GAMMA INTERFERON REGULATION OF VITAMIN D ACTION

1,25-二羟基维生素D [1,25D]，激素形式的维生素D是钙稳态的有效调节剂。为了维持正常的血清钙，1,25 d通过抑制肾脏和肾外的1 α -羟化酶以及诱导24-羟化酶来严格控制自身的血清钙水平。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的人巨噬细胞暴露于γ -干扰素（γ - ifn）在体外重现。γ - IFN显著增强1,25(OH)2D3的产生，但拮抗1,25 d对1 α -和24-羟化酶的调节。显然，γ - IFN损害了1,25 d对自身合成和分解代谢的控制。为了阐明γ - ifn抑制1,25 d作用的机制，我们利用人类单核细胞系THP-1。THP-1细胞在1,25 d和γ - ifn的作用下模拟人巨噬细胞的1,25 d合成和1 α -和24-羟化酶的调节。我们主要研究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可损害1,25 d对24-羟化酶mRNA的诱导。γ -IFN既不影响1,25D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1,25D诱导的24-羟化酶基因转录。大多数对γ - ifn的反应需要激活Stat1。γ - ifn与其受体结合激活Janus激酶，使酪氨酸磷酸化Stat1。Stat1随后同二聚并易位到细胞核，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的转录激活是通过Stat1独立通路进行的。为了诱导24-羟化酶的表达，1,25与细胞质VDR结合，VDR易位到细胞核并与类视黄醇X受体（RXR）异二聚。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响应元件（VDREs）相互作用可增强基因转录。在THP-1细胞中，γ - ifn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1,25 d转录活性。因此，我们假设，γ - ifn激活Stat1（或Stat1样蛋白）通过与VDR/RXR的相互作用（损害VDRE结合）和与VDR/RXR竞争必需的核共激活因子，拮抗1,25 d诱导的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，我们建议研究：(1)JAK-Stat1和/或Stat1独立通路对γ - ifn抑制1,25 d诱导24-羟化酶基因转录的作用的贡献；(2) γ - IFN对VDR/RXR蛋白-蛋白和蛋白- dna相互作用的拮抗作用对1,25 d转录活性至关重要；(3) γ - IFN/ 1,25d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.