Bone morphogenic proteins (BMPs) stimulate bone formation in vivo and osteoblast differentiation in vitro via a Smad signaling pathway. Recent findings revealed that the activation of nuclear factor-κB (NF-κB) inhibits BMP-induced osteoblast differentiation. Here, we show that NF-κB inhibits BMP signaling by directly targeting the Smad pathway. A selective inhibitor of the classic NF-κB pathway, BAY11-770682, enhanced BMP2-induced ectopic bone formation in vivo. In mouse embryonic fibroblasts (MEFs) prepared from mice deficient in p65, the main subunit of NF-κB, BMP2, induced osteoblastic differentiation via the Smad complex to a greater extent than that in wild-type MEFs. In p65(-/-) MEFs, the BMP2-activated Smad complex bound much more stably to the target element than that in wild-type MEFs without affecting the phosphorylation levels of Smad1/5/8. Overexpression of p65 inhibited BMP2 activity by decreasing the DNA binding of the Smad complex. The C-terminal region, including the TA2 domain, of p65 was essential for inhibiting the BMP-Smad pathway. The C-terminal TA2 domain of p65 associated with the MH1 domain of Smad4 but not Smad1. Taken together, our results suggest that p65 inhibits BMP signaling by blocking the DNA binding of the Smad complex via an interaction with Smad4. Our study also suggests that targeting the association between p65 and Smad4 may help to promote bone regeneration in the treatment of bone diseases.
Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF-κB) suppresses BMP-induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF-κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4-binding domain (SBD), an amino-terminal region of TA2 that associates with the MH1 domain of Smad4. Cell-permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2-induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF-κB signaling. SBD peptide enhanced the binding of the BMP2-inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id-1) compared with control peptide. Although SBD peptide did not affect BMP2-induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2-induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2-induced bone regeneration without inhibiting NF-κB activity.
Protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) are major members of the protein serine/threonine phosphatase families. We have identified PP1 and PP2A as interacting partners of PRIP (phospholipase C-related but catalytically inactive protein), a protein isolated in our laboratory. We first investigated the interaction of PRIP with two phosphatases, using purified recombinant proteins. PRIP immobilized on beads pulled down the catalytic subunits of both PP1 and PP2A, indicating that the interactions were in a direct manner, and the binding of PP1 and the binding of PP2A to PRIP were mutually exclusive. Site-directed mutagenesis experiments revealed that the binding sites for PP1 and PP2A on PRIP were not identical, but similar. Phosphorylation of PRIP by protein kinase A (PKA) resulted in the weakened binding of PP1, but not PP2A. Rather, the dissociation of PP1 from PRIP by phosphorylation accompanied the strengthened binding of PP2A in in vitro experiments. This regulation of binding of PP1 and PP2A to PRIP by PKA-dependent phosphorylation was also observed in living cells treated with forskolin or isoproterenol. These results suggested that PRIP directly interacts with the catalytic subunits of two distinct phosphatases in a mutually exclusive manner and the interactions are regulated by phosphorylation, thus functioning as a scaffold to regulate the activities and subcellular localizations of both PP1 and PP2A in phospho-dependent cellular signaling.
Oral squamous cell carcinoma (OSCC) cells display significantly augmented nuclear factor-κB (NF-κB) activity, and inhibiting this activity suppresses malignant tumor characteristics. Thus, we evaluated the effect of IMD-0560, a novel inhibitor of IκB kinase (IKK) β that is under assessment in a clinical trial of rheumatoid arthritis, on bone invasion by the mouse OSCC cell line SCCVII. We examined the inhibitory effects of IMD-0560 on NF-κB activity and tumor invasion using human OSCC cell lines and SCCVII cells in vitro. Using a mouse model of jaw bone invasion by SCCVII cells, we assessed the inhibitory effect of IMD-0560 on jaw bone invasion, tumor growth, and matrix degradation in vivo. IMD-0560 suppressed the nuclear translocation of NF-κB and the degradation of IκBα in OSCC cells. IMD-0560 also inhibited invasion by suppressing matrix metalloproteinase-9 (MMP-9) production in OSCC cells. IMD-0560 protected against zygoma and mandible destruction by SCCVII cells, reduced the number of osteoclasts by inhibiting receptor activator of NF-κB ligand (RANKL) expression in osteoblastic cells and SCCVII cells, increased SCCVII cell death and suppressed cell proliferation and MMP-9 production in SCCVII cells. Based on these results, IMD-0560 may represent a new therapeutic agent for bone invasion by OSCC cells.
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