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).