Tumor Necrosis Factor α Represses Bone Morphogenetic Protein (BMP) Signaling by Interfering with the DNA Binding of Smads through the Activation of NF-κB^*

Reagents

Purified recombinant human BMP2 was provided by Wyeth Pharmaceuticals (Madison, NJ). Recombinant human TNFα was purchased from PeproTech Inc. (Rocky Hill, NJ). Anti-Smad1 (sc-7965), anti-Smad4 (sc-7154), anti-IκBα (sc-371), anti-HDAC1 (sc-7872), anti-β-actin (sc-8432), anti-IgG antibodies p65siRNA (sc-29411), and siRNA transfection reagent (sc-2528) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). BAY11-7082 and anti-p65 antibodies and anti-phosphorylated-Smad1,5,8 antibodies, were obtained from Biomol (Plymouth Meeting, PA) and Cell Signaling (Beverly, MA), respectively.

Cell Culture Conditions

Mouse osteoblastic cell line MC3T3-E1 cells were maintained in an α-modified minimum essential medium (α-MEM) with 10% heat-inactivated fetal bovine serum (FBS) with 100 units/ml penicillin and 100 μg/ml streptomycin. p65-deficient mouse embryonic fibroblasts (p65^−/− MEFs) were prepared from p65-deficient mice (20) and maintained in Dulbecco's modified Eagle's medium with 5% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. These cells were cultured under 5% CO₂ at 37 °C.

Alkaline Phosphatase (ALP) Activity and Staining

MC3T3-E1 cells were seeded at a density of 1.0 × 10⁴ cells/well in 96-well plates with α-MEM containing 10% FBS the day before treatment. The cells were pretreated with TNFα (10 ng/ml) for 30 min or left untreated and were subsequently treated with BMP2 (100 ng/ml) for 72 h. After the 72-h treatment, the cells were fixed with an acetone/ethanol mixture (50:50, v/v) and then incubated with a substrate solution (0.1 m diethanolamine, 1 mm MgCl₂, and 10 mg/ml p-nitrophenyl phosphate). The reaction was terminated by adding 5 m NaOH, and then the absorbance was measured at 405 nm using a microplate reader (Bio-Rad). To determine ALP activity histochemically, cells were stained for enzyme activity as previously described (5).

Transfection Conditions and Luciferase Assay

MC3T3-E1 cells and p65^−/− MEFs were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Total amounts of transfected plasmids in each group were equalized by the addition of an empty vector. BMP signaling was monitored using the IdWT4F-luciferase reporter plasmid, which expresses a luciferase protein under the control of a BMP-responsive element in the human ID1 gene, as described previously (21). The pNF-κB luciferase plasmid was purchased from Promega (Madison, WI). Luciferase activities were measured using a dual luciferase system (Promega).

Immunoprecipitation and Immunoblotting

Cells were lysed in TnT buffer (20 mm Tris-HCl, pH 7.5, 200 mm NaCl, 1% Triton X-100, 1 mm dithiothreitol) containing protease inhibitors (Roche, Basel, Switzerland). Cytosolic and nuclear fractions were prepared according to methods described by Dignam et al. (21). For co-precipitation experiments, whole cell extracts were incubated for 6 h at 4 °C with anti-Smad1, anti-Smad4, or anti-p65 antibodies coupled to A/G-Sepharose beads. The immune complex was extensively washed with TnT buffer, and then the samples were boiled and analyzed by immunoblotting. Protein content was measured with Pierce reagent following the manufacturer's protocol. Twenty micrograms of protein were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to a polyvinylidene difluoride membrane at 100 V for 1 h at 4 °C. These membranes were incubated with antibodies at dilutions of 1:500 to 1:1,000 in a 5% dry milk solution containing 0.01% azide overnight at 4 °C. Subsequently, the blots were washed in TTBS (10 mm Tris-HCl, 50 mm NaCl, 0.25% Tween 20) and incubated with a horseradish peroxidase-conjugated secondary antibody. The immunoreactive proteins were visualized using ECL (Amersham Biosciences, Piscataway, NJ).

Chromatin Immunoprecipitation (ChIP) Assays

ChIP was performed with a ChIP Assay Kit (Upstate Biotechnology, Waltham, MA), according to the manufacturer's instructions, using antibodies against phosphorylated Smad1,5,8, p65 antibodies or normal IgG. The purified DNA was analyzed by PCR using primers that amplify sequences containing the Id1 promoter, which harbors a BMP2-responsive element (BRE) to which Smad proteins bind (21). The primer pairs for the Id1, Smad6, and IκBα promoters were 5′-TAAGTTGACCCTTGGTCAGC-3′ (forward) and 5′-GACGTCACCCATTCATAAAAC-3′ (reverse), 5′-CATCCCTAGTGTATCCAACAAAGAG-3′ (forward) and 3′-AGCTCAAGACGGTCACGTACTC-3′ (reverse), and 5′-GCTCATCAAAAAGTTCCCTGTGC-3′ (forward) and 5′-TGGCGAGGTCTGACTGTTGTGG-3′ (reverse), respectively.

Electrophoresis Mobility Shift Assay

EMSAs were performed using MC3T3-E1 cells pretreated with, or without, TNFα (10 ng/ml) for 30 min and were subsequently treated with BMP2 (100 ng/ml) for 30 min. Nuclear extracts were prepared from the pretreated cells. The Smad DNA binding activity in the nuclear fractions was measured by EMSA. The double-stranded BRE probe and the κB probe were radiolabeled using T4 polynucleotide kinase and [γ-³²P]ATP (New England Nuclear, Boston, MA) using a labeling kit (Takara Shuzo Co.). The sequences of the oligo DNAs we used were as follows: 5′-TCTCCATGGCGACCGCCCGCGCGGCGCCAGCCT-3′ (BRE probe) and 5′-GATCAGAGGGGACTTTCCGAGG-3′ (κB probe). Six micrograms of the nuclear extracts were incubated with the labeled BRE probe. For the supershift experiments, antibodies were added prior to the addition of the probe, and the mixture was incubated for 15 min at room temperature. Antibodies against Smad1 (sc-7154X) and Smad4 (sc-6201X) were obtained from Santa Cruz Biotechnology. The reaction mixture was loaded onto a 5% polyacrylamide gel in 0.5 × TBE (44.5 mm Tris base, 44.5 mm boric acid, and 1 mm EDTA) and resolved by electrophoresis.

RT-PCR

For semi-quantitative RT-PCR, total RNA form MC3T3-E1 cells prepared with TRIzol (Invitrogen) was amplified by Superscript II and Taq polymerase (Invitrogen). Primer sequences were as follows: Id1: 5′-TCCTGCAGCATGTAATCGAC-3′ (forward) and 5′-GAGAGGGTGAGGCTCTGTTG-3′ (reverse), ALP: 5′-ACTGCTGATCATTCCCACGTT-3′ and (forward), 5′-GAACAGGGTGCGTAGGGAGA-3′ (reverse), osterix: 5′-GCGTCCTCTCTGCTTGA-3′ (forward) and 5′-TGTGTTGCCTGGACCTGGTG-3′ (reverse), and osteocalcin : 5′-AAGCAGGAGGGCAATAAGGT-3′ (forward) and 5′-TTTGTAGGCGGTCTTCAAGC-3′ (reverse); GAPDH: 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′ (forward) and 5′-CATGTAGGCCATGAGGTCCACCAC-3′ (reverse).

Statistical Analysis

Statistical significance was determined using the Student's t test. A p value of less than 0.05 was considered significant.

TNFα Inhibited BMP2-induced Osteoblast Differentiation

We first examined the effect of TNFα on the BMP2-induced ALP activity, a typical marker of osteoblast differentiation, in MC3T3-E1 cells. BMP2 induced a ∼7-fold increase in ALP activity, and there were many more ALP-positive cells compared with cells cultured with the control levels of BMP2. Consistent with previous reports (10, 11, 22), this induction was completely abolished by pretreatment with TNFα (Fig. 1, A and B).

To precisely determine whether TNFα inhibits BMP signaling via Smad-dependent or Smad-independent mechanisms, we examined the inhibitory effect of TNFα on BMP2-induced Id1-luciferase activity. The Id1-luciferase reporter construct contains a consensus Smad binding site that drives the expression of the luciferase gene (21). BMP2 induced a ∼3–4-fold increase in Id1-luciferase activity compared with controls, and pretreatment with TNFα strongly inhibited the luciferase activity induced by BMP2 (Fig. 1C). These results demonstrate that TNFα inhibits osteoblast differentiation and BMP2-induced Id1-luciferase activity by inhibiting the Smad-dependent pathway.

TNFα Did Not Affect the Phosphorylation or Nuclear Translocation of Smad1,5,8 Induced by BMP2

Upon binding of the BMP ligand to the type I and type II receptor complexes, the activated type I receptor phosphorylates Smad1,5,8, which then assemble into complexes with Smad4 and translocate into the nucleus. In the nucleus, the Smad complexes regulate the expression of genes related to osteoblast differentiation, such as ALP and osteocalcin (24, 25). The previous report demonstrated that TNFα inhibited BMP-induced Smad1,5,8 phosphorylation in C2C12 cells (23). In this study, as shown in Fig. 2A, phosphorylation of Smad1,5,8 was first detected within 10 min and declined thereafter (Fig. 2A). Pretreatment with TNFα did not affect the Smad1,5,8 phosphorylation induced by BMP2 (Fig. 2A). Furthermore, the translocation of phosphorylated Smad1,5,8 was not inhibited by pretreatment with TNFα, although TNFα led to nuclear translocation of p65, an NF-κB subunit (Fig. 2B).

Activation of NF-κB Inhibited BMP2-induced Id1-luciferase Activity in Both MC3T3-E1 Cells and p65^−/− MEFs

We hypothesized that TNFα inhibits BMP/Smad signaling through NF-κB activation because NF-κB regulates the expression of several genes related to inflammation induced by TNFα (15, 16). The most abundant form of NF-κB is a heterodimer of the p50 and p65 subunits that contains a transcriptional activator domain (15, 16). p65^−/− MEFs transfected with Id1-luc or pNF-κB-luc were cotransfected together with or without p65 and were treated with BMP2 for 24 h. The transcriptional activity of NF-κB was evaluated in a p65 dose-dependent manner (Fig. 3A). The inhibition of BMP2-induced Id1-luciferase activity coincided with the activation of NF-κB in p65^−/− MEFs (Fig. 3B). To further confirm the possibility that NF-κB activation inhibits BMP-induced Id1-luc activity, we inhibited NF-κB activity in MC3T3-E1 cells by two different approaches: using p65 siRNA to knockdown p65 expression or using the pharmacological NF-κB-specific inhibitor BAY11-7082. BMP2-induced Id1-luc activity was reduced by pretreatment with TNFα in MC3T3-E1 cells (Fig. 3C, lanes 3 and 4). By contrast, TNFα failed to suppress BMP2-induced Id1-luc activity in MC3T3-E1 cells that had been transfected with p65 siRNA (Fig. 3C, lanes 4 and 8). NF-κB suppression by BAY11-7082 also led to a recovery of Id1-luc activity (Fig. 3C, lanes 4 and 12).

Furthermore, we cotransfected cells with expression constructs for Id1-luc, a constitutively active form of FLAG-Smad1 (FLAG-Smad1(DVD)) that exchanges two distal serines that are part of the C-terminal SSXS motif for aspartic acids (4), FLAG-Smad4, and/or p65, with or without an expression vector for a dominant-negative form of IκBα (IκBαDN). The overexpression of p65 completely abrogated the Id1-luciferase activity induced by FLAG-Smad1(DVD) and FLAG-Smad4. By contrast, the transient overexpression of IκBαDN, which inhibits NF-κB in the cytoplasm, rescued this inhibition (Fig. 4A). TNFα signaling can activate MAPK pathway members, including MEK, ERK, and JNK (14). Recently, MAPK phosphorylation sites were identified in Smad1 at Ser-187, Ser-195, Ser-206, and Ser-214. Subsequently, the phosphorylation of these sites by MAPK negatively regulates BMP signaling (26). Therefore, we mutated all of these serines to alanines to get the FLAG-Smad1(MAPKmut-DVD) construct. The overexpression of p65 strongly inhibited the Id1-luciferase activity induced by FLAG-Smad1(MAPKmut-DVD) and FLAG-Smad4. Furthermore, the transient overexpression of IκBαDN again rescued this inhibition (Fig. 4B). These results suggest that cross-talk between NF-κB and BMP/Smad signaling influences BMP2-induced osteoblast differentiation induced by pretreatment with TNFα.

TNFα Did Not Disrupt the Smad1 and Smad4 Complex but Did Activate p65 through the Association of TNFα with Smad1 and Smad4

To confirm the possibility that TNFα interferes with the formation of a Smad1/Smad4 complex, we examined the interactions between Smad1 and Smad4 in cells pretreated with or without TNFα using co-immunoprecipitation assays. Cell extracts were subjected to immunoprecipitation with anti-Smad1, anti-Smad4, or anti-p65 antibodies, followed by immunoblot analysis with individual antibodies. Smad1 and Smad4 formed a complex in a ligand-dependent fashion (Fig. 5A, lanes 2 and 7). Pretreatment with TNFα failed to disrupt the association of Smad1 and Smad4. However, p65 activated by TNFα did associate with the complex of Smad1 and Smad4 (Fig. 5A, lanes 3, 8, and 13). Pretreatment with BAY11-7082 interfered with the association of p65 with Smad1 and/or Smad4 but did not interfere with the complex of Smad1 and Smad4 without affecting the expression levels of Smad1, Smad4, and p65 (Fig. 5, A, lanes 4, 5, 9, 10, 14, and 15 and B).

Activation of p65 by TNFα Inhibits the DNA Binding Activities of Smad Proteins Induced by BMP2

We next examined whether TNFα inhibited the DNA binding of Smad complexes induced by BMP2. MC3T3-E1 cells pretreated with or without TNFα were treated with BMP2 for the indicated time. Following pretreatment, genomic DNA was immunoprecipitated with anti-phosphorylated Smad1,5,8 antibodies, or control IgG, and was subsequently subjected to PCR amplification using primers that amplify the Id1 promoter region harboring the BMP2-responsive element. ChIP analysis showed that phosphorylated Smad1,5,8 was recruited to the Id1 promoter after 30 min of BMP2 stimulation, reached a maximum level at 1 h, and declined thereafter (Fig. 6A). When cells were pretreated with TNFα, the recruitment of phosphorylated Smad1,5,8 to the Id1 promoter was strongly suppressed compared with BMP2 treatment alone (Fig. 6A). p65 still bound to the Id1 promoter under these conditions (Fig. 6A). Similar results were obtained using Smad6, a BMP target gene (27), promoter (Fig. 6B).

To further confirm whether the inhibitory effect of TNFα on the activation of Smad-DNA binding in response to BMP2 is mediated by p65, we performed EMSAs using nuclear extracts prepared from MC3T3-E1 cells. BMP2 induced the formation of DNA-binding protein complexes in MC3T3-E1 cells (Fig. 7A, lane 3). Consistent with the ChIP analysis, TNFα suppressed BMP2-induced DNA-binding protein complexes (Fig. 7A, lanes 3 and 4). The addition of anti-Smad1 or anti-Smad4 antibodies to the BMP2-treated nuclear extracts reduced the amount of the complex and induced supershifted bands (Fig. 7B, lanes 3 and 4). Moreover, when both the anti-Smad1 and anti-Smad4 antibodies were added together, a slower-migrating supershifted band appeared (Fig. 7B). We further performed EMSAs using nuclear extracts prepared from p65^−/− MEFs. BMP2 induced Smad DNA binding in wild-type and p65^−/− MEFs (Fig. 7C, lanes 2 and 5). TNFα pretreatment in wild-type strongly suppressed the DNA binding activities of Smad proteins induced by BMP2 (Fig. 7C, lanes 3 and 6). Although TNFα slightly suppressed Smad DNA binding activity, large amount of Smads still bound to DNA in p65^−/− MEFs compared with wild-type cells (Fig. 7C, lanes 5 and 6). TNFα induced NF-κB DNA binding in wild-type but not p65^−/− MEFs (Fig. 7C, lanes 9 and 12). These results suggest that the activation of p65 by TNFα inhibits the DNA binding of Smad proteins normally induced by BMP2.

A Specific Inhibitor of NF-κB Signaling Restored the TNFα-induced Inhibition of the DNA Binding Activities of Smad Proteins and the ALP Activity Induced by BMP2

Because NF-κB signaling was found to antagonize Smad activation by BMP2, we next examined the effect of BAY11-7082 on the TNFα-induced inhibition of the DNA binding activities of Smad proteins by BMP2. NF-κB suppression by BAY11-7082 led to a recovery of the binding of phosphorylated Smad1,5,8 to the Id1 promoter (Fig. 8A). By contrast, TNFα recruited p65 to the IκBα promoter, a target gene of NF-κB, but BAY11-7082 suppressed this recruitment in a dose-dependent manner (Fig. 8A). BAY11-7082 also rescued the inhibitory effect of TNFα on BMP2-induced Smad DNA binding in a dose-dependent manner (Fig. 7A, lanes 4–6). We next examined the expression of osteoblast differentiation marker genes in MC3T3-E1 cells. BMP2 induced Id1, ALP, osterix, and osteocalcin mRNA expression within 12 h, whereas TNFα pretreatment strongly suppressed the expression of these genes. BAY11-7082 restored TNFα-mediated inhibition of gene expression in a dose-dependent manner (Fig. 8B). In addition, the TNFα-mediated inhibition of ALP activity and the staining induced by BMP2 were also restored by pretreatment with BAY11-7082 (Fig. 8, C and D). These results strongly suggest that TNFα represses BMP signaling by interfering with the DNA binding of Smads through the activation of NF-κB.