u-0126 and triptolide

Since its isolation from Tripterygium wilfordii in 1972, triptolide has been shown to possess potent anticancer activity against a variety of cancers, and has entered phase I clinical trial. It is a diterpenoid triepoxide that acts through multiple molecular targets and signaling pathways. The mitogen-activated protein kinases are well known for their modulation of cell survival and proliferation. In particular, the ERK pathway has a dual role in cell proliferation and cell death. Thus far, data on the effect of triptolide on ERK signaling remain limited. In our current study, we have shown for the first time that ERK activation rather than inhibition occurred in a dose- and time-dependent manner following triptolide treatment in MDA-MB-231 breast cancer cells. ERK activation was crucial in mediating triptolide-induced caspase-dependent apoptosis. Tritpolide-induced ERK activation modulated the expression of the Bcl-2 protein family member Bax but was not involved in the downregulation of Bcl-xL expression. Signals acted upstream of ERK activation included generation of reactive oxygen species (ROS) and endoplasmic reticulum stress predominantly via the PERK‑eIF2α pathway, as the MEK inhibitor U0126 did not inhibit the phosphorylation of PERK and eIF2α or the generation of ROS.

Topics: Apoptosis; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Cell Survival; Clinical Trials as Topic; Diterpenes; eIF-2 Kinase; Endoplasmic Reticulum Stress; Epoxy Compounds; Eukaryotic Initiation Factor-2; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinases; Nitriles; Oxidative Stress; Phenanthrenes; Reactive Oxygen Species; Signal Transduction

We examined the expression of dual specificity phosphatase 1 (DUSP1) by gonadotropin-releasing hormone (GnRH) stimulation and investigated the role of DUSP1 on gonadotropin gene expression using LbetaT2 gonadotroph cell line. DUSP1 expression was markedly increased 60 min after GnRH stimulation, and mitogen-activated protein kinase 3/1 (MAPK3/1) activation was gradually decreased after 60 min. GnRH-induced MAPK3/1 activation was completely inhibited by U0126, a MEK inhibitor, whereas GnRH-induced DUSP1 expression was partially inhibited by U0126. GnRH-induced DUSP1 induction was inhibited by triptolide, a diterpenoid triepoxide. In contrast, this compound potentiated MAPK3/1 activation. U0126 prevented GnRH-stimulated gonadotropin subunit promoter activation dose dependently, and 10 muM of U0126 reduced the effects of GnRH on the Lhb and Fshb promoters to 79.15% and 55.66%, respectively. GnRH-stimulated activation of Lhb and Fshb promoters as well as serum response factor (Srf) promoters were almost completely inhibited by triptolide, suggesting that this component had a nonspecific effect to the cells. Dusp1 siRNA reduced the expression of DUSP1 and augmented MAPK3/1 phosphorylation, but it did not increase of gonadotropin promoters. By overexpression of DUSP1, both GnRH-stimulated Lhb and Fshb promoters were significantly reduced. We have previously shown that insulin-like growth factor 1 (IGF1) increases MAPK3/1 but does not activate gonadotropin subunit promoters. IGF1 failed to induce DUSP1 expression. In addition, under pulsatile GnRH stimulation, DUSP1 expression was observed following high-frequency GnRH pulses but not following low-frequency pulses. Our study demonstrated that DUSP1, induced by GnRH, functions not only as an MAPK3/1-inactivating phosphatase but also as an important mediator in gonadotropin subunit gene expression regulation.

Topics: Animals; Butadienes; Cell Line; Diterpenes; Dual Specificity Phosphatase 1; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Epoxy Compounds; Follicle Stimulating Hormone, beta Subunit; Gene Expression; Gonadotropin-Releasing Hormone; Gonadotropins, Pituitary; Kinetics; Luteinizing Hormone, beta Subunit; Mice; Mitogen-Activated Protein Kinase 3; Nitriles; Phenanthrenes; Pituitary Gland; Promoter Regions, Genetic

The role of extracellular signal-regulated kinase (ERK) in mediating the ability of thyrotropin-releasing hormone (TRH) to stimulate the prolactin gene has been well elucidated. ERK is inactivated by a dual specificity phosphatase, mitogen-activated protein kinase phosphatase (MKP). In this study, we examined the induction of MKP-1 protein by thyrotropin-releasing hormone (TRH) in pituitary GH3 cells, and investigated the possible role for MKP-1 in TRH-induced prolactin gene expression. MKP-1 protein was induced significantly from 60 min after TRH stimulation, and remained elevated at 4h. The effect of TRH on MKP-1 expression was completely prevented in the presence of the MEK inhibitor, U0126. In the experiments using triptolide, a potent blocker for MKP-1, MKP-1 induction by TRH was completely inhibited in a dose-dependent manner. TRH-induced ERK activation was significantly enhanced in this condition. Prolactin promoter activity, activated by TRH, was reduced to the control level in the presence of triptolide in a dose-dependent manner. In GH3 cells, which were transfected with MKP-1 specific siRNA, both the basal and TRH-stimulated activities of the prolactin promoter were significantly reduced compared to the cells transfected with negative control siRNA. Our present results support a critical role of MKP-1 in TRH-induced, ERK-dependent, prolactin gene expression.

Topics: Animals; Butadienes; Cell Line; Diterpenes; Dual Specificity Phosphatase 1; Enzyme Inhibitors; Epoxy Compounds; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Gene Expression Regulation; Nitriles; Phenanthrenes; Prolactin; Rats; RNA, Small Interfering; Thyrotropin-Releasing Hormone