Gelatinases Cleave Dentin Sialoprotein Intracellularly.

Zhi Chen,Xiaohui Gou,Guohua Yuan,Huiwen Zheng,Guobin Yang,Shuo Chen,Yifan Xue

doi:10.3389/fphys.2020.00686

Abstract

Dentin sialoprotein (DSP), the NH2-terminal fragment of dentin sialophosphoprotein (DSPP), is essential for dentin formation and further processed into small fragments inside the odontoblasts. Gelatinases, including matrix metalloproteinases 9 (MMP9) and MMP2, were able to cleave DSP(P) in tooth structures. We hypothesized that gelatinases may also cleave DSP intracellularly in the odontoblasts. In this study, the co-expression and physical interaction between DSP and gelatinases were proved by double immunofluorescence and in situ proximity ligation assay (PLA). Intracellular enzymatic activity of gelatinases was verified by gelatin zymography and in situ zymography. To confirm whether DSP was cleaved by active gelatinases intracellularly, lysates of wild-type (WT) odontoblastic cells treated with a MMP2 inhibitor or a MMP9 inhibitor or a MMP general inhibitor and of Mmp9–/– odontoblastic cells were analyzed by western blotting. Compared with the WT odontoblastic cells without inhibitor treatment, all these groups exhibited significantly higher ratios of high molecular weight to low molecular weight band density. FURIN was verified to be co-localized and physically interacted with MMP9 by double immunofluorescence and in situ PLA. The ratio of proMMP9 to activated MMP9 inside the odontoblastic cells were increased when function of endogenous FURIN was inhibited. And overexpressed proMMP9 was intracellularly cleaved by FURIN in the HEK293E cells, which was completely blocked by the mutation of proMMP9 with R96TPR99 substituted by A96AAA99. Taken together, these results indicate that DSP is intracellularly processed by gelatinases, and FURIN is involved in the intracellular activation of proMMP9 through cleavage of its R96TPR99 motif.

Highlights

Dentin is a major mineralized tissue of tooth and dentinogenesis is an important process in tooth development
To determine whether dentin sialoprotein (DSP) was processed by gelatinases intracellularly, first, their co-expression in odontoblasts and odontoblastic cells was detected by double immunofluorescence
The results showed that DSP, matrix metalloproteinases 9 (MMP9), and MMP2 were all highly expressed in the pre-odontoblasts, secretory odontoblasts, and mature odontoblasts of mouse molars at PN1, 3, and 7 (Figures 1A,B)

Summary

Introduction

Dentin is a major mineralized tissue of tooth and dentinogenesis is an important process in tooth development. Multiple non-collagenous proteins (NCPs) secreted by odontoblasts are involved in dentinogenesis and biomineralization (Linde, 1989; Butler and Ritchie, 1995; Butler, 1998; Goldberg et al, 2011) Among these NCPs, dentin sialophosphoprotein (DSPP), is regarded as a key factor which exerts influence over the odontoblasts differentiation, maturation, secretion, and mineralization of dentin matrix (Prasad et al, 2010; Jia et al, 2015; Chen et al, 2016). The NH2terminal fragments of DSP were enriched in the non-mineralized predentin matrix but weak in the mineralized dentin, while the COOH-terminal fragments were mainly distributed in the mineralized dentin (Yuan et al, 2012) These results suggested that the majority of full-length DSP may be processed into smaller fragments by some unknown enzymes in the cytoplasm of odontoblasts before being transported into dentin matrix (Yuan et al, 2012)

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