TRP14 inhibits osteoclast differentiation via its catalytic activity

Abstract

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.

FIG 1

Augmentation of RANKL-induced OC differentiation in TRP14-depleted cells. (A) NIH 3T3 cells were transfected for 60 h with empty vector (Mock) or one of three mouse TRP14 shRNA expression vectors (pSUPER-mTRP14-1, -2, and -3) by using FuGene6, and the cell lysates were subjected to immunoblot analysis using antibodies specific for TRP14 and β-actin. (B) RAW 264.7 cells were transfected with either an empty vector or pSuperior.puro-mTRP14 using an Amaxa transfection kit (reagent V) according to the manufacturer's manual. After 60 h, the cells were incubated in medium containing 1.5 μg/ml puromycin, and puromycin-resistant clones were isolated and subjected to immunoblot analysis using antibodies specific for TRP14 and β-actin. (C) RAW 264.7 cells stably transfected with either empty vector or pSuperior.puro-mTRP14 (line 7) were treated with 100 ng/ml of RANKL. At the indicated times, cells were stained with TRAP solution (left), and TRAP-positive multinucleated cells containing more than 3 nuclei (TRAP⁺ MNCs) were counted (right). Scale bars, 200 μm. Data represent means ± SD (n = 3). *, P < 0.05; **, P < 0.005.

FIG 2

Promotion of RANKL-induced actin ring formation and bone resorption in TRP14-depleted cells. (A) After incubation of RAW 264.7 cells with 100 ng/ml RANKL for 5 days, the cells were fixed with 3.7% formaldehyde solution in PBS, permeabilized with 0.1% Triton X-100, and incubated with Alexa Fluor 488-phalloidin for 20 min. After washing with PBS, cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 2 min and then photographed under a fluorescence microscope. Scale bars, 200 μm. (B) The RAW 264.7 cells on dentine discs were incubated with RANKL (100 ng/ml) for 9 days. (Left) The cells were removed from the dentine discs, and the resorption pits were visualized by staining with hematoxylin. Scale bars, 100 μm. (Right) The values for pit area represent means ± SD (n = 4). *, P < 0.005.

FIG 3

Reduction of the increased intracellular ROS level and OC differentiation in TRP14-depleted cells by antioxidant. (A) RAW 264.7 cells were treated with RANKL (100 ng/ml) for 10 min, washed with α-MEM lacking phenol red, and then incubated with 5 μM 5-(and-6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate in the dark for 5 min. (Left) The cells were analyzed with a flow cytometer. R indicates RANKL treatment. (Right) The values represent means ± SD (n = 4). *, P < 0.005. (B) RAW 264.7 cells were incubated with RANKL (100 ng/ml) in the presence of 5 mM NAC or 0.1 mM DPI for 4 days. The cells were stained for TRAP (left), and the TRAP-positive MNCs containing more than 3 nuclei were counted (right) as described in the legend to Fig. 1C. Scale bar, 200 μm. The values are means ± SD (n = 3). Con, control; **, P < 0.0005. (C) After preincubation of RAW 264.7 cells with 5 mM NAC or 0.1 mM DPI for 60 min, the cells were treated with 100 ng/ml of RANKL for 10 min and the intracellular ROS levels were measured as described for panel A. The values represent means ± SD (n = 4). **, P < 0.0005.

FIG 4

Augmentation of RANKL-induced NF-κB activation by TRP14 depletion. (A) RAW 264.7 cells were exposed to RANKL (100 ng/ml) for the indicated times, and the cell lysates were subjected to immunoblot analysis using antibodies specific for phospho-IκBα (p-IκBα) and IκBα (top). The chemiluminescence signals for phospho-IκBα and IκBα were quantified and normalized based on the signals of β-actin (bottom). The values represent means ± SD (n = 3). *, P < 0.05. (B) RAW 264.7 cells were incubated with RANKL (100 ng/ml) for 10 min and subjected to nuclear fractionation as described in Materials and Methods. The proteins (10 μg) in the cytosolic and nuclear fraction were analyzed with antibodies specific to p65. Tubulin and TATA binding protein (TBP) were used as markers for the cytosol and nucleus, respectively. (C) RAW 264.7 cells were incubated with RANKL (100 ng/ml) for 4 h. Total RNA was extracted from the cells and used in real-time PCR to quantify the mRNA levels of SOD2 and COX2 genes. The relative levels of individual mRNAs were normalized to those of β-actin mRNA and are presented as fold induction values. The values represent means ± SD (n = 3). **, P < 0.001; ***, P < 0.0005. (D) After preincubation of RAW 264.7 cells with 5 mM NAC or 100 nM DPI for 60 min, the cells were treated with 100 ng/ml of RANKL for 6 min. The cell lysates were subjected to immunoblot analysis as described for panel A.

FIG 5

Enhancement of RANKL-induced MAPK activation by TRP14 depletion. (A) RAW 264.7 cells were exposed to 100 ng/ml of RANKL for the indicated times. The cell lysates were then subjected to immunoblot analysis using antibodies against phospho-JNK and JNK1, phospho-ERK and ERK2, or phospho-p38 and p38, and representative immunoblots are shown. (B) The chemiluminescence signals for phospho-JNK, phospho-ERK, and phospho-p38 were quantified and normalized based on the signals of JNK1, ERK2, and p38, respectively. The values represent means ± SD (n = 3). *, P < 0.05; **, P < 0.001. (C) After preincubation of RAW 264.7 cells with 5 mM NAC or 0.1 mM DPI for 60 min, the cells were treated with 100 ng/ml RANKL for 15 min. The cell lysates were subjected to immunoblot analysis as described for panel A.

FIG 6

Increase of RANKL-induced expression of c-Fos and NFATc1 in TRP14-depleted cells. (A) RAW 264.7 cells were incubated with RANKL (100 ng/ml) for 8 h (c-Fos), 1 day (TRAF6), or 2 days (NFATc1, MMP9, and CK). Total RNA was extracted from the cells and used in real-time PCR to quantify the mRNA levels. The relative levels of individual mRNAs were normalized to those of β-actin mRNA and are presented as fold induction values. The values represent means ± SD (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.0005. (B and C) RAW 264.7 cells were treated with 100 ng/ml of RANKL for the indicated times. Immunoblot analysis of c-Fos (B) and NFATc1 (C) was performed. (D) RAW 264.7 cells were incubated with RANKL (100 ng/ml) for 3 days. TRAP activity was determined as described in Materials and Methods. The values represent means ± SD (n = 3). *, P < 0.05. (E and F) After preincubation of RAW 264.7 cells with 5 mM NAC or 50 nM DPI for 60 min, the cells were treated with 100 ng/ml of RANKL for 8 h (E) or 2 days (F). The cell lysates were subjected to immunoblot analysis of c-Fos and NFATc1.

FIG 7

Effects of ectopic expression of wild-type TRP14 or its catalytically inactive mutant on RANKL-induced OC differentiation of RAW 264.7 cells. (A) RAW 264.7 cells were transfected with pCR3.1 vector (Mock), pCR-mTRP14 (WT), or pCR-mTRP14-C43S (C43S), and G418-resistant cell lines were isolated. The abundance of TRP14 was assessed by immunoblot analysis. (B and C) Cells stably transfected with pCR3.1 vector (Mock), pCR-mTRP14 (line 9), or pCR-mTRP14-C43S (line 40) were treated with 100 ng/ml of RANKL. At the indicated times, cells were stained with TRAP solution (B, left), TRAP-positive multinucleated cells containing more than 3 nuclei (TRAP⁺ MNCs) were counted (B, right), and TRAP activity was measured (C). The values represent means ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.005. (D) RAW 264.7 cells were treated with RANKL (100 ng/ml) for 10 min, washed with α-MEM lacking phenol red, and incubated with 5 μM 5-(and-6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate in the dark for 5 min. The cells were analyzed with a flow cytometer. The values represent means ± SD (n = 3). *, P < 0.01; **, P < 0.0005. (E) RAW 264.7 cells were treated with 100 ng/ml of RANKL for 6 min, and the cell lysates were subjected to immunoblot analysis using antibodies specific for phospho-IκBα and IκBα.