Yu Minjun, Moreno Jose L, Stains Joseph P, Keegan Achsah D
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
J Biol Chem. 2009 Nov 20;284(47):32968-79. doi: 10.1074/jbc.M109.001016. Epub 2009 Sep 28.
Interleukin 4 (IL-4) inhibits receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast formation and functional activity in a STAT6-dependent manner. IL-4 down-regulates expression of tartrate-resistant acid phosphatase (TRAP) in mature osteoclasts. To determine whether IL-4 regulates TRAP promoter activity, RAW264.7 cells were transfected with a TRAP promoter-luciferase reporter. Treatment with IL-4 alone modestly enhanced TRAP luciferase activity. However, IL-4 suppressed the ability of RANKL to up-regulate TRAP-luciferase activity, suggesting that IL-4 has multiple effects on TRAP transcription. IL-4 also reduced the RANKL-induced association of RNA polymerase II with the TRAP gene in osteoclasts. The TRAP promoter contains a STAT6-binding motif, and STAT6 bound to the endogenous TRAP promoter after IL-4 treatment. To determine the impact of STAT6 binding, we transfected cells with STAT6VT, a constitutively active STAT6 mutant. STAT6VT alone up-regulated TRAP-luciferase activity; this effect was abrogated by mutating the STAT6 binding site in the minimal TRAP promoter. STAT6VT did not inhibit the potent up-regulation of TRAP promoter activity caused by overexpression of NFATc1, PU.1, and microphthalmia transcription factor, downstream targets of macrophage colony-stimulating factor and RANKL. IL-4 down-regulated the expression of c-Fos and NFATc1 in mature osteoclasts. Knockdown of NFATc1 by short interfering RNA caused TRAP expression to be down-regulated, and ectopic expression of NFATc1 abrogated the IL-4-induced down-regulation of TRAP. These results suggest that STAT6 plays two distinct roles in TRAP expression. The IL-4-induced activation of STAT6 mediates suppression of the RANKL-induced TRAP promoter activity indirectly by inhibiting NFATc1 expression. However, in the absence of RANKL and osteoclast differentiation, STAT6 binds the TRAP promoter after IL-4 treatment and directly enhances TRAP expression.
白细胞介素4（IL-4）以依赖STAT6的方式抑制核因子κB受体激活剂配体（RANKL）诱导的破骨细胞形成和功能活性。IL-4下调成熟破骨细胞中抗酒石酸酸性磷酸酶（TRAP）的表达。为了确定IL-4是否调节TRAP启动子活性，将RAW264.7细胞用TRAP启动子-荧光素酶报告基因转染。单独用IL-4处理适度增强了TRAP荧光素酶活性。然而，IL-4抑制了RANKL上调TRAP-荧光素酶活性的能力，表明IL-4对TRAP转录有多种作用。IL-4还减少了RANKL诱导的破骨细胞中RNA聚合酶II与TRAP基因的结合。TRAP启动子包含一个STAT6结合基序，并且在IL-4处理后STAT6与内源性TRAP启动子结合。为了确定STAT6结合的影响，我们用组成型活性STAT6突变体STAT6VT转染细胞。单独的STAT6VT上调TRAP-荧光素酶活性；通过在最小TRAP启动子中突变STAT6结合位点，这种作用被消除。STAT6VT不抑制由巨噬细胞集落刺激因子和RANKL的下游靶标NFATc1、PU.1和小眼转录因子过表达引起的TRAP启动子活性的强力上调。IL-4下调成熟破骨细胞中c-Fos和NFATc1的表达。用小干扰RNA敲低NFATc1导致TRAP表达下调，并且NFATc1的异位表达消除了IL-4诱导的TRAP下调。这些结果表明STAT6在TRAP表达中发挥两种不同的作用。IL-4诱导的STAT6激活通过抑制NFATc1表达间接介导对RANKL诱导的TRAP启动子活性的抑制。然而，在没有RANKL和破骨细胞分化的情况下，IL-4处理后STAT6与TRAP启动子结合并直接增强TRAP表达。
