Major contribution of MEK1 to the activation of ERK1/ERK2 and to the growth of LS174T colon carcinoma cells

Mammalian cells express two closely related MEK isoforms, MEK1 and MEK2, upstream of the ERK1/ERK2 MAPK module. Although genetic studies have suggested that MEK1 and MEK2 do not have overlapping functions in vivo, little is known about their specific contribution to the activation of ERKs and to tumor cell proliferation. We used Tet-inducible shRNA to investigate the independent role of MEK1 and MEK2 for the oncogenic and the serum-induced activation of ERK1 and ERK2 in LS174T colon carcinoma cells. We show that MEK1 is the main activator of both ERK1 and ERK2. MEK2 removal has no impact by itself but it can cooperate with MEK1 ablation for the inhibition of ERK1/2 activity. In addition, we show that MEK1 is the critical isoform regulating tumor cell proliferation in vitro and in vivo.     

Inhibition of MEK1,2/ERK mitogenic pathway by estrogen with antiproliferative properties in rat aortic smooth muscle cells

Proliferation and migration of vascular smooth muscle cells (VSMCs) are believed to contribute significantly to intimal thickening in atheroscleosis, restenosis, and venous bypass graft disease. Estrogen inhibits proliferation and migration of VSMCs. However, antiproliferative mechanisms of estrogen were not well elucidated yet. In this study, we investigated the antiproliferative effect of estrogen to determine whether the transduction signals and protooncogenes were affected in rat aortic smooth muscle cells (RASMCs). Estrogen inhibited the proliferative response stimulated by 5% fetal bovine serum (FBS) dose-dependently in RASMCs (IC50: 40 nM). In 0.5% serum-treated RASMCs, estrogen dramatically inhibited the activity of extracellular signal-regulated kinases (ERK) followed by inhibition of MEK1,2 activity in dose-dependent manner without affecting the other mitogen-activating protein kinases (MAPKs), c-jun N-terminal kinases (JNK) and p38. Induction of Elk-1 mRNA was significantly reduced dose-dependently up to 100 nM of estrogen. These results indicate that the antiproliferative effects of estrogen in RASMCs involved ERK inhibition followed by the inactivation of MEK1,2 and downregulation of Elk-1 expression.     

Antiproliferative effect of panaxynol on RASMCs via inhibition of ERK1/2 and CREB

Panaxynol (PNN) occurs in many foods such as carrot, celery, and several reports have shown that it has neuritogenic and neuroprotective properties. In this study, we have investigated the antiproliferative effect and the mechanism of PNN on platelet-derived growth factor (PDGF)-BB-induced proliferation of rat aortic vascular smooth muscle cells (RASMCs). PNN significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of RASMCs in a concentration-dependent manner. Flow cytometry analysis showed that PNN blocked the cell cycle progression at the G(1)/S phase. Preincubation of RASMCs with 9 microM PNN resulted in a significant inhibition of PDGF-BB-induced extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation expression and PDGF-BB-induced CREB phosphorylation expression. The results indicated that the inhibitory effect of PNN on the PDGF-BB-induced proliferation of RASMCs might be mediated by blocking phosphorylation of ERK1/2 and that of CREB.     

Adhesion stimulates direct PAK1/ERK2 association and leads to ERK-dependent PAK1 Thr212 phosphorylation

The Rac1/Cdc42 effector p21-activated kinase (PAK) is activated by various signaling cascades including receptor-tyrosine kinases and integrins and regulates a number of processes such as cell proliferation and motility. PAK activity has been shown to be required for maximal activation of the canonical Ras/Raf/MEK/ERK Map kinase signaling cascade, likely because of PAK co-activation of Raf and MEK. Herein, we found that adhesion signaling also stimulates an association between PAK1 and ERK1/2. PAK1 and ERK1/2 co-immunoprecipitated from rat aortic smooth muscle cells (SMC) plated on fibronectin, and the two proteins co-localized in membrane ruffles and adhesion complexes following PDGF-BB or sphingosine 1-phosphate treatment, respectively. Far Western analysis demonstrated a direct association between the two proteins, and peptide mapping identified an ERK2 binding site within the autoinhibitory domain of PAK1. Interestingly, deletion of a major ERK binding site in PAK attenuates activation of an ERK-dependent serum-responsive element (SRE)-luciferase reporter gene, indicating that association between PAK and ERK is required to facilitate ERK signaling. We also show that ERK2 phosphorylates PAK1 on Thr(212) in vitro and that Thr(212) is phosphorylated in smooth muscle cells following PDGF-BB treatment in an adhesion- and MEK/ERK-dependent fashion. Expression of a phosphomimic variant, PAK-T212E, does not alter ERK association, but markedly attenuates downstream ERK signaling. Taken together, these data suggest that PAK1 may facilitate ERK signaling by serving as a scaffold to recruit Raf, MEK, and ERK to adhesion complexes, and that subsequent growth factor-stimulated phosphorylation of PAK-Thr(212) by ERK may serve to provide a negative feedback signal to control coordinate activation of ERK by growth factor- and matrix-induced signals.     

Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [³H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5 min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.     

Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

Previously, we demonstrated that progesterone (P4) at physiologic levels (5-500 nM) inhibited proliferation in cultured rat aortic smooth muscle cells (RASMCs) through a P4 receptor (PR)-dependent pathway. We also showed that P4-induced cell cycle arrest in RASMCs occurs when the cyclin-CDK2 system is inhibited just as p21^cip1 and p27^kip1 protein levels are augmented. In the present study, we further investigated the molecular mechanism underlying P4-induced up-regulations of p21^cip1 and p27^kip1 in RASMCs. We used pharmacological inhibitors as well as dominant negative constructs and conducted Western blot analyses to delineate the signaling pathway involved. Our data suggest that P4 up-regulated the expression of p21^cip1 and p27^kip1 in RASMCs through increasing the level of p53 protein mediated by activating the cSrc/Kras/Raf-1/AKT/ERK/p38/IκBα/NFκB pathway. The findings of the present study highlight the molecular mechanism underlying P4-induced up-regulations in p21^cip1 and p27^kip1 in RASMCs.     

Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation

ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS^G12C/G13D or BRAF^V600E. Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.     

PDGF-BB-induced MT1-MMP expression regulates proliferation and invasion of mesenchymal stem cells in 3-dimensional collagen via MEK/ERK1/2 and PI3K/AKT signaling

Mesenchymal stem cells (MSCs) mobilize membrane type-1 matrix metalloproteinase (MT1-MMP) to traffic through both 3-dimensional (3D) collagen as well as basement membrane barriers, but factors capable of regulating the expression and activity of the protease remain unidentified. Herein, we report that the MT1-MMP-dependent invasive activities of rat MSCs are controlled by PDGF-BB. Furthermore, PDGF-BB also stimulates MSC proliferation in 3D type I collagen via an MT1-MMP-dependent process that is linked to pericellular collagen degradation. PDGF-BB stimulates MT1-MMP expression at both the mRNA and protein levels in concert with ERK1/2 and PI3K/AKT activation. Inhibition of ERK1/2 or PI3K/AKT activity potently suppresses both MT1-MMP-dependent invasive and proliferative activities. Basement membrane invasion is likewise stimulated by PDGF-BB in an MT1-MMP-dependent manner via ERK1/2 and PI3K/AKT signaling. Taken together, these data serve to identify PDGF-BB as an important MSC agonist that controls invasive and proliferative activities via MT1-MMP-dependent processes that are regulated by the ERK1/2 and PI3K/AKT signaling pathways.     

Fluvastatin attenuates IGF-1-induced ERK1/2 activation and cell proliferation by mevalonic acid depletion in human mesangial cells

AimsInsulin-like growth factor (IGF)-1 is a major mitogenic growth factor for mesangial cells (MCs). Statins slow the progression of chronic kidney disease by affecting inflammatory cell signaling pathways, in addition to improving lipid profile, however, no studies have investigated the effects of fluvastatin on mitogen-activated protein (MAP) kinase activity or MC proliferation in kidney cells. We investigated the effects of fluvastatin on IGF-1-induced activation of intracellular signal pathways and MC proliferation, and examined the inhibitory mechanisms of fluvastatin.Main methodsWestern blotting and cell proliferation assay were used.Key findingsIGF-1 induced phosphorylation of extracellular-related kinase (ERK)1/2, MAP or ERK kinase (MEK)1/2, and Akt, expression of cyclin D1, and MC proliferation in cultured human MCs. Fluvastatin or PD98059, an MEK1 inhibitor, completely abolished IGF-1-induced MEK1/2 and ERK1/2 phosphorylation and MC proliferation, whereas inhibition of Akt had no effect on MC proliferation. Mevalonic acid prevented fluvastatin inhibition of IGF-1-induced MEK1/2 and ERK1/2 phosphorylation, cyclin D1 expression, and MC proliferation.SignificanceFluvastatin inhibits IGF-1-induced activation of the MAP kinase pathway and MC proliferation by mevalonic acid depletion, and might have renoprotective effects by inhibiting IGF-1-mediated MC proliferation.     

The 2″-O-glucosylation of vitexin and isovitexin in petals of Silene alba is catalysed by two different enzymes

Two separate genes, Fg and Vg, which govern the presence of isovitexin 2″-O-glucoside and vitexin 2″-O-glucoside respectively in the petals of Silene alba control different glucosyltransferases. In Vg/Vg,fg/fg plants no isovitexin 2″-O-glucosyltransferase was present and in vg/vg,Fg/Fg plants no vitexin 2″-O-glucosyltransferase activity could be detected. The Fg-controlled UDP-glucose: isovitexin 2″-O-glucosyltransferase has a pH optimum of8.5, while the Vg-controlled vitexin 2″- O-glucosyltransferase has a pH optimum of7.5. Both glucosyltransferases are stimulated by the divalent cations Ca²⁺, Co²⁺, Mn²⁺ and Mg²⁺. For isovitexin 2″-O-glucosylation, however, much higher concentrations are needed than for vitexin 2″-O-glucosylation.For UDP-glucose a ‘true K_m’ value of0.3 mM with the Fg-controlled and of 0.2 mM with the Vg-controlled enzyme was found. For isovitexin and vitexin these values are respectively 0.09 and 0.01 mM.     

Impact of Glutathione Peroxidase-1 Deficiency on Macrophage Foam Cell Formation and Proliferation: Implications for Atherogenesis

Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE^−/− mice. However, the distribution of GPx-1 within the atherosclerotic lesion as well as the mechanisms leading to increased macrophage numbers in lesions is still unknown. Accordingly, the aims of the present study were (1) to analyze which cells express GPx-1 within atherosclerotic lesions and (2) to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both in situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE^−/− mice after 12 weeks on a Western type diet revealed that both macrophages and – even though to a less extent – smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated for 3 days with macrophage-colony-stimulating factor (MCSF), GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL) induced foam cell formation and led to increased proliferative activity of peritoneal macrophages. The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1^−/−ApoE^−/− mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK. Representative effects of GPx-1 deficiency on both macrophage proliferation and MAPK phosphorylation could be abolished by the GPx mimic ebselen. The present study demonstrates that GPx-1 deficiency has a significant impact on macrophage foam cell formation and proliferation via the p44/42 MAPK (ERK1/2) pathway encouraging further studies on new therapeutic strategies against atherosclerosis.     

白藜芦醇通过下调MEK/ERK/c-Jun信号通路抑制H2O2诱导的肺癌细胞增殖

目的:探讨白藜芦醇(Res)是否通过下调ERK激酶/胞外信号调节激酶/原癌基因(MEK/ERK/c-Jun)信号通路抑制小剂量过氧化氢(H2O2)诱导肺癌细胞增殖。方法:采用MTS实验检测小剂量20μM H2O2以及分别加入MEK阻断剂U0126和Res后H2O2对肺癌细胞NCI-H1395增殖的影响,采用Western Blot检测H2O2对ERK1/2和Akt蛋白磷酸化水平以及加入Res后H2O2对MEK、ERK1/2和c-Jun蛋白磷酸化水平的影响。结果:小剂量H2O2对肺癌细胞NCI-H1395具有促增殖作用,H2O2通过活化ERK1/2和Akt蛋白的磷酸化水平促进肺癌细胞NCI-H1395增殖,加入MEK阻断剂U0126后H2O2对肺癌细胞NCI-H1395增殖作用降低(P<0.05)。Res可抑制H2O2诱导的肺癌细胞NCI-H1395增殖,加入Res后,H2O2引起的MEK、ERK1/2和c-Jun蛋白磷酸化水平均降低(P<0.05)。结论:小剂量H2O2对肺癌细胞NCI-H1395具有促增殖作用,Res通过抑制MEK/ERK/c-Jun信号通路来抑制H2O2对肺癌细胞NCI-H1395的促增殖作用,其具体机制还需进一步研究。     

DMF inhibits PDGF-BB induced airway smooth muscle cell proliferation through induction of heme-oxygenase-1

Background

Airway wall remodelling is an important pathology of asthma. Growth factor induced airway smooth muscle cell (ASMC) proliferation is thought to be the major cause of airway wall thickening in asthma. Earlier we reported that Dimethylfumarate (DMF) inhibits platelet-derived growth factor (PDGF)-BB induced mitogen and stress activated kinase (MSK)-1 and CREB activity as well as IL-6 secretion by ASMC. In addition, DMF altered intracellular glutathione levels and thereby reduced proliferation of other cell types.

Methods

We investigated the effect of DMF on PDGF-BB induced ASMC proliferation, on mitogen activated protein kinase (MAPK) activation; and on heme oxygenase (HO)-1 expression. ASMC were pre-incubated for 1 hour with DMF and/or glutathione ethylester (GSH-OEt), SB203580, hemin, cobalt-protoporphyrin (CoPP), or siRNA specific to HO-1 before stimulation with PDGF-BB (10 ng/ml).

Results

PDGF-BB induced ASMC proliferation was inhibited in a dose-dependant manner by DMF. PDGF-BB induced the phosphorylation of ERK1/2 and p38 MAPK, but not of JNK. DMF enhanced the PDGF-BB induced phosphorylation of p38 MAPK and there by up-regulated the expression of HO-1. HO-1 induction inhibited the proliferative effect of PDGF-BB. HO-1 expression was reversed by GSH-OEt, or p38 MAPK inhibition, or HO-1 siRNA, which all reversed the anti-proliferative effect of DMF.

Conclusion

Our data indicate that DMF inhibits ASMC proliferation by reducing the intracellular GSH level with subsequent activation of p38 MAPK and induction of HO-1. Thus, DMF might reduce ASMC and airway remodelling processes in asthma.     

Identification et dosage des constituants amers des racines de Gentiana lutea L.

The bitter constituents of Gentiana lutea L. roots were isolated and identified by mass spectrometry. Gentiopicroside, swertiamarin, amarogentin were characterized and their abundance evaluated. Amarogentin was shown to be the main bitter constituent of the roots. By cultivation of Gentiana lutea, roots with a high content in amarogentin can be obtained in 2 yr.     

Ellagic acid inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and prevents atheroma formation in streptozotocin-induced diabetic rats

Plant-derived polyphenolic compounds have beneficial health effects. In the present study, we determined the ability of ellagic acid (EA) to prevent platelet-derived growth factor-BB (PDGF-BB)-induced proliferation of primary cultures of rat aortic smooth muscle cells (RASMCs). We also determined the ability of EA to prevent atherosclerosis in streptozotocin-induced diabetic rats. Proliferation of cells was measured via Alamar Blue assay and through propidium iodide-based cell cycle analysis in flow cytometer. Reactive oxygen species (ROS) were measured via 2′,7′-dichlorofluorescin diacetate and Amplex red methods. Expression of proliferation markers and activation of kinases were assessed by immunoblot analysis. Cotreatment of primary cultures of RASMCs with 25 μmol/L of EA significantly reduced PDGF-BB (20 ng/ml)-induced proliferation by blocking S-phase entry. EA effectively blocked PDGF receptor-β (PDGFR-β) tyrosine phosphorylation, generation of intracellular ROS and downstream activation of extracellular signal-regulated kinase 1/2. It also blocked PDGF-BB-induced expression of cyclin D1. Computational molecular docking of EA with the PDGFR-β–PDGF-BB complex revealed two putative inhibitor binding sites which showed similar binding energies with the known PDGFR-β inhibitor AG1295. In diabetic rats, supplementation of diet with 2% EA significantly blocked diabetes-induced medial thickness, and lipid and collagen deposition in the arch of aorta. These were assessed through haematoxylin and eosin, Oil Red O and Masson’s trichome staining, respectively. EA treatment also blocked cyclin D1 expression in medial smooth muscle cells in experimental animals. Thus, EA is effective in reducing atherosclerotic process by blocking proliferation of vascular smooth muscle cells.     

Role of platelet-derived growth factor-BB (PDGF-BB) in human pulmonary artery smooth muscle cell proliferation

Abstract

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) and suppressed apoptosis. Platelet-derived growth factor (PDGF) is a potent mitogen involved in cell proliferation and migration. PDGF-BB induces the proliferation and migration of PASMCs and has been proposed to be a key mediator in the progression of PAH. Previous studies have shown that PDGF and its receptor are substantially elevated in lung tissues and PASMCs isolated from patients and animals with PAH, but the underlying mechanisms are still poorly manifested. MAP kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun NH2-terminal kinase1/2 (JNK1/2), and p38 are the key intracellular signals for stimuli-induced cell proliferation, survival, and apoptosis. Therefore, the purpose of this study is to determine whether PDGF-BB on cell proliferation process is mediated through the MAP kinases pathway in human PASMCs (HPASMCs). Our results showed PDGF-BB-induced proliferating cell nuclear antigen (PCNA), Cyclin A and Cyclin E expression in a concentration-dependent manner. The expression levels of phosphorylated JNK (p-JNK) was upregulated with 20?ng/ml PDGF-BB treatment, while PDGF-BB could not increase phosphorylated ERK1/2 (p-ERK1/2) and p-38 (p-p38) expression. The effects of PDGF-BB on cell proliferation and survival were weakened after the administration of antagonist of the JNK pathway or si-JNK. In addition, PDGF-BB protected against the loss of mitochondrial membrane potentials evoked by serum deprivation (SD) in a JNK-dependent manner. These results suggest that PDGF-BB promotes HPASMCs proliferation and survival, which is likely to be mediated via the JNK pathway.     

Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway

The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells.     

T-cell activation decreases miRNA-15a/16 levels to promote MEK1–ERK1/2–Elk1 signaling and proliferative capacity

While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post–T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3′-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal–regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1–ERK1/2–Elk1 signaling required for optimal proliferation.