J Am Soc Nephrol 13: 394–399, 2002 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.

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

The X-linked hypophosphatemia (XLH) is an X-linked domi- the amino acid level and is expressed in tissues, including bone nant mendelian disorder that is characterized by growth retar- (8,9). Since its discovery, a variety of loss-of-function muta- dation, rachitic and osteomalacic bone disease, hypophos- tions in the PHEX gene have been described in XLH patients phatemia, and defects in renal phosphate (Pi) reabsorption and (10,11). These findings raise the possibility that the defect in vitamin D metabolism (1). Since its first description by Al- PHEX function may play a central role in the pathogenesis of bright et al. (2) in 1937, the pathogenesis of XLH has remained XLH. To test this possibility in our current study, we have incompletely understood. However, significant progress has examined the effect of disrupting PHEX expression in a human been made over the last two decades, aided particularly by the osteoblast cell line (MG-63 cells) by using antisense strategy. finding of a murine homologue, i.e., the hypophosphatemic Our results show that a decrease in PHEX expression in anti- (Hyp) mice (3). Extensive studies performed with Hyp mice sense-transfected MG-63 cells caused an impaired mineraliza- have led to the current understanding that the defect in bones tion and led to the release of factors into culture media that are may lead to the release of humoral factors that affect both bone capable of inhibiting osteoblast cell mineralization and proxi- mineralization and proximal tubule Pi reabsorption and con- mal tubular cell phosphate uptake. tribute 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 Materials and Methods osteoblast cells can release factors into culture media capable Cell Cultures of inhibiting bone mineralization (5) and proximal tubular cell The human osteoblast cell line (MG-63 cells) was obtained from Pi transport (6). Another important progress made recently in American Type Culture Collection and maintained in Eagle’s mini- our understanding of XLH was the discovery of the candidate mum essential medium (MEM) supplemented with 7% fetal calf gene, which has been designated PHEX (Phosphate regulating serum, ␤-glycerolphosphate (10 mM), and ascorbate (0.28 mM). The gene with homology to Endopeptidases on the X chromosome) opossum kidney (OK) cells (originally provided by Dr. D. M. (7). The PHEX gene contains significant homology to the Shoback, UCSF, San Francisco, CA) were maintained in Dulbecco’s family of metalloproteinase genes (NEP, KELL, and ECE-1) at modified Eagle’s medium (DMEM)–Ham’s F12 1:1 mix culture me- dium 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 Received March 7, 2001. Accepted October 5, 2001. 2ϩ 2ϩ Correspondence to Dr. Norimoto Yanagawa, Nephrology Division (111R), trypsinized with 0.1% trypsin in Ca -free and Mg -free phosphate- Sepulveda VA Medical Center, 16111 Plummer Street, Sepulveda, CA 91343. buffered saline containing 0.5 mM ethylenediaminetetraacetic acid Phone: 818-891-7711 ext. 7520; Fax: 818-895-9402; E-mail: [email protected] (EDTA) and plated in culture plates of appropriate sizes. Cells were 1046-6673/1302-0394 used at 2 wk after seeding and were serum-deprived for 24 h before Journal of the American Society of Nephrology experiments by changing the culture medium to serum-free medium Copyright © 2002 by the American Society of Nephrology the day before the experiment. J Am Soc Nephrol 13: 394–399, 2002 PHEX Antisense in MG63 Cells 395

RNA Isolation Mineralization Assays in MG-63 Cells RNA was isolated from MG-63 cells by using RNAzol (Biotecx The mineralization of MG-63 cells was assayed by measuring 45Ca Laboratories, Inc., Houston, TX), and mRNA was further prepared by incorporated within the cell layer and matrix and by determining the using an mRNA isolation kit (Quiagen, Inc., Valencia, CA). These formation of mineralization nodules. To measure 45Ca incorporation, preparations were quantified by the absorbance at 260 nm, and their MG-63 cells were incubated at 37°C and 5% CO2–95% air for 48 h in ␮ 45 purity was determined by the 260/280 nm absorbance ratio. culture medium that contained 0.5 Ci/ml CaCl2. 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 Plasmid Construction liquid scintillation spectroscopy. The formation of mineralization To construct PHEX antisense vector, the PHEX cDNA spanning nodules was determined by alizarin red-S histochemical staining (5). from Ϫ62 to ϩ206 of human PHEX sequence (Genebank accession Cell layers were fixed for 24 h in 1:1:1.5 solution of 10% formalin, number U87284) was obtained by reverse transcriptase–PCR (RT- methanol, and water. The fixative was removed, and the fixed cells PCR) by using mRNA isolated from MG-63 cells and PHEX-specific and matrices were stained for 15 min with a 2% (wt/vol) solution of primers, PHEXϪ62F (5'-GAGACCAGCCACCAAACCAC- alizarin red-S at pH 4.0. The stained samples were washed with water GAAAAGT-3') and PHEXϩ206R (5'-TTACTTAAGATGGCAG- and air-dried. CAGCC-3'). As the marker for the expression of exogenous construct, rabbit ␤-globin was used as described previously (12). For this pur- Production of PHEX Antisera pose, the second intron containing partial exon 2 and exon 3 of the A PHEX cDNA encoding a 63–amino acid polypeptide from the c rabbit ␤-globin gene (Genebank accession number V00878) was terminal region that shares the least homology to the same regions of obtained by PCR amplification of rabbit genomic DNA with ␤-glo- other closely related metalloendopeptidase family members (NEP, bin–specific primers ␤-GBϩ377F (5'-GATCCTGAGAACT- KELL, and ECE-1) was obtained from MG63 cell mRNA by using TCAGGG-3') and ␤-GBϩ958R (5'-CCCAGGAGCTGTAGGAAA- RT-PCR with PHEX-specific primers, PHEXϩ2058F (5'-GAGCT- 3'). The PHEX-rabbit ␤-globin construct was then produced by CAAGTTATGCTCATGTGAGGTGC-3') and PHEX ϩ2247R (5'- ligating the rabbit ␤-globin PCR product into the ScaI site (ϩ158) of AAATAAGAGCTCCAGAGTCGACAGGAGTCCA-3'). This PCR PHEX cDNA, and the resultant PHEX-rabbit ␤-globin construct was product was sequence-verified, cloned into pMal-c2x vector (New cloned into pcDNA 3.1 vector (Invitrogen, Carlsbad, CA) in either England BioLabs, Beverly, MA), and introduced into Escherichia coli sense or antisense orientation, designated as pcDNA 3.1/PHEX·S and (TOP10). The fusion protein (maltose-binding protein-PHEX) thus pcDNA 3.1/PHEX·AS, respectively. 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- Cell Transfection and Selection PHEX antisera thus produced was confirmed by their lack of reactiv- To produce sense and antisense cell lines, MG-63 cells were stably ity against the similar 63–amino acid polypeptides produced from the transfected with pcDNA 3.1/PHEX·S or pcDNA 3.1/PHEX·AS by c termini of other closely related metalloendopeptidase family mem- using SuperFect reagent (Qiagen, Inc., Valentia, CA) according to bers (NEP, KELL, and ECE-1). manufacturer’s instruction. Stable transfectants were selected by sup- plementing the medium with 300 ␮g/ml G418 48 h after transfection, Northern and Western Blot Analyses and the G418-resistant cells were cloned to establish individual cell For Northern blots, cellular mRNA was size-fractioned on a 1.0% lines. The sense and antisense cells were further confirmed by the 299 formaldehyde/agarose gel in 1 ϫ MOPS buffer (20 mM 3-(N-mor- bp of RT-PCR amplification product from the cellular RNA with pholino)-propanesulfonic acid [MOPS], 8 mM sodium acetate, 1 mM ϩ Ϫ PHEX 206R and PHEX 62F primers, respectively. The extra 33 bp EDTA, pH 7.0) and transferred to nylon membranes (Pierce, Rock- ␤ from -globin exons in these RT-PCR products served to identify the ford, IL). The blots were probed with a biotinyl-labeled PHEX RNA expression of the exogenous construct from the original DNA probe and detected by chemiluminescence according to manufactur- construct. er’s instruction (Pierce). The PHEX RNA probe spanned from ϩ1419 to ϩ2281 of the published PHEX cDNA sequence. This region was chosen so that the overexpressed partial PHEX mRNA in sense- 32 3 Pi and 3-O-methyl-[ H]glucose Uptake transfected cells (from Ϫ62 to ϩ206) will not interfere with the Measurements in OK Cells detection of intrinsic PHEX mRNA. To make the PHEX RNA probe, 32Pi and 3-O-methyl-[3H]glucose uptake by OK cells was mea- a PCR product of PHEX cDNA (ϩ1419 to ϩ2281) was first obtained sured by using the uptake medium containing 137 mM NaCl, 5.4 mM by using PHEXϩ1419 sense primer (5'-TTGGCAAAAGTTGGC- ϩ KCl, 1.2 mM MgSO4, 1.5 mM CaCl2, 14 mM Tris-(hydroxymethyl) TATCCAG-3') and PHEX 2281 antisense primer (5'-GTCTCAG- 32 aminomethane-base (pH 7.5) and 0.1 mM K2H PO4 or 3-O-methyl- GATGCCATAAACCAGC-3'). This PCR product was cloned into [3H]glucose (0.2 ␮Ci/ml). Cell uptake was terminated by washing pGEM-T easy (Promega, Medison, MI), sequence-verified, and then with substrate-free uptake medium (4°C) three times and solubilized used to produce the RNA probe by using a North2South in vitro in 0.2 N NaOH. Aliquots of each sample were counted for 32Pi or 3H transcription kit (Pierce) according to manufacturer’s instruction. As activity by liquid scintillation spectroscopy counter (1600-TR; Pack- a control, another biotinyl-labeled RNA probe was produced in a ard, Downers Grove, IL) or analyzed for total protein content by using similar fashion from a cDNA template of human glyceraldehyde-3- Coomassie brilliant blue G250 with bovine serum albumin as the phosphate dehydrogenase (GAPDH) (Ambion, Austin, TX). For standard (13). Naϩ-independent uptake was determined by replacing Western blots, cell protein samples were extracted in a solution NaCl in the uptake medium with 137 mM choline chloride, and the containing 10 mM Tris-HCL (pH 8.0), 1 mM EDTA, 150 mM NaCl, Naϩ-dependent uptake was calculated as the difference between up- 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sul- takes in the presence and absence of Naϩ. fate (SDS), 0.7 mM p-methylsulfonylfluorid, and 20 ␮g/ml leupeptin. 396 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 394–399, 2002

Protein samples were denatured by boiling in 2% SDS, separated on seeding was analyzed by Northern and Western blot analyses, 4to15%–gradient SDS-PAGE gels, and transferred to supported respectively. For Northern blot analysis, cell mRNA was nitrocellulose membranes (Millipore Co., Bedford, MA). The cellu- probed with an RNA probe against a region (PHEXϩ1419 to lose membranes were blocked with 3% bovine serum albumin and PHEXϩ2281) downstream from the antisense region probed with rabbit anti-PHEX antisera. After washing with Tris-based (PHEXϪ62 to PHEXϩ206). For Western blot analysis, cell saline with 0.05% Tween 20, membranes were probed with a second- protein samples were probed with rabbit anti-PHEX antisera, ary alkaline phosphatase conjugated mouse monoclonal anti-rabbit ϩ ϩ antibody (Bio-Rad Laboratories, Hercules, CA), and the signal of the which detect the c terminal region ( 2058 to 2247) of the secondary antibody was visualized by chemiluminescence according PHEX protein. Results from a representative cell line are to manufacturer’s manual. shown in Figure 2, where antisense-transfected cells showed a decrease in PHEX expression at mRNA (Northern blot, left) Materials and protein (Western blot, right) levels as compared with The culture media (MEM, DMEM, Ham’s F12) and fetal calf sense-transfected cells. serum were purchased from Irvine Scientific (Santa Ana, CA). Tissue culture plates were purchased from (Nunc Interlab, Thousand Oaks, Impaired Mineralization in Antisense Cells CA). Radioisotopes were purchased from ICN Biochemicals Inc. The mineralization of sense- and antisense-transfected (Irvine, CA). Other chemicals were purchased from Sigma (St. Louis, MG-63 cells at 2 wk after seeding was assessed by deter- MO). mining the rate of 45Ca incorporation and the formation of Statistical Analyses. At least three determinations were obtained calcification nodules. As shown in Figure 3, the 45Ca incor- for each data point, and the experimental data are expressed as mean poration into cell layers and matrix was significantly less in Ϯ SEM. The significance of differences was analyzed by t test for antisense-transfected cells as compared with sense-trans- paired or unpaired data or by one-way ANOVA, with individual elements analyzed by the Scheffe method. fected cells. Similarly, the extent of mineralization nodule formation was also significantly lower in antisense-trans- Results fected cells (Figure 3). Characterization of PHEX Expression in Antisense Cells Effects of Conditioned Culture Media from Antisense The PHEX mRNA expression in MG-63 cells was first Cells confirmed by RT-PCR with PHEX-specific primers, Conditioned culture media were collected from sense- and ϩ antisense-transfected cells and were tested for their effects PHEX 360F (5'-AGAATCAATCAGTAGAAGCCG-3') and 45 ϩ on both Ca incorporation into nontransfected MG-63 cells PHEX 929R (5'-GGAATCTAGCACTCAGTTCAGA-3'). 32 45 MG-63 cell mRNA was harvested at 1, 2, and 3 wk after and P uptake by OK cells. To test the effect on Ca seeding, and the RT-PCR products were fractionated on 1.0% incorporation, MG-63 cells were incubated with conditioned ␮ 45 agarose gel and visualized with ethidium bromide staining. As culture media containing 0.5 Ci/ml of CaCl2 for 48 h, and the rate of 45Ca incorporation was determined. The shown in Figure 1, the expected 569-bp fragment was detected 32 from the first week and continued up to 3 wk after seeding. effect of conditioned culture media on OK cell P uptake MG-63 cells were then transfected with sense and antisense was also determined after OK cells were preincubated with plasmids and selected by G418. We have obtained four clones conditioned culture media for 48 h before uptake measure- each of G418 resistant sense and antisense cells. The abun- dance of PHEX mRNA and protein in these cells at 2 wk after

Figure 2. Decreased PHEX expression in antisense cells. The levels of PHEX mRNA (left) and protein (right) expression in antisense-trans- fected MG-63 cells were decreased as compared with sense-trans- fected cells. For these studies, cells were used at 2 wk after seeding. Figure 1. PHEX expression in MG-63 cells. MG-63 cell mRNA was For Northern blot analysis, cellular mRNA was probed with RNA harvested at 1, 2, and 3 wk after seeding, and the level of PHEX probes against PHEX at a region downstream from the antisense mRNA expression was assessed by a reverse transcriptase–PCR (RT- region and against GAPDH. For Western blot analysis, cellular pro- PCR), using PHEX-specific primers, PHEXϩ360F and PHEXϩ929R. tein samples were probed with rabbit anti-PHEX antisera. Results The expected 569-bp fragment was detected in all samples. from a representative cell line are shown. J Am Soc Nephrol 13: 394–399, 2002 PHEX Antisense in MG63 Cells 397

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 ϭ 4; * P Ͻ 0.05.

ments. As shown in Figure 4, conditioned culture media Ϯ 0.08 versus 1.14 Ϯ 0.12 nmol/mg protein per 5 min; n ϭ from antisense-transfected cells caused a significant de- 5; P Ͼ 0.7). crease in MG-63 cell 45Ca incorporation and OK cell 32P uptake, as compared with that collected from sense-trans- Discussion fected cells. These inhibitory effects of the conditioned culture media derived from antisense-transfected cells were With the availability of Hyp mice, extensive studies have specific because they did not affect other transport activities been performed and have led to the current understanding in these cells, i.e., 32Pi uptake by MG-63 cells (0.29 Ϯ 0.01 that bone-derived humoral factors may participate in the versus 0.31 Ϯ 0.01 nmol/mg protein per 5 min; n ϭ 5; P Ͼ pathogenesis of XLH. The existence of putative circulating 0.3) and 3-O-methyl-[3H]glucose uptake by OK cells (1.12 phosphaturic factor(s) was first suggested by parabiosis

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 ϭ 4; * P Ͻ 0.05. 398 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 394–399, 2002 studies in which normal mice developed phosphaturia and dia collected from these antisense-transfected cells exhibited hypophosphatemia after being surgically parabiosed with inhibitory activities on both osteoblast cell 45Ca incorporation Hyp mice (14). Subsequent studies showed that this factor and proximal tubular cell 32P uptake (Figure 4). These inhib- was not parathyroid hormone (15) and was not produced by itory effects were specific, because the conditioned media the kidney (16). The possibility that bones may contribute to the derived from antisense-transfected cells did not affect other production of this putative phosphaturic factor was suggested by transport functions of these cells, such as 32Pi uptake in MG-63 the demonstration that the conditioned culture media derived from cells and 3-O-methyl-[3H]glucose uptake in OK cells. cultured Hyp mouse osteoblast cells were capable of inhibiting The results of our study therefore provide strong evidence in proximal tubular cell Pi transport (6). support of the link between PHEX abnormality and the devel- Although studies in mice (17) and in human patients (18) opment of bone defects with the release of factors affecting indicated that both hypophosphatemia and aberrant vitamin D bone mineralization and proximal tubular Pi transport. The metabolism play an important role in XLH bone defect, there expression of PHEX was reduced but not totally abolished in is growing evidence that suggests that intrinsic abnormalities in the antisense-transfected cells; it is, therefore, likely that a bones may also contribute to the bone defect in XLH. Thus, it decrease in PHEX expression at this level is sufficient to was found that the mineralization defect of Hyp mouse bones induce detectable effects on cell mineralization and on the persisted after transplantation into normal mice (19) and that release of humoral factors into the conditioned media. Addi- correcting hypophosphatemia by Pi supplementation in Hyp tional evidence in support of the pathogenic role of PHEX mice improved, but did not completely correct, the bone min- abnormality was also provided by a recent study in which the eralization defect (20). Furthermore, Hyp mouse osteoblast maturational regulation of PHEX in cultured mouse osteoblast cells showed defective mineralization even after prolonged in cells was found to coordinate with the inhibitory activity of the vitro culture (5). Similar to the proximal tubular defect, recent conditioned culture media on proximal tubular phosphate studies have also demonstrated that Hyp mouse osteoblast cells transport (22). Combined, it is likely that the inactivating release humoral factors into the culture media that are capable mutations of PHEX in XLH patients may play an important of blocking normal mouse osteoblast cell mineralization (5). It role in the phenotypic expression of the disease. The exact thus appears that the intrinsic bone defect can contribute to the function of the PHEX protein and the identity of these osteo- pathogenesis of XLH through the release of factor(s), which blast-derived pathogenic factors remain to be identified. affects bone mineralization locally and inhibits proximal tubule phosphate transport when delivered to the kidney through Acknowledgments circulation. This work was supported by grants from the Department of Vet- Consistent with this schema of events, the candidate gene for erans Affairs, NIH (ROIDK/AR58886), and American Society of XLH, i.e., PHEX, was found to share high homology with Nephrology. endopeptidase genes (7) and expressed in a variety of tissues, including the bone (21). These findings raise the possibility References that the loss of PHEX protein function in XLH patients may 1. Rasmussen H, Tenenhouse HS: Hypophosphatemias. 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