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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Endocrinology Study Section (END)
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
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Tully, D B; Allgood, V E; Cidlowski, J A (1994) Modulation of steroid receptor-mediated gene expression by vitamin B6. FASEB J 8:343-9
Tully, D B; Cidlowski, J A (1993) Protein-blotting procedures to evaluate interactions of steroid receptors with DNA. Methods Enzymol 218:535-51
Allgood, V E; Oakley, R H; Cidlowski, J A (1993) Modulation by vitamin B6 of glucocorticoid receptor-mediated gene expression requires transcription factors in addition to the glucocorticoid receptor. J Biol Chem 268:20870-6
Silva, C M; Cidlowski, J A (1992) The effect of oxidation/reduction on the charge heterogeneity of the human glucocorticoid receptor. J Steroid Biochem Mol Biol 41:1-10
Allgood, V E; Cidlowski, J A (1992) Vitamin B6 modulates transcriptional activation by multiple members of the steroid hormone receptor superfamily. J Biol Chem 267:3819-24
Burnstein, K L; Cidlowski, J A (1992) The down side of glucocorticoid receptor regulation. Mol Cell Endocrinol 83:C1-8
Burnstein, K L; Bellingham, D L; Jewell, C M et al. (1991) Autoregulation of glucocorticoid receptor gene expression. Steroids 56:52-8
Burnstein, K L; Jewell, C M; Cidlowski, J A (1991) Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized human glucocorticoid receptor. Mol Endocrinol 5:1013-22
Allgood, V E; Powell-Oliver, F E; Cidlowski, J A (1990) Vitamin B6 influences glucocorticoid receptor-dependent gene expression. J Biol Chem 265:12424-33
Tully, D B; Cidlowski, J A (1990) Affinity of interactions between human glucocorticoid receptors and DNA: at physiologic ionic strength, stable binding occurs only with DNAs containing partially symmetric glucocorticoid response elements. Biochemistry 29:6662-70

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