GAMMA INTERFERON REGULATION OF VITAMIN D ACTION

1，25-二羟基维生素D [1，25 D]，维生素D的激素形式是钙稳态的有效调节剂。 为了维持正常的血清钙，1，25 D通过抑制肾和肾外1 α-羟化酶和诱导24-羟化酶来严格控制其自身的血清水平。 在结节病和结核病中，1，25 D调节其合成和降解的能力丧失。 这种现象可以通过将正常人巨噬细胞暴露于γ-干扰素（γ-IFN）在体外再现。 γ-干扰素能显著促进1，25（OH）_2D_3的产生，但能拮抗1，25 D对1 α-和24-羟化酶的调节作用。 显然，γ- IFN损害1，25 D对其自身合成和催化的控制。为了阐明介导γ-IFN抑制1，25 D作用的机制，我们利用人单核细胞系THP-1。 THP-1细胞在1，25 D合成和响应1，25 D和γ-IFN的1 α-和24-羟化酶的调节中模拟人巨噬细胞。 我们重点研究了1，25 D对24-羟化酶的诱导作用。在THP-1细胞和正常单核细胞中，γ-IFN损害24-羟化酶mRNA的1，25 D诱导。 γ-IFN既不影响1，25 D与维生素D受体（VDR）的结合，也不影响24-羟化酶mRNA的稳定性，这表明γ- IFN可能直接损害1，25 D对24-羟化酶基因转录的诱导。 对γ-IFN的大多数应答需要Stat 1激活。 γ-IFN与其受体结合激活Janus激酶使Stat 1酪氨酸磷酸化。 然后Stat 1同源二聚化并易位到细胞核，在那里它直接与γ-IFN激活序列和核共激活因子CBP/p300相互作用以调节转录。 然而，最近的研究表明，γ-IFN转录激活通过Stat 1独立的途径。 为了诱导24-羟化酶的表达，1，25与胞质VDR结合，其易位至细胞核并与类维生素A X受体（RXR）异源二聚化。 VDR/RXR与人24-羟化酶启动子和核受体共激活子中的维生素D应答元件（VDRE）的相互作用增强基因转录。在THP-1细胞中，γ-IFN通过活性Stat 1与VDR/RXR的相互作用损害VDR/RXR与两个VDRE的结合，并降低VDRE处的1，25 D转录活性。 因此，我们推测，γ-IFN激活Stat 1（或Stat 1样蛋白）通过与VDR/RXR相互作用（损害VDRE结合）和与VDR/RXR竞争必需的核辅激活因子拮抗1，25 D诱导24-羟化酶。 此外，γ-IFN对1，25 D转录活性的拮抗作用导致炎症过程中1，25 D稳态异常。 为了验证这些假设，我们拟研究：（1）JAK-Stat 1和/或Stat 1非依赖性途径在γ-IFN抑制1，25 D诱导24-羟化酶基因转录中的作用;（2）γ- IFN拮抗VDR/RXR的蛋白-蛋白和蛋白-DNA相互作用，VDR/RXR在1，25 D转录活性中起关键作用;（3）γ- IFN/1，25 D对1 α-羟化酶表达的拮抗作用。

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.