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

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.