A study of APin-protein interactions using protein microarray

Article information

Restor Dent Endod. 2007;32(5):459-468

Publication date (electronic) : 2007 September 30

doi : https://doi.org/10.5395/JKACD.2007.32.5.459

Joo-Cheol Park ¹, Sun-Hwa Park ¹, Heung-Joong Kim ¹, Jong-Tae Park ¹, Seong-Ho Youn ¹, Ji-Woong Kim ¹, Tae-Yeon Lee ², Ho-Hyun Son ²

¹Department of Oral Histology, College of Dentistry, Chosun University, Korea.

²Department of Conservative Dentistry, School of Dentistry, Seoul National University, Korea.

Corresponding Author: Ho-Hyun Son. Department of Conservative Dentistry, School of Dentistry, Seoul National University 22 Yeongun-dong, Chongro-gu, Seoul, 110-749, Korea. Tel: 82-2-2072-2652, Fax: 82-2-2072-3859, hhson@snu.ac.kr

Received 2007 July 11; Revised 2007 August 11; Accepted 2007 August 16.

Abstract

Protein microarray or protein chips is potentially powerful tools for analysis of protein-protein interactions. APin cDNA was previously identified and cloned from a rat odontoblast cDNA library. The purpose of this study was to investigate the APin-protein interactions during ameloblast differentiation. Protein microarray was carried with recombinant APin protein and MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein were selected among 74 interacting proteins. Immortalized ameloblast cells (ALCs) were transfected with pCMV-APin construct and U6-APin siRNA construct. After transfection, the expression of the mRNAs for four proteins selected by protein micoarrays were assessed by RT-PCR.

The results were as follows:

1. APin expression was increased and decreased markedly after its over-expression and inactivation, respectively.

2. Over-expression of the APin in the ALCs markedly down-regulated the expression of MEF2 and Aurora kinase A, whereas their expression remained unchanged by its inactivation.

3. Expression of BMPR-IB and EF-hand calcium binding protein were markedly increased by the overexpression of the APin in the ALCs, whereas expression of BMPR-IB remained unchanged and expression of EF-hand calcium binding protein was markedly decreased by its inactivation.

These results suggest that APin plays an important role in ameloblast differentiation and mineralization by regulating the expression of MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein.

Keywords: APin; Ameloblast; Protein microarray; Mineralization; Interaction; Differentiation

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Figure 1

Detection of the APin protein using protein microarray.

Figure 2

Relative affinity ranking with the APin protein.

Figure 3

RT-PCR amplification of the APin in ameloblast cell line after overexpression with CMV-APin plasmid and inactivation with U6-APin siRNA.

Figure 4

RT-PCR amplification of MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein ameloblast cell line after over-expression with CMV-Apin protein plasmid and inactivation with U6-Apin protein siRNA.

Figure 5

The amplification for the APin reactions in control, over-expression, inactivation and mock groups. The relative amounts of MEF2 (A), Aurora kinase A (B), BMPR-IB (C) and EF-hand calcium binding (D) mRNA after normalization with the amounts of the GAPDH mRNA. The results were represented as means ± SD of three independent transfections. Con, normal ALC; Over, APin overexpression; Inact, APin inactivation; Mock, ALC expressing empty vector.

Table 1

Nucleotide sequences of the primers used for RT-PCR

Table 2

Four high expression proteins are related to the functional characteristics, such as tumorigenesis, organ development and mineralization