- Email: [email protected]
Molecular cloning of the murine PHEX gene promoter1
Biochimica et Biophysica Acta 1493 (2000) 333^336
www.elsevier.com/locate/bba
Promoter paper
Molecular cloning of the murine PHEX gene promoter1 Eric R. Hines, James F. Collins, Fayez K. Ghishan * Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, 1501 N. Campbell Avenue, Tucson, AZ 85724, USA Received 9 May 2000; accepted 25 July 2000
Abstract We report the novel cloning of the murine PHEX promoter, the gene that is mutated in X-linked hypophosphatemic rickets (XLH). Four promoter/reporter gene constructs, 3133/+104, 3542/+104, 31061/+104, and 32866/+104, showed significant luciferase activity (4.9^13.2fold over background) when transfected into rat osteogenic sarcoma (UMR-106) cells. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : PEX; X-linked rickets; UMR-106 cells; Hyp mouse
X-Linked hypophosphatemic rickets (XLH) is a dominant, genetic disorder with a prevalence of 1 in 20 000 live births. Phenotypical characteristics can vary widely and include rickets, which does not respond to vitamin D treatment, growth retardation, and lower body skeletal abnormalities [1]. Biochemical manifestations include high serum alkaline phosphatase levels [1] and hypophosphatemia resulting from decreased phosphate reabsorption in the renal proximal tubules [2^5]. Positional cloning of the gene responsible for XLH identi¢ed PHEX (Phosphate regulating gene with Homologies to Endopeptidases on the X chromosome ; formerly PEX), in chromosomal region Xp22.1 [6]. The human and murine PHEX cDNAs have been cloned [7^12] and they are believed to encode membrane bound proteins with homology to members of the neutral endopeptidase (NEP) family [6]. Further evidence suggesting that PHEX is a member of the NEP family was provided by Lipman et al. [11], who showed that PHEX mediated degradation of human parathyroid hormone (PTH1^38 and PTH1^34), and that it was localized to the cell membrane. Precise tissue distribution of PHEX is unclear; however, reverse transcriptase^polymerase chain reaction (RT^ PCR) and Northern blot experiments have shown expression predominantly in bone with lower levels in lung [7^
* Corresponding author. Fax: +1-520-626-4141; E-mail : [email protected] 1 Sequence data from this study has been deposited in the GenBank data base under accession number AF142648.
9,11]. PHEX has been further localized to the osteoblasts [10,13^15], which is in agreement with the defective bone mineralization seen in patients with XLH and in Hyp mice (the animal homolog of XLH). Moreover, PHEX has been shown in primary osteoblast cultures to be down regulated by 1,25-(OH)2 vitamin D3 [13], which is known to inhibit bone matrix mineralization and to play a role in phosphate homeostasis. Although PHEX has been identi¢ed as the gene responsible for XLH, it is still uncertain how its loss of function results in the XLH phenotype or what its normal physiological role is in bone metabolism and phosphate homeostasis. To gain further insight into the physiological and pathophysiological role of PHEX, we cloned 2985 bases of the 5P £anking DNA of the murine PHEX gene, identi¢ed the transcriptional start site and showed that this DNA region contains a functional promoter. The mouse genomic BAC clone 308A23, previously shown to contain exon 1 of the mouse PHEX gene [16], was obtained from Research Genetics (Huntsville, AL). Previous studies on this BAC clone suggested that a 4.3 kb BamHI fragment likely contains the 5P PHEX gene sequence [16]. We subcloned this DNA fragment into a plasmid vector and used sequence information from this clone to identify a 6 kb PstI fragment by Southern blot analysis. This fragment was also subcloned into a plasmid vector, and PHEX gene upstream sequence was determined on both strands by standard sequencing methods. Sequencing of both the BamHI and PstI subclones revealed 2985 bps of unique 5P £anking DNA (GenBank accession no. AF142648) (Fig. 1).
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 1 8 8 - 3
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E.R. Hines et al. / Biochimica et Biophysica Acta 1493 (2000) 333^336
Fig. 1. Nucleotide sequence of the 5P £anking region of the murine PHEX gene (GenBank accession no. AF142648). Depicted in this ¢gure is the ¢rst 800 bp of 5P £anking DNA of the murine PHEX gene, which includes 104 bp of novel 5P cDNA (underlined) and 696 bp of the putative promoter region. The transcriptional start site (+1) is indicated by a square. Putative cis-elements as determined by MatInspector are indicated in gray with the exception of an AP4 site (underlined) that overlaps a GRE and CREB site. Asterisks indicate cis-elements on the complementary strand.
A GenBank BLAST homology search of our 2985 bases of £anking DNA showed 92% identity with the ¢rst 162 bases of exon 1 of the human PHEX gene (EMBL YO8111) and 67% identity with 323 bases of human genomic DNA (EMBL Y10196) immediately upstream of human PHEX exon 1. Closer inspection of the murine and human sequences revealed 85% homology between the ¢rst 162 bp of exon 1 and 75 bp of 5P £anking sequence of the human gene and bp 3133 to +104 of our murine sequence. These ¢ndings suggest similar basal promoter regulation between species. Further computer analysis of the PHEX promoter, bp 31 to bp 32881, was conducted to detect putative cisacting elements using MatInspector (Genomatix, Munich, Germany; http://genomatix.gsf.de/). Numerous potential cis-acting elements were identi¢ed including, but not limited to, binding sites for: AP1 (activator protein 1), AP4 (activator protein 4), CREB (cAMP response element binding protein), ER (estrogen receptor), and GR (glucocorticoid receptor). At this point, it is unclear which of these putative elements may play a role in transcriptional regulation of PHEX. However, we have preliminary data suggesting that this promoter is regulated by glucocorticoids [17], although it is currently uncertain which cis-element(s) is responsible for these observations. We then used primer extension to determine the transcriptional start site [18]. Brie£y, a synthetic oligonucleotide (called PEX244r; 5P-AGTCCCTCTCAAGCAATTGTTTGGC-3P) complementary to bp 219^244 of the murine PHEX cDNA [11] was end-labeled with [Q32 P]ATP (ICN,
Costa Mesa, CA). 200 fmol of labeled primer was annealed to 30 Wg of mouse bone total RNA, isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD), in a 12-Wl reaction by heating at 90³C for 2 min then cooling to 58³C at 1³C/min. The annealing reaction was held at 58³C for 30 min then snap-chilled on ice. Annealed primers were extended at 42³C for 2 h by the addition of 200 units of Superscript II reverse transcriptase (Life Technologies), 1 Wl RNasin, 1 Wl 10 mM dNTPs, 4 Wl 5U¢rststrand reaction bu¡er, and nuclease-free H2 O to 20 Wl. The reaction was terminated by the addition of 3 Wl of 0.2 M EDTA (pH 8.0), and the RNA was degraded by the addition of 0.9 Wg of DNase-free RNase A followed by incubation at 37³C for 30 min. The primer extension reaction was then EtOH precipitated, and the pellet was resuspended in 5 Wl loading dye/TE bu¡er (1:1). Samples were heated at 75³C for 10 min prior to loading them on a sequencing gel. Dried gels were exposed to Kodak X-omat Blue XB-1 ¢lm at 370³C. The experiment was repeated twice with RNA samples isolated from di¡erent groups of animals. A sequencing reaction for comparison with the primer extension product was performed with the Thermo Sequenase Cycle Sequencing kit (USB, Cleveland, OH) according to the manufacturer's protocol. We used a synthetic oligonucleotide (called PEX244seq ; 5P-AGTCCCTCTCAAGCAATTGT-3P) complementary to bp 224^244 of the murine PHEX cDNA [14] and the 6 kb PstI PHEX subclone as a template. Primer extension indicated that the PHEX transcrip-
BBAEXP 91456 20-9-00
E.R. Hines et al. / Biochimica et Biophysica Acta 1493 (2000) 333^336
Fig. 2. Primer extension analysis of PHEX. The arrow indicates the primer extension product obtained using primer PEX244r and mouse bone total RNA as template. This extension product aligns with a guanidine residue, indicated in bold (Top-ACGTTCCTAAAAT-Bottom) as determined by sequencing the 6 kb PstI subclone with primer PEX244seq.
tional initiation site corresponds to a G residue 104 bases 5P of the longest murine PHEX cDNA [11] (Fig. 2). The transcriptional initiation site was designated +1 and upstream bases were numbered negatively. Both repetitions showed the same result. To identify a suitable cell line in which to study PHEX promoter activity, we tested two cell lines for PHEX expression by Northern blot analyses [19]. mRNA was isolated with the Micro-FastTrack kit (Invitrogen, Carlsbad, CA), from newly con£uent mouse preosteoblast cells (MC3T3-E1; from Riken, Tsukuba Science City, Japan) and rat osteogenic sarcoma cells (UMR-106; from ATCC). RNA gels were run, blotted onto nitrocellulose and processed under high-stringency conditions with the NorthernMax kit (Ambion, Austin, TX). The blot was probed for PHEX mRNA using radiolabeled, cDNA-speci¢c probes corresponding to bp 420^2835 of the murine PHEX cDNA [11]. Furthermore, a commercially available GAPDH probe was used as an internal standard. Fig. 3 shows the results of the Northern blot in which a single band at approximately 6.3 kb is seen only in the UMR-106 cells. These data were also con¢rmed by RT^ PCR (not shown). The absence of expression in MC3T3E1 cells is consistent with previous studies that have shown that PHEX is expressed in these cells only after several days of con£uency [9,12]. These data suggested that UMR-106 cells would be a more appropriate model for promoter analysis due to their earlier expression of PHEX mRNA. Furthermore UMR-106 cells possess the characteristics of fully di¡erentiated osteoblasts, including high alkaline phosphatase activity and PTH induced adenylate cyclase activation [20]. Therefore, the UMR-106 cell line was selected for these studies and it presents itself as a useful model for the in vitro study of PHEX gene regulation. PHEX promoter constructs of increasing size were made by PCR and subcloned into the KpnI and NheI restriction sites of the ¢re£y luciferase reporter vector, pGL-3 basic (Promega, Madison, WI). The three smaller constructs were ampli¢ed with Taq polymerase, while the 32866/ +104 construct was ampli¢ed with Pfu polymerase. All
335
constructs were created with the same reverse primer 5P-AGCCAATCTAGATGTCTGAACTGTC-3P with an XbaI site engineered into the sequence. The following forward primers with an engineered KpnI site were used: 5PCTCACATATGGTACCCTTCGTCTT-3P for the 3133 construct, 5P-CACAGGTACCTAGCAATTCACG-3P for the 3542 construct, 5P-ACTACTGGTACCATTAAGCTCACAC-3P for the 31061 construct and 5P-TGACCAGGTACCCCACGTTC-3P for the 32866 construct. Note that NheI and XbaI have compatible overhanging ends. All constructs were sequenced to con¢rm ¢delity. In order to study PHEX promoter activity, UMR-106 cells (passages 4^8) seeded into 24-well plates were cotransfected, via liposome mediated transfection, at 60^ 70% con£uency with 0.5 Wg of reporter vector DNA and 0.015 Wg of pRL-TK vector (encoding renilla luciferase; used as an internal standard) (Promega) in serum-free media. Twelve hours after transfection, media was changed to normal media, and cell extracts prepared 48 h later. Then, protein was quantitated and dual luciferase assays were performed with equal amounts of cellular protein. These experiments were repeated at least three times with separate cell populations on di¡erent days. In each experiment, three dishes were considered as n = 1 and the results were averaged. Additionally, ¢re£y luciferase activity was normalized for renilla luciferase activity in each cell extract. Highest luciferase activity was seen with the 31061/ +104 construct and lower activities were seen with the 3133/+104, 3542/+104 and 32866/+104 constructs (P 6 0.0001 for 31061/+104 versus all others) (Fig. 4). All constructs showed a signi¢cant increase in luciferase activity over the negative control pGL3 basic (P 9 0.006). Activity of the PHEX promoter showed a 4.7-fold increase over background for the 3133/+104 construct, 5.4-fold for 3542/+104, 12.7-fold for 31061/+104, and a 5.6-fold increase for the 32866/+104 construct. There was no signi¢-
Fig. 3. Northern blot analysis of PHEX in newly con£uent MC3T3-E1 and UMR-106 cells. Northern bolts showed an approximate 6.3 kb hybridization band only for UMR-106 cells. The GAPDH signal reveals equal loading of the samples.
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References
Fig. 4. The x-axis indicates the promoter construct assayed and the yaxis shows relative luciferase activity. *Indicates a signi¢cant di¡erence between that construct and the negative control pGL3-basic (P90.006; n = 4^6). **Indicates a signi¢cant di¡erence between 31061/+104 and pGL3-basic, and between 31061/+104 and the other three promoter constructs (P 6 0.001; n = 3).
cant di¡erence between expression from the 3133/+104 and 3542/+104 constructs, suggesting that the basal promoter may be located near the 5P end of the shortest construct. Between the 3542/+104 and 31061/+104 constructs a 2.3-fold increase in promoter activity was seen. Additionally, promoter activity decreased from the 31061/ +104 to the 32866/+104 construct, suggesting the possibility of a negative regulatory element(s) in this region. In conclusion, we have made the novel observation that the 5P £anking DNA of the murine PHEX gene contains a functional promoter. The cloning of this promoter will provide a useful tool for understanding the regulation of PHEX and will contribute new insight into its role in phosphate homeostasis and XLH. We have also developed UMR-106 rat osteogenic sarcoma cells as a useful in vitro model for studying PHEX gene regulation. We thank Tameson Snyder for her technical assistance. This study was supported by National Institutes of Health Grant R37DK33209 and the W.M. Keck Foundation.
[1] H. Rasmussen, H.S. Tenenhous, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic Basis of Inherited Disease, McGraw-Hill, New York, 1989, pp. 2581^2604. [2] N. Nakagawa, N. Arab, F.K. Ghishan, J. Biol. Chem. 266 (1991) 13616^13620. [3] N. Nakagawa, F.K. Ghishan, Proc. Soc. Exp. Biol. Med. 203 (1993) 328^335. [4] H.S. Tenenhouse, C.R. Scriver, R.R. McInnes, F.H. Glorieux, Kidney Int. 14 (1978) 236^244. [5] J.F. Collins, F.K. Ghishan, FASEB J. 8 (1994) 862^868. [6] HYP Consortium, Nat. Genet. (1995), 11, 130^136 [7] L. Beck, Y. Soumounou, J. Martel, G. Krishnamurthy, C. Gauthier, C.G. Goodyer, H.S. Tenenhouse, J. Clin. Invest. 99 (1997) 1200^ 1209. [8] L. Du, M. Desbarats, J. Viel, F.H. Glorieux, C. Cawthorn, B. Ecarot, Genomics 36 (1996) 22^28. [9] M. Grie¡, S. Mumm, P. Waeltz, R. Mazzarella, M.P. Whyte, R.V. Thakker, D. Schlessinger, Biochem. Biophys. Res. Commun. 231 (1997) 635^639. [10] R. Guo, L.D. Quarles, J. Bone Miner. Res. 12 (1997) 1009^1017. [11] M.L. Lipman, D. Panda, H.P. Bennett, J.E. Henderson, E. Shane, Y. Shen, D. Goltzman, A.C. Karaplis, J. Biol. Chem. 273 (1998) 13729^ 13737. [12] T.M. Strom, F. Francis, B. Lorenz, A. Boddrich, M.J. Econs, H. Lehrach, T. Meitinger, Hum. Mol. Genet. 6 (1997) 165^171. [13] B. Ecarot, M. Desbarats, Endocrinology 140 (1999) 1192^1199. [14] A.F. Ruchon, M. Marcinkiewicz, G. Siegfried, H.S. Tenenhouse, L. DesGroseillers, P. Crine, G. Boileau, J. Histochem. Cytochem. 46 (1998) 459^468. [15] Z.S. Xiao, M. Crenshaw, R. Guo, T. Nesbitt, M.K. Drezner, L.D. Quarles, Am. J. Physiol. 275 (1998) E700^E708. [16] B. Lorenz, F. Francis, K. Gempel, A. Boddrich, M. Josten, W. Schmahl, J. Schmidt, H. Lehrach, T. Meitinger, T.M. Strom, Hum. Mol. Genet. 7 (1998) 541^547. [17] E.R. Hines, P.R. Kiela, J.F. Collins, F.K. Ghishan, Gastroenterology 118 (2000) A289. [18] L. Muller, J.F. Collins, L. Bai, H. Xu, F.K. Ghishan, Biochim. Biophys. Acta 1442 (1998) 314^319. [19] J.F. Collins, N.M. Bulus, F.K. Ghishan, Am. J. Physiol. 268 (1995) G917^G924. [20] N.C. Partridge, D. Alcorn, V.P. Michelangeli, G. Ryan, T.J. Martin, Cancer Res. 43 (1983) 4308^4314.
BBAEXP 91456 20-9-00
www.elsevier.com/locate/bba
Promoter paper
Molecular cloning of the murine PHEX gene promoter1 Eric R. Hines, James F. Collins, Fayez K. Ghishan * Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, 1501 N. Campbell Avenue, Tucson, AZ 85724, USA Received 9 May 2000; accepted 25 July 2000
Abstract We report the novel cloning of the murine PHEX promoter, the gene that is mutated in X-linked hypophosphatemic rickets (XLH). Four promoter/reporter gene constructs, 3133/+104, 3542/+104, 31061/+104, and 32866/+104, showed significant luciferase activity (4.9^13.2fold over background) when transfected into rat osteogenic sarcoma (UMR-106) cells. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : PEX; X-linked rickets; UMR-106 cells; Hyp mouse
X-Linked hypophosphatemic rickets (XLH) is a dominant, genetic disorder with a prevalence of 1 in 20 000 live births. Phenotypical characteristics can vary widely and include rickets, which does not respond to vitamin D treatment, growth retardation, and lower body skeletal abnormalities [1]. Biochemical manifestations include high serum alkaline phosphatase levels [1] and hypophosphatemia resulting from decreased phosphate reabsorption in the renal proximal tubules [2^5]. Positional cloning of the gene responsible for XLH identi¢ed PHEX (Phosphate regulating gene with Homologies to Endopeptidases on the X chromosome ; formerly PEX), in chromosomal region Xp22.1 [6]. The human and murine PHEX cDNAs have been cloned [7^12] and they are believed to encode membrane bound proteins with homology to members of the neutral endopeptidase (NEP) family [6]. Further evidence suggesting that PHEX is a member of the NEP family was provided by Lipman et al. [11], who showed that PHEX mediated degradation of human parathyroid hormone (PTH1^38 and PTH1^34), and that it was localized to the cell membrane. Precise tissue distribution of PHEX is unclear; however, reverse transcriptase^polymerase chain reaction (RT^ PCR) and Northern blot experiments have shown expression predominantly in bone with lower levels in lung [7^
* Corresponding author. Fax: +1-520-626-4141; E-mail : [email protected] 1 Sequence data from this study has been deposited in the GenBank data base under accession number AF142648.
9,11]. PHEX has been further localized to the osteoblasts [10,13^15], which is in agreement with the defective bone mineralization seen in patients with XLH and in Hyp mice (the animal homolog of XLH). Moreover, PHEX has been shown in primary osteoblast cultures to be down regulated by 1,25-(OH)2 vitamin D3 [13], which is known to inhibit bone matrix mineralization and to play a role in phosphate homeostasis. Although PHEX has been identi¢ed as the gene responsible for XLH, it is still uncertain how its loss of function results in the XLH phenotype or what its normal physiological role is in bone metabolism and phosphate homeostasis. To gain further insight into the physiological and pathophysiological role of PHEX, we cloned 2985 bases of the 5P £anking DNA of the murine PHEX gene, identi¢ed the transcriptional start site and showed that this DNA region contains a functional promoter. The mouse genomic BAC clone 308A23, previously shown to contain exon 1 of the mouse PHEX gene [16], was obtained from Research Genetics (Huntsville, AL). Previous studies on this BAC clone suggested that a 4.3 kb BamHI fragment likely contains the 5P PHEX gene sequence [16]. We subcloned this DNA fragment into a plasmid vector and used sequence information from this clone to identify a 6 kb PstI fragment by Southern blot analysis. This fragment was also subcloned into a plasmid vector, and PHEX gene upstream sequence was determined on both strands by standard sequencing methods. Sequencing of both the BamHI and PstI subclones revealed 2985 bps of unique 5P £anking DNA (GenBank accession no. AF142648) (Fig. 1).
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 1 8 8 - 3
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Fig. 1. Nucleotide sequence of the 5P £anking region of the murine PHEX gene (GenBank accession no. AF142648). Depicted in this ¢gure is the ¢rst 800 bp of 5P £anking DNA of the murine PHEX gene, which includes 104 bp of novel 5P cDNA (underlined) and 696 bp of the putative promoter region. The transcriptional start site (+1) is indicated by a square. Putative cis-elements as determined by MatInspector are indicated in gray with the exception of an AP4 site (underlined) that overlaps a GRE and CREB site. Asterisks indicate cis-elements on the complementary strand.
A GenBank BLAST homology search of our 2985 bases of £anking DNA showed 92% identity with the ¢rst 162 bases of exon 1 of the human PHEX gene (EMBL YO8111) and 67% identity with 323 bases of human genomic DNA (EMBL Y10196) immediately upstream of human PHEX exon 1. Closer inspection of the murine and human sequences revealed 85% homology between the ¢rst 162 bp of exon 1 and 75 bp of 5P £anking sequence of the human gene and bp 3133 to +104 of our murine sequence. These ¢ndings suggest similar basal promoter regulation between species. Further computer analysis of the PHEX promoter, bp 31 to bp 32881, was conducted to detect putative cisacting elements using MatInspector (Genomatix, Munich, Germany; http://genomatix.gsf.de/). Numerous potential cis-acting elements were identi¢ed including, but not limited to, binding sites for: AP1 (activator protein 1), AP4 (activator protein 4), CREB (cAMP response element binding protein), ER (estrogen receptor), and GR (glucocorticoid receptor). At this point, it is unclear which of these putative elements may play a role in transcriptional regulation of PHEX. However, we have preliminary data suggesting that this promoter is regulated by glucocorticoids [17], although it is currently uncertain which cis-element(s) is responsible for these observations. We then used primer extension to determine the transcriptional start site [18]. Brie£y, a synthetic oligonucleotide (called PEX244r; 5P-AGTCCCTCTCAAGCAATTGTTTGGC-3P) complementary to bp 219^244 of the murine PHEX cDNA [11] was end-labeled with [Q32 P]ATP (ICN,
Costa Mesa, CA). 200 fmol of labeled primer was annealed to 30 Wg of mouse bone total RNA, isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD), in a 12-Wl reaction by heating at 90³C for 2 min then cooling to 58³C at 1³C/min. The annealing reaction was held at 58³C for 30 min then snap-chilled on ice. Annealed primers were extended at 42³C for 2 h by the addition of 200 units of Superscript II reverse transcriptase (Life Technologies), 1 Wl RNasin, 1 Wl 10 mM dNTPs, 4 Wl 5U¢rststrand reaction bu¡er, and nuclease-free H2 O to 20 Wl. The reaction was terminated by the addition of 3 Wl of 0.2 M EDTA (pH 8.0), and the RNA was degraded by the addition of 0.9 Wg of DNase-free RNase A followed by incubation at 37³C for 30 min. The primer extension reaction was then EtOH precipitated, and the pellet was resuspended in 5 Wl loading dye/TE bu¡er (1:1). Samples were heated at 75³C for 10 min prior to loading them on a sequencing gel. Dried gels were exposed to Kodak X-omat Blue XB-1 ¢lm at 370³C. The experiment was repeated twice with RNA samples isolated from di¡erent groups of animals. A sequencing reaction for comparison with the primer extension product was performed with the Thermo Sequenase Cycle Sequencing kit (USB, Cleveland, OH) according to the manufacturer's protocol. We used a synthetic oligonucleotide (called PEX244seq ; 5P-AGTCCCTCTCAAGCAATTGT-3P) complementary to bp 224^244 of the murine PHEX cDNA [14] and the 6 kb PstI PHEX subclone as a template. Primer extension indicated that the PHEX transcrip-
BBAEXP 91456 20-9-00
E.R. Hines et al. / Biochimica et Biophysica Acta 1493 (2000) 333^336
Fig. 2. Primer extension analysis of PHEX. The arrow indicates the primer extension product obtained using primer PEX244r and mouse bone total RNA as template. This extension product aligns with a guanidine residue, indicated in bold (Top-ACGTTCCTAAAAT-Bottom) as determined by sequencing the 6 kb PstI subclone with primer PEX244seq.
tional initiation site corresponds to a G residue 104 bases 5P of the longest murine PHEX cDNA [11] (Fig. 2). The transcriptional initiation site was designated +1 and upstream bases were numbered negatively. Both repetitions showed the same result. To identify a suitable cell line in which to study PHEX promoter activity, we tested two cell lines for PHEX expression by Northern blot analyses [19]. mRNA was isolated with the Micro-FastTrack kit (Invitrogen, Carlsbad, CA), from newly con£uent mouse preosteoblast cells (MC3T3-E1; from Riken, Tsukuba Science City, Japan) and rat osteogenic sarcoma cells (UMR-106; from ATCC). RNA gels were run, blotted onto nitrocellulose and processed under high-stringency conditions with the NorthernMax kit (Ambion, Austin, TX). The blot was probed for PHEX mRNA using radiolabeled, cDNA-speci¢c probes corresponding to bp 420^2835 of the murine PHEX cDNA [11]. Furthermore, a commercially available GAPDH probe was used as an internal standard. Fig. 3 shows the results of the Northern blot in which a single band at approximately 6.3 kb is seen only in the UMR-106 cells. These data were also con¢rmed by RT^ PCR (not shown). The absence of expression in MC3T3E1 cells is consistent with previous studies that have shown that PHEX is expressed in these cells only after several days of con£uency [9,12]. These data suggested that UMR-106 cells would be a more appropriate model for promoter analysis due to their earlier expression of PHEX mRNA. Furthermore UMR-106 cells possess the characteristics of fully di¡erentiated osteoblasts, including high alkaline phosphatase activity and PTH induced adenylate cyclase activation [20]. Therefore, the UMR-106 cell line was selected for these studies and it presents itself as a useful model for the in vitro study of PHEX gene regulation. PHEX promoter constructs of increasing size were made by PCR and subcloned into the KpnI and NheI restriction sites of the ¢re£y luciferase reporter vector, pGL-3 basic (Promega, Madison, WI). The three smaller constructs were ampli¢ed with Taq polymerase, while the 32866/ +104 construct was ampli¢ed with Pfu polymerase. All
335
constructs were created with the same reverse primer 5P-AGCCAATCTAGATGTCTGAACTGTC-3P with an XbaI site engineered into the sequence. The following forward primers with an engineered KpnI site were used: 5PCTCACATATGGTACCCTTCGTCTT-3P for the 3133 construct, 5P-CACAGGTACCTAGCAATTCACG-3P for the 3542 construct, 5P-ACTACTGGTACCATTAAGCTCACAC-3P for the 31061 construct and 5P-TGACCAGGTACCCCACGTTC-3P for the 32866 construct. Note that NheI and XbaI have compatible overhanging ends. All constructs were sequenced to con¢rm ¢delity. In order to study PHEX promoter activity, UMR-106 cells (passages 4^8) seeded into 24-well plates were cotransfected, via liposome mediated transfection, at 60^ 70% con£uency with 0.5 Wg of reporter vector DNA and 0.015 Wg of pRL-TK vector (encoding renilla luciferase; used as an internal standard) (Promega) in serum-free media. Twelve hours after transfection, media was changed to normal media, and cell extracts prepared 48 h later. Then, protein was quantitated and dual luciferase assays were performed with equal amounts of cellular protein. These experiments were repeated at least three times with separate cell populations on di¡erent days. In each experiment, three dishes were considered as n = 1 and the results were averaged. Additionally, ¢re£y luciferase activity was normalized for renilla luciferase activity in each cell extract. Highest luciferase activity was seen with the 31061/ +104 construct and lower activities were seen with the 3133/+104, 3542/+104 and 32866/+104 constructs (P 6 0.0001 for 31061/+104 versus all others) (Fig. 4). All constructs showed a signi¢cant increase in luciferase activity over the negative control pGL3 basic (P 9 0.006). Activity of the PHEX promoter showed a 4.7-fold increase over background for the 3133/+104 construct, 5.4-fold for 3542/+104, 12.7-fold for 31061/+104, and a 5.6-fold increase for the 32866/+104 construct. There was no signi¢-
Fig. 3. Northern blot analysis of PHEX in newly con£uent MC3T3-E1 and UMR-106 cells. Northern bolts showed an approximate 6.3 kb hybridization band only for UMR-106 cells. The GAPDH signal reveals equal loading of the samples.
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References
Fig. 4. The x-axis indicates the promoter construct assayed and the yaxis shows relative luciferase activity. *Indicates a signi¢cant di¡erence between that construct and the negative control pGL3-basic (P90.006; n = 4^6). **Indicates a signi¢cant di¡erence between 31061/+104 and pGL3-basic, and between 31061/+104 and the other three promoter constructs (P 6 0.001; n = 3).
cant di¡erence between expression from the 3133/+104 and 3542/+104 constructs, suggesting that the basal promoter may be located near the 5P end of the shortest construct. Between the 3542/+104 and 31061/+104 constructs a 2.3-fold increase in promoter activity was seen. Additionally, promoter activity decreased from the 31061/ +104 to the 32866/+104 construct, suggesting the possibility of a negative regulatory element(s) in this region. In conclusion, we have made the novel observation that the 5P £anking DNA of the murine PHEX gene contains a functional promoter. The cloning of this promoter will provide a useful tool for understanding the regulation of PHEX and will contribute new insight into its role in phosphate homeostasis and XLH. We have also developed UMR-106 rat osteogenic sarcoma cells as a useful in vitro model for studying PHEX gene regulation. We thank Tameson Snyder for her technical assistance. This study was supported by National Institutes of Health Grant R37DK33209 and the W.M. Keck Foundation.
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