A peptide that blocks the interaction of NF‐κB p65 subunit with Smad4 enhances BMP2‐induced osteogenesis

Abstract

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.

<p>The Role of Tantalum Nanoparticles in Bone Regeneration Involves the BMP2/Smad4/Runx2 Signaling Pathway</p>

“…Smad4 is the only common Smad involved in BMP2 signaling. 24,25 Conditional deletion of Smad4 in osteoblasts leads to reduced bone mineral density, decreased bone volume, a decreased bone formation rate, and a reduced number of osteoblasts. 26 Controlling Smad4 is a good way to regulate bone formation.…”

“…30 Postnatal deletion of Smad4 in preosteoblasts increases the mitosis rate of cells on trabecular bone surfaces and in primary osteoblast cultures and delays differentiation and matrix mineralization by primary osteoblasts. 25 In summary, Smad4 has multiple roles in osteogenic differentiation and bone regeneration.…”

The Role of NF-κB in Physiological Bone Development and Inflammatory Bone Diseases: Is NF-κB Inhibition “Killing Two Birds with One Stone”?

“…Furthermore, administration of SBD peptide together with BMP2 induced ectopic thick cortical bone formation in vivo. The SBD peptide did not affect the activation of the classical NF-κB pathway by TNF-α stimulation [94]. Based on these results, it is possible that peptides targeting the association site of NF-κB, p65 and Smad4 may be useful for promoting bone formation by BMP with few side effects (Figure 3).…”

“…It is currently used as one of the therapeutic agents for RA and has been reported to be effective [56]. Thus, targeting NF-κB is effective at maintaining bone mass during inflammation [13][14][15][34][35][36][37][49][50][51][52][53][54][55][56][58][59][60][61][62][63][64][65][66][67][68][69]85,[91][92][93][94], and there is a possibility for "killing two birds with one stone". However, embryonic lethality has been reported in mice where molecules involved in the NF-κB signaling have been knocked out [26][27][28][29][30][31], and it is necessary to consider the possibility of causing serious side effects simply by inhibiting NF-κB.…”

“…Therefore, when the association sites of p65 and Smad4 were examined, the transactivation domain 2 (TA2) of p65 and the mad homology (MH) 1 region of Smad1 were directly associated. We further narrowed the association site to the amino acid level and found that the 16 amino acid sites on the N-terminal side of the TA domain of p65 were critical for binding to Smad4; we named the site the Smad binding domain (SBD) [94]. We synthesized the SBD peptide to compete with the interaction of p65 with Smad4.…”