Specific Amelogenin Gene Splice Products Have Signaling Effects on Cells in Culture and in Implants in Vivo

Shure-Min Jengh,Kevin Tompkins,Xue Song Wang,Kuiru Wei,Kevin E. Healy,Arthur Veis,Lin Wang,Anna G. Brownell,Keith Alvares

doi:10.1074/jbc.m002308200

Abstract

Low molecular mass amelogenin-related polypeptides extracted from mineralized dentin have the ability to affect the differentiation pathway of embryonic muscle fibroblasts in culture and lead to the formation of mineralized matrix in in vivo implants. The objective of the present study was to determine whether the bioactive peptides could have been amelogenin protein degradation products or specific amelogenin gene splice products. Thus, the splice products were prepared, and their activities were determined in vitro and in vivo. A rat incisor tooth odontoblast pulp cDNA library was screened using probes based on the peptide amino acid sequencing data. Two specific cDNAs comprised from amelogenin gene exons 2,3,4,5,6d,7 and 2,3,5,6d, 7 were identified. The corresponding recombinant proteins, designated r[A+4] (8.1 kDa) and r[A-4] (6.9 kDa), were produced. Both peptides enhanced in vitro sulfate incorporation into proteoglycan, the induction of type II collagen, and Sox9 or Cbfa1 mRNA expression. In vivo implant assays demonstrated implant mineralization accompanied by vascularization and the presence of the bone matrix proteins, BSP and BAG-75. We postulate that during tooth development these specific amelogenin gene splice products, [A+4] and [A-4], may have a role in preodontoblast maturation. The [A+4] and [A-4] may thus be tissue-specific epithelial mesenchymal signaling molecules.

Highlights

Members of the BMP/VGR family of proteins have the ability to induce osteogenesis when implanted in appropriate carriers at nonbone sites in vivo [1, 2]
Preparation of Amelogenin Peptides from Rat Incisor cDNA—When the PCR primers P1 and P2 were used to probe the mRNA isolated from fresh rat incisor odontoblast pulp complex (Fig. 1A), two PCR products were detected (Fig. 2A) and sequenced
These data established that differentially spliced amelogenin mRNAs, containing exons 2, 3, and 5 and 2–5, respectively, were present in the presumed odontoblast pulp tissue

Summary

Introduction

Members of the BMP/VGR family of proteins have the ability to induce osteogenesis when implanted in appropriate carriers at nonbone sites in vivo [1, 2]. Amelogenin amino acid sequences are highly conserved across all species, the human and bovine have amelogenin genes on the X and Y chromosomes, whereas rat and murine amelogenin genes reside only on the Y chromosome These genes yield distinct sets of splice product isoforms [12, 15, 16]. The “active” peptide described by Nebgen et al [11] was characterized only by amino-terminal sequencing It was not determined whether it was an amelogenin degradation product or an intact polypeptide transcribed and translated as a specific enamel gene splice product. This is a very important distinction relative to the function and regulation of the potential in vivo

Objectives

Methods

Results

Conclusion