Journal: The Journal of Neuroscience

Article Title: AP-1 Transcription Factors Mediate BDNF-Positive Feedback Loop in Cortical Neurons

doi: 10.1523/JNEUROSCI.3360-15.2016

Figure Lengend Snippet: TrkB signaling induces rBDNF mRNA expression in primary cortical neurons. A, Expression of all measured BDNF transcripts is induced by TrkB signaling. At 7 DIV, rat primary cortical neurons were treated with 50 ng/ml BDNF for the time indicated; endogenous BDNF mRNA levels were measured using RT-qPCR with primers specific for transcripts with the respective 5′ exons, or primers against coding region to measure total BDNF mRNA. mRNA levels are shown as fold induction relative to the levels of the respective transcripts in untreated cells (CTRL). B, C, BDNF induction in response to TrkB signaling is mediated mainly by the MAPK pathway. Inhibitors of different signaling pathways (10 μm U0126, 10 μm Bix02189, 1 μm PD184352, 25 nm Az-23, 10 μm Go6983, 100 nm Wortmannin, 1 μm U73122) or vehicle (0.1% DMSO) was added to the culture medium 30 min before treating primary neurons with 50 ng/ml BDNF for 3 h at 7 DIV. BDNF expression was measured using RT-qPCR. For each transcript, mRNA levels are shown relative to the respective transcript's mRNA expression in cells treated with vehicle and not treated with BDNF. Error bars represent SEM of at least three independent experiments in A–C. Asterisks indicate statistical significance relative to the respective transcript's untreated control (A), or relative to the respective transcript levels in cells treated with DMSO and either left untreated or treated with BDNF for 3 h, respectively (B, C). *p < 0.05, **p < 0.01, ***p < 0.001; t test on log-transformed and autoscaled data.

Article Snippet: Final concentrations of the compounds were as follows: 0.1% DMSO (Sigma-Aldirch), 10 μ m U0126 (Tocris Bioscience), 10 μ m Bix02189 (Axon Medchem), 1 μ m PD184352 (Sigma-Aldrich), 25 n m Az-23 (Axon Medchem), 10 μ m Go6983 (Tocris Bioscience), 100 n m Wortmannin (Millipore), 1 μ m {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}} U73122 (Cayman Europe).

Techniques: Expressing, Quantitative RT-PCR, Transformation Assay

Journal: Nature Communications

Article Title: Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors

doi: 10.1038/s41467-020-15031-3

Figure Lengend Snippet: a Schematic diagram of ERK5 (full length) and ERK5 ΔTAD, which lacks the C-terminal extension. Functional domains with amino acid positions are: cytosolic targeting domain (1–77), kinase domain (48–383), activation-loop TEY motif (219–221), proline rich domain (PR) 1 (434–485) and 2 (578–701), nuclear localisation signal (NLS) (505–539), minimal transactivation domain (TAD) (664–789), and the N-terminal interaction domain (740-816). b Chemical structures of ERK5i: compounds 25 , 26 and AX15836. c Schematic representation of the ERK5:MEF2D luciferase assay. d Chemical structure of the MEK5i, BIX02189. Structures were drawn using ChemDraw v16.0.

Article Snippet: BIX02189 is from Selleckchem.

Techniques: Functional Assay, Activation Assay, Luciferase

Journal: Nature Communications

Article Title: Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors

doi: 10.1038/s41467-020-15031-3

Figure Lengend Snippet: a , b , c and e HEK293 cells were transfected with GAL4-MEF2D, GAL4:LUC and CMV:Renilla, together with either wild-type HA-ERK5 (full length) or HA-ERK5 ΔTAD and EGFP-MEK5D or EGFP (control) as indicated. Four hours post transfection, cells were treated with either DMSO (control) or compound 26 ( a ), compound 25 ( b ), AX15836 ( c ) or BIX02189 ( e ) at concentrations indicated. Twenty-four post transfection, cells were lysed and firefly luciferase activity was measured and normalised to Renilla. The results are presented as the mean of three independent experiments ± SEM. Source data are provided as a Source Data file. d HEK293 cells were transfected as in ( a ). Twenty-four hours post transfection cells were lysed and processed as in a . f HEK293 cells were transfected with GAL4-MEF2D, GAL4:LUC and CMV:Renilla together with either wild-type HA-ERK5 (full length) or HA-ERK5 kinase dead and EGFP-MEK5D or EGFP (control) as indicated. Twenty-four hours post transfection cells were lysed and processed as in a . g and h HEK293 cells were transfected as in ( f ). Four hours post transfection, cells were treated with either DMSO (control) or compound 26 ( g ) or AX15836 ( h ) at concentrations indicated. Twenty-four hours post transfection cells were lysed and processed as in ( a ).

Article Snippet: BIX02189 is from Selleckchem.

Techniques: Transfection, Control, Luciferase, Activity Assay

Journal: Journal of pineal research

Article Title: Biological effects of melatonin on osteoblast/osteoclast cocultures, bone, and quality of life: Implications of a role for MT2 melatonin receptors, MEK1/2, and MEK5 in melatonin-mediated osteoblastogenesis.

doi: 10.1111/jpi.12465

Figure Lengend Snippet: Figure 6 illustrates the effect of melatonin on the expres- sion of the metabolic proteins, PPARγ, GLUT4, and insulin Rß in transwell osteoblasts, due to their potential impact on bone cell differentiation and activity.60-62 Exposure to mela- tonin in osteogenic media (Os+/Mel) significantly reduced PPARγ expression in both cocultures—transwell and layered (Figure 6A, Table 1 and Figure 9F, respectively). In tran- swell osteoblasts, the addition of 4P-PDOT trended toward blocking melatonin’s inhibitory effect on PPARγ. Except for SC-1-151, which increased PPARγ expression in transwell osteoblasts, no other effects of the inhibitors alone—MEK1/2 or MEK5—were observed. When coadministered with SC-1- 151, melatonin trended toward a decrease in PPARγ expres- sion (Figure 6A). Regarding GLUT4 expression, melatonin was without effect on its expression in transwell osteoblasts (Figures 6B and 9G); however, when added in combination with 4P- PDOT, BIX02189, or PD98959, GLUT4 levels increased. Both BIX02189 and SC-1-151 increased GLUT4 levels on their own in transwell osteoblasts. In layered cocul- tures, melatonin decreased GLUT4 expression (Table 1; Figure 9G). For insulin receptor ß, melatonin was without effect in either coculture (Figure 6C, Table 2 and Figure 9H, respec- tively); however, when combined with BIX02189, PD98059, or SC-1-151, insulin receptor ß levels increased in transwell osteoblasts. Except for SC-1-151, which increased insulin re- ceptor ß expression alone, no other effects of the inhibitors were observed (Figure 6C).

Article Snippet: For the inhibitor studies, cells were treated with 1 μmol/L of the MT2 melatonin receptor antagonist, 4P- PDOT (Tocris), 3 μmol/L of the MEK1/2 inhibitor, PD898059 (SantaCruz Biotechnology, USA), 10 μmol/L of the MEK5 inhibitor, BIX02189 (SantaCruz Biotechnology, Dallas, TX, USA), or 10 μmol/L of SC- 1- 151, a MEK1/2 and MEK5 inhibitor (generous gift from Dr. Patrick Flaherty; https://www.google.com/patents/ WO2015038743A1?cl=en) either alone or in combination with melatonin.

Techniques: Cell Differentiation, Activity Assay, Expressing, Blocking Assay

Journal: Journal of pineal research

Article Title: Biological effects of melatonin on osteoblast/osteoclast cocultures, bone, and quality of life: Implications of a role for MT2 melatonin receptors, MEK1/2, and MEK5 in melatonin-mediated osteoblastogenesis.

doi: 10.1111/jpi.12465

Figure Lengend Snippet: FIGURE 11 Proposed mechanisms underlying melatonin’s actions on osteoblastogenesis and osteoclastogenesis. As shown, melatonin, via MT2 melatonin receptors, modulates osteoblastogenesis by coupling to both MEK1/2 and MEK5. Stimulation of MT2 melatonin receptors by melatonin in osteoblasts leads to activation of MEK5 resulting in increases in pERK5, RUNX2, NFκB, and GLUT4. MT2 melatonin receptor-mediated activation of MEK1/2 leads to increases in pERK1/2 and decreases in PPARγ, GLUT4, and IRβ. It is proposed that cross talk between MEK1/2 and MEK5 may occur where MEK5 negative regulates MEK1/2 only when both kinases are inhibited simultaneously. In MSCs, melatonin/MT2R-mediated decreases in PPARγ levels shift MSCs away from adipogenesis and toward osteogenesis. Following MSC differentiation into osteoblasts by melatonin, the mature osteoblasts begin secreting OPG to inhibit osteoclastogenesis from PBMCs

Article Snippet: For the inhibitor studies, cells were treated with 1 μmol/L of the MT2 melatonin receptor antagonist, 4P- PDOT (Tocris), 3 μmol/L of the MEK1/2 inhibitor, PD898059 (SantaCruz Biotechnology, USA), 10 μmol/L of the MEK5 inhibitor, BIX02189 (SantaCruz Biotechnology, Dallas, TX, USA), or 10 μmol/L of SC- 1- 151, a MEK1/2 and MEK5 inhibitor (generous gift from Dr. Patrick Flaherty; https://www.google.com/patents/ WO2015038743A1?cl=en) either alone or in combination with melatonin.

Techniques: Activation Assay

Journal: Cells

Article Title: Mechanosensitive Channel PIEZO1 Senses Shear Force to Induce KLF2/4 Expression via CaMKII/MEKK3/ERK5 Axis in Endothelial Cells

doi: 10.3390/cells11142191

Figure Lengend Snippet: MEKK3 deletion in endothelial cells restrains PIEZO1-induced KLF2/4 expression. ( A ) MBMECs were exposed to differential shear stress for 5 days, and cell lysates were probed with the indicated antibodies. ( B ) MBMECs were treated with Yoda1 with the indicated concentration for 2 h, and cell lysates were probed with the indicated antibodies. ( C , D ) Control and Mekk3 -dificient MBMECs were exposed to differential shear stress for 5 days. KLF4 and MEKK3 proteins were monitored by western blotting ( C ), and mRNA levels of Klf2 and Klf4 were measured by quantitative RT-PCR ( D ). ( E , F ) Control and Mekk3 -dificient MBMECs were treated with Yoda1 (5 μM) for the indicated time, and KLF4 proteins ( E ) and mRNA levels of Klf2 and Klf4 ( F ) were monitored by western blotting and quantitative RT-PCR respectively. ( G , H ) MBMECs were pretreated with MEK5 inhibitor (BIX02189, 10 μM) for 12 h and then treated with Yoda1 (5 μM) for the indicated time. KLF4 proteins ( G ) and mRNA levels of Klf2 and Klf4 ( H ) were monitored by western blotting and quantitative RT-PCR, respectively. Data are representative of three independent experiments and are presented as mean ± SEM of three technical replicates by an unpaired Student’s t -test ( D , F , H ). The immunoblot was measured using ImageJ to determine the relative intensities of the indicated bands, which were normalized using the internal loading control proteins. The relative intensities are shown as numbers. ST: static culture; UF: unidirectional laminar flow.

Article Snippet: After serum starvation overnight, MBMECs were treated with agonists, including Yoda1 (TargetMol), 2-APB (100 μM; TargetMol), GSK1016790A (10 μM; TargetMol) and ICILIN (10 μM; TargetMol) or pretreated with inhibitors, including KN-93 (10 μM, 2 h, TargetMol), TAE226 (10 μM, 2 h, TargetMol), Bisindolylmaleimide I (10 μM, 2 h, TargetMol), BIX02189 (10 μM, 12 h, TargetMol), BAPTA-AM (10 μM, 2 h, TargetMol) and Ruthenium Red (RR, 10 μM, 12 h, TargetMol).

Techniques: Expressing, Concentration Assay, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Physiology

Article Title: Identification of Basic Fibroblast Growth Factor as the Dominant Protector of Laminar Shear Medium from the Modified Shear Device in Tumor Necrosis Factor-α Induced Endothelial Dysfunction

doi: 10.3389/fphys.2017.01095

Figure Lengend Snippet: Identification of cytoprotective mechanisms of bFGF. (A) Gene expression of KLF2 was increased significantly by 1.2-fold by TNF-α, compared to the control group. In the TNF-α+LSM, TNF-α+LSM+IgG, and TNF-α+rbFGF groups, the gene expression of KLF2 was further enhanced by 1.41-, 1.57-, and 1.53- fold compared to the control groups. The KLF2 level in the TNF-α+LSM+Ab group was not significantly different from that in the TNF-α group. Data are expressed as the mean ± S.E.M. ( n = 4). * p < 0.05 and # p < 0.05 indicate a significant different relative to the control group and TNF-α group, respectively. (B) Compared to the TNF-α group, ICAM-1 expression was significantly decreased to 0.71-fold in the TNF-α+bFGF group. However, the anti-inflammatory effect of bFGF on ICAM-1 expression was reversed to 0.99- and 1.35-fold compared to in the TNF-α group by pretreatment with SB203580 (10 μM) and BIX02189 (10 μM), respectively. (C) Compared to the TNF-α group, the PAI-1 expression level was significantly decreased to 0.76-fold in the TNF-α+bFGF group. However, the anti-thrombotic effect of bFGF on PAI-1 gene expression was reversed to 0.93- and 1.05-fold compared to in the TNF-α group by pretreatment with SB203580 and BIX02189, respectively. T: TNF-α (B,C) Data are expressed as the mean ± S.E.M. ( n = 4). # p < 0.05 indicates a significant difference relative to the TNF-α group and $ p < 0.05 indicates a significant difference relative to the TNF-α+rbFGF group. (D) Compared with the TNF-α group, ICAM-1 and PAI-1 protein levels were significantly decreased in the TNF-α+rbFGF group and were reverted by pretreatment with SB203580 and BIX02189, respectively ( n = 3). # p < 0.05 indicates a significant difference relative to the TNF-α group and $ p < 0.05 indicates a significant difference relative to the TNF-α+rbFGF group.

Article Snippet: The cells were pretreated with 10 μM SB203580 (specific inhibitor of p38) and 10 μM BIX02189 (specific inhibitor of MEK5) (Cayman Chemical, Ann Arbor, MI, USA) for 1 h, followed by addition of TNF-α (10 ng/mL) and rbFGF (10 ng/mL) for 6 h. Gene expression was determined by q-PCR as described above.

Techniques: Gene Expression, Control, Expressing