Biomedical subjects

Justin Silver

Publications and source records attributed to Justin Silver.

17 recordsLinked to original sources

PubMed2006

Lanthanum carbonate decreases PTH gene expression with no hepatotoxicity in uraemic rats.

BACKGROUND: Lanthanum (La) carbonate is an effective phosphate-binder, used to reverse hyperphosphataemia due to chronic kidney disease. Some recent studies in rodents have cast safety uncertainties. The aims of this study were to examine the effects of La on parathyroid hormone (PTH) gene expression and hepatic toxicity. METHODS: Uraemia was induced in rats by a 0.3% adenine-containing diet for 2 weeks. Thereafter, rats were maintained for 4 weeks on an adenine diet with 1.5 or 3% La. Normal, uraemic, and uraemic rats fed a low-phosphorus (P) diet served as controls. RESULTS: Azotaemia developed in all adenine-fed rats. La of 3%, but not 1.5% La, decreased serum P to normal in uraemic rats. Both La 1.5 and 3% decreased urine P excretion. Plasma PTH was tripled in uraemic compared with normal rats. Both the 3.0% La and the low-P diet decreased PTH to normal. PTH mRNA content was increased 2-fold in uraemic compared with normal rats. The 3% La or the low-P diet decreased PTH mRNA levels to normal in uraemic rats. Liver enzymes were normal in all groups. Adenine-induced uraemia decreased liver weight with no additional effect of La. Liver weights corrected for body-weights were the same in all study groups, including the La group. Therefore, La had no effect upon absolute or corrected liver weight. Liver magnetic resonance imaging and microscopy did not reveal toxic changes due to La. CONCLUSIONS: These findings confirm that in rats with adenine-induced uraemia, the P-binder La reverses the hyperphosphataemia and hyperparathyroidism. They are the first demonstration that La decreases PTH gene expression. Importantly, we found no evidence of drug-induced liver toxicity.

Animals↗

PubMed2006

Mutational analysis of the PTH 3'-untranslated region in parathyroid disorders.

OBJECTIVE: Sequence alterations in untranslated regions (UTRs) of genes are important contributors to human diseases, including hereditary thrombophilia, hereditary hyperferritinaemia-cataract and fragile X mental retardation syndromes. Recently, functional studies of the 3'-UTR of the PTH gene, encoding parathyroid hormone, have highlighted it as a potential target for pathogenic mutations in patients with parathyroid dysfunction. Regulation of PTH gene expression occurs in part through protein binding to a specific 26 nucleotide instability element in the 3'-UTR of PTH mRNA, in a sequence-dependent manner. Thus, the PTH 3'-UTR has emerged as an important potential contributor to parathyroid dysfunction. Therefore, we sought to rigorously examine the PTH 3'-UTR in patients with primary and secondary parathyroid disorders, including primary parathyroid hyperplasia, secondary parathyroid hyperplasia, sporadic parathyroid adenoma and familial hypoparathyroidism of unknown genetic basis. PATIENTS AND DESIGN: Twenty-one parathyroid glands from 14 patients with primary parathyroid hyperplasia, 40 sporadic parathyroid adenomas from 40 patients, 42 parathyroid glands from 29 patients with secondary parathyroid hyperplasia and peripheral blood leucocytes from 24 affected members of eight kindreds with familial hypoparathyroidism of unknown genetic basis were examined for mutations in the 3'-UTR of the PTH gene. RESULTS: No alterations from the normal sequence were detected in any of the 127 samples examined. CONCLUSIONS: Based on the absence of identifiable DNA sequence alterations in these forms of parathyroid dysfunction, it is unlikely that mutation of the PTH 3'-UTR contributes frequently to their pathogenesis.

3' Untranslated Regions↗

PubMed2005

Increased parathyroid hormone gene expression in secondary hyperparathyroidism of experimental uremia is reversed by calcimimetics: correlation with posttranslational modification of the trans acting factor AUF1.

Most patients with chronic kidney disease develop secondary hyperparathyroidism with disabling systemic complications. Calcimimetic agents are effective tools in the management of secondary hyperparathyroidism, acting through allosteric modification of the calcium-sensing receptor (CaR) on the parathyroid gland (PT) to decrease parathyroid hormone (PTH) secretion and PT cell proliferation. This study showed that rats that were fed an adenine high-phosphorus diet had increased serum PTH and PTH mRNA levels at 7 and 21 d. For studying the effect of activation of the CaR by the calcimimetics R-568 on PTH gene expression, R-568 was given by gavage to uremic rats for the last 4 d of a 7-d adenine high-phosphorus diet. R-568 decreased both PTH mRNA and serum PTH levels. The effect of the calcimimetic on PTH gene expression was posttranscriptional and correlated with differences in protein-RNA binding and posttranslational modifications of the trans acting factor AUF1 in the PT. The AUF1 modifications as a result of uremia were reversed by treatment with R-568 to those of normal rats. Therefore, uremia and activation of the CaR mediated by calcimimetics modify AUF1 posttranslationally. These modifications in AUF1 correlate with changes in protein-PTH mRNA binding and PTH mRNA levels.

Aniline Compounds↗

PubMed2005

A novel mutation in fibroblast growth factor 23 gene as a cause of tumoral calcinosis.

CONTEXT: Tumoral calcinosis is a disease characterized by ectopic calcification and hyperphosphatemia due to enhanced renal tubular phosphate reabsorption. Fibroblast growth factor (FGF)23 was identified as a responsible factor in hypophosphatemic diseases caused by renal phosphate leak. OBJECTIVE: The objective of the study was to analyze the involvement of FGF23 in the development of tumoral calcinosis. DESIGN: Serum FGF23 level was evaluated in a patient with tumoral calcinosis by two kinds of ELISA: full-length assay that detects only full-length FGF23 with phosphate-lowering activity and C-terminal assay that measures full-length as well as C-terminal fragment of FGF23. FGF23 gene was analyzed by direct sequencing of PCR products, and mutant FGF23 was analyzed by Western blotting after expression in mammalian cells. PATIENTS: A family of tumoral calcinosis patients were studied. RESULTS: Serum FGF23 was extremely high when measured by C-terminal assay. In contrast, it was low normal by full-length assay. Analysis of FGF23 gene detected a serine to phenylalanine mutation in codon 129. No wild-type allele of this codon was found in the patient. The brother of the proband showed the same base change. When this mutant FGF23 was expressed in vitro, full-length and N-terminal fragments were barely detectable by Western blotting, whereas C-terminal fragment with the same molecular weight as that from wild-type FGF23 could be detected. CONCLUSION: The production and serum level of C-terminal fragment of FGF23 are increased in this patient with tumoral calcinosis. Together with the recent similar report of FGF23 mutation, impaired action of full-length FGF23 seems to result in tumoral calcinosis.

Adult↗

PubMed2005

Regulation of PTH synthesis and secretion relevant to the management of secondary hyperparathyroidism in chronic kidney disease.

Regulation of PTH synthesis and secretion relevant to the management of secondary hyperparathyroidism in chronic kidney disease. Small decreases in serum Ca(++) and more prolonged increases in serum phosphate (P) stimulate the parathyroid (PT) to secrete parathyroid hormone (PTH), while 1,25(OH)(2)-vitamin D(3) decreases PTH synthesis and secretion. A prolonged decrease in serum Ca(++) and 1,25(OH)(2)D(3), or increase in serum P, such as in patients with chronic renal failure, leads to the appropriate secondary increase in serum PTH. This secondary hyperparathyroidism involves increases in PTH gene expression, synthesis, and secretion, and, if chronic, to proliferation of the parathyroid cells. A low serum Ca(++) leads to an increase in PTH secretion, PTH mRNA stability, and parathyroid cell proliferation. Pi also regulates the parathyroid in a similar manner. The effect of Ca(++) on the parathyroid is mediated by a membrane Ca(2+) receptor (CaR). 1,25(OH)(2)D(3) decreases PTH gene transcription. Ca(2+) and P regulate the PTH gene post-transcriptionally by regulating the binding of parathyroid cytosolic proteins, trans factors, to a defined cis sequence in the PTH mRNA 3'-untranslated region (UTR), thereby determining the stability of the transcript. The parathyroid trans factors and cis elements have been defined.

Animals↗

PubMed2004

Identification and characterization of cis-acting elements in the human and bovine PTH mRNA 3'-untranslated region.

UNLABELLED: The human PTH mRNA 3'-UTR has a cis element homologous to the rat cis-acting instability element and a more proximal element identical to the single binding element identified in bovine PTH mRNA 3'-UTR. The function of the elements was shown in vitro. INTRODUCTION: In the rat, Ca(2+) and phosphate regulate PTH mRNA stability by the interaction of trans-acting proteins with a defined cis-acting instability element in the distal region of the PTH mRNA 3'-untranslated region (UTR). This element has been characterized in the rat and is conserved in human, canine, feline, and murine 3'-UTRs but not in bovine and porcine 3'-UTRs. MATERIALS AND METHODS: Parathyroid protein-binding assays to the PTH mRNA transcripts were performed. Functionality was studied in reporter genes that were transiently transfected into HEK293 cells. RESULTS: Protein-RNA binding experiments identified an element in bovine PTH mRNA at the proximal end of the 3'-UTR that is different from the rat protein-binding element. The human 3'-UTR contains both elements, but only the distal element binds proteins. Functional studies with HEK293 cells transiently transfected with reporter genes containing the different elements and flanking nucleotides (nt) showed that the human distal element destabilized a reporter mRNA similar to the effect of this element in the rat. A reporter mRNA containing the single bovine PTH mRNA protein-binding element was also destabilized, and this was prevented by coexpression of AU-rich element binding factor 1 (AUF1). CONCLUSION: Our results identify a new protein-binding element in the PTH mRNA 3'-UTR. In bovine PTH mRNA, it is the only element, and it is functional in destabilizing a reporter gene. It is also present in other species, including human PTH mRNA, where it is not functional, possibly because of differences in flanking sequences. The human PTH mRNA 3'-UTR distal element is highly homologous to the rat cis-acting instability element and destabilized a reporter gene, indicating its functionality. Therefore, different species have alternative cis-acting protein-binding elements that may determine the regulation of PTH mRNA stability in response to changes in serum calcium and phosphate.

3' Untranslated Regions↗

PubMed2004

Pathogenesis of parathyroid dysfunction in end-stage kidney disease.

Small decreases in serum calcium (Ca(2+)) and more-prolonged increases in serum phosphate (Pi) stimulate the parathyroid (PT) to secrete parathyroid hormone (PTH). 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2) D(3)] decreases PTH synthesis and secretion. The prolonged decrease in serum Ca(2+) and 1,25(OH)(2) D(3), or increase in serum Pi, observed in patients with chronic renal failure leads to a secondary increase in serum PTH. This secondary hyperparathyroidism involves increases in PTH gene expression, synthesis, and secretion and, if chronic, to proliferation of the PT cells. A low serum Ca(2+) leads to an increase in PTH secretion, PTH mRNA stability, and PT cell proliferation. Pi also regulates the PT in a similar manner. The effect of Ca(2+) on the PT is mediated by a membrane Ca(2+) receptor. 1,25(OH)(2) D(3) decreases PTH gene transcription. Ca(2+) and Pi regulate the PTH gene post transcriptionally by regulating the binding of PT cytosolic proteins, trans factors, to a defined cis sequence in the PTH mRNA 3'-untranslated region, thereby determining the stability of the transcript. The PT trans factors and cis elements have been defined.

Animals↗

PubMed2004

The protein phosphatase calcineurin determines basal parathyroid hormone gene expression.

Calcium and phosphate regulate PTH mRNA stability through differences in binding of parathyroid (PT) proteins to a minimal 63-nucleotide (nt) cis-acting instability element in its 3'-untranslated region. One of these proteins is adenosine-uridine-rich binding factor (AUF1), whose levels are not regulated in PT extracts from rats fed the different diets. However, two-dimensional gels showed posttranslational modification of AUF1 that included phosphorylation. There is no PT cell line, but in HEK 293 cells the 63-nt element is recognized as an instability element, and RNA interference for AUF1 decreased human PTH secretion in cotransfection experiments. Stably transfected cells with a chimeric GH gene containing the PTH 63-nt cis-acting element were used to study the signal transduction pathway that regulates AUF1 modification and chimeric gene mRNA stability. Cyclosporine A, the calcineurin inhibitor, regulated AUF1 posttranslationally, and this correlated with an increase in the stability of GH-PTH 63-nt mRNA but not of the control GH mRNA. Mice with genetic deletion of the calcineurin Abeta gene had markedly increased PTH mRNA levels that were still regulated by low calcium and phosphorus diets. Therefore, calcineurin regulates AUF1 posttranslationally in vitro and PTH gene expression in vivo but still allows its physiological regulation by calcium and phosphate.

3' Untranslated Regions↗

PubMed2004

Calcineurin Abeta is central to the expression of the renal type II Na/Pi co-transporter gene and to the regulation of renal phosphate transport.

The sensing and response to extracellular phosphate (Pi) concentration is preserved from prokaryotes to mammals and ensures an adequate supply of Pi in the face of large differences in its availability. In mammals, the kidneys are central to Pi homeostasis. Renal Pi reabsorption is mediated by a Na/Pi co-transporter that is regulated by a renal Pi sensing system and humoral factors. The signal transduction by which Pi regulates type II Na/Pi activity is largely unknown. It is shown that calcineurin inhibitors specifically and dramatically decrease type II Na/Pi gene expression in a proximal tubule cell line and in vivo. Mice with genetic deletion of the calcineurin Abeta gene had a marked decrease in type II Na/Pi mRNA levels and remarkably did not show the expected increase in type II Na/Pi mRNA levels after the challenge of a low-Pi diet. In contrast, the regulation of renal 25(OH)-vitamin D 1alpha-hydroxylase gene expression by Pi was intact. This is the first demonstration that calcineurin has a crucial role in the signal transduction pathway regulating renal Pi homeostasis both in vitro and in vivo. These results suggest that the use of calcineurin inhibitors contributes to the renal Pi wasting seen in renal transplant patients.

Animals↗

PubMed2004

Harnessing the parathyroids to create stronger bones.

PURPOSE OF REVIEW: Parathyroid hormone is anabolic to bone but when secreted in excess it is catabolic. It is important, therefore, to understand the mechanisms that determine the normal circadian rhythm of parathyroid hormone secretion and whether the cellular response to it will be anabolic or catabolic. This may lead to new strategies for the treatment of osteoporosis and the low turnover bone disease of some dialysis patients. RECENT FINDINGS: The parathyroid plays a central role in normal mineral and bone homeostasis by acting on its receptor, the PTH/PTHrP receptor (PTH1R). In fact there is more than one receptor - the PTH2 receptor and a putative carboxy-terminal PTH receptor. The latter, in particular, may be particularly relevant to our understanding of the role of parathyroid hormone and its use in pharmacology. Parathyroid hormone in excess destroys bone, as in most patients with chronic renal failure, and when it is lacking this may result in low turnover bone disease. At a more subtle level, patients with postmenopausal osteoporosis may have a blunting of the normal circadian rhythm of parathyroid hormone, with its peak at night and nadir in the morning. Insights as to what determines whether parathyroid hormone will be anabolic or catabolic to bone are reviewed. SUMMARY: Attempting to correct the circadian rhythm in osteoporotic patients by calcilytic drugs or perhaps physiological equivalents may have a role in the future in treating osteoporosis. In the meantime, the administration of recombinant parathyroid hormone is an effective agent in the management of osteoporosis.

Bone Diseases↗

PubMed2004

A form of Jansen's metaphyseal chondrodysplasia with limited metabolic and skeletal abnormalities is caused by a novel activating parathyroid hormone (PTH)/PTH-related peptide receptor mutation.

A novel heterozygous PTH/PTHrP receptor missense mutation (T410R) was identified in a male and his two sons who are all affected by a less severe form of Jansen's metaphyseal chondrodysplasia (JMC). JMC is a rare disorder that is typically characterized by severe growth plate abnormalities that lead to short-limbed dwarfism. Furthermore, affected individuals usually show significant hypercalcemia, despite normal or undetectable levels of PTH and PTHrP. In contrast, the three affected members of this new family showed only mild skeletal dysplasia, comparatively normal stature, and blood calcium concentrations either within or at the upper end of the normal range. However, PTH levels were suppressed, and urinary calcium excretion was elevated, which led to nephrolithiasis in both children. When expressed in COS-7 cells, the PTH/PTHrP receptor with the T410R mutation led to agonist-independent cAMP formation, which was less pronounced than that observed with the previously identified T410P mutant. Our findings indicate that a mild form of JMC has been identified that is characterized by less pronounced skeletal and laboratory abnormalities.

Adult↗

PubMed2003

The parathyroid hormone mRNA 3'-untranslated region AU-rich element is an unstructured functional element.

Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally by hypocalcemia and hypophosphatemia. This regulation is dependent upon binding of protective trans-acting factors to a specific element in the PTH mRNA 3'-untranslated region (UTR). We have previously demonstrated that a 63-nucleotide (nt) AU-rich PTH mRNA element is sufficient to confer regulation of RNA stability by calcium and phosphate in an in vitro degradation assay (IVDA). The 63-nt element consists of a core 26-nt minimal binding sequence and flanking regions. We have now studied the functionality of this element in HEK293 cells using reporter genes and showed that it destabilizes mRNAs for green fluorescent protein (GFP) and growth hormone, similar to its effect in the IVDA. To understand how the cis-element functions as an instability element, we have analyzed its structure by RNase H, primer extension, and computer modeling. The results indicate that the PTH mRNA 3'-UTR and in particular the region of the cis-element are dominated by significant open regions with little folded base pairing. Mutation analysis of the 26-nt core element demonstrated the importance of defined nucleotides for protein-RNA binding. In the GFP reporter system, the same mutations that prevented binding were also ineffective in destabilizing GFP mRNA in HEK293 cells. This is the first study of an AU-rich element that relates function to structure. The PTH mRNA 3'-UTR cis-acting element is an open region that utilizes the distinct sequence pattern to determine mRNA stability by its interaction with trans-acting factors.

3' Untranslated Regions↗

PubMed2003

Sensing phosphate across the kingdoms.

PURPOSE OF REVIEW: The present review summarizes recent findings that may help in understanding how the cell senses changes in serum phosphate. RECENT FINDINGS: The sensing of phosphate determines the organism's response to change in supply of this essential nutrient. Phosphate depletion or surfeit results in homeostatic responses that involve changes in transcription, transcript stability, transporter recruitment or breakdown, and cell replication. These responses are shared across the biological kingdoms, and lessons from unicellular organisms may be relevant to multicellular mammals. An understanding of nutrient sensing in general may help in determining how the cell senses changes in phosphate concentration. SUMMARY: Research has yielded important advances in unravelling phosphate sensing and the response to nutrient phosphate supply. However, the actual sensing event for phosphate and most other nutrients must still be defined. Lessons may be learned from those examples in which the sensing event is known, and these are summarized here.

Animals↗

PubMed2002

Characterization of cis-acting element in renal NaPi-2 cotransporter mRNA that determines mRNA stability.

Hypophosphatemia leads to an increase in Na(+)-P(i) cotransporter (NaPi-2) mRNA levels. This increase is posttranscriptional and correlates with a more stable transcript mediated by the terminal 698 nt of the NaPi-2 mRNA. A 71-nt binding element was identified with renal proteins from rats fed control and low-P(i) (-P(i)) diet. The binding of -P(i) renal proteins to this transcript was increased compared with control proteins. The functionality of the cis element was demonstrated by an in vitro degradation assay. -P(i) renal proteins stabilized transcripts that included the cis element compared with control renal extracts. The full-length NaPi-2 transcript, but not control transcripts, was stabilized by -P(i) extracts. Insertion of the binding element into green fluorescent protein (GFP) as a reporter gene decreased chimeric GFP mRNA levels in transfection experiments. Our results suggest that the protein-binding region of the NaPi-2 mRNA functions as a cis-acting instability element. In hypophosphatemia there is increased binding to the cis-acting element and subsequent stabilization of NaPi-2 mRNA.

Animals↗

PubMed2002

Cis and trans acting factors in the regulation of parathyroid hormone (PTH) mRNA stability by calcium and phosphate.

Calcium and phosphate regulate parathyroid hormone (PTH) mRNA stability through differences in binding of parathyroid proteins to an element in its 3'-untranslated region. One of the proteins is AUF1 (A+U-rich element binding factor 1). An in vitro degradation assay showed that transcripts for PTH and chimeric growth hormone (GH)-PTH 63 nt, but not for native GH, were stabilized by PT proteins from rats on low calcium diets and destabilized by proteins from rats on low phosphate diets, correlating with PTH mRNA levels in vivo. In transfection experiments the 63 nt binding element destabilized mRNAs of reporter genes and this was prevented by over-expression of AUF1. Our results identified a functional cis element in PTH mRNA. Differences in protein binding to this element determine PTH mRNA stability and its regulation by calcium and phosphate.

3' Untranslated Regions↗

PubMed2002

Pathogenesis of parathyroid dysfunction in end-stage renal disease.

The parathyroid functions to maintain normal calcium and phosphate homeostasis and is central to normal bone physiology. In end-stage renal disease (ESRD), there is a failure of these normal homeostatic mechanisms with the frequent development of secondary hyperparathyroidism, which contributes to the pathogenesis of renal bone disease. The phosphate retention of ESRD, together with the reduced serum calcium and 1,25-dihydroxycholecalciferol vitamin D(3) (1,25[OH](2)D(3)) concentrations are the known factors that determine the progression to secondary hyperparathyroidism. 1,25(OH)(2)D(3) markedly decreases parathyroid hormone (PTH) gene transcription, whereas the effects of calcium and phosphate are on PTH mRNA stability, PTH secretion, and parathyroid cell proliferation. The mechanisms of these effects are discussed in this review.

Calcium↗

PubMed2002

Mechanisms of secondary hyperparathyroidism.

Small decreases in serum Ca(2+) and more prolonged increases in serum phosphate (P(i)) stimulate the parathyroid (PT) to secrete parathyroid hormone (PTH), and 1,25(OH)(2)D(3) decreases PTH synthesis and secretion. A prolonged decrease in serum Ca(2+) and 1,25(OH)(2)D(3), or increase in serum P(i), such as in patients with chronic renal failure, leads to the appropriate secondary increase in serum PTH. This secondary hyperparathyroidism involves increases in PTH gene expression, synthesis, and secretion, and if chronic, to proliferation of the PT cells. Low serum Ca(2+) leads to an increase in PTH secretion, PTH mRNA stability, and PT cell proliferation. P(i) also regulates the PT in a similar manner. The effect of Ca(2+) on the PT is mediated by a membrane Ca(2+) receptor. 1,25(OH)(2)D(3) decreases PTH gene transcription. Ca(2+) and P(i) regulate the PTH gene posttranscriptionally by regulating the binding of PT cytosolic proteins, trans factors, to a defined cis sequence in the PTH mRNA 3'-untranslated region, thereby determining the stability of the transcript. PT trans factors and cis elements have been defined.

Animals↗