Osteoinductive LIM mineralization protein-1 suppresses activation of NF-kappaB and selectively regulates MAPK pathways in pre-osteoclasts

Abstract

LIM mineralization protein-1 (LMP-1) is an intracellular regulator of bone formation and has been shown to be osteoinductive in vitro and in vivo. The effect of LMP-1 on other aspects of bone homeostasis has not been previously studied. In a pilot study we observed that LMP-1 decreased nitric oxide (NO) production in pre-osteoclasts. Here we report a new anti-inflammatory effect of LMP-1 and define its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 pre-osteoclasts. We found that LMP-1 significantly inhibited LPS-induced NO production. LMP-1 also effectively inhibited the expression of inducible nitric oxide synthase (iNOS), potently suppressed the transcriptional activity and nuclear translocation of nuclear factor kappa B (NF-kappaB), and prevented the phosphorylation of inhibitor of kappa B (IkappaB). Interestingly, LMP-1 had no effect on Receptor-Activator of Nuclear Factor B Ligand (RANKL)-induced activation of NF-kappaB. Furthermore, LMP-1 had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it did attenuate the phosphorylation of c-Jun NH2-terminal kinase (JNK) while enhancing phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK). These results suggest that LMP-1 has an anti-inflammatory effect, and this effect is, at least in part, due to the inhibition of NO production by the suppression of NF-kappaB activation and selective regulation of mitogen-activated protein kinase (MAPK) pathways.

Fig.1

Evaluation of the cytotoxic effect of TAT-LMP-1 alone and LPS plus TAT-LMP-1 on RAW 264.7 macrophages/pre-osteoclasts. The MTT assay was used to assess cytotoxic effects 4 h and 24 h after TAT-LMP-1 or LPS treatment. (A) No significant difference in cell viability was observed in cultures treated with low doses of TAT-LMP-1 compared to the no treatment group. (B) No significant difference in cell viability was observed in cultures treated with LPS plus low doses of TAT-LMP-1 compared to the no treatment group. Values are mean ± SEM of one representative experiment out of three independent experiments performed in triplicate. (*P > 0.05)

Fig.2

LMP-1 decreases NO production and iNOS expression in LPS-treated RAW 264.7 macrophages/pre-osteoclasts. (A) A Nitrite assay was used to assess NO production. TAT-LMP-1 inhibited LPS-induced NO production in a concentration-dependent manner 24 h after LPS treatment. Data are presented as mean ± SEM from one representative experiment out of three independent experiments performed in triplicate. (B) Expression of iNOS protein was determined by Western blot using specific antibody. β-actin antibody was used to show equal protein loading. TAT-LMP-1 inhibited LPS-induced iNOS protein expression in a concentration-dependent manner. The image presented is from one representative experiment out of three independent experiments. Dentitometric quantification and statistical analysis include the results from three independent experiments. (C) The level of iNOS mRNA was detected by real-time RT-PCR. TAT-LMP-1 inhibited LPS-induced iNOS mRNA expression in a concentration-dependent manner. Data are presented as mean ± SEM of the fold change in mRNA levels from one representative experiment out of three independent experiments performed in triplicate. (*P < 0.05)

Fig.3

LMP-1 decreases LPS-induced iNOS promoter activity in RAW 264.7 macrophages/pre-osteoclasts. A Luciferase reporter assay was used to assess iNOS promoter activity. Luciferase activity was normalized to Renilla luciferase activity as relative luciferase units (RLU). TAT-LMP-1 inhibited LPS-induced iNOS promoter activity in a concentration-dependent manner. Data are presented as mean ± SEM of the fold change in RLU from one representative experiment out of three independent experiments performed in triplicate. (*P < 0.05)

Fig.4

LMP-1 decreases LPS-induced NF-κB transcriptional activity in RAW 264.7 macrophages/pre-osteoclasts. A Luciferase reporter assay was used to assess NF-κB transcriptional activity. Luciferase activity was normalized to Renilla luciferase activity as relative luciferase units (RLU). TAT-LMP-1 inhibited LPS-induced NF-κB transcriptional activity in a concentration-dependent manner. Data are presented as mean ± SEM of the fold change in RLU from one representative experiment out of three independent experiments performed in triplicate. (*P < 0.05)

Fig.5

LMP-1 prevents LPS-induced nuclear translocation of NF-κB in RAW 264.7 macrophages/pre-osteoclasts. The protein level of the NF-κB subunit, p65, in nuclear extracts was determined by Western blot 3 h after LPS treatment. Lamin B antibody was used to show equal protein loading. TAT-LMP-1 inhibited LPS-induced p65 protein levels in the nucleus in a concentration-dependent manner. The image presented is from one representative experiment out of three independent experiments. Densitometric quantification and statistical analysis include the results from three independent experiments. (*P < 0.05)

Fig.6

LMP-1 prevents LPS-induced phosphorylation of IκB in RAW 264.7 macrophages/pre-osteoclasts. Protein levels of phospho-IκB and IκB were determined by Western blot. (A) Time course of LPS-induced phosphorylation of IκB in RAW 264.7 cells. (B) TAT-LMP-1 inhibited LPS-induced IκB phosphorylation (9 min) in a concentration-dependent manner. Detection of the IκB level served as a loading control. The image presented is from one representative experiment out of three independent experiments. Densitometric quantification and statistical analysis include the results from three independent experiments. (*P < 0.05)

Fig.7

LMP-1 does not inhibit activation of NF-κB in RANKL-induced RAW 264.7 macrophages/pre-osteoclasts. A Luciferase reporter assay was used to assess NF-κB transcriptional activity. Luciferase activity was normalized to Renilla luciferase activity as relative luciferase units (RLU). TAT-LMP-1 inhibited LPS-induced (24 h) NF-κB transcriptional activity in a concentration-dependent manner. Data are presented as mean ± SEM of the fold change in RLU from one representative experiment out of three independent experiments performed in triplicate. (*P < 0.05, ^#P < 0.05)

Fig.8

LMP-1 selectively regulates LPS-induced activation of MAPKs in RAW 264.7 macrophages/pre-osteoclasts. Protein levels of phospho-MAPKs were by Western blot after 30 min LPS treatment. (A) LMP-1 enhanced activation of p38 induced by LPS. (B) LMP-1 had no effect on activation of ERK1/2 induced by LPS. (C) LMP-1 suppressed activation of JNK induced by LPS. Detection of levels of the p38, ERK1/2 and JNK served as the loading control. The image presented is from one representative experiment out of three independent experiments. Densitometric quantification and statistical analysis include the results from three independent experiments. (*P < 0.05)