Carboxy-Terminal Cementum Protein 1-Derived Peptide 4 (Cemp1-P4) Promotes Mineralization Through Wnt/Beta-Catenin Signaling In Human Oral Mucosa Stem Cells

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1's secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1's short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1's C-terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/beta-catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/beta-catenin pathway. CEMP1-p4 stimulation upregulated the expression of beta-catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of beta-catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a "mineralizing-like" phenotype by activating the beta-catenin signaling cascade.