The Dentin Sialoprotein (DSP) Domain Regulates Dental Mesenchymal Cell Differentiation through a Novel Surface Receptor.

Chunyan Wan,Zhi Chen,Huan Liu,Lei Chen,Shuo Chen,Heng Lin,Guohua Yuan,Daoshu Luo,Lu Zhang,Guobin Yang

doi:10.1038/srep29666

Abstract

Dentin sialophosphoprotein (DSPP) is a dentin extracellular matrix protein that is processed into dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP). DSP is mainly expressed in odontoblasts. We hypothesized that DSP interacts with cell surface receptors and subsequently activates intracellular signaling. Using DSP as bait for screening a protein library, we demonstrate that DSP acts as a ligand and binds to integrin β6. The 36 amino acid residues of DSP are sufficient to bind to integrin β6. This peptide promoted cell attachment, migration, differentiation and mineralization of dental mesenchymal cells. In addition, DSP aa183-219 stimulated phosphorylation of ERK1/2 and P38 kinases. This activation was inhibited by an anti-integrin β6 antibody and siRNA. Furthermore, we demonstrate that this DSP fragment induces SMAD1/5/8 phosphorylation and nuclear translocation via ERK1/2 and P38 signaling. SMAD1/5/8 binds to SMAD binding elements (SBEs) in the DSPP gene promoter. SBE mutations result in a decrease in DSPP transcriptional activity. Endogenous DSPP expression was up-regulated by DSP aa183-219 in dental mesenchymal cells. The data in the current study demonstrate for the first time that this DSP domain acts as a ligand in a RGD-independent manner and is involved in intracellular signaling via interacting with integrin β6. The DSP domain regulates DSPP expression and odontoblast homeostasis via a positive feedback loop.

Highlights

During the process of dentinogenesis, highly controlled extracellular events occur
To further identify the specific dentin sialoprotein (DSP) domain interacting with integrin β6, the NH2-terminal-DSP aa[9-190] and COOH-terminal-DSP aa[183-456] domains were expressed, purified and confirmed by Coomassie blue staining and western blot assays (Fig. 1D,E)
Protein-protein interaction assays revealed that integrin β6 could bind to the COOH-terminal fragment of DSP aa[183-456], but not the NH2terminal domain aa[9-190] (Fig. 1F)

Summary

Introduction

During the process of dentinogenesis, highly controlled extracellular events occur. This process is tightly controlled by odontoblasts, which secrete extracellular matrix (ECM) proteins and regulate dentin mineralization. DSPP is a member of the SIBLING (Small Integrin-Binding Ligand N-linked Glycoproteins) family, consisting of bone sialoprotein (BSP), dentin matrix protein[1] (DMP1), DSPP, osteopontin (OPN), and matrix extracellular phosphoglycoprotein (MEPE). These SIBLING genes are clustered on human chromosome 415–20 and share an Arg-Gly-Asp (RGD) sequence that facilitates cell attachment, migration, differentiation and triggers intracellular signal transduction via binding to cell surface receptors, such as integrin[21]. In mouse DSPP, RGD is located within the DPP domain, and DPP activates MAPK and SMAD pathways and triggers intracellular signals by directly interacting with integrin[27,28]. SMAD1/5/8 combined with SMAD4 binds to SMAD binding elements (SBEs) in the DSPP gene regulatory region and activates DSPP gene transcription and cell behaviors

Methods

Results

Conclusion