Measles virus nucleocapsid protein, a key contributor to Paget's disease, increases IL-6 expression via down-regulation of FoxO3/Sirt1 signaling

Abstract

Measles virus plays an important role as an environmental factor in the pathogenesis of Paget's disease (PD). Previous studies have shown that IL-6 is increased in the bone marrow of Paget's patients and that measles virus nucleocapsid protein (MVNP) induces IL-6 secretion by pagetic osteoclasts. Further, IL-6 plays a critical role in the development of pagetic osteoclasts and bone lesions induced by PD, but the mechanisms regulating IL-6 production by MVNP remain unclear. Our current studies revealed that MVNP expression in osteoclast precursors down-regulated Sirt1 mRNA and protein, a negative regulator of NF-κB activity, which is a key factor for IL-6 expression. MVNP expression in NIH3T3 cells also elevated Il-6 transcription and impaired the expression of Sirt1 mRNA both under basal conditions and upon activation of the Sirt1 upstream regulator FoxO3 by LY294002 (a PI3K/AKT inhibitor). Luciferase activity assays showed that constitutively active FoxO3 abolished the repressive effect of MVNP on reporters driven by either FoxO3 response elements or the Sirt1 promoter. Further, protein stability assays revealed that FoxO3 was degraded more rapidly in MVNP-expressing cells than in control cells following the addition of cycloheximide. Similarly, co-transfection of MVNP and FoxO3 into HEK293 cells demonstrated that MVNP decreased the protein levels of over-expressed FoxO3 in a dose-dependent manner. Treatment with the proteasome inhibitor, MG132, blocked the MVNP-triggered decrease of FoxO3, and the treatment with the serine/threonine phosphatase inhibitor, calyculin A, revealed that MVNP increased phosphorylation of FoxO3. Further, over-expression of Sirt1 or treatment with the Sirt1 activator resveratrol blocked the increase in Il-6 transcription by MVNP. Finally, resveratrol reduced the numbers of TRAP positive multi-nuclear cells in bone marrow cultures from TRAP-MVNP transgenic mice to wild type levels. These results indicate that MVNP decreases FoxO3/Sirt1 signaling to enhance the levels of IL-6, which in part mediate MVNP's contribution to the development of Paget's disease.

Figure 1. Expression of Sirt1 in MVNP expressing cells

(A) Detection of Il-6 mRNA levels in EV- and MVNP-NIH3T3 cells by real-time PCR. *P < 0.01 as determined by student’s t-test. (B) Cells were treated with 2 μg/ml Actinomycin D for indicated times before cells were harvested for the analysis of Il-6 mRNA levels. (C) EV-CFU-GM and MVNP-CFU-GM co-transfected with a NF-κB reporter plasmid and a β-gal expression vector were treated with TNF-α for 24 hr. Cell lysate firefly luciferase values were divided by β-gal activity values for normalization in each sample for quadruplicate determinations. The means and standard deviations of the fold increases for each treatment set were plotted. *P < 0.01 as determined by as determined by student’s t-test. (D) HEK293R cells co-transfected with a NF-κB reporter and either an empty (EV) or MVNP-expression plasmid were stimulated with either vehicle (none), or 2 ng/ml IL-1β, or TNF-α for 24 hr. The relative luciferase units/μg protein for each of the triplicate samples were divided by the mean of the EV-transfected un-stimulated triplicates to derive a fold increase for each sample. The means and standard deviations of the fold increases for each treatment set (EV/MVNP) were plotted (None: 1.0 ± 0.1/2.4 ± 0.2; IL-1β: 8.3 ± 1.3/22.4 ± 1.9; TNF-α: 14.2 ± 1.8/58.3 ± 6.5). *P < 0.05 as determined by student’s t-test. (E) Bone marrow cells were derived from wild-type and TRAP-MVNP transgenic mice. After treatment with M-CSF for 3 days, osteoclast (OCL) precursors were harvested for total RNA and protein. Real-time PCR or Western blotting was performed to analyze the mRNA (left) or protein (right) levels of Sirt1, respectively. *P < 0.05 as determined by student’s t-test. (F) Expression of Sirt1 mRNA (left) and protein (right) was detected in EV- and MVNP-NIH3T3 cells. *P < 0.01 as determined by student’s t-test.

Figure 2. FoxO3 regulation of Sirt1 mRNA was down-regulated by MVNP

(A) Both EV-and MVNP-NIH3T3 cells were treated with 20 μM LY294002 for 2.5 hours. Cells were harvested and proteins in the cytosolic (cyt) and nuclear (nuc) compartments were isolated. FoxO3 protein levels were analyzed by Western blotting. PARP and-α-tubulin were used as controls for proteins from cytosolic and nuclear compartments, respectively. (B) After the treatment of LY294002 for 5 hours, cells were harvested for RNA extraction. Real-time PCR was performed to analyze the level of Sirt1 mRNA. The level of Sirt1 mRNA in EV or MVNP cells with DMSO treatment was set as 1.*P < 0.05 as determined by student’s t-test. (C) HEK293 cells were co-transfected with either FHRE-luciferase or Sirt1 promoter (−202) luciferase reporter together with the renilla luciferase construct (pRL-CMV, Promega). At 48 hours post-transfection, cells were harvested for dual-luciferase assays. Firefly luciferase values were divided by renilla luciferase values for normalization in each sample. Empty vector pCMV-Tag2C or pCDNA3.1 was used as control construct for MVNP (500 ng) or FoxO3^™ (300 ng), respectively. A representative experiment is shown. Similar results were observed in 2 independent experiments. *P < 0.01 or **P < 0.05 as determined by student’s t-test.

Figure 3. MVNP induced the degradation of FoxO3

(A and B) The stability of FoxO3 protein was assayed by cycloheximide (CHX) chase in EV- and MVNP-NIH3T3 cells. Cells were harvested and protein lysates prepared at 0.5, 1, 2, 4, 6 hours (h) after addition of CHX. (A) Western blotting was performed to detect the FoxO3 levels at each time point. (B) Data represent densitometric analysis of the results in (A). (C) HEK293 cells were transfected with a Flag-FoxO3 plasmid and increasing amount of MVNP plasmid. At 36 hours post-transfection, cells were harvested and analyzed for the expression of Flag-FoxO3 and MVNP with their respective antibodies. (D) HEK293 cells were transfected with a Flag-FoxO3 plasmid and MVNP plasmid (500 ng of each). At 36 hours post-transfection, cells were harvested after 10 μM MG132 treatment for 2 hours or 100 nM calyculin A treatment for 40 minutes. Cell lysates were analyzed by immunoprecipitation and immunoblotting. A representative experiment is shown. Similar results were observed in 2 independent experiments. (E and F) Both EV- and MVNP-NIH3T3 cells were treated with 10 μM MG132 for 5 hours. Cells were then harvested for RNA and protein extraction. (E) Western blotting was performed to analyze the protein levels of FoxO3. (F) Real-time PCR was performed to analyze the levels of Il-6 mRNA. *P < 0.01 or **P < 0.05 as determined by student’s t-test.

Figure 4. MVNP increased Il-6 through down-regulation of Sirt1

(A) An IL-6 promoter (−225) luciferase reporter was transfected into EV- or MVNP-NIH3T3 cells together with a Sirt1 construct or an empty vector. *P < 0.01 as determined by student’s t-test. (B) Both EV- and MVNP-NIH3T3 cells were treated with resveratrol or the vehicle (DMSO) for 24 hours and harvested for Il-6 mRNA determination by real-time PCR. *P < 0.01 as determined by student’s t-test. (C) EV-NIH3T3 cells were treated with 10 ng/mL recombinant human Il-6 or left untreated for 4 hours. Western blotting or real-time PCR was performed to analyze the protein levels of Stat3, FoxO3, and Sirt1 (left) or mRNA levels of Sirt1 (right), respectively. *P < 0.05 as determined by student’s t-test.

Figure 5. Resveratrol inhibited abnormal OCL differentiation in the presence of MVNP

Nonadherent bone marrow cells were derived from wild-type and TRAP-MVNP mice long bone. After 3 days of pretreatment of M-CSF, the cells were treated with RANKL and different doses of resveratrol (0, 10 μM, 25 μM, and 50 μM). After 3 days of further culture, cells were fixed and processed for TRAP staining. TRAP positive multinuclear cells (MNC) were counted under a microscope. *P < 0.05 as determined by two-way ANOVA with Bonferroni post-tests; ns, not significant.

Figure 6. Schematic representation of IL-6 gene regulation by MVNP modulation of FoxO3 and Sirt1

MVNP-increased phosphorylation of FoxO3, perhaps by members of the IKK family, leads to degradation of FoxO3 and down-regulation of Sirt1 expression. Decreased Sirt1 leads to more active NF-κB and increased IL6 gene expression.