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. 2009 Dec 18;284(51):35987-95.
doi: 10.1074/jbc.M109.070540.

Tumor necrosis factor alpha represses bone morphogenetic protein (BMP) signaling by interfering with the DNA binding of Smads through the activation of NF-kappaB

Masato Yamazaki  1 , Hidefumi Fukushima, Masashi Shin, Takenobu Katagiri, Takahiro Doi, Tetsu Takahashi, Eijiro Jimi
Affiliations

Tumor necrosis factor alpha represses bone morphogenetic protein (BMP) signaling by interfering with the DNA binding of Smads through the activation of NF-kappaB

Masato Yamazaki et al. J Biol Chem. .

Abstract

Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor alpha (TNFalpha) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFalpha inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFalpha had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-kappaB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-kappaB by the overexpression of the dominant negative IkappaBalpha. Although TNFalpha failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFalpha suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-kappaB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFalpha inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-kappaB.

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Figures

FIGURE 1.
FIGURE 1.
TNFα inhibited BMP2-induced osteoblast differentiation in MC3T3-E1 cells. A, MC3T3-E1 cells were pretreated with or without TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for 72 h. The cells were fixed with an acetone/ethanol mixture (50:50, v/v) and were then incubated with a substrate solution (0.1 m diethanolamine, 1 mm MgCl2, 10 mg/ml p-nitrophenyl phosphate). ALP activity was then determined. B, cells were stained for ALP activity. C, MC3T3-E1 cells were transiently transfected with the Id1-luc reporter plasmid. After 24 h, the cells were pretreated with or without TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for 24 h. Luciferase activity was determined. Data are means + S.E. (n = 3). Similar results were obtained in three independent experiments. *, p < 0.01.
FIGURE 2.
FIGURE 2.
TNFα did not affect the phosphorylation or the nuclear translocation of Smad1,5,8 induced by BMP2. A, MC3T3-E1 cells were pretreated with or without TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for the indicated time. Total cell lysates were immunoblotted with anti-phosphorylated Smad1,5,8 antibodies, and anti-β-actin was used as a loading control. B, MC3T3-E1 cells were pretreated with or without TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for 1 h. Cytosolic and nuclear fractions were prepared, and then lysates were immunoblotted with anti-phosphorylated Smad1,5,8, and anti-p65 antibodies. Anti-IκBα antibodies and anti-HDAC antibodies were used to probe the cytosolic fraction and nuclear fraction, respectively. Similar results were obtained in three independent experiments.
FIGURE 3.
FIGURE 3.
Activation of NF-κB inhibited BMP2-induced Id1-luciferase activity in MC3T3-E1 cells and p65−/− MEFs. A, p65−/− MEFs were transiently transfected with a pNF-κB-luc reporter and increasing concentrations of a p65 expression plasmid and were then assayed for luciferase activity after 24 h. B, p65−/− MEFs were transiently transfected with an Id1-luc reporter with or without a p65 expression plasmid for 24 h. The cells were then treated with BMP2 (100 ng/ml) and assayed for luciferase activity after 24 h. C, NF-κB activation in MC3T3-E1 cells was inhibited by either p65 siRNA or BAY11-7082, and then the cells were treated with BMP2 (100 ng/ml) with or without pretreatment with TNFα (10 ng/ml). The cells were assayed for Id1- or NF-κB- luciferase activity after 24 h. The expression level of p65 was examined by immunoblotting using anti-p65 antibodies.
FIGURE 4.
FIGURE 4.
Overexpression of p65 inhibited Id1-luciferase activity induced by the constitutively active form of Smad1-induced in p65−/− MEFs. p65−/− MEFs were transiently transfected with Id1-luc, FLAG-Smad1(DVD) (A) or with FLAF-Smad1(MAPKmut-DVD) (B), FLAG-Smad4, p65, and IκBαDN expression plasmids and were assayed for luciferase activity after 24 h. Data are means + S.E. (n = 3). Similar results were obtained in three independent experiments. *, p < 0.01.
FIGURE 5.
FIGURE 5.
TNFα did not disrupt the Smad1 and Smad4 complex but activated p65 by TNFα associated with Smad1 and Smad4. A, MC3T3-E1 cells were pretreated with or without TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for 1 h in the presence or absence of increasing amounts of BAY11-7082. The whole cell extract was immunoprecipitated with anti-Smad1, anti-Smad4, or anti-p65 antibodies that were coupled to A/G-Sepharose beads; the whole cell extract was then processed for immunoblotting with the indicated antibodies. B, part of the whole cell extract was immunoblotted with the indicated antibodies to examine that all treatments did not affect the expression levels of Smad1, Smad4, and p65 and anti-β-actin was used as a loading control. Similar results were obtained in three independent experiments.
FIGURE 6.
FIGURE 6.
Activation of p65 by TNFα inhibited the BMP-induced Smad1 recruitment to the Id1 and Smad6 promoters. MC3T3-E1 cells were pretreated with or without TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for the indicated time. Chromatin from individual samples was precipitated using the indicated antibodies or with control IgG. The Id1 promoter (A) or the Smad6 promoter (B) was amplified by PCR from the precipitated DNA.
FIGURE 7.
FIGURE 7.
Activation of p65 by TNFα inhibited the DNA-binding activities of Smad proteins induced by BMP2. A, MC3T3-E1 cells were pretreated with or without increasing amounts of BAY11-7082 and TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for 30 min. The BMP2-induced DNA binding activity in the nuclear fractions was measured by EMSA using a BRE probe. B, subunit composition of the BMP2-induced DNA-binding proteins in MC3T3-E1 cells. The nuclear extracts were pretreated at 4 °C for 60 min with vehicle (lanes 1 and 2) or with 1 μl of the following polyclonal antibodies: anti-Smad1 (lane 3), anti-Smad4 (lane 4), anti-Smad1 and anti-Smad4 (lane 5), and control IgG (lane 6) antibodies. EMSAs were performed on these pretreated nuclear extracts. C, wild-type or p65−/− MEFs were pretreated with or without TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for 30 min. The Smad and NF-κB DNA binding activities in the nuclear fractions were measured by EMSA using a BRE probe and a κB probe, respectively. Similar results were obtained in three independent experiments.
FIGURE 8.
FIGURE 8.
A specific inhibitor of NF-κB signaling restored the TNFα-induced inhibition of the DNA binding activities of Smad proteins, expression of osteoblast marker genes, and ALP activity, which were all induced by BMP2. A, MC3T3-E1 cells were pretreated with BAY11-7082 and TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for the indicated time. Chromatin from individual samples was precipitated using the indicated antibodies or control IgG. The Id1 or IκBα promoter was amplified by PCR from the precipitated DNA. B, MC3T3-E1 cells were pretreated with BAY11-7082 and TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for 12 h. Total RNA was extracted and subjected to reverse transcription with a random primer. The first strand cDNAs were submitted to PCR analysis for Id-1, ALP, osterix, osteocalcin, and GAPDH. C, MC3T3-E1 cells were pretreated with BAY11-7082 and TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for 72 h. The cells were fixed with an acetone/ethanol mixture (50:50, v/v) and were incubated with a substrate solution (0.1 m diethanolamine, 1 mm MgCl2, 10 mg/ml p-nitrophenyl phosphate). ALP activity was then determined. Data are means + S.E. (n = 3). Similar results were obtained in three independent experiments. *, p < 0.01. D, The cells were stained for ALP activity.
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