Effects of Prostaglandin E2 on the Differentiation and Gene Expression of Human Dental Pulp Cells
Date Issued
2011
Date
2011
Author(s)
Wang, Yu-Ting
Abstract
Aim: During pulpal inflammation, PGE2 level was elevated and known to be an important mediator to regulate the function of dental pulp cells. This can be due to binding of PGE2 to cell surface receptors such as EP1, EP2 and EP3 prostaglandin receptor to stimulate downstream signaling. The aim of the study is to investigate the effect of PGE2 and EP4 receptor activation on the differentiation and gene expression in human dental pulp and its signal transduction pathway.
Materials and Methods: Primary-cultured human dental pulp cells were treated with PGE2 and it agonists (19ROH-PGE2, EP2 agonist; sulprostone, EP1/EP3 agonist; CAY10598, EP4 agonist) with different concentration and then observed the morphological change of pulp cell under contrast microscope. Cell proliferation was evaluated by MTT assay. Effects of PGE2 and CAY10598 on cell differentiation and mineralization during different stimulation periods or with different concentrations were evaluated by alkaline phosphatase (ALP) staining and reverse-transcriptase polymerase chain reaction(RT-PCR). We observed both the AP-1 transcription factors (c-fos, FosB, c-jun, JunB) and the mineralization markers (ALP, Runx2, Osterix, osteocalcin) mRNA change in human dental pulp after stimulation. Cell were pretreated with H89 (PKA inhibitor), dorsomorphin (AMPK inhibitor) 30 minutes before adding PGE2. Signaling of PGE2 was investigated by RT-PCR.
Results: The cell morphology didn’t affect by PGE2、19ROH-PGE2 and Sulprostone. However, the nuclei turned into lager and hyperchromatic after stimulation by CAY10598 10 uM. MTT assay also indicated CAY10598 inhibited the growth of pulp cells at high concentration (5μM-10μM). Cells under the treatment of PGE2 and its agonists all showed an increase in ALP activity at lower concentration and decreased at higher concentration. In 5 days cultured group, PGE2 (5μM-10μM) can induce mineralization markers (ALP, Runx2, Osterix, Osteocalcin) expression, which was consistent with the expression of AP-1 transcription factors (c-fos, FosB, JunB). PGE2 also can induce expression of c-fos, FosB and JunB during 120 mins, while c-jun decreased time dependently. In 30mins culture group, PGE2 and CAY10598 induce c-fos、FosB at lower concentration (0.1μM-0.5μM) compared with 5 days cultured group. Interestingly, the expression of junB were always enhanced at higher concentration or longer stimulation period, but c-jun decreased dose-dependently and time-dependently. H89 (PKA inhibitor) and dorsomorphin (AMPK inhibitor) attenuated the effects of PGE2 .
Conclusion: PGE2 may be involved in dental pulp inflammationand repair process via induction of AP-1 transduction of AP-1 transcription factors and mineralization markers via EP2 and EP4 receptors through PKA/AMPK singaling pathway. EP1/3 agonist (sulprostone)、EP2 agonist (19ROH-PGE2) and EP4 agonist(CAY10598) can increase the ALP activity of dental pulp cells. The responsiveness of human dental pulp cells to PGE2 may change during the culture period. Although EP4 is the least receptor in human dental pulp, but its activation by CAY10598 still has significant effects on dentinogenesis.
Subjects
Dental pulp cells
PGE2
ALP
Runx2
Osterix
Osteocalcin
AP-1 transcription factors
Type
thesis
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