The long term goal of our research is to understand more completely the relationship between cellular nutrition and glucocorticoid hormone action. Specifically we will continue to explore the modulatory role of the dietary cofactor Vitamin B6 (pyridoxal phosphate) in the mechanisms of glucocorticoid receptor action in cultured human cells. Findings made during the past granting period have revealed that physiological concentrations of Vitamin B6 present in most cell culture media suppress or attenuate the responsiveness of cells to glucocorticoids. Correspondingly, glucocorticoid receptor dependent gene transcription is enhanced by Vitamin B6 deficiency. These modulatory effects of Vitamin B6 occur without significant alterations in the cellular content of glucocorticoid receptor protein. Furthermore, we have presented evidence which demonstrates that glucocorticoid receptors are subject to pyridoxylation in vivo. Based on these observations, we propose to test the hypothesis that Vitamin B6 is a physiological modulator of steroid receptor dependent gene expression and, further, that the effects of Vitamin B6 result from a direct interaction of pyridoxal phosphate with the steroid receptor protein. To formally test this hypothesis we propose the following specific aims: I. To evaluate in detail the influence which Vitamin B6 has on both glucocorticoid receptor dependent and independent gene expression. II. To genetically identify at the amino acid level the functional pyridoxal phosphate binding sites on human glucocorticoid receptors. III. To engineer mutant human glucocorticoid receptors that bear altered pyridoxal phosphate binding sites and to evaluate the consequences of these mutations on steroid receptor mechanisms. Given the resources and technology currently available, we are now in a position to determine where pyridoxal phosphate interacts with steroid receptors in vivo and to elucidate the mechanism(s) by which this essential vitamin alters the ability of cells to respond to steroid hormones.
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