Inhibitory Effects Of U0126 Research Articles

MAT2A/2B promote porcine intramuscular preadipocyte proliferation through ERK signaling pathway.

Intramuscular fat (IMF) content has been identified as a crucial factor of porcine meat quality. MAT2A and MAT2B coordinately catalyzes the synthesis of the major biological methyl donor S-adenosylmethionine (SAMe). However, the regulatory effect of MAT2A and MAT2B on porcine intramuscular preadipocyte proliferation has not been clarified. In this study, we investigated the effect of MAT2A and MAT2B and its potential mechanism during porcine intramuscular proliferation. We demonstrated that overexpression of MAT2A and MAT2B promoted the cell cycle progression of porcine preadipocyte by flow cytometry and EdU-labeling assay, as well as promoted the expression of cell cycle marker genes including Cyclin B, Cyclin D, and Cyclin-dependent kinase 4, but reduced the expression of cell cycle inhibitor P27. Consistently, knockdown of MAT2A and MAT2B inhibited cell cycle progression and downregulated the mRNA and protein levels of the above genes. Furthermore, overexpression of MAT2A and MAT2B activated the phosphorylation of ERK1/2. Moreover, the inhibitory effect of U0126 (a specific ERK1/2 inhibitor) on the ERK1/2 activities was partially recovered by overexpression of MAT2A and MAT2B in porcine intramuscular preadipocytes. Taken together, our findings suggested that MAT2A and MAT2B promote porcine preadipocyte proliferation by ERK1/2 signaling pathway.

Inhibition of ERK1/2 downregulates triglyceride and palmitic acid accumulation in cashmere goat foetal fibroblasts

ABSTRACTAn extracellular signal-regulated kinase (ERK) is a mitogen-activated protein kinase (MAPK) in mammalian cells that interact with the mechanistic target of rapamycin (mTOR) signalling pathway. ERK signalling integrates signals from growth factors, nutrients, and mitogens to regulate cell proliferation, differentiation, and the cell cycle. The purpose of this study was to examine the inhibitory effects of U0126, a specific MEK/ERK inhibitor, on ERK and mTOR signalling, Lipin 1 protein level, PPARγ activation, and triglyceride (TAG) and palmitic acid (PA) accumulation in Cashmere goat foetal fibroblasts (GFbs). The proliferation of GFbs was significantly impeded by U0126, the IC50 of which was 1.54 µM. U0126 prevented the phosphorylation of ERK1/2 (Thr202/Tyr204), S6 (Ser240/244), and 4EBP1 (Thr37/46), the latter 2 of which are mTOR downstream effectors. Moreover, the cytoplasmic level of Lipin 1 and the nuclear PPARγ protein abundance were attenuated by U0126 while the concentration of TAG and PA was significantly decreased (p < .05) in GFbs. These data indicate that ERK1/2 have important functions in GFb proliferation, governing the activation of PPARγ and Lipin 1 level and the accumulation of TAG and PA.

Oncogenic KRAS Activates Hedgehog Signaling Pathway in Pancreatic Cancer Cells

Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAF(E600), but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression.

Renal upregulation of HO-1 reduces albumin-driven MCP-1 production: implications for chronic kidney disease

Proteinuria contributes to chronic kidney disease by stimulating renal tubular epithelial cells to produce cytokines such as monocyte chemoattractant protein-1 (MCP-1). The present study determined whether cellular overexpression of heme oxygenase-1 (HO-1) can influence albumin-stimulated MCP-1 production. In response to bovine serum albumin, NRK-52E cells constitutively overexpressing HO-1 (HO-1 OE cells) exhibit less induction of MCP-1 mRNA and less production of MCP-1 protein compared with similarly treated, control NRK-52E cells (CON cells). In wild-type NRK-52E cells, and under these conditions, we demonstrate that the induction of MCP-1 is critically dependent on intact NF-kappaB binding sites in the MCP-1 promoter. In response to albumin, CON cells exhibit activation of NF-kappaB, and this is reduced in HO-1 OE cells. Albumin also activates ERK1/2 and increases ERK activity, both of which are exaggerated in HO-1 OE cells. Studies with an inhibitor of MAPK/ERK kinase (U0126) demonstrate that the inhibitory effects of U0126 on MCP-1 production are attenuated in HO-1 OE cells. We conclude that HO-1 overexpression in the proximal tubule reduces MCP-1 production in response to albumin, and this occurs, at least in part, by inhibiting an ERK-dependent, NF-kappaB-dependent pathway at a site that is distal to the activation of ERK. These findings suggest that the induction of HO-1 in the proximal tubule, as occurs in chronic kidney disease, may be a countervailing response that reduces albumin-stimulated production of cytokines such as MCP-1.

P21Cip-1/SDI-1/WAF-1 expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21Cip-1/SDI-1/WAF-1, is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.

Requirement of Mitogen-Activated Protein Kinase Activation for the Meiotic Resumption of Porcine Oocytes

The requirement of mitogen-activated protein kinase (MAPK) for the initiation of meiotic maturation in porcine oocytes was examined using U0126, an inhibitor of MAPK activator. Immunoblot analysis and kinase assay showed that U0126 inhibited the phosphorylation and activation of MAPK incompletely in porcine oocytes. In this condition, maturation/M-phase promoting factor (MPF) activity was also inhibited and the rate of germinal vesicle breakdown (GVBD, 95%) was reduced to 42%. The classification of oocytes remaining at germinal vesicle stage (GV) and reached to second metaphase stage (M2) after 48h culture with U0126 revealed that GV oocytes had low MAPK and MPF activities while M2 oocytes had high activities in both kinases, suggesting the indispensability of MAPK activation for meiotic resumption of porcine oocytes. In mouse oocytes, however, the inhibition of MAPK activation by U0126 had no effect on GVBD nor MPF activation, consistent with previous reports using mos deficient mice. Additionally, we found that the inhibitory effects of U0126 needed the presence of cumulus cells because of the ineffectiveness of U0126 on denuded porcine oocytes. These results suggest that MAPK act as indispensable mediator of MPF activation and GVBD in porcine oocytes, like in Xenopus oocytes but not in mouse oocytes.