrations between 5 x 10-13 and 10-8 M had no significant effect on Phex mRNA levels. However, 16 h incubation with higher PTHrP(1–34) concentrations (10-7 and 10-6 M) significantly decreased Phex mRNA levels in a dose-dependent way (65 ± 3% and 37 ± 5% of control, respectively; n = 6) (Fig. 1AGo). The effect of PTHrP(1–34) (10-6 M) was time dependent, showing statistically significant decrease at 11 h (63 ± 3%, n = 6) and a maximal effect at 16–24 h (47 ± 3% and 37 ± 7%, respectively, n = 6) (Fig. 1BGo). Under the same treatment conditions, mRNA levels of NEP, another member of the M13 family of peptidases present in osteoblasts (41), were not affected significantly, suggesting a specific effect of the hormone on Phex mRNA (Fig. 1Go, A and B).



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  		FIG. 1. Dose response and time course of PTHrP(1–34) effects on Phex and NEP mRNA levels in UMR-106 cells. Cells were incubated with either various concentrations of PTHrP(1–34) for 16 h (A) or 10-6 M PTHrP(1–34) for the time indicated (B). RNA was extracted and Phex or NEP mRNA levels determined by RT-PCR as indicated in Materials and Methods. Data are presented as the percent of Phex or NEP mRNA levels in controls (n = 6). Phex mRNA levels were normalized by comparison with ß-actin or G3PDH mRNA levels and NEP mRNA levels with ß-actin mRNA levels. Data were analyzed by one-way ANOVA followed by Duncan’s multiple-range test: *, P < 0.01, compared with control. Insets in A and B show the results of a representative RT-PCR amplification in each experimental condition. Solid circles, Phex/ß-actin ratio; open triangles, NEP/ ß-actin ratio; solid squares, Phex/G3PDH ratio.

 
PTHrP(1–34) and PTH(1–34) effects on osteoblasts are mediated by the common type-1 PTH/PTHrP receptor (42, 43, 44), suggesting that PTH(1–34) could also reduce Phex expression. As expected, treatment of UMR-106 cells with rPTH(1–34), bPTH(1–34), or human PTH(1–34) (10-7 M) for 24 h significantly reduced Phex mRNA levels (Table 1Go).


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  		TABLE 1. Effects of PTHrP(1–34), PTHrP(107–111), PTH(1–34), and PTH fragments on Phex mRNA levels in UMR-106 cells

 
To determine whether Phex protein levels were also affected by treatment of cells with PTHrP(1–34), UMR-106 cells treated for 24 h with vehicle alone or with 10-7 M PTHrP(1–34) were extracted and the cell extracts analyzed by immunoblotting. Consistent with the decrease in mRNA levels, the presence of the hormone in the culture medium decreased the levels of Phex protein (Fig. 2Go).



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  		FIG. 2. PTHrP(1–34), forskolin, and TPA regulate Phex protein levels in UMR-106 cells. Cells were incubated for 24 h with either PTHrP(1–34) (10-7 M), TPA (10-8 M), forskolin (10-6 M), or vehicle alone. Proteins were extracted as described in Material and Methods and 50 µg protein were resolved by SDS-PAGE on a 7.5% polyacrylamide gel. Phex protein was revealed using an anti-PHEX antibody.

 
Because PTHrP(1–34) and PTH(1–34) are known to activate the PKC and PKA signaling pathways through the common type-1 PTH/PTHrP receptor (42, 43, 44), we next determined the effects of activating independently either pathway on Phex expression.

The PKC pathway up-regulates Phex expression in UMR-106 cells
To determine whether PKC regulates Phex mRNA levels, cells were incubated for 16 h with increasing concentrations of TPA (from 10-10 to 10-6 M), a PKC activator. TPA induced a concentration-dependent increase in Phex mRNA levels, maximum (218 ± 17%, n = 6) at 10-8 M (Fig. 3AGo). TPA (10-8 M) caused a time-dependent increase of Phex expression (Fig. 3BGo). The maximal effect occurred at 16 h (238 ± 18%, n = 6); after 24 h of TPA exposure, mRNA levels returned toward control values (125 ± 22%, n = 6). Incubation of cells for 16 h with 4{alpha}-PDD (10-8 M), an inactive analog of TPA, had no significant effect on Phex mRNA levels (Table 2Go; experiment A). Treatment with TPA (10-8 M) for 16 h had no significant effect on NEP mRNA levels (not shown). As for PTHrP(1–34), the effect of TPA added to the culture medium at a concentration of 10-8 M for 24 h was also observed at the protein level (Fig. 2Go).



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  		FIG. 3. Dose response and time course of TPA effects on Phex mRNA levels in UMR-106 cells. Cells were incubated with either various concentrations of TPA for 16 h (A) or 10-8 M TPA for the time indicated (B). Phex mRNA level determination and data analysis were as described in Fig. 1Go (n = 3). *, P < 0.01, compared with control. Insets in A and B show the results of a representative RT-PCR amplification in each experimental condition.

 

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  		TABLE 2. Effects of PKC pathway on Phex mRNA levels in UMR-106 cells

 
To demonstrate that PKC is involved in Phex mRNA up-regulation by TPA, cells were incubated for 16 h with the PKC inhibitor bisindolylmaleimide I (10-6 M), alone or in combination with 10-8 M TPA. Bisindolylmaleimide I did not modify significantly the basal Phex mRNA levels but interfered with the TPA-induced up-regulation (Table 2Go; experiment B). These data demonstrate that PKC activation up-regulates Phex expression.

The cAMP pathway down-regulates Phex expression in UMR-106 cells
The involvement of the cAMP pathway in Phex expression was evaluated with forskolin, an adenylate cyclase activator. Forskolin present for 16 h down-regulated Phex mRNA levels in a dose-dependent manner, with a maximal effect (47 ± 6%, n = 6) at 10-6 M (Fig. 4AGo). A time-dependent decrease of Phex mRNA was observed when cells were incubated with 10-6 M forskolin (Fig. 4BGo). A significant decrease (75 ± 2%, n = 6) occurred at 6 h, and a maximal effect was observed at 16–24 h (32 ± 5% and 20 ± 5%, respectively, n = 6) (Fig. 4BGo). Treatment of cells for 16 h with an inactive analog of forskolin, 1,9-dideoxyforskolin (10-6 M), did not change significantly Phex mRNA levels (Table 3Go; experiment A). Forskolin at 10-6 M had no significant effect on NEP mRNA levels (not shown). Forskolin present at a concentration of 10-6 M for 24 h also had an inhibitory effect at the protein level (Fig. 2Go).



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  		FIG. 4. Dose response and time course of forskolin effects on Phex mRNA levels in UMR-106 cells. Cells were incubated with either various concentrations of forskolin for 16 h (A) or 10-6 M forskolin for the time indicated (B). Phex mRNA level determination and data analysis were as described in Fig. 1Go (n = 3). *, P < 0.01, compared with control. Insets in A and B show the results of a representative RT-PCR amplification in each experimental condition.

 

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  		TABLE 3. Effects of cAMP pathway on Phex mRNA levels in UMR-106 cells

 
Adenylate cyclase activation augments intracellular cAMP levels. Incubation of cells for 16 h with the permeable cAMP analog (Bu)2cAMP (10-3 M) resulted in a decrease in Phex mRNA levels (Table 3Go; experiment B). PKA is one of the major targets of cAMP. To determine whether (Bu)2cAMP regulated Phex mRNA levels through PKA activation, cells were treated for 16 h with the PKA inhibitor H89, alone or in combination with (Bu)2cAMP. H89 (10-8 M) had no significant effect on basal Phex mRNA levels but blocked the effect of 10-3 M (Bu)2cAMP (Table 3Go; experiment B), demonstrating that increased cAMP levels down-regulate Phex expression by PKA activation.

Interaction between the PKC and PKA pathways in the regulation of Phex mRNA levels in UMR-106 cells
Because diverse transduction pathways may interact at many levels, we determined whether the PKC and PKA pathways interact to regulate Phex mRNA levels. Cotreatment for 16 h with maximal effective doses of TPA (10-8 M) and increasing concentrations of forskolin (10-10 to 10-6 M) caused a dose-dependent decrease in Phex mRNA levels. Compared with TPA (10-8 M), significant effects of forskolin were observed at 10-8 M (60 ± 3%), 10-7 M (35 ± 2%), and 10-6 M (27 ± 8%). These values are very similar to those obtained with forskolin alone (Fig. 5Go). This suggests that PKA activation efficiently inhibits the effect of PKC on Phex mRNA levels.



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  		FIG. 5. Forskolin blocks TPA effect on Phex mRNA levels in UMR-106 cells. Cells were treated with increasing concentrations of forskolin alone (open circles) or in combination with 10-8 M TPA (solid circles) for 16 h. Phex mRNA level determination and data analysis were as described in Fig. 1Go. Dashed line shows the levels of Phex expression in cells treated with 10-8 M TPA. Numbers in parentheses indicate the number of determinations for each point (solid circles). *, P < 0.01 with respect to 10-8 M TPA alone.

 
PTHrP(1–34) does not regulate Phex mRNA levels through PKC activation in UMR-106 cells
PTHrP(1–34) causes two peaks of PKC activation in rat osteosarcoma cells: one peak is observed at 10-12 to 10-11 M and the other at 10-9 to 10-8 M; a peak of adenylate cyclase activation is obtained at 10-7 M (42). Because a significant effect of PTHrP(1–34) was not detected in the pico- or nanomolar range and its effect in the micromolar range was opposite to that of PKC activation, the data suggested that PKC activation by PTHrP(1–34) did not regulate Phex expression. This result may be explained by an interference of the PKA pathway with the PKC pathway and/or by the fact that the isozyme(s) activated by TPA to regulate Phex expression may differ from that used by PTHrP(1–34). To test the possibility that activation of the cAMP pathway may have masked PKC activation, cells were treated with bPTH(3–34), an agonist of the type-1 PTH/PTHrP receptor that activates PKC but not PKA (45, 46, 47). Incubation of cells for 16 h with bPTH (3–34) at concentrations between 10-10 and 10-7 M (data not shown) or for 24 h with 10-7 M bPTH (3–34) (Table 1Go) did not significantly alter Phex mRNA levels. This observation suggests that PTHrP(1–34) (or PTH1–34) does not activate the isozyme of PKC that increases Phex mRNA levels. UMR-106 cells are known to express several PKC isozymes (48), but only the PKC-{delta} was reported activated by PTH(1–34) (49).

To test the effects of TPA and PTHrP(1–34) on activation of PKC-{alpha} and PKC-{delta}, recruitment of these isozymes to the plasma membrane and nucleus, respectively, was determined by immunoblotting of fractionated extracts from control UMR-106 cells or cells treated with 10-8 M TPA or 10-7 M PTHrP(1–34). TPA but not PTHrP(1–34) provoked a displacement of PKC-{alpha} to the plasma membrane (Fig. 6AGo). As reported previously (49), TPA provoked a decrease in PKC-{delta} protein levels but no significant recruitment of this isozyme to the nucleus. In contrast, PTHrP(1–34) activated PKC-{delta} isozyme (Fig. 6BGo). These results using PTHrP(1–34) as agonist of the type-1 PTH/PTHrP receptor are consistent with a previous report by Erclic and Mitchell (49) showing activation of PKC-{delta} but not PKC-{alpha} by PTH(1–34), and clearly show that PTHrP(1–34) and TPA do not activate the same PKC isozymes.



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  		FIG. 6. Activation of PKC isozymes in UMR-106 cells by TPA and PTHrP(1–34). UMR-106 control cells or cells treated with 10-7 M PTHrP(1–34) or 10-8 M TPA were fractionated and subcellular fractions analyzed by immunoblotting with an anti-PKC-{alpha} antibody (A) or an anti-PKC-{delta} antibody (B).

 
In osteoblasts and osteoblastic cell lines, including UMR-106 cells, PTHrP(107–111) activates PKC through a receptor different from the type-1 PTH/PTHrP receptor (50, 51). PTHrP(107–111) (10-7 M) did not significantly modify Phex mRNA levels at 24 h (Table 1Go). These results indicate that activation of an adequate PKC isozyme is necessary to up-regulate Phex mRNA levels. As expected, treatment of cells with bisindolylmaleimide I (10-6 M) before incubation with 10-7 M PTHrP(1–34) for 16 h did not prevent down-regulation of Phex mRNA levels (Table 2Go; experiment C).

PTHrP(1–34) down-regulates Phex mRNA levels through the PKA pathway in UMR-106 cells
Because PTHrP(1–34) effects were similar to those of forskolin and (Bu)2cAMP, it is possible that PTHrP(1–34) down-regulates Phex mRNA levels through PKA. This was supported by the findings that human PTH(1–31) (10-7 M, for 24 h), a fragment of PTH that activates PKA but not PKC (44), decreased Phex mRNA levels even more efficiently than PTHrP(1–34) (10-7 M) (Table 1Go). To test directly this hypothesis, cAMP accumulation was measured in control UMR-106 cells and cells treated with 10-6 M forskolin or 10-7 M PTHrP(1–34). Both treatments produced a significant increase in intracellular cAMP accumulation that can be correlated with Phex mRNA down-regulation (Fig. 7Go, A and B). In addition, co treatment for 16 h with near maximal effective doses of forskolin (10-6 M) and PTHrP(1–34) (10-6 M) did not produce a significantly additive effect on Phex mRNA levels (Fig. 7BGo), further suggesting that both act through the same pathway. Finally, preincubation of cells with H89 (10-6 M) inhibited the effect of 10-7 M PTHrP(1–34) at 16 h (Table 3Go; experiment C).



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  		FIG. 7. PTHrP(1–34) and forskolin increase intracellular cAMP and their effects on Phex mRNA levels are not additive in UMR-106 cells. A, Cells were treated with 10-6 M forskolin or 10-7 M PTHrP(1–34) and the ratio [3H]cAMP:([3H]ATP+[3H]cAMP) was determined to assess the activation of the cAMP pathway. B, Cells were treated with 10-7 M PTHrP(1–34) or 10-6 M forskolin alone or in combination with PTHrP(1–34) for 16 h. Phex mRNA level determination and data analysis were as described in Fig. 1Go (n = 9 in A; n = 3 in B). *, P < 0.01, compared with control.

 
PTHrP(1–34) and forskolin but not TPA affect Phex mRNA levels in cultures of rat calvaria cells
To determine whether PTHrP(1–34), TPA, and forskolin also regulate Phex gene expression in differentiating osteoblasts, we isolated calvaria cells from fetal rats and treated the cultures with the hormone or drugs at d 7, 12, 16, 19, and 22 after seeding. Incubation of cells with both PTHrP(1–34) at 10-7 M and forskolin at 10-6 M for 16 h down-regulated Phex mRNA levels at d 7 (55 ± 5% and 59 ± 6%, respectively; n = 3) (Fig. 8Go) of culture but not significantly at later stages (not shown). In contrast, incubation of cells with TPA at a concentration of 10-8 M for 16 h did not significantly affect Phex gene expression (Fig. 8Go).



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  		FIG. 8. PTHrP(1–34) and forskolin down-regulate Phex mRNA levels in calvaria cells. Calvaria cells at d 7 of culture were treated for 16 h with PTHrP(1–34) (10-7 M), TPA (10-8 M), forskolin (10-6 M), or vehicle alone (control). Phex mRNA level determination and data analysis were as described in Fig. 1Go (n = 3). *, P < 0.01, compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several studies have confirmed that PHEX/Phex has an important role in the regulation of bone mineralization (3, 16, 19, 24, 25, 26, 27, 28, 29, 30, 31, 52, 53, 54). Therefore, the way Phex expression is controlled should profoundly influence bone mineralization. Accordingly, recent studies have shown that vitamin D3, glucocorticoids, IGF and GH can regulate Phex expression in osteoblasts (27, 32, 33, 34). In the present work, we demonstrated that PTHrP(1–34), a hormone involved in regulating bone development, down-regulates Phex expression at the mRNA and protein levels in UMR-106 cells. This effect is not restricted to this cell line because PTHrP(1–34) also down-regulates Phex expression in primary cultures of fetal rat calvaria cells. PTHrP(1–34) as well as PTH(1–34) are known to affect osteoblast metabolism through the common type-1 PTH/PTHrP receptor (42, 43, 44). In accordance, we have observed that ligands of the receptor reduced Phex mRNA levels; in contrast, a ligand (PTHrP107–139) that activates PKC and regulates osteoblast function through a distinct receptor did not alter Phex mRNA levels. These data suggest that the common type-1 PTH/PTHrP receptor is the receptor used by PTHrP(1–34) to regulate Phex expression.

In osteoblasts, the common type-1 PTH/PTHrP receptor is coupled to Gs and Gq proteins that activate the PKA and PKC transduction pathways, respectively. Activation of PKC activity presents two peaks: the first is reached at 10-12 to 10-11 M of ligand and the second at 10-9 to 10-8 M (42, 43). In contrast, 10-7 M of ligand is needed to observe a maximal increase in intracellular cAMP levels and PKA activation (42, 43). The following evidences suggest that PTHrP(1–34) activates PKA: 1) PTHrP(1–34) down-regulated Phex expression only in the 10-7 to 10-6 M range; 2) human PTH(1–31), a ligand of the common type-1 PTH/PTHrP receptor that activates PKA but not PKC, also decreased Phex mRNA levels; 3) forskolin and (Bu)2cAMP mimicked PTHrP(1–34) effects on Phex regulation but 1–9-dideoxyforskolin, an analog that mimics the adenylate-cyclase-independent effects of forskolin (55, 56), had no effect on Phex expression; 4) Phex down-regulation correlated with increased intracellular cAMP accumulation provoked by PTHrP(1–34) and forskolin; 5) cotreatment with near maximal effective doses of forskolin and PTHrP(1–34) did not produce an additive effect on Phex mRNA levels, indicating that both factors may act by the same pathway; and 6) finally, the target of increased intracellular cAMP accumulation is probably PKA because a specific inhibitor of PKA, H89, blocked the effects of PTHrP(1–34) and (Bu)2cAMP. All these data indicate that PTHrP(1–34) uses the cAMP pathway to down-regulate Phex expression through PKA activation.

We observed down-regulation of Phex expression only at high PTHrP(1–34) concentrations (10-7 and 10-6 M). This suggests that PTHrP(1–34), produced locally by osteoblasts, acts in a paracrine/autocrine pathway. It probably also excludes a role for PTH(1–34), produced by the parathyroid glands, in regulating Phex gene expression, because this hormone is found circulating at much lower levels (approximately 10-12 M) (57).

We also observed that incubation of UMR-106 cells with a phorbol ester that activates PKC (TPA) increased Phex expression. However, when PTHrP(1–34) was used in the concentration range (5 x 10-13 to 10-9 M) that activates PKC but not the cAMP pathway, Phex mRNA levels were not modified. Similar results were obtained when cells were incubated with bPTH(3–34) that binds the common type-1 PTH/PTHrP receptor and activates the PKC but not the cAMP pathway. One possibility to explain this discrepancy is to postulate that, because PKC is a superfamily of isozymes with different regulatory properties (58) and because normal osteoblasts and many osteoblastic cell lines, including UMR-106 cells, express several isozymes (48), the PKC isozyme(s) that PTHrP(1–34) or PTH(1–34) activates in UMR-106 cells is distinct from the isozyme(s) induced by TPA that regulates Phex expression. Our results indicate that in UMR-106 cells TPA and PTHrP(1–34) do indeed activate different PKC isozymes: the {alpha} and {delta} isozymes, respectively. These findings suggest that PKC-{alpha} can regulate Phex gene expression, whereas PKC-{delta}, which is coupled to the type-1 PTH/PTHrP receptor, has no effect on Phex expression.

In osteoblasts, downstream signaling from PKA and PKC involves transcription factors that regulate gene expression. The murine Phex gene promoter has several potential cis-acting elements including binding sites for cAMP response element-binding protein, activator protein 1 (AP1), glucocorticoid receptor, and estrogen receptor (59). Transactivation of cAMP response element-binding protein induced by PTH(1–34) is mediated by PKA but not PKC or calcium in osteoblastic cells (60, 61). PTH(1–34) also regulates the expression of c-fos and c-jun, components of the AP1 transcription factor (60). Our results indicate that cAMP response and/or AP1 elements may mediate the effects on Phex expression of PTHrP(1–34) and TPA, respectively.

Interestingly, the effects of PTHrP(1–34) on primary cultures of rat calvaria cells were restricted to an early stage of culture: Inhibition of Phex mRNA levels was observed at d 7 but not later. This observation is best explained by a down-regulation of the type-1 PTH/PTHrP receptor as cells progress toward final differentiation. This phenomenon has been well documented in primary cultures of rat calvaria cells (62). In addition, higher levels of PTHrP expression are found in less differentiated cells (63, 64). In contrast to the expression patterns of PTHrP and its receptor, we observed that Phex expression in rat calvaria cells is weak at d 7, starts to increase around d 10, and reaches a maximum at d 15, corresponding with mineralization (results not shown). Thus, there is an inverse correlation between the expression of Phex and that of PTHrP(1–34) and its receptor. We suggest that PTHrP(1–34) acting in an autocrine or paracrine way down-regulates Phex expression in osteoprogenitor cells and preosteoblasts.

We see no effect of TPA on Phex expression in primary cultures of rat calvaria cells. This can be explained by the absence in these cells of the isozyme(s) responsible for Phex gene activation. Alternatively, the presence in these cultures of autocrine/paracrine factor(s) activating the PKA pathway may mask the effect of TPA, as we observed in this study when increasing concentrations of forskolin were added to TPA-stimulated cells.

In conclusion, the work presented in this paper demonstrates that PTHrP(1–34) by binding to the common type-1 PTH/PTHrP receptor down-regulates Phex expression through activation of the PKA transduction pathway. This role of PTHrP(1–34) may be important for the timing of Phex expression in differentiating osteoprogenitor cells. It would be interesting to determine whether other hormones that activate PKA or PKC and regulate the mineralization of bone also regulate the expression of Phex.


   Acknowledgments
 
We are grateful to Demian Barbas for his help in measuring cAMP levels.


   Footnotes
 
This work was supported by Grants NRF13052 from the Canadian Institutes of Health Research and ER-73358 from Fonds pour la Formation des Chercheurs et l’Aide à la Recherche (FCAR).

Abbreviations: AP1, Activator protein 1; bPTH, bovine PTH; (Bu)2cAMP, N6,2'-O-dibutiryladenosine-3', 5'-cyclic monophosphate; FBS, fetal bovine serum; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; NEP, neprilysin; 4{alpha}-PDD, 4{alpha}-phorbol 12,13-didecanoate; Phex, a phosphate-regulating gene with homologies to endopeptidases on the X chromosome; PKA, protein kinase A; PKC, protein kinase C; TPA, 12-O-tetradecanoyl phorbol-13-acetate, XLH, X-linked hypophosphatemia.

Received February 25, 2003.

Accepted for publication July 28, 2003.


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