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J Am Soc Nephrol 13:394-399, 2002
© 2002 American Society of Nephrology

Effects of PHEX Antisense in Human Osteoblast Cells

Nengjen Remi Shih, Oak D. Jo and Norimoto Yanagawa

Medical and Research Services, Sepulveda Veterans Administration Medical Center, Sepulveda, California; and Department of Medicine, School of Medicine, University of California at Los Angeles, Los Angeles, California.

Correspondence to Dr. Norimoto Yanagawa, Nephrology Division (111R), Sepulveda VA Medical Center, 16111 Plummer Street, Sepulveda, CA 91343. Phone: 818-891-7711 ext. 7520; Fax: 818-895-9402; E-mail: nori{at}ucla.edu


   Abstract
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 Abstract
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 Materials and Methods
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 Discussion
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ABSTRACT. X-linked hypophosphatemia (XLH) is an X-linked dominant disorder that is characterized by rachitic bone disease and hypophosphatemia due to renal phosphate transport defect. The candidate gene for XLH, PHEX, has recently been identified and found to share high homology with endopeptidases. PHEX is expressed in various tissues, including bones, and the available evidence today indicates that bones can release abnormal humoral factors that affect bone mineralization and proximal tubule phosphate transport in XLH. It was, therefore, hypothesized that the inactivating mutations of PHEX in bone may lead to the release of humoral factors and contribute to the phenotypic expression of the disease. To test this possibility, clones of MG-63 cells, a human osteoblast cell line, were produced and stably transfected with PHEX-antisense vectors, resulting in a decrease in PHEX expression at mRNA and protein levels. It was found that these antisense-transfected cells had impaired mineralization, with a decrease in 45Ca incorporation and calcification nodule formation. It was also found that the conditioned culture media collected from these antisense-transfected cells exhibited inhibitory activities on 45Ca incorporation by the nontransfected MG-63 cells and 32P uptake by the opossum kidney proximal tubular cells. The results of the study, therefore, provide strong evidence that supports the link between PHEX mutations and the pathogenesis of XLH.


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 Introduction
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The X-linked hypophosphatemia (XLH) is an X-linked dominant mendelian disorder that is characterized by growth retardation, rachitic and osteomalacic bone disease, hypophosphatemia, and defects in renal phosphate (Pi) reabsorption and vitamin D metabolism (1). Since its first description by Albright et al. (2) in 1937, the pathogenesis of XLH has remained incompletely understood. However, significant progress has been made over the last two decades, aided particularly by the finding of a murine homologue, i.e., the hypophosphatemic (Hyp) mice (3). Extensive studies performed with Hyp mice have led to the current understanding that the defect in bones may lead to the release of humoral factors that affect both bone mineralization and proximal tubule Pi reabsorption and contribute to the phenotypic expression of XLH (4). The direct evidence in support of this notion was provided by recent studies demonstrating that the in vitro cultured Hyp mouse osteoblast cells can release factors into culture media capable of inhibiting bone mineralization (5) and proximal tubular cell Pi transport (6). Another important progress made recently in our understanding of XLH was the discovery of the candidate gene, which has been designated PHEX (Phosphate regulating gene with homology to Endopeptidases on the X chromosome) (7). The PHEX gene contains significant homology to the family of metalloproteinase genes (NEP, KELL, and ECE-1) at the amino acid level and is expressed in tissues, including bone (8,9). Since its discovery, a variety of loss-of-function mutations in the PHEX gene have been described in XLH patients (10,11). These findings raise the possibility that the defect in PHEX function may play a central role in the pathogenesis of XLH. To test this possibility in our current study, we have examined the effect of disrupting PHEX expression in a human osteoblast cell line (MG-63 cells) by using antisense strategy. Our results show that a decrease in PHEX expression in antisense-transfected MG-63 cells caused an impaired mineralization and led to the release of factors into culture media that are capable of inhibiting osteoblast cell mineralization and proximal tubular cell phosphate uptake.


   Materials and Methods
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Cell Cultures
The human osteoblast cell line (MG-63 cells) was obtained from American Type Culture Collection and maintained in Eagle’s minimum essential medium (MEM) supplemented with 7% fetal calf serum, ß-glycerolphosphate (10 mM), and ascorbate (0.28 mM). The opossum kidney (OK) cells (originally provided by Dr. D. M. Shoback, UCSF, San Francisco, CA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM)–Ham’s F12 1:1 mix culture medium supplemented with 10% fetal calf serum. Cells were maintained under a humidified atmosphere of 5% CO2–95% air, and the culture medium was exchanged every 3 to 4 d. For serial passages, cells were trypsinized with 0.1% trypsin in Ca2+-free and Mg2+-free phosphate-buffered saline containing 0.5 mM ethylenediaminetetraacetic acid (EDTA) and plated in culture plates of appropriate sizes. Cells were used at 2 wk after seeding and were serum-deprived for 24 h before experiments by changing the culture medium to serum-free medium the day before the experiment.

RNA Isolation
RNA was isolated from MG-63 cells by using RNAzol (Biotecx Laboratories, Inc., Houston, TX), and mRNA was further prepared by using an mRNA isolation kit (Quiagen, Inc., Valencia, CA). These preparations were quantified by the absorbance at 260 nm, and their purity was determined by the 260/280 nm absorbance ratio.

Plasmid Construction
To construct PHEX antisense vector, the PHEX cDNA spanning from -62 to +206 of human PHEX sequence (Genebank accession number U87284) was obtained by reverse transcriptase–PCR (RT-PCR) by using mRNA isolated from MG-63 cells and PHEX-specific primers, PHEX-62F (5'-GAGACCAGCCACCAAACCACGAAAAGT-3') and PHEX+206R (5'-TTACTTAAGATGGCAGCAGCC-3'). As the marker for the expression of exogenous construct, rabbit ß-globin was used as described previously (12). For this purpose, the second intron containing partial exon 2 and exon 3 of the rabbit ß-globin gene (Genebank accession number V00878) was obtained by PCR amplification of rabbit genomic DNA with ß-globin–specific primers ß-GB+377F (5'-GATCCTGAGAACTTCAGGG-3') and ß-GB+958R (5'-CCCAGGAGCTGTAGGAAA-3'). The PHEX-rabbit ß-globin construct was then produced by ligating the rabbit ß-globin PCR product into the ScaI site (+158) of PHEX cDNA, and the resultant PHEX-rabbit ß-globin construct was cloned into pcDNA 3.1 vector (Invitrogen, Carlsbad, CA) in either sense or antisense orientation, designated as pcDNA 3.1/PHEX·S and pcDNA 3.1/PHEX·AS, respectively.

Cell Transfection and Selection
To produce sense and antisense cell lines, MG-63 cells were stably transfected with pcDNA 3.1/PHEX·S or pcDNA 3.1/PHEX·AS by using SuperFect reagent (Qiagen, Inc., Valentia, CA) according to manufacturer’s instruction. Stable transfectants were selected by supplementing the medium with 300 µg/ml G418 48 h after transfection, and the G418-resistant cells were cloned to establish individual cell lines. The sense and antisense cells were further confirmed by the 299 bp of RT-PCR amplification product from the cellular RNA with PHEX+206R and PHEX-62F primers, respectively. The extra 33 bp from ß-globin exons in these RT-PCR products served to identify the expression of the exogenous construct from the original DNA construct.

32Pi and 3-O-methyl-[3H]glucose Uptake Measurements in OK Cells
32Pi and 3-O-methyl-[3H]glucose uptake by OK cells was measured by using the uptake medium containing 137 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO4, 1.5 mM CaCl2, 14 mM Tris-(hydroxymethyl) aminomethane-base (pH 7.5) and 0.1 mM K2H32PO4 or 3-O-methyl-[3H]glucose (0.2 µCi/ml). Cell uptake was terminated by washing with substrate-free uptake medium (4°C) three times and solubilized in 0.2 N NaOH. Aliquots of each sample were counted for 32Pi or 3H activity by liquid scintillation spectroscopy counter (1600-TR; Packard, Downers Grove, IL) or analyzed for total protein content by using Coomassie brilliant blue G250 with bovine serum albumin as the standard (13). Na+-independent uptake was determined by replacing NaCl in the uptake medium with 137 mM choline chloride, and the Na+-dependent uptake was calculated as the difference between uptakes in the presence and absence of Na+.

Mineralization Assays in MG-63 Cells
The mineralization of MG-63 cells was assayed by measuring 45Ca incorporated within the cell layer and matrix and by determining the formation of mineralization nodules. To measure 45Ca incorporation, MG-63 cells were incubated at 37°C and 5% CO2–95% air for 48 h in culture medium that contained 0.5 µCi/ml 45CaCl2. After incubation, cell layers were washed with Hank’s balanced salt solution and digested in 0.2 N NaOH. Aliquots were counted for 45Ca activity by liquid scintillation spectroscopy. The formation of mineralization nodules was determined by alizarin red-S histochemical staining (5). Cell layers were fixed for 24 h in 1:1:1.5 solution of 10% formalin, methanol, and water. The fixative was removed, and the fixed cells and matrices were stained for 15 min with a 2% (wt/vol) solution of alizarin red-S at pH 4.0. The stained samples were washed with water and air-dried.

Production of PHEX Antisera
A PHEX cDNA encoding a 63–amino acid polypeptide from the c terminal region that shares the least homology to the same regions of other closely related metalloendopeptidase family members (NEP, KELL, and ECE-1) was obtained from MG63 cell mRNA by using RT-PCR with PHEX-specific primers, PHEX+2058F (5'-GAGCTCAAGTTATGCTCATGTGAGGTGC-3') and PHEX +2247R (5'-AAATAAGAGCTCCAGAGTCGACAGGAGTCCA-3'). This PCR product was sequence-verified, cloned into pMal-c2x vector (New England BioLabs, Beverly, MA), and introduced into Escherichia coli (TOP10). The fusion protein (maltose-binding protein-PHEX) thus produced was purified by affinity-column chromatography (Amylose; New England BioLabs) according to manufacturer’s manual and used to produce rabbit anti-PHEX antisera. The specificity of the anti-PHEX antisera thus produced was confirmed by their lack of reactivity against the similar 63–amino acid polypeptides produced from the c termini of other closely related metalloendopeptidase family members (NEP, KELL, and ECE-1).

Northern and Western Blot Analyses
For Northern blots, cellular mRNA was size-fractioned on a 1.0% formaldehyde/agarose gel in 1 x MOPS buffer (20 mM 3-(N-morpholino)-propanesulfonic acid [MOPS], 8 mM sodium acetate, 1 mM EDTA, pH 7.0) and transferred to nylon membranes (Pierce, Rockford, IL). The blots were probed with a biotinyl-labeled PHEX RNA probe and detected by chemiluminescence according to manufacturer’s instruction (Pierce). The PHEX RNA probe spanned from +1419 to +2281 of the published PHEX cDNA sequence. This region was chosen so that the overexpressed partial PHEX mRNA in sense-transfected cells (from -62 to +206) will not interfere with the detection of intrinsic PHEX mRNA. To make the PHEX RNA probe, a PCR product of PHEX cDNA (+1419 to +2281) was first obtained by using PHEX+1419 sense primer (5'-TTGGCAAAAGTTGGCTATCCAG-3') and PHEX+2281 antisense primer (5'-GTCTCAGGATGCCATAAACCAGC-3'). This PCR product was cloned into pGEM-T easy (Promega, Medison, MI), sequence-verified, and then used to produce the RNA probe by using a North2South in vitro transcription kit (Pierce) according to manufacturer’s instruction. As a control, another biotinyl-labeled RNA probe was produced in a similar fashion from a cDNA template of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, TX). For Western blots, cell protein samples were extracted in a solution containing 10 mM Tris-HCL (pH 8.0), 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.7 mM p-methylsulfonylfluorid, and 20 µg/ml leupeptin. Protein samples were denatured by boiling in 2% SDS, separated on 4 to 15%–gradient SDS-PAGE gels, and transferred to supported nitrocellulose membranes (Millipore Co., Bedford, MA). The cellulose membranes were blocked with 3% bovine serum albumin and probed with rabbit anti-PHEX antisera. After washing with Tris-based saline with 0.05% Tween 20, membranes were probed with a secondary alkaline phosphatase con'ugated mouse monoclonal anti-rabbit antibody (Bio-Rad Laboratories, Hercules, CA), and the signal of the secondary antibody was visualized by chemiluminescence according to manufacturer’s manual.

Materials
The culture media (MEM, DMEM, Ham’s F12) and fetal calf serum were purchased from Irvine Scientific (Santa Ana, CA). Tissue culture plates were purchased from (Nunc Interlab, Thousand Oaks, CA). Radioisotopes were purchased from ICN Biochemicals Inc. (Irvine, CA). Other chemicals were purchased from Sigma (St. Louis, MO).

Statistical Analyses.
At least three determinations were obtained for each data point, and the experimental data are expressed as mean ± SEM. The significance of differences was analyzed by t test for paired or unpaired data or by one-way ANOVA, with individual elements analyzed by the Scheffe method.


   Results
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Discussion
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Characterization of PHEX Expression in Antisense Cells
The PHEX mRNA expression in MG-63 cells was first confirmed by RT-PCR with PHEX-specific primers, PHEX+360F (5'-AGAATCAATCAGTAGAAGCCG-3') and PHEX+929R (5'-GGAATCTAGCACTCAGTTCAGA-3'). MG-63 cell mRNA was harvested at 1, 2, and 3 wk after seeding, and the RT-PCR products were fractionated on 1.0% agarose gel and visualized with ethidium bromide staining. As shown in Figure 1, the expected 569-bp fragment was detected from the first week and continued up to 3 wk after seeding. MG-63 cells were then transfected with sense and antisense plasmids and selected by G418. We have obtained four clones each of G418 resistant sense and antisense cells. The abundance of PHEX mRNA and protein in these cells at 2 wk after seeding was analyzed by Northern and Western blot analyses, respectively. For Northern blot analysis, cell mRNA was probed with an RNA probe against a region (PHEX+1419 to PHEX+2281) downstream from the antisense region (PHEX-62 to PHEX+206). For Western blot analysis, cell protein samples were probed with rabbit anti-PHEX antisera, which detect the c terminal region (+2058 to +2247) of the PHEX protein. Results from a representative cell line are shown in Figure 2, where antisense-transfected cells showed a decrease in PHEX expression at mRNA (Northern blot, left) and protein (Western blot, right) levels as compared with sense-transfected cells.



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  		Figure 1. PHEX expression in MG-63 cells. MG-63 cell mRNA was harvested at 1, 2, and 3 wk after seeding, and the level of PHEX mRNA expression was assessed by a reverse transcriptase–PCR (RT-PCR), using PHEX-specific primers, PHEX+360F and PHEX+929R. The expected 569-bp fragment was detected in all samples.

 


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  		Figure 2. Decreased PHEX expression in antisense cells. The levels of PHEX mRNA (left) and protein (right) expression in antisense-transfected MG-63 cells were decreased as compared with sense-transfected cells. For these studies, cells were used at 2 wk after seeding. For Northern blot analysis, cellular mRNA was probed with RNA probes against PHEX at a region downstream from the antisense region and against GAPDH. For Western blot analysis, cellular protein samples were probed with rabbit anti-PHEX antisera. Results from a representative cell line are shown.

 
Impaired Mineralization in Antisense Cells
The mineralization of sense- and antisense-transfected MG-63 cells at 2 wk after seeding was assessed by determining the rate of 45Ca incorporation and the formation of calcification nodules. As shown in Figure 3, the 45Ca incorporation into cell layers and matrix was significantly less in antisense-transfected cells as compared with sense-transfected cells. Similarly, the extent of mineralization nodule formation was also significantly lower in antisense-transfected cells (Figure 3).



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  		Figure 3. Impaired mineralization in antisense cells. MG-63 cell mineralization was assayed by measuring 45Ca incorporation within the cell layer and matrix (left) and by determining the formation of mineralization nodules by alizarin red-S histochemical staining (right). Both assays showed an impaired mineralization in antisense-transfected cells as compared with sense-transfected cells. For these studies, cells were used at 2 wk after seeding. For mineralization nodule staining, results from a representative cell line are shown. n = 4; * P < 0.05.

 
Effects of Conditioned Culture Media from Antisense Cells
Conditioned culture media were collected from sense- and antisense-transfected cells and were tested for their effects on both 45Ca incorporation into nontransfected MG-63 cells and 32P uptake by OK cells. To test the effect on 45Ca incorporation, MG-63 cells were incubated with conditioned culture media containing 0.5 µCi/ml of 45CaCl2 for 48 h, and the rate of 45Ca incorporation was determined. The effect of conditioned culture media on OK cell 32P uptake was also determined after OK cells were preincubated with conditioned culture media for 48 h before uptake measurements. As shown in Figure 4, conditioned culture media from antisense-transfected cells caused a significant decrease in MG-63 cell 45Ca incorporation and OK cell 32P uptake, as compared with that collected from sense-transfected cells. These inhibitory effects of the conditioned culture media derived from antisense-transfected cells were specific because they did not affect other transport activities in these cells, i.e., 32Pi uptake by MG-63 cells (0.29 ± 0.01 versus 0.31 ± 0.01 nmol/mg protein per 5 min; n = 5; P > 0.3) and 3-O-methyl-[3H]glucose uptake by OK cells (1.12 ± 0.08 versus 1.14 ± 0.12 nmol/mg protein per 5 min; n = 5; P > 0.7).



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  		Figure 4. Inhibitory activities of conditioned culture media from antisense cells. As compared with sense-transfected cells, conditioned culture media collected from antisense-transfected cells exhibited inhibitory activities on both 45Ca incorporation into nontransfected MG-63 cells (left) and 32P uptake by opossum kidney (OK) cells (right). n = 4; * P < 0.05.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
With the availability of Hyp mice, extensive studies have been performed and have led to the current understanding that bone-derived humoral factors may participate in the pathogenesis of XLH. The existence of putative circulating phosphaturic factor(s) was first suggested by parabiosis studies in which normal mice developed phosphaturia and hypophosphatemia after being surgically parabiosed with Hyp mice (14). Subsequent studies showed that this factor was not parathyroid hormone (15) and was not produced by the kidney (16). The possibility that bones may contribute to the production of this putative phosphaturic factor was suggested by the demonstration that the conditioned culture media derived from cultured Hyp mouse osteoblast cells were capable of inhibiting proximal tubular cell Pi transport (6).

Although studies in mice (17) and in human patients (18) indicated that both hypophosphatemia and aberrant vitamin D metabolism play an important role in XLH bone defect, there is growing evidence that suggests that intrinsic abnormalities in bones may also contribute to the bone defect in XLH. Thus, it was found that the mineralization defect of Hyp mouse bones persisted after transplantation into normal mice (19) and that correcting hypophosphatemia by Pi supplementation in Hyp mice improved, but did not completely correct, the bone mineralization defect (20). Furthermore, Hyp mouse osteoblast cells showed defective mineralization even after prolonged in vitro culture (5). Similar to the proximal tubular defect, recent studies have also demonstrated that Hyp mouse osteoblast cells release humoral factors into the culture media that are capable of blocking normal mouse osteoblast cell mineralization (5). It thus appears that the intrinsic bone defect can contribute to the pathogenesis of XLH through the release of factor(s), which affects bone mineralization locally and inhibits proximal tubule phosphate transport when delivered to the kidney through circulation.

Consistent with this schema of events, the candidate gene for XLH, i.e., PHEX, was found to share high homology with endopeptidase genes (7) and expressed in a variety of tissues, including the bone (21). These findings raise the possibility that the loss of PHEX protein function in XLH patients may result in a failure to inactivate a host of humoral factors, and the release of these pathogenic factors may contribute to the phenotypic manifestation of the disease (8). However, the exact function of PHEX protein remains unknown, and the evidence linking the abnormal PHEX protein function to the development of these disease phenotypes remains absent.

We thus conducted our current study to examine the effect of reducing PHEX expression in MG-63 cells by using antisense strategy. We stably transfected MG-63 cells with vectors containing PHEX-antisense nucleotides in the region of -62 to +206 of PHEX cDNA and obtained clones of MG-63 cells where the expression of PHEX mRNA and protein was suppressed (Figure 2). As a control, we also obtained clones of MG-63 cells stably transfected with vectors that contained the same nucleotide insert but in sense orientation. These sense-transfected cells showed no difference from nontransfected cells or cells transfected with vector alone in terms of their PHEX expression levels, mineralization, or the effect of the conditioned media derived therefrom. However, as shown in Figure 3, the antisense-transfected cells exhibited impaired mineralization with a decrease in 45Ca incorporation and calcification nodule formation as compared with the sense-transfected control cells. Furthermore, the conditioned culture media collected from these antisense-transfected cells exhibited inhibitory activities on both osteoblast cell 45Ca incorporation and proximal tubular cell 32P uptake (Figure 4). These inhibitory effects were specific, because the conditioned media derived from antisense-transfected cells did not affect other transport functions of these cells, such as 32Pi uptake in MG-63 cells and 3-O-methyl-[3H]glucose uptake in OK cells.

The results of our study therefore provide strong evidence in support of the link between PHEX abnormality and the development of bone defects with the release of factors affecting bone mineralization and proximal tubular Pi transport. The expression of PHEX was reduced but not totally abolished in the antisense-transfected cells; it is, therefore, likely that a decrease in PHEX expression at this level is sufficient to induce detectable effects on cell mineralization and on the release of humoral factors into the conditioned media. Additional evidence in support of the pathogenic role of PHEX abnormality was also provided by a recent study in which the maturational regulation of PHEX in cultured mouse osteoblast cells was found to coordinate with the inhibitory activity of the conditioned culture media on proximal tubular phosphate transport (22). Combined, it is likely that the inactivating mutations of PHEX in XLH patients may play an important role in the phenotypic expression of the disease. The exact function of the PHEX protein and the identity of these osteoblast-derived pathogenic factors remain to be identified.


   Acknowledgments
 
This work was supported by grants from the Department of Veterans Affairs, NIH (ROIDK/AR58886), and American Society of Nephrology.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication March 7, 2001. Accepted for publication October 5, 2001.




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