GAMMA INTERFERON REGULATION OF VITAMIN D ACTION

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1,25-dihydroxyvitamin D [1,25D], the hormonal form of vitamin D is a potent regulator of calcium homeostasis. To maintain normal serum calcium, 1,25D tightly controls its own serum levels by suppressing renal and extrarenal 1alpha-hydroxylases and by inducing 24-hydroxylase. In sarcoidosis and tuberculosis, the capacity of 1,25D to regulate its synthesis and degradation is lost. This phenomenon can be reproduced in vitro by exposing normal human macrophages to gamma-interferon (gamma-IFN). Gamma- IFN markedly enhances 1,25(OH)2D3 production but antagonizes 1,25D regulation of 1alpha- and 24-hydroxylases. Clearly, gamma- IFN impairs 1,25D control of its own synthesis and catabolism. To clarify the mechanisms mediating gamma-IFN inhibition of 1,25D action, we utilized the human monocytic cell line THP-1. THP-1 cells mimic human macrophages in 1,25D synthesis and in the regulation of 1alpha- and 24-hydroxylases in response to 1,25D and gamma-IFN. We focused on 1,25D induction of 24-hydroxylase. In THP-1 cells and normal monocytes, gamma-IFN impairs 1,25D induction of 24-hydroxylase mRNA. Gamma-IFN does not affect either the binding of 1,25D to the vitamin D receptor (VDR) or the stability of the 24-hydroxylase mRNA suggesting that gamma- IFN may directly impair 1,25D-induction of 24-hydroxylase gene transcription. Most responses to gamma-IFN require Stat1 activation. Gamma-IFN binding to its receptor activates Janus kinases to tyrosine phosphorylate Stat1. Stat1 then homodimerizes and translocates to the nucleus where it interacts directly with a gamma-IFN activation sequence and with the nuclear co-activators CBP/p300 to regulate transcription. Recent studies, however, demonstrate gamma-IFN transcriptional activation through Stat1 independent pathways. To induce 24- hydroxylase expression, 1,25 binds to cytosolic VDR, which translocates to the nucleus and heterodimerizes with the retinoid X receptor (RXR). VDR/RXR interactions with both vitamin D responsive elements (VDREs) in the human 24-hydroxylase promoter and nuclear receptor co-activators enhance gene transcription. In THP-1 cells, gamma-IFN impairs VDR/RXR binding to both VDREs through interactions of active Stat1 with the VDR/RXR, and reduces 1,25D-transcriptional activity at the VDRE. We hypothesize, therefore, that gamma-IFN activation of Stat1 (or a Stat1 like protein) antagonizes 1,25D induction of 24-hydroxylase through both interactions with the VDR/RXR that impair VDRE binding and competition with the VDR/RXR for essential nuclear coactivators. Also, gamma-IFN antagonism on 1,25D transcriptional activity causes abnormal 1,25D homeostasis in inflammatory processes. To test these hypotheses, we propose to examine: (1) The contribution of JAK-Stat1 and/or Stat1- independent pathways to the inhibitory effects of gamma-IFN on 1,25D induction of 24-hydroxylase gene transcription; (2) gamma- IFN antagonism on protein-protein and protein-DNA interactions of VDR/RXR critical in 1,25D transcriptional activity; (3) gamma- IFN/1,25D antagonism on 1alpha-hydroxylase expression.