Gou Xiaohui, Xue Yifan, Zheng Huiwen, Yang Guobin, Chen Shuo, Chen Zhi, Yuan Guohua
The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China.
Department of Developmental Dentistry, University of Texas Health Science Center, San Antonio, TX, United States.
Front Physiol. 2020 Jun 25;11:686. doi: 10.3389/fphys.2020.00686. eCollection 2020.
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 proximity ligation assay (PLA). Intracellular enzymatic activity of gelatinases was verified by gelatin zymography and zymography. To confirm whether DSP was cleaved by active gelatinases intracellularly, lysates of odontoblastic cells treated with a MMP2 inhibitor or a MMP9 inhibitor or a MMP general inhibitor and of odontoblastic cells were analyzed by western blotting. Compared with the 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 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 RTPR substituted by AAAA. 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 RTPR motif.
牙本质涎蛋白（DSP）是牙本质涎磷蛋白（DSPP）的氨基末端片段，对牙本质形成至关重要，并在成牙本质细胞内进一步加工成小片段。包括基质金属蛋白酶9（MMP9）和MMP2在内的明胶酶能够在牙齿结构中切割DSP（P）。我们推测明胶酶也可能在成牙本质细胞内切割DSP。在本研究中，通过双重免疫荧光和邻近连接分析（PLA）证实了DSP与明胶酶之间的共表达和物理相互作用。通过明胶酶谱法和酶谱法验证了明胶酶的细胞内酶活性。为了确认DSP是否在细胞内被活性明胶酶切割，通过蛋白质免疫印迹法分析了用MMP2抑制剂或MMP9抑制剂或MMP通用抑制剂处理的成牙本质细胞裂解物和成牙本质细胞的裂解物。与未用抑制剂处理的成牙本质细胞相比，所有这些组均显示出高分子量与低分子量条带密度的比率显著更高。通过双重免疫荧光和PLA证实FURIN与MMP9共定位并发生物理相互作用。当内源性FURIN的功能受到抑制时，成牙本质细胞内proMMP9与活化MMP9的比率增加。并且在HEK293E细胞中，过表达的proMMP9被FURIN在细胞内切割，而将proMMP9的RTPR突变为AAAA则完全阻断了这种切割。综上所述，这些结果表明DSP在细胞内被明胶酶加工，并且FURIN通过切割其RTPR基序参与proMMP9的细胞内活化。
