Fibroblast growth factor 23 (FGF23) is a key regulator of mineral ion homeostasis. Genetic ablati... more Fibroblast growth factor 23 (FGF23) is a key regulator of mineral ion homeostasis. Genetic ablation of Fgf23 in mice leads to severe biochemical disorders including elevated serum 1,25-dihydroxyvitamin D [1,25(OH)2D], hypercalcemia, hyperphosphatemia, and marked decreased PTH levels. Because PTH stimulates 1,25(OH)2D production and increases serum calcium levels, we hypothesized that ablation of PTH from the Fgf23 knockout (Fgf23-/-) mice could suppress these affects, thus ameliorating the soft tissue and skeletal anomalies in these animals. In this study, we generated a genetic mouse model with dual ablation of the Fgf23/PTH genes. The data show that deletion of PTH does suppress the markedly higher serum 1,25(OH)2D and calcium levels observed in Fgf23-/- mice and results in much larger, heavier, and more active double-knockout mice with improved soft tissue and skeletal phenotypes. On the contrary, when we infused PTH (1-34) peptide into Fgf23-/- mice using osmotic minipumps, serum 1,25(OH)2D and calcium levels were increased even further, leading to marked reduction in trabecular bone. These results indicate that PTH is able to modulate the anomalies of Fgf23-/- mice by controlling serum 1,25(OH)2D and calcium levels.
The PTH/PTHrP receptor gene is expressed in bone and kidney as well as in many other tissues. Usi... more The PTH/PTHrP receptor gene is expressed in bone and kidney as well as in many other tissues. Using primer extension followed by rapid cloning of amplified complementary DNA ends, we have isolated new PTH/PTHrP receptor complementary DNAs with different splicing patterns and have characterized a new upstream transcription start site. Three 5' nontranslated exons, U3, U2 and U1, located 4.8, 2.5, and 1.2 kb upstream of the exon that encodes the putative signal peptide of the classical receptor (exon S), have been characterized. Four types of splicing patterns were recognized. Type I splicing pattern is transcribed from exon U1 and is spliced to exons S and E1; this pattern was found in most tissues tested. Types II, III, and IV splicing patterns are transcribed from exon U3 and have a restricted tissue distribution. Type II splice pattern, containing exons U3, U2, and S and type III splicing pattern, containing exon U3, U2, and E1 (skipping exon S), was found only in kidney. Type IV splice pattern, containing exon U3 and S was found both in kidney and ovary. Because the type III splice variant skips exon S, translation of this splice variant initiates at a different AUG codon. The type III splice variant was weakly expressed on the cell surface of COS-7 cells, as assessed by double antibody binding assay, and no detectable ligand binding was observed on intact cells. The type III splice variant, however, increased cAMP accumulation in COS-7 cells when challenged with PTH(1-34), PTH(1-84) and hPTHrP(1-36) with EC50s that are similar to those observed in COS-7 cells expressing the type I variant but with a maximum stimulation that was lower than that observed in COS-7 cells expressing the type I variant. These data indicate low levels of cell surface expression of the type III splice variant. Treatment of COS-7 cells with tunicamycin decreased the size of the type I splice variant from a broad band of 85 kDa to a compact band of about 60 kDa. The type III splice variant did not change in size in COS-7 cells treated with tunicamycin, indicating that the type III splice variant did not undergo any glycosylation step. In conclusion, the PTH/PTHrP receptor gene uses alternate promoters in a tissue-specific manner that results in several tissue-specific alternatively spliced transcripts. One of these transcripts, the type III splice variant, is expressed in kidney and lacks the signal peptide.
Biochemical and Biophysical Research Communications, 1994
The organization of the PTH/PTHrP receptor gene is highly homologous in three mammalian species, ... more The organization of the PTH/PTHrP receptor gene is highly homologous in three mammalian species, rat, human and mouse. This gene extends over 22 kb and contains at least 15 exons and 14 introns. The most 5' exon we have identified (exon U) is followed by an approximately 1kb intron. The second exon (exon S) encodes the initiator methionine and the putative signal peptide and is followed by the largest intron of this gene (about 11 kb). The amino-terminal extracellular region is encoded by 4 exons (E1, E2, E3 and G); exon G contains all 4 potential glycosylation sites. Membrane-spanning domains 1-4 and portions of their connecting intracellular and extracellular loops are encoded by 4 exons (M1, M2, M3 and M4). The second extracellular loop and portions of 4th and 5th membrane-spanning domains are encoded by one exon, EL2. The 5th membrane-spanning domain and portion of the 3rd intracellular loop are encoded by one exon, M5. The 6th membrane-spanning domain, the 3rd extracellular loop and the proximal part of the 7th membrane-spanning domain are encoded by one single exon (M6/7); the remaining sequence of the 7th membrane-spanning domain is encoded by a short exon, M7. The carboxy-terminal tail of the receptor and the 3' untranslated region are encoded by one single exon, exon T. The 3' untranslated region does not contain the classical polyadenylation signal, AATAAA. Expression in COS-7 cells of a minigene constructed of a 5' rat cDNA fragment (1.3 Kb) ligated in-frame to a 3' genomic fragment at the NsiI site, which is located in exon M6/7 resulted in a transcript that was translated into a functional receptor; it bound PTH and showed PTH-stimulated accumulation of intracellular cAMP. Therefore, the PTH/PTHrP receptor gene contains alternative 3' sequences that allow cleavage and polyadenylation of its transcript.
Fibroblast growth factor 23 (FGF23) is a key regulator of mineral ion homeostasis. Genetic ablati... more Fibroblast growth factor 23 (FGF23) is a key regulator of mineral ion homeostasis. Genetic ablation of Fgf23 in mice leads to severe biochemical disorders including elevated serum 1,25-dihydroxyvitamin D [1,25(OH)2D], hypercalcemia, hyperphosphatemia, and marked decreased PTH levels. Because PTH stimulates 1,25(OH)2D production and increases serum calcium levels, we hypothesized that ablation of PTH from the Fgf23 knockout (Fgf23-/-) mice could suppress these affects, thus ameliorating the soft tissue and skeletal anomalies in these animals. In this study, we generated a genetic mouse model with dual ablation of the Fgf23/PTH genes. The data show that deletion of PTH does suppress the markedly higher serum 1,25(OH)2D and calcium levels observed in Fgf23-/- mice and results in much larger, heavier, and more active double-knockout mice with improved soft tissue and skeletal phenotypes. On the contrary, when we infused PTH (1-34) peptide into Fgf23-/- mice using osmotic minipumps, serum 1,25(OH)2D and calcium levels were increased even further, leading to marked reduction in trabecular bone. These results indicate that PTH is able to modulate the anomalies of Fgf23-/- mice by controlling serum 1,25(OH)2D and calcium levels.
The PTH/PTHrP receptor gene is expressed in bone and kidney as well as in many other tissues. Usi... more The PTH/PTHrP receptor gene is expressed in bone and kidney as well as in many other tissues. Using primer extension followed by rapid cloning of amplified complementary DNA ends, we have isolated new PTH/PTHrP receptor complementary DNAs with different splicing patterns and have characterized a new upstream transcription start site. Three 5' nontranslated exons, U3, U2 and U1, located 4.8, 2.5, and 1.2 kb upstream of the exon that encodes the putative signal peptide of the classical receptor (exon S), have been characterized. Four types of splicing patterns were recognized. Type I splicing pattern is transcribed from exon U1 and is spliced to exons S and E1; this pattern was found in most tissues tested. Types II, III, and IV splicing patterns are transcribed from exon U3 and have a restricted tissue distribution. Type II splice pattern, containing exons U3, U2, and S and type III splicing pattern, containing exon U3, U2, and E1 (skipping exon S), was found only in kidney. Type IV splice pattern, containing exon U3 and S was found both in kidney and ovary. Because the type III splice variant skips exon S, translation of this splice variant initiates at a different AUG codon. The type III splice variant was weakly expressed on the cell surface of COS-7 cells, as assessed by double antibody binding assay, and no detectable ligand binding was observed on intact cells. The type III splice variant, however, increased cAMP accumulation in COS-7 cells when challenged with PTH(1-34), PTH(1-84) and hPTHrP(1-36) with EC50s that are similar to those observed in COS-7 cells expressing the type I variant but with a maximum stimulation that was lower than that observed in COS-7 cells expressing the type I variant. These data indicate low levels of cell surface expression of the type III splice variant. Treatment of COS-7 cells with tunicamycin decreased the size of the type I splice variant from a broad band of 85 kDa to a compact band of about 60 kDa. The type III splice variant did not change in size in COS-7 cells treated with tunicamycin, indicating that the type III splice variant did not undergo any glycosylation step. In conclusion, the PTH/PTHrP receptor gene uses alternate promoters in a tissue-specific manner that results in several tissue-specific alternatively spliced transcripts. One of these transcripts, the type III splice variant, is expressed in kidney and lacks the signal peptide.
Biochemical and Biophysical Research Communications, 1994
The organization of the PTH/PTHrP receptor gene is highly homologous in three mammalian species, ... more The organization of the PTH/PTHrP receptor gene is highly homologous in three mammalian species, rat, human and mouse. This gene extends over 22 kb and contains at least 15 exons and 14 introns. The most 5' exon we have identified (exon U) is followed by an approximately 1kb intron. The second exon (exon S) encodes the initiator methionine and the putative signal peptide and is followed by the largest intron of this gene (about 11 kb). The amino-terminal extracellular region is encoded by 4 exons (E1, E2, E3 and G); exon G contains all 4 potential glycosylation sites. Membrane-spanning domains 1-4 and portions of their connecting intracellular and extracellular loops are encoded by 4 exons (M1, M2, M3 and M4). The second extracellular loop and portions of 4th and 5th membrane-spanning domains are encoded by one exon, EL2. The 5th membrane-spanning domain and portion of the 3rd intracellular loop are encoded by one exon, M5. The 6th membrane-spanning domain, the 3rd extracellular loop and the proximal part of the 7th membrane-spanning domain are encoded by one single exon (M6/7); the remaining sequence of the 7th membrane-spanning domain is encoded by a short exon, M7. The carboxy-terminal tail of the receptor and the 3' untranslated region are encoded by one single exon, exon T. The 3' untranslated region does not contain the classical polyadenylation signal, AATAAA. Expression in COS-7 cells of a minigene constructed of a 5' rat cDNA fragment (1.3 Kb) ligated in-frame to a 3' genomic fragment at the NsiI site, which is located in exon M6/7 resulted in a transcript that was translated into a functional receptor; it bound PTH and showed PTH-stimulated accumulation of intracellular cAMP. Therefore, the PTH/PTHrP receptor gene contains alternative 3' sequences that allow cleavage and polyadenylation of its transcript.
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Papers by B. Lanske