Effects of Notch signaling pathway on the dentin differentiation of human dental pulp stem cells in hypoxic environment

doi:10.3877/cma.j.issn.1674-1366.2020.04.003

Abstract

Abstract:

Objective

To investigate whether and how hypoxia affects the differentiation capacity of human dental pulp stem cells (HDPSCs) via Notch signaling.

Methods

Primary cultured HDPSCs were stimulated by CoCl₂, which could induce a chemical hypoxic environment. HIF-1α protein level was detected by Western blot. The mRNA expression levels of Notch and dentin related genes were detected by RT-PCR. Alizarin red staining was used to detect the ability of HDPSCs to form mineralized nodules. Notch signaling pathway specific blocker GSI was further added to observe the changes of the indicators above. Reporter gene method was then used to detect the regulation of HIF-1α gene on Notch signaling pathway. R software (version 3.5.3) was used for statistical analysis. All measurement data were done by normality test. One-Way ANOVA (homogeneity of variance) or Kruskal-Wallis H test (heterogeneity of variance) was used for the multi-group comparison, and LSD-t test (homogeneity of variance) or Mann-Whitney U test (heterogeneity of variance) was used for the pair-wise comparison.

Results

(1) HIF-1α protein level was up-regulated by the chemical hypoxic environment induced by CoCl₂, and then reduced when GSI was added. The mRNA expression level of Notch target gene Hes1 was also significantly increased from 1.06 ± 0.09 to 1.46 ± 0.12 (t = -4.64, P = 0.012) under the hypoxic condition, and then significantly decreased to 0.82 ± 0.14 when GSI was added (t = 5.98, P = 0.004) . Moreover, the same resultsthat the mRNA expression level of Hes1 was significantly decreased from 1.06 ± 0.09 to 0.30 ± 0.09 when GSI was added (t = 10.08, P = 0.001) , were observed under a normoxic condition. (2) The results of alizarin red staining showed that the dentin differentiative potential of HDPSCs was decreased under the hypoxic environment, and then enhanced when GSI was added. The mRNA expression levels of osteogenic/dentin related genes BSP, OCN and DSPP were 1.21 ± 0.12, 1.08 ± 0.19 and 1.03 ± 0.13 under the normoxic environment, and 0.53 ± 0.14, 0.43 ± 0.20 and 0.48 ± 0.11 under the hypoxic environment, where there was a statistically significant difference between the two conditions (t_BSP = 6.30, P_BSP = 0.003; t_OCN = 4.07, P_OCN = 0.015; t_DSPP = 5.67, P_DSPP = 0.005) . When GSI was added, the mRNA expression levels of BSP, OCN and DSPP under the hypoxic environment were then significantly increased to 0.99 ± 0.13, 1.09 ± 0.13 and 1.09 ± 0.13 (t_BSP = -4.17, P_BSP = 0.014; t_OCN = -4.83, P_OCN = 0.012; t_DSPP = -4.30, PDSPP = 0.017) . Moreover, the same results that the mRNA expression level of BSP, OCN and DSPP were significantly increased to 1.73 ± 0.20, 1.55 ± 0.08 and 1.52 ± 0.14 respectively after GSI was added (t_BSP = -3.84, P_BSP = 0.027; t_OCN = -3.99, P_OCN = 0.035; t_DSPP = -4.43, P_DSPP = 0.011) , were observed under the normoxic condition. (3) The results of luciferase reporter gene experiment showed that the activity of Notch signaling pathway was significantly increased from 2.09 ± 0.15 to 5.37 ± 0.12 after the addition of HIF-1α (t = -28.92, P<0.001) .

Conclusion

Hypoxia caused an increase of HIF-1α in HDPSCs and HIF-1α may inhibit the dentin differentiative potential of HDPSCs by activating the Notch signaling pathway.

Key words: Dental pulp, Stem cells, Anoxia, Hypoxia inducible factors, Notch signaling pathway, Odontogenic differentiation

Lina Guan, Fan Yang, Dongfeng Yin, Zigeng Yang, Dunhong Wei, Kefu Luo, Rui Wang. Effects of Notch signaling pathway on the dentin differentiation of human dental pulp stem cells in hypoxic environment[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2020, 14(04): 214-220.

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Figures/Tables 8

References 21

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组别	CoCl₂	DMSO	GSI
常氧组	-	+	-
常氧+GSI组	-	-	+
低氧组	+	+	-
低氧+GSI组	+	-	+

组别	CoCl₂	DMSO	GSI
常氧组	-	+	-
常氧+GSI组	-	-	+
低氧组	+	+	-
低氧+GSI组	+	-	+

基因	引物序列
Hes1	上游：5′-TGGAAATGACAGTGAAGCACCTC-3′
?	下游：5′-TCGTTCATGCACTCGCTGAAG-3′
BSP	上游：5′-CTGGCACAGGGTATACAGGGTTAG -3′
?	下游：5′-GCCTCTGTGCTGTTGGTACTGGT-3′
OCN	上游：5′-CATGAGAGCCCTCACA-3′
?	下游：5′-AGAGCGACACCCTAGAC-3′
DSPP	上游：5′-ATATTGAGGGCTGGAATGGGGA-3′
?	下游：5′-TTTGTGGCTCCAGCATTGTCA-3′
β-actin	上游：5′-TGGCACCCAGCACAATGAA-3′
?	下游：5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′

基因	引物序列
Hes1	上游：5′-TGGAAATGACAGTGAAGCACCTC-3′
?	下游：5′-TCGTTCATGCACTCGCTGAAG-3′
BSP	上游：5′-CTGGCACAGGGTATACAGGGTTAG -3′
?	下游：5′-GCCTCTGTGCTGTTGGTACTGGT-3′
OCN	上游：5′-CATGAGAGCCCTCACA-3′
?	下游：5′-AGAGCGACACCCTAGAC-3′
DSPP	上游：5′-ATATTGAGGGCTGGAATGGGGA-3′
?	下游：5′-TTTGTGGCTCCAGCATTGTCA-3′
β-actin	上游：5′-TGGCACCCAGCACAATGAA-3′
?	下游：5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′

组别	BSP	OCN	DSPP
常氧组	1.21 ± 0.12	1.08 ± 0.19	1.03 ± 0.13
常氧+GSI组	1.73 ± 0.20^a	1.55 ± 0.08^a	1.52 ± 0.14^a
低氧组	0.53 ± 0.14^a	0.43 ± 0.20^a	0.48 ± 0.11^a
低氧+GSI组	0.99 ± 0.13^b	1.09 ± 0.13^b	0.95 ± 0.16^b

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