-Original-

Dentin Matrix Protein 1 Regulates Mineralization of MC3T3-E1 Cells via the TNAP-ANK-ENPP1 Axis

Jianmin Liu^1,2,#, Juhua Zhao^1,#, Zhi Li¹, Hongmei Wang³, Binbin Wang¹, Wei Liu¹ and Lei Zhou¹

¹Allergy and Clinical Immunology Research Centre, the First Affiliated Hospital of Jinzhou Medical University, Liaoning, P. R. China
²Jinzhou Palmtop Cloud Biotechnology Co., Ltd., Liaoning, P. R. China
³Department of Clinical Laboratory, The First Affiliated Hospital of Jinzhou Medical University, Liaoning, P. R. China
^#Contributed equally

Background: Dentin matrix protein 1 (DMP1) is central to matrix mineralization. Clarification of the function of DMP1 is crucial to understanding normal bone formation and pathological calcification. The tissue-nonspecific alkaline phosphatase (TNAP)-progressive ankylosing enzyme (ANK)-extracellular nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) axis induces deposition of hydroxyapatite (HA) and pyrophosphate dehydrate (CPPD) by regulating pyrophosphate (PPi). Here, we investigated the mechanism by which DMP1 and the TNAP-ANK-ENPP1 axis participate in mineralization.
Methods: Expression of DMP1, TNAP, NPP1, and ANK genes in MC3T3-E1 cells was detected by RT-qPCR before and after treatment with DMP1 siRNA. An enzyme-linked immunosorbent assay was used to determine expression of DMP1 protein, TNAP activity was detected by SIGMAFAST p-nitrophenyl phosphate tablets, and mineralization of osteoblasts was determined by alizarin red staining. PPi levels were determined radiometrically and equalized for cell DNA. Levels of calcium, inorganic phosphate, zinc, and magnesium were assessed by standard laboratory techniques.
Results: After DMP1 gene silencing, expressions of TNAP, ENPP1, and ANK were correspondingly reduced. DMP1 altered extravesicular and intravesicular ion levels through the TNAP-ENPP1-ANK axis in MC3T3-E1 cells.
Conclusions: DMP1 regulated mineralization of MC3T3-E1 cells via the TNAP-ANK-ENPP1 axis and affected TNAP activity by two processes-rapid regulation of the Zn²⁺ transporter (ZnT) and transcriptional regulation of hysteresis. However, DMP1 may affect expression of ENPP1 and ANK only via hysteresis transcriptional regulation. DMP1, as a calcium trap or catalytic enzyme, appears to have a role in collagen mineralization.

J Nippon Med Sch 2023; 90: 262-271

Keywords
DMP1, TNAP-ANK-ENPP1 axis, mineralization, Pi/PPi-TNAP-positive feedback

Correspondence to
Lei Zhou, Allergy and Clinical Immunology Research Centre, the First Affiliated Hospital of Jinzhou Medical University, No. 2, Section 5 Renmin Street, Guta District, Jinzhou, Liaoning 121001, People's Republic of China
Angela_xiaolei@163.com

Received, October 9, 2022
Accepted, January 26, 2023