Triptolide Inhibits the Proliferation of Immortalized HT22 Hippocampal Cells Via Persistent Activation of Extracellular Signal-Regulated Kinase-1/2 by Down-Regulating Mitogen-Activated Protein Kinase Phosphatase-1 Expression

Abstract

Objective: Triptolide (TP) has been reported to suppress the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), of which main function is to inactivate the extracellular signal-regulated kinase-1/2 (ERK-1/2), the p38 MAPK and the c-Jun N-terminal kinase-1/2 (JNK-1/2), and to exert antiproliferative and pro-apoptotic activities. However, the mechanisms underlying antiproliferative and pro-apoptotic activities of TP are not fully understood. The purpose of this study was to examine whether the down-regulation of MKP-1 expression by TP would account for antiproliferative activity of TP in immortalized HT22 hippocampal cells.

Methods: MKP-1 expression and MAPK phosphorylation were analyzed by Western blot. Cell proliferation was assessed by (3)H-thymidine incorporation. Small interfering RNA (siRNA) against MKP-1, vanadate (a phosphatase inhibitor), U0126 (a specific inhibitor for ERK-1/2), SB203580 (a specific inhibitor for p38 MAPK), and SP600125 (a specific inhibitor for JNK-1/2) were employed to evaluate a possible mechanism of antiproliferative action of TP.

Results: At its non-cytotoxic dose, TP suppressed MKP-1 expression, reduced cell growth, and induced persistent ERK-1/2 activation. Similar growth inhibition and ERK-1/2 activation were observed when MKP-1 expression was blocked by MKP-1 siRNA and its activity was inhibited by vanadate. The antiproliferative effects of TP, MKP-1 siRNA, and vanadate were significantly abolished by U0126, but not by SB203580 or SP600125.

Conclusion: Our findings suggest that TP inhibits the growth of immortalized HT22 hippocampal cells via persistent ERK-1/2 activation by suppressing MKP-1 expression. Additionally, this study provides evidence supporting that MKP-1 may play an important role in regulation of neuronal cell growth.

Keywords: Extracellular signal-regulated kinase-1/2; HT22 hippocampal cell; Mitogen-activated protein kinase; Mitogen-activated protein kinase phosphatase-1; Proliferation; Triptolide.

Fig. 1

Effect of triptolide (TP) on the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) in immortalized HT22 cells. Cells were treated with indicated concentrations of TP for 6 h. The cells that were transiently transfected with either MKP-1 small interfering RNA (MKP-1 siRNA; Lane 5) or MKP-1 gene (Lane 6) served as the controls for MKP-1 expression, and were cultured for 6 h. Expression of MKP-1 was examined by Western blot analysis with anti-MKP-1 antibody, as described in materials and methods. Representative Western blots of three independent experiments are shown.

Fig. 2

Effect of triptolide (TP) on necrosis and apoptosis of immortalized HT22 cells. A : Immortalized HT22 cells were treated with indicated concentrations of TP or 300 µM of H₂O₂ for 24 h. For the analysis of necrosis, the release of lactate dehydrogenase (LDH) into the culture medium was determined by a LDH assay kit, as described in materials and methods. Values represent mean ± SE of four replicates in three different experiments. ^*p < 0.05 versus untreated control cells. B : Immortalized HT22 cells were treated with 0.5 µM of TP or 0.5 µM of paclitaxel (positive control) for 24 h. For the analysis of apoptosis, the nuclei of HT22 cells were stained with 4',6-diamidine-2-phenylindole, and observed under a fluorescent microscope. To visualize cell morphology, cytoplasm was stained with rhodamin 6G, a cell-permeable fluorescence dye. The fragmented nuclei that are characteristics of apoptotic cells are shown only in paclitaxel-treated cells.

Fig. 3

Effect of triptolide (TP) on proliferation of immortalized HT22 cells. Cells were treated with indicated concentrations of TP for 24 h. The cells that were transiently transfected with either mitogen-activated protein kinase phosphatase-1 (MKP-1) small interfering RNA (MKP-1 siRNA; column 5) or MKP-1 gene (column 6) served as the controls for MKP-1-dependent proliferation, and were cultured for 24 h. Cell proliferation was determined by ³H-thymidine incorporation, as described in materials and methods. Values represent mean ± SE of four replicates in three different experiments. ^*p < 0.05 versus untreated control cells.

Fig. 4

Effect of triptolide (TP) on phosphorylation of mitogen-activated protein kinases (MAPKs) in immortalized HT22 cells. A : Cells were treated with 0.5 µM of TP for 6 h. The cells that were transiently transfected with MAPK phosphatase-1 (MKP-1) small interfering RNA (MKP-1 siRNA; lane 3) served as the control for MKP-1-dependent phosphorylation of MAPKs, and were cultured for 6 h. B : Cells were treated with 0.5 µM of TP for indicated times. Phosphorylation and expression of the extracellular signal-regulated kinase-1/2 (ERK-1/2), the p38 MAPK and the c-Jun N-terminal kinase-1/2 (JNK-1/2) were examined by Western blot analysis, as described in materials and methods. Representative Western blots of three independent experiments are shown.

Fig. 5

Effect of the phosphatase inhibitor vanadate on the extracellular signal-regulated kinase-1/2 (ERK-1/2) phosphorylation and proliferation of immortalized HT22 cells. A : Cells were treated with 10 µM of vanadate for indicated times. Phosphorylation and expression of ERK-1/2 were examined by Western blot analysis, as described in materials and methods. Representative Western blots of three independent experiments are shown. B : Cells were treated with 0.5 µM of triptolide or 10 µM of vanadate for 24 h. Cell proliferation was determined by 3H-thymidine incorporation, as described in materials and methods. Values represent mean ± SE of four replicates in three different experiments. ^*p < 0.05 versus untreated control cells.

Fig. 6

Effect of U0126 on cell growth inhibition by triptolide (TP), vanadate, and mitogen-activated protein kinase phosphatase-1 (MKP-1) small interfering RNA (siRNA). A : Immortalized HT22 cells were transiently transfected with MKP-1 siRNA, or treated for 6 h with 0.5 µM of TP or 10 µM of vanadate in the presence or absence of 10 µM of U0126. Phosphorylation and expression of the extracellular signal-regulated kinase-1/2 were examined by Western blot analysis as described in materials and methods. Representative Western blots of three independent experiments are shown. B : Immortalized HT22 cells were transfected with MKP-1 siRNA or treated for 24 h with 0.5 µM of TP or 10 µM of vanadate in the presence or absence of 10 µM of U0126. Cell proliferation was determined by ³H-thymidine incorporation, as described in materials and methods. Values represent mean ± SE of four replicates in three different experiments. ^*p < 0.05.