GAMMA INTERFERON REGULATION OF VITAMIN D ACTION

1，25-二羟基维生素D[1，25D]，维生素D的激素形式是钙稳态的有效调节剂。为了维持正常的血钙，1，25D通过抑制肾和肾外的1α-羟基酶和诱导24-羟基酶来严格控制自己的血清水平。在结节病和结核病中，1，25D调节其合成和降解的能力丧失。这种现象可以在体外通过将正常的人巨噬细胞暴露于伽马-干扰素(伽马-干扰素)来复制。γ-干扰素显著增强1，25(OH)2D3的产生，但拮抗1，25D对1α和24-羟基酶的调节。显然，γ-干扰素损害了对自身合成和分解代谢的1，25D控制。为了阐明γ-干扰素抑制1，25D作用的机制，我们利用了人单核细胞系THP-1。THP-1细胞模拟人巨噬细胞1，25D的合成，以及对1，25D和伽马-干扰素反应的1α和24-羟基酶的调节。重点研究了1，25D对24-羟基酶的诱导作用。在THP-1细胞和正常单核细胞中，γ-干扰素可抑制1，25D对24-羟基酶mRNA的诱导。γ-干扰素既不影响1，25D与维生素D受体(VDR)的结合，也不影响24-羟基酶基因转录的稳定性，提示γ-干扰素可能直接削弱1，25D诱导的24-羟基酶基因转录。大多数对γ-干扰素的反应需要STAT1的激活。γ-干扰素与其受体结合可激活Janus激酶，使STAT1酪氨酸磷酸化。然后，STAT1同源二聚体并移位到细胞核，在那里它直接与伽马-干扰素激活序列相互作用，并与核共激活因子CBP/p300相互作用来调节转录。然而，最近的研究表明，γ-干扰素的转录激活是通过STAT1非依赖的途径实现的。为了诱导24-羟基酶的表达，1，25与胞浆中的VDR结合，后者转位到细胞核，并与维甲酸X受体(RXR)异源二聚体。VDR/RXR与人类24-羟基酶启动子和核受体共激活子中的维生素D反应元件(VDRE)相互作用促进基因转录。在THP-1细胞中，γ-干扰素通过活性STAT1与VDR/RXR的相互作用损害VDR/RXR与两个VDRE的结合，并降低VDRE的1，25D转录活性。因此，我们假设，STAT1(或STAT1样蛋白)的伽马-干扰素激活通过与VDR/RXR的相互作用拮抗1，25D对24-羟基酶的诱导，从而削弱VDRE的结合，并与VDR/RXR竞争基本的核共激活因子。此外，γ-干扰素对1，25D转录活性的拮抗作用导致炎症过程中异常的1，25D动态平衡。为了验证这些假说，我们建议检验：(1)JAK-STAT1和/或STAT1非依赖途径对伽玛-干扰素抑制1，25D诱导的24-羟基酶基因转录的作用；(2)伽玛-干扰素对1，25D转录活性关键的VDR/RXR的蛋白质-蛋白质和蛋白质-DNA相互作用的拮抗作用；(3)伽玛-干扰素/1，25D拮抗1α-羟基酶的表达。

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.