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Regulation of parathyroid hormone gene expression by calcium and 1,25-dihydroxyvitamin D3

Hawa, Nigar Sultana; (1994) Regulation of parathyroid hormone gene expression by calcium and 1,25-dihydroxyvitamin D3. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The two major regulators of parathyroid hormone (PTH) synthesis are calcium and the steroid hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the aim of this study was to investigate the molecular mechanisms of regulation by these modulators. Low extracellular calcium (0.4 mM) had no effect on steady-state preproPTH mRNA levels, but increased preproPTH mRNA levels associated with membrane-bound polysomes by 200%. Actinomycin D did not abolish this rise in polysomal preproPTH mRNA but increased mRNA levels by 1.6-fold in cells incubated in 0.4 and 1.0 mM calcium. Sucrose density gradient ultracentrifugation demonstrated that low calcium had no effect on polysomal size and, in addition, there was no evidence of a pool of non-ribosomal preproPTH mRNA. These data indicate that low calcium regulates PTH synthesis post-transcriptionally possibly by increasing the apparent half-life of preproPTH mRNA on polysomes. The binding sites for the vitamin D3 receptor (VDR) were localised using Southwestern and gel mobility shift assays which indicated two binding sites within the -451 to -348 bp and -668 to -452 bp fragments of the bovine PTH gene. To investigate functional activity of these putative VDRE, plasmids containing fragments of the -668 to +50 bp region were linked to the reporter gene, chloramphenicol acetyltransferase (CAT). The effect of 1,25(OH)2D3 on these constructs was investigated by transient transfection of oppossum kidney cells. 1,25(OH)2D3 suppressed CAT activity of the construct containing the -668 to +50 bp fragment by 22%, of the -668 to -452 bp construct by 27% and of the -451 to -348 bp construct was reduced by 25%. However, 1,25(OH)2D3 did not affect the activity of the construct containing the -347 to +50 bp fragment. These functional assays confirm the presence of two binding sites within the region -485 to -348 bp. The study presented demonstrated that low calcium regulates PTH synthesis post-transcriptionally and VDR binding sites were localised on the bovine PTH gene.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Regulation of parathyroid hormone gene expression by calcium and 1,25-dihydroxyvitamin D3
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Gene expression; Parathyroid
URI: https://discovery.ucl.ac.uk/id/eprint/10107025
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