Complex Regulation of Tartrate-resistant Acid Phosphatase (TRAP) Expression by Interleukin 4 (IL-4)

Minjun Yu,Jose L Moreno,Joseph P Stains,Achsah D Keegan

doi:10.1074/jbc.m109.001016

Abstract

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.

Highlights

TRAP2 is a di-iron-containing metalloenzyme that is expressed in osteoclasts and in subsets of tissue macrophages and dendritic cells [1]
Interleukin 4 (IL-4) Down-regulates RANKL-induced tartrate-resistant acid phosphatase (TRAP) Expression in Mature Osteoclasts—We previously showed that IL-4 suppressed the formation of osteoclasts induced by the treatment of the RAW264.7 cell line with M-CSF and RANKL [29]
IL-4 did not have an effect on osteoclast numbers, we observed less TRAP staining in the RANK-RAW cells treated with IL-4 and RANKL compared with cells treated with RANKL alone (Fig. 1A)

Summary

Introduction

TRAP2 is a di-iron-containing metalloenzyme that is expressed in osteoclasts and in subsets of tissue macrophages and dendritic cells [1]. In the absence of RANKL and osteoclast differentiation, STAT6 binds the TRAP promoter after IL-4 treatment and directly enhances TRAP expression.

Results

Conclusion