SLAT negatively regulates RANKL-induced osteoclast differentiation - PubMed (original) (raw)

SLAT negatively regulates RANKL-induced osteoclast differentiation

Bang Ung Youn et al. Mol Cells. 2013 Sep.

Abstract

RANKL induces the formation of osteoclasts, which are responsible for bone resorption. Herein, we investigated the role of SWAP-70-like adapter of T cells (SLAT) in RANKL-induced osteoclastogenesis. Expression levels of SLAT were reduced during RANKL-induced osteoclastogenesis. Overexpression of SLAT in BMMs inhibited TRAP-positive multinuclear osteoclast formation and attenuated the expression of NFATc1, which is an important modulator in osteoclastogenesis. Furthermore, silencing of SLAT by RNA interference enhanced osteoclast formation as well as NFATc1 expression. In addition, SLAT was involved in RANKL-induced JNK activation in osteoclasts. Taken together, our data suggest that SLAT acts as a negative modulator of RANKL-induced osteoclastogenesis.

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Figures

Fig. 1.

Fig. 1.

SLAT gene expression during osteoclastogenesis. (A, B) BMMs were cultured with M-CSF and RANKL for the indicated times. Total RNA was collected at each time point. (A) RT-PCR was performed to detect expression of the indicated genes. (B) Real-time PCR analysis was performed for the expression of SLAT and GAPDH. (C, D) BMMs were cultured with M-CSF and RANKL for 2 days in the presence of various inhibitors; Mock (DMSO), BAY (20 μM), LY294002 (10 μM), PD98059 (20 μM), SB203580 (20 μM), SP600125 (5 μM), TPCK (5 μM). RT-PCR (C) and Real-time PCR (D) were performed for the expression of SLAT and GAPDH.

Fig. 2.

Fig. 2.

Overexpression of SLAT in BMMs inhibits osteoclast formation. (A, B) BMMs were transduced with pMX-IRES-EGFP (control) or SLAT retrovirus and cultured for 4 days with M-CSF alone or M-CSF and various concentrations of RANKL as indicated. (A) Cultured cells were fixed and stained for TRAP. (B) Numbers of TRAP+ multinucleated cells were counted (#P < 0.05, *P < 0.01, ** P < 0.005 vs Control. ns, not significant). (C-F) BMMs were transduced with pMX-IRES-EGFP (control) or SLAT retrovirus and cultured with MCSF and RANKL for the indicated times. Realtime PCR analysis was performed to detect the indicated genes in control or SLAT-over-expressing samples.

Fig. 3.

Fig. 3.

Silencing of SLAT enhances osteoclast formation and induction of NFATc1. (A) BMMs transfected with GFP or SLAT siRNA were cultured for 2 days in the absence or presence of RANKL. RTPCR was performed. (B) BMMs transfected with GFP or SLAT siRNA were cultured for 5 days in the presence of M-CSF and RANKL. Cultured cells were fixed and stained for TRAP. (C) TRAP(+) MNCs were counted as osteoclasts. (D-G) BMMs transfected with GFP or SLAT siRNA were cultured with M-CSF and RANKL for the indicated times. Real-time PCR analysis was performed with the primers specific for SLAT, NFATc1, OSCAR, TRAP, and GAPDH (control). #P < 0.05, *P < 0.01 vs control.

Fig. 4.

Fig. 4.

SLAT is involved in RANKL-induced JNK activation. (A) BMMs were transduced with control (pMX-IRES-EGFP) or SLAT retrovirus. (B) BMMs transfected with GFP or SLAT siRNA were cultured for 2 days in the presence of M-CSF. (A, B) Cells were stimulated with RANKL (500 ng/ml) for the indicated times. Whole cell extracts were subjected to Western blot analysis with specific antibodies as indicated. (C, D) BMMs transfected with control or SLAT siRNA were cultured with M-CSF and RANKL in the absence or presence of SP600125 (5 μM). (C) Cultured cells were fixed and stained for TRAP. (D) Real-time PCR analysis was performed with the primers specific for NF ATc1 and G APDH. * P < 0.05 vs control.

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