The effect of inhibiting p38 MAPK on the expression of genes related to enamel development in mice

doi:10.12016/j.issn.2096-1456.2021.08.004

Abstract

Abstract:

Objective To study the effect of p38 mitogen activated protein kinase (p38 MAPK) on the expression of genes related to enamel development in the enamel epithelium and to provide a basis for the study of the molecular mechanism of enamel development. Methods The p38 MAPK-specific inhibitor SB203580 dissolved in DMSO was added to the culture medium of mouse mandibular molar tooth germs in vitro as experiment group, and mouse mandibular molar tooth germs treated with the same amount of DMSO were used as control group. Western blot was used to detect the protein expression level of phosphorylated p38 (p-p38) in the enamel epithelium. Real-time PCR was used to detect the mRNA expression levels of runt-related transcription factor 2 (Runx2), osteoblast-specific transcription factor (Osx), ameloblast markers odontogenic ameloblast associated protein (ODAM), amelotin (AMTN), matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4) in the enamel epithelium. Results Western blot results showed that under the action of the inhibitor SB203580, the phosphorylation level of p38 MAPK in mouse enamel epithelium decreased, and the difference was statistically significant (P < 0.05). Real-time PCR results showed that the expression levels of the transcription factors Runx2 and Osx and the ameloblast markers ODAM, AMTN, MMP20, and KLK4 in the SB203580 group were lower than those in the control group, and the difference was statistically significant (P < 0.05). Conclusion The p38 MAPK signaling pathway can mediate enamel development by regulating the expression of the transcription factors Runx2 and Osx and the ameloblast markers ODAM, AMTN, MMP20 and KLK4 in the mouse enamel epithelium.

Key words: p38 MAPK, SB203580, enamel, tooth germ, enamel epithelium, enamel development, signaling pathway, runt-related transcription factor 2, osterix, ameloblast markers

摘要：

目的研究p38丝裂原活化蛋白激酶（p38 mitogen activated protein kinase,p38 MAPK）对成釉上皮中釉质发育相关基因表达的影响,为釉质发育分子机制的研究提供基础。方法在体外小鼠下颌磨牙牙胚的培养基中加入以DMSO溶解的p38 MAPK特异性抑制剂SB203580为实验组,以培养基中加入等量DMSO处理的牙胚为对照组,利用Western blot检测成釉上皮中磷酸化p38（p-p38）的蛋白表达水平,实时定量PCR检测成釉上皮中Runt相关转录因子2（runt-related transcription factor 2,Runx2）、成骨细胞特异性转录因子（osterix,Osx）以及成釉细胞标志物牙成釉细胞相关蛋白（odontogenic ameloblast associated protein,ODAM）、釉成熟蛋白（amelotin,AMTN）、基质金属蛋白酶20（matrix metalloproteinase 20,MMP20）和激肽释放酶4（kallikrein 4,KLK4）的mRNA表达水平。结果 Western blot结果显示,在抑制剂SB203580的作用下,小鼠成釉上皮中p38 MAPK的磷酸化水平降低,差异有统计学意义（P < 0.05）。实时定量PCR结果表明,SB203580组的转录因子Runx2和Osx以及成釉细胞标志物ODAM、AMTN、MMP20、KLK4的表达均低于对照组,差异有统计学意义（P < 0.05）。结论 p38 MAPK信号通路可能通过调控转录因子Runx2、Osx及成釉细胞标志物ODAM、AMTN、MMP20和KLK4的表达介导釉质发育。

关键词: p38 MAPK, SB203580, 牙釉质, 牙胚, 成釉上皮, 釉质发育, 信号通路, Runt相关转录因子2, 成骨细胞特异性转录因子, 成釉细胞标志物

CLC Number:

LUO Xiaona,LIU Xianghui,WANG Bo,LIU Xin,XIE Xiaohua. The effect of inhibiting p38 MAPK on the expression of genes related to enamel development in mice[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2021, 29(8): 529-534.

罗晓娜,刘向晖,王博,刘昕,谢晓华. 抑制p38 MAPK对小鼠釉质发育相关基因表达的影响[J]. 口腔疾病防治, 2021, 29(8): 529-534.

Figures/Tables 5

Figure 1 The appearance of the tooth germ of the mouse mandibular first molar 2 days after birth observed under a stereomicroscope a: the arrow points to the enamel epithelium of the tooth germ; b: the area enclosed by the solid yellow line is the enamel epithelium of the mandibular first molar tooth germ

Table 1 Primers for real-time PCR

Primers	Primer sequence,5′-3′
Runx2	Forward： GAAATGCCTCCGCTGTTATG
Runx2	Reverse： GCTTCTGTCTGTGCCTTCTTG
Osx	Forward： GGCGTCCTCTCTGCTTGAG
Osx	Reverse： GGGCTGAAAGGTCAGCGTAT
ODAM	Forward： GCAATAGTTGGATCTATCCCAGA
ODAM	Reverse： GAATGCCAGTAGAAGGAAGCC
AMTN	Forward： TGAAGATTTGGGAGGCTAACG
AMTN	Reverse： CTGGGACCACTGAATGGACAG
MMP20	Forward： TGTCTAAGCTCAAGGTGCCCTGTT
MMP20	Reverse： TAAGTTGTCCATGTGGGTGCTGGA
KLK4	Forward： AACCTAAGGGACAGGGCAGT
KLK4	Reverse： GACAGTATCGGCCTCAGGAA
GAPDH	Forward： TCCAGAACATCATCCCTGCCTCTA
GAPDH	Reverse： ACAAAGTGGTCGTTGAGGGCAATG

Figure 2 The phosphorylation of p38 in SB203580 treated enamel epithelium was detected by Western blot The phosphorylation level of p38 in the enamel epithelium of the SB203580 group was lower than that in the control group; p-p38: phosphorylated p38; **: P < 0.01 vs. the control group

Table 2 Real-time PCR to detect the expression levels of Runx2 and Osx in the enamel epithelium treated with SB203580 $\overline x$ ± s,n=3

Group	Runx2	Osx
Control	1.00 ± 0.04	1.00 ± 0.10
SB203580	0.74 ± 0.07	0.60 ± 0.07
t	5.89	5.64
P	0.004	0.005

Table 3 Real-time PCR to detect the expression levels of ODAM, AMTN, MMP20 and KLK4 in the enamel epithelium treated with SB203580 $\overline x$ ± s,n=3

Group	ODAM	AMTN	MMP20	KLK4
Control	1.00 ± 0.10	1.00 ± 0.01	1.00 ± 0.04	1.00 ± 0.11
SB203580	0.39 ± 0.05	0.13 ± 0.01	0.52 ± 0.09	0.44 ± 0.01
t	7.445	69.87	8.629	8.847
P	0.018	< 0.001	0.001	0.001

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