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. 2017 Aug:101:236-244.
doi: 10.1016/j.bone.2017.05.013. Epub 2017 May 12.

NFAM1 signaling enhances osteoclast formation and bone resorption activity in Paget's disease of bone

Yuvaraj Sambandam  1 , Kumaran Sundaram  1 , Takamitsu Saigusa  2 , Sundaravadivel Balasubramanian  3 , Sakamuri V Reddy  4
Affiliations

NFAM1 signaling enhances osteoclast formation and bone resorption activity in Paget's disease of bone

Yuvaraj Sambandam et al. Bone. 2017 Aug.

Abstract

Paget's disease of bone (PDB) is marked by the focal activity of abnormal osteoclasts (OCLs) with excess bone resorption. We previously detected measles virus nucleocapsid protein (MVNP) transcripts in OCLs from patients with PDB. Also, MVNP stimulates pagetic OCL formation in vitro and in vivo. However, the mechanism by which MVNP induces excess OCLs/bone resorption activity in PDB is unclear. Microarray analysis identified MVNP induction of NFAM1 (NFAT activating protein with ITAM motif 1) expression. Therefore, we hypothesize that MVNP induction of NFAM1 enhances OCL differentiation and bone resorption in PDB. MVNP transduced normal human PBMC showed an increased NFAM1 mRNA expression without RANKL treatment. Further, bone marrow cells from patients with PDB demonstrated elevated levels of NFAM1 mRNA expression. Interestingly, shRNA suppression of NFAM1 inhibits MVNP induced OCL differentiation and bone resorption activity in mouse bone marrow cultures. Live cell widefield fluorescence microscopy analysis revealed that MVNP induced intracellular Ca2+ oscillations and levels were significantly reduced in NFAM1 suppressed preosteoclasts. Further, western blot analysis demonstrates that shRNA against NFAM1 inhibits MVNP stimulated PLCγ, calcineurin, and Syk activation in preosteoclast cells. Furthermore, NFAM1 expression controls NFATc1, a critical transcription factor expression and nuclear translocation in MVNP transuded preosteoclast cells. Thus, our results suggest that MVNP modulation of the NFAM1 signaling axis plays an essential role in pagetic OCL formation and bone resorption activity.

Keywords: Bone resorption; MVNP; NFAM1; Osteoclast; Paget's disease of bone.

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Figures

Fig. 1
Fig. 1
MVNP enhances NFAM1mRNA expression. (A) Human peripheral blood monocytes (PBMC) were transduced with EV and MVNP expression plasmids. Cells were stimulated with M-CSF (10 ng/mL) and RANKL (100 ng/mL) for 48 h. Total RNA isolated was subjected to real-time RT-PCR analysis for NFAM1 mRNA expression. The relative level of mRNA expression was normalized by GAPDH amplification in these cells. (B) Human PBMC were transduced with EV or MVNP and stimulated with RANKL (100 ng/mL) for 48 h. Total cell lysates were subjected to western blot analysis for NFAM1 expression using specific antibody. β-actin expression served as control. NFAM1 expression in PDB patients' bone marrow cells. (C) Total RNA isolated from normal and PDB patient's (n= 8) bone marrow cells were subjected to real-time RT-PCR analysis for NFAM1 expression. The relative level of mRNA expression was normalized by GAPDH amplification in these cells. Each bar represents the mean ± SD of three independent experiments (P < 0.05).
Fig. 2
Fig. 2
NFAM1 shRNA inhibits MVNP stimulated osteoclast differentiation and bone resorption activity. (A) Mouse bone marrow derived non-adherent cells (1.5 × 106/mL) were transduced with EV, EV + NFAM1 shRNA, MVNP and MVNP + NFAM1 shRNA and stimulated with RANKL (100 ng/mL) and M-CSF (10 ng/mL) for 7 days. TRAP-positive multinucleated OCLs formed in these cultures were scored. Each bar represents the mean ± SD of three independent experiments (P < 0.05). (B) Mouse bone marrow derived non-adherent cells (1.5 × 106/mL) transduced with EV/MVNP with and without NFAM1 shRNA were cultured on dentine slices in the presence of M-CSF and RANKL for 10 days. The percentage of the resorbed area was calculated relative to the total dentine area. Each bar represents the mean ± SD of three independent experiments (P < 0.05). *Compared to EV transduced culture; #Compared to MVNP transduced culture. (C) Vitamin D3 stimulation of NFAM1 expression in preosteoclast cells. RAW 264.7 cells were treated with different concentration (0–5 ng/mL) of vitamin D3 [1,25 (OH)2D] for 24 h. Total RNA isolated from these cells were subjected to real-time RT-PCR analysis for NFAM1 expression. The relative level of mRNA expression was normalized by GAPDH amplification. Each bar represents the mean ± SD of three independent experiments (P < 0.05). *Compared to unstimulated cells.
Fig. 3
Fig. 3
NFAM1mediates RANKL induced intracellular Ca2+ levels in MVNP transduced preosteoclast cells. RAW 264.7 cells transduced with EV,MVNP and MVNP+NFAM1 shRNA were cultured on 30 mm clear glass bottom plate and were serum-starved for 1 h. Calcium (Ca2+) levels were analyzed by live cell widefield microscopy and fluorescence was continuously recorded for 20 min after addition of RANKL (50 ng/mL) at 340 and 380 nm emission. (A) Immunofluorescence image of RAW 264.7 cells loaded with Fura-2 for 30 min (in green). (B) Representative time-lapse changes (20 min) in calcium level (Δ340/380 ratio) after RANKL treatment given at baseline (Time 0). (C) Changes in Ca2+ level from baseline in each group, after RANKL treatment (20 min). Each bar represents the mean ± SD of three independent experiments (*P < 0.05).
Fig. 4
Fig. 4
NFAM1modulation of signaling molecules in MVNP transduced preosteoclast cells. (A) RAW246.7 cells were transduced with EV, EV + NFAM1 shRNA, MVNP or MVNP + NFAM1 shRNA and stimulated with M-CSF (10 ng/mL) and RANKL (100 ng/mL) for 60 min. Total-cell lysates were subjected to western blot analysis for p-Syk and Syk. The band intensity was quantified by NIH ImageJ. The relative band intensity was normalized to Syk expression and compared with EV. (B)Mouse bone marrow derived non-adherent cells transduced with EV or MVNP and stimulated with RANKL (100 ng/mL) for 60min. Total-cell lysates were subjected to western blot analysis for p-Syk expression using specific antibody. Syk expression served as control. (C) Confocal microscopy analysis for p-Syk expression in RAW cells. Cells were transduced EV, EV + NFAM1 shRNA,MVNP, MVNP + NFAM1 shRNA and stimulated with M-CSF (10 ng/mL) and RANKL (100 ng/mL) for 60 min. Immunostaining for p-Syk as detected by Alexa 568–conjugated anti-rabbit antibody. Nuclear staining was performed with DRAQ5. EV, MVNP and MVNP + NFAM1 shRNA transduced preosteoclast cells stimulated with M-CSF (10 ng/mL) and RANKL (100 ng/mL) for 24 h. Total-cell lysates were subjected to western blot analysis for (D) PLCγ and (F) calcineurin expression. The relative band intensity was normalized to β-actin expression and compared with EV. (E) Mouse bone marrow derived non-adherent cells transduced with EV or MVNP and stimulated with RANKL (100 ng/mL) for 24 h. Total-cell lysates were subjected to western blot analysis for using an antibody against PLCγ. β-actin expression served as control.
Fig. 5
Fig. 5
NFAM1 shRNA suppresses MVNP induced NFATc1 expression and nuclear localization. (A) RAW 246.7 cells were transduced with EV, MVNP and MVNP + NFAM1 shRNA and stimulated with M-CSF (10 ng/mL), RANKL (100 ng/mL) for 24 h. Total-cell lysates were subjected to western blot analysis for NFATc1 expression. β-actin expression served as control. (B) Confocal microscopy analysis of NFATc1 localization in RAW 264.7 cells transduced with EV or MVNP in the presence and absence of NFAM1 shRNA and stimulated with RANKL (100 ng/mL). Immunostaining for NFATc1 shown at 24 h as detected by Alexa 488–conjugated anti-mouse antibody. Nuclear staining was performed with DRAQ5.
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