Abstract
We have examined the influence of intracellular vitamin B6 concentration on glucocorticoid receptor function in HeLa S3 cells transfected with a glucocorticoid-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid. CAT activity is induced from this plasmid specifically by glucocorticoid hormones in a glucocorticoid receptor-dependent manner. The intracellular concentration of pyridoxal phosphate, the physiologically active form of the vitamin, was elevated by supplementation of the culture medium with the synthesis precursor pyridoxine and lowered by exposure to the pyridoxal phosphate synthesis inhibitor 4-deoxypyridoxine. Analysis of glucocorticoid responsiveness revealed that elevated concentrations of intracellular pyridoxal phosphate suppressed the amount of glucocorticoid-induced CAT activity whereas moderate deficiency enhanced the level of glucocorticoid receptor-mediated gene expression. In contrast, modulation of the intracellular pyridoxal phosphate concentration had no effect on either basal CAT activity derived from cells not stimulated with dexamethasone or on CAT activity derived from two glucocorticoid-insensitive reporter plasmids. The modulatory effects of pyridoxal phosphate concentration occur without changes in glucocorticoid receptor mRNA levels, glucocorticoid receptor protein concentration, or the steroid binding capacity of the receptor. These observations demonstrate that vitamin B6 selectively influences glucocorticoid receptor-dependent gene expression through a novel mechanism that does not involve alterations in glucocorticoid receptor concentration or ligand binding capacity.
Highlights
We have examined the influence of intracellular vitamin Bs concentration on glucocorticoid receptor function in HeLa Sa cells transfected with a glucocorticoid-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid
Half-maximal induction of CAT activity is achieved at a dexamethasone concentration of 50 nM; this concentration is consistent with that reported for induction of mouse mammary tumor virus RNA [40, 41] and for regulation of enzymatic activity encoded by another glucocorticoid-responsive gene, alkaline phosphatase, in HeLa cells [26]
The intracellular concentration of vitamin Bs is known to influence two parameters of steroid receptors which are requisite for regulation of target gene expression, namely the subcellular localization of the receptor and its DNA binding capacity
Summary
We have examined the influence of intracellular vitamin Bs concentration on glucocorticoid receptor function in HeLa Sa cells transfected with a glucocorticoid-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid. DiSorbo and Litwack [20] have investigated the effect of vitamin Bs on the induction of tyrosine aminotransferase by glucocorticoids and have reported that the amount of hormone-induced tyrosine aminotransferase activity is slightly increased after restriction of the vitamin and is decreased following exposure to pharmacological doses of the vitamin Interpretation of these data, is complicated by the fact that tyrosine aminotransferase is itself a vitamin Bgdependent enzyme [21], and these studies relied on the detection of enzymatic activity. We have utilized a system for analyzing gene expression which is positively regulated by glucocorticoid hormones in a glucocorticoid receptor-dependent manner and demonstrate that alteration of the intracellular vitamin Bg concentration has profound effects on glucocorticoid receptor-mediated gene expression These findings provide direct evidence in support of a role for vitamin Bs in the mechanism(s) of glucocorticoid hormone action in uiuo
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