Inhibition of NFAM1 suppresses phospho-SAPK/JNK signaling during osteoclast differentiation and bone resorption

Abstract

We have recently demonstrated NFAT activating protein with ITAM motif 1 (NFAM1) signaling increases osteoclast (OCL) formation/bone resorption associated with the Paget's disease of bone, however, the underlying molecular mechanisms of the NFAM1 regulation of OCL differentiation and bone resorption remains unclear. Here, we showed that RANK ligand stimulation enhances NFAM1 expression in preosteoclast cells. Conditioned media collected from RANKL stimulated RAW264.7 NFAM1 knockdown (KD) stable cells showed inhibition of interleukin-6 (2.5-fold), tumour necrosis factor-α (2.2-fold) and CXCL-5 (3-fold) levels compared to wild-type (WT) cells. Further, RANKL stimulation significantly increased p-STAT6 expression (5.5-fold) in WT cells, but no significant effect was observed in NFAM1-KD cells. However, no changes were detected in signal transducer and activator of transcription 3 levels in either of cell groups. Interestingly, NFAM1-KD suppressed the RANKL stimulated c-fos, p-c-Jun and c-Jun N-terminal kinase (JNK) activity in preosteoclasts. We further showed that the suppression of JNK activity is through inhibition of p-SAPK/JNK in these cells. In addition, NFATc1 expression, a critical transcription factor associated with osteoclastogenesis is significantly inhibited in NFAM1-KD preosteoclast cells. Interestingly, NFAM1 inhibition suppressed the OCL differentiation and bone resorption capacity in mouse bone marrow cell cultures. We also demonstrated inhibition of tartrate-resistant acid phosphatase expression in RANKL stimulated NFAM1-KD preosteoclast cells. Thus, our results suggest that NFAM1 control SAPK/JNK signaling to modulate osteoclast differentiation and bone resorption.

Keywords: JNK activity; NFAM1; NFATc1; Osteoclast; c-fos; p-SAPK/JNK.

Figure 1:. RANKL enhances NFAM1 expression in preosteoclast cells.

(A) RAW264.7 cells stimulated with RANKL (80 ng/mL) for 5 days, cell lysates obtained on each day was analyzed by western blot for NFAM1 expression. (B) RAW264.7 cells stimulated with RANKL (0–80 ng/mL) for 48 h. Total cell lysates obtained were subjected to immunoblotting for NFAM1 expression. β-actin was used as a loading control. (C&D) Total RNA isolated was subjected to quantitative real-time PCR analysis of NFAM1 gene expression. (E&F) RAW264.7 cells transduced with GIPZ Lentiviral shRNA particles against NFAM1 (NFAM1-KD) or empty vector (WT) and multiple selections were done for neomycin (500 μg/mL) resistance. Total RNA isolated was subjected to quantitative real-time PCR analysis of NFAM1 gene expression. GAPDH amplification was normalized with a relative level of mRNA expression. The bar graph represents triplicate of experiments and data shown as mean ± SD (*p<0.05).

Figure 2:. NFAM1 deficiency inhibit cytokines production and p-STAT6 expression.

(A-C)RAW264.7 WT&NFAM1-KD cells were stimulated with and without RANKL (80 ng/mL) for 48 h. The conditioned media obtained were measured for the levels of IL-6, TNF-α and CXCL5 by ELISA. Data are shown as mean ± SD (*p<0.05) in triplicate experiments. (D) Total cell lysates obtained were subjected to immunoblotting for STAT3,6 & p-STAT3,6 expression. β-actin was used as a loading control.

Figure 3:. NFAM1 knock-down (NFAM1-KD) inhibits p-SAPK/JNK and c-fos expression.

(A&D) Immunoprecipitation analysis for JNK activity and p-c-Jun was determined in the WT & NFAM1-KD cells stimulated with RANKL for 48 h. Mouse IgG1 was used as control. (B&C) RAW264.7 WT & NFAM1-KD cells were stimulated with RANKL (80 ng/mL) for 48 h. Total cell lysates obtained were subjected to immunoblotting for c-Jun, c-fos expression and p-SAPK/JNK expression. β-actin was used as a loading control.

Figure 4:. NFAM1-KD suppress NFATc1 expression.

(A) RAW264.7 WT&NFAM1-KD cells were stimulated with RANKL (80 ng/mL) for 48 h. Total cell lysates obtained were subjected to immunoblotting for NFATc1 & c2 expression. β-actin was used as a loading control. (B) Total RNA isolated was subjected to quantitative real-time PCR analysis of NFATc1 gene expression. GAPDH amplification was normalized with a relative level of mRNA expression.

Figure 5:. NFAM1 inhibition suppresses osteoclast (OCL) formation and bone resorption.

(A) Mouse bone marrow derived non-adherent monocytes from control WT & shRNA inhibition of NFAM1 were stimulated with RANKL (80 ng/mL) and M-CSF (10 ng/mL) for 7 days. TRAP positive multinucleated (MNC) OCLs formed in these cultures were scored (upper panel). Scale bar 50 μM. Mouse bone marrow derived non-adherent monocytes from WT&NFAM1 shRNA transduced cells were cultured on dentine slices for 10 days in the presence of RANKL & M-CSF. The percentage of resorbed areas was calculated relative to the total dentine area. Scale bar 50 μM (lower panel) (A-i&ii) Data shown in bar graphs represent triplicate experiments as mean ± SD (*p<0.05) for TRAP+ MNC formed per well and per cent bone resorption area, respectively. (B) WT&NFAM1-KD preosteoclast cells were stimulated with RANKL (80 ng/mL) for 48 h. Total cell lysates obtained were subjected to immunoblotting for TRAP expression. β-actin was used as a loading control.

Figure 6:. Schematic illustration of NFAM1 functional role in osteoclast formation and bone resorption.

NFAM1 knock-down (NFAM1-KD) suppresses p-SAPK/JNK, AP-1 (c-fos and p-c-Jun) transcription factor complex, NFATc1, p-STAT6, TRAP gene expression, osteoclast formation and bone resorption. PLC-γ: phospholipase γ; CN: calcineurin. Dotted lines represent NFAM1-KD suppression of RANKL signaling molecules. Red arrow represents the down- regulation of gene expression.