TRP14 inhibits osteoclast differentiation via its catalytic activity.

We previously reported the inhibitory role of thioredoxin-related protein of 14 kDa (TRP14), a novel disulfide reductase, in nuclear factor-κB (NF-κB) activation, but its biological function has remained to be explored. Here, we evaluated the role of TRP14 in the differentiation and function of osteoclasts (OCs), for which NF-κB and cellular redox regulation have been known to be crucial, using RAW 264.7 macrophage cells expressing wild-type TRP14 or a catalytically inactive mutant, as well as its small interfering RNA. TRP14 depletion enhanced OC differentiation, actin ring formation, and bone resorption, as well as the accumulation of reactive oxygen species (ROS). TRP14 depletion promoted the activation of NF-κB, c-Jun NH2-terminal kinase, and p38, the expression of c-Fos, and the consequent induction of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1), a key determinant of osteoclastogenesis. However, pretreatment with N-acetylcysteine or diphenylene iodonium significantly reduced the OC differentiation, as well as the ROS accumulation and NF-κB activation, that were enhanced by TRP14 depletion. Furthermore, receptor activator of NF-κB ligand (RANKL)-induced ROS accumulation, NF-κB activation, and OC differentiation were inhibited by the ectopic expression of wild-type TRP14 but not by its catalytically inactive mutant. These results suggest that TRP14 regulates OC differentiation and bone resorption through its catalytic activity and that enhancing TRP14 may present a new strategy for preventing bone resorption diseases.

A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement.

2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H(2)O(2)). 2-Cys Prxs are inactivated when their active site cysteine residue is hyperoxidized to sulfinic acid. Sulfiredoxin (Srx) is an enzyme that catalyzes reduction of hyperoxidized 2-Cys Prxs in the presence of ATP, Mg(2+), and thiol equivalent. Therefore, Srx activity is crucial for cellular function of 2-Cys Prxs. The method currently available for the determination of Srx activity relies on immunoblot detection using antibodies to hyperoxidized enzymes. Here we introduce a simple quantitative assay for Srx activity based on the colorimetric determination of inorganic phosphate released in Srx-dependent reduction of hyperoxidized Prx using the malachite green. The colorimetric assay was used for high-throughput screening of 25,000 chemicals to find Srx inhibitors.