lbt613  (Novartis)

Journal: Environmental Health Perspectives

Article Title: Diesel Exhaust Particles Activate the Matrix-Metalloproteinase-1 Gene in Human Bronchial Epithelia in a β-Arrestin–Dependent Manner via Activation of RAS

doi: 10.1289/ehp.0800311

Figure Lengend Snippet: MAP kinase signaling via the RAS-RAF-MEK-ERK1/2 pathway is necessary for the DEP MMP-1 response in HBE. (A–C) MEK inhibition. ( A and B ) Dependence of the DEP MMP-1 response on MEK (and ERK1/2): transcriptional activation of MMP-1 at 2 hr ( A ; left) and 24 hr ( A ; right) in relative units (RU), and secretion of MMP-1 at the same time points ( B ). Both responses depend on MAP kinase signaling by MEK-ERK1/2, evidenced by the effect of UO126 and PD98059 (both 10 μM). (A and B; right) For transcription, differences reach statistical significance for the −1607GG MMP-1 polymorphism (A; right) For secretion of MMP-1, differences caused by MEK inhibitors are statistically signifi-cant. DEP stimulation was with 100 μg/mL; experiments conducted in triplicate with two independent experiments. ( C ) Effect of MEK inhibitors UO126 and PD98059 on primary HBE cells. Note the complete prevention of MMP-1 secretion. Experiment carried out in quadruplicate, with 100 μg/mL DEPs. ( D–F ) RAF inhibition with AAL881 and LBT613 down-regulated transcription of MMP-1 fLUC reporter genes. ( D ) Down-regulation strongest for the −1607GG polymorphism. ( E ) Secretion of MMP-1 protein from BEAS-2B cells. ( F ) Secretion of MMP-1 protein from primary airway epithelia. Experiments conducted in triplicate, with at least two independent experiments, using 100 μg/mL DEPs. ( G and H ) RAS inhibition [transient transfection of a dominant-negative (dom-neg) RAS] down-regulated the DEP MMP-1 response. ( G ) Transcriptional activation of MMP-1 and secretion of MMP-1 after 24 hr measured using MMP-1 –fLUC assays (4.4-kb MMP-1 promoter, –1607G and –1607GG). Cells transfected with the –1607G polymorphism showed no effect; further reduction of transcription for the –1607GG polymorphism was statistically significant, yet incomplete. ( H ) Transcriptional activation of MMP-1 and secretion of MMP-1 after 24 hr measured using MMP-1 ELISA. Transfection of dominant-negative RAS without DEP stimulation had no effect on MMP-1 transcription. Dominant-negative RAS virtually eliminated MMP-1. Experiment conducted in triplicate with two sets of independent experiments. * p < 0.05, ** p < 0.01, and # p < 0.001, indicate significant inhibition of the increase caused by DEP. ## p < 0.05, † p < 0.01, and †† p < 0.001 indicate statistically significant differences from control.

Article Snippet: We chose the concentrations based on recommendations of the supplier (Tocris) or those commonly used in previous studies ( ; ), usually 5–10 μM, except for LBT613, which we used at 1 μM ( ).

Techniques: Inhibition, Activation Assay, Transfection, Dominant Negative Mutation, Enzyme-linked Immunosorbent Assay, Control

Journal: Environmental Health Perspectives

Article Title: Diesel Exhaust Particles Activate the Matrix-Metalloproteinase-1 Gene in Human Bronchial Epithelia in a β-Arrestin–Dependent Manner via Activation of RAS

doi: 10.1289/ehp.0800311

Figure Lengend Snippet: Phospho-ERK1/2 is trafficked to the nucleus as an early signaling event of the DEP MMP-1 response. ( A ) Time course of nuclear phospho-ERK1/2 after stimulation of BEAS-2B cells with DEPs (100 μg/mL). Nuclear phospho-ERK was detected by immunofluorescence and its abundance was evaluated by densitometry of the nucleus (cell numbers indicated beneath each coordinate). ( B ) Representative micrographs after DEP exposure of nuclear phospho-ERK immunofluorescence. Arrows indicate DEPs in direct contact with a cell. Western blots below show increased abundance of phospho-ERK in whole-cell lysate. ( C ) Nuclear phospho-ERK1/2 at 0 min and 30 min. A significant increase for DEP stimulation (100 μg/mL) was absent in controls (media, 0.2% DMSO). Chemical inhibition of MEK and RAF eliminated generation of a nuclear signal for phospho-ERK (MEK: UO126, PD98059, both used at 10 μM; RAF: 1 μM LBT613 and 10 μM AAL881). Eighty cells were analyzed per condition after DEP stimulation, and 40 before stimulation. ( D ) Knockdown of β-arrestins also eliminated generation of a nuclear signal for phospho-ERK. The graph (left) shows quantitation of control siRNA versus anti–pan-arrestin siRNA. The confocal micrographs (right) show representative findings. Eighty cells were analyzed for each condition after DEP stimulation, and 40 before stimulation. ( E ) Western blot as shown in ( C ) plus the corresponding Western blot for whole-cell phospho-ERK. Cell lysates sampled at 60 min. *** p < 0.001 difference from time point 0 min. # p < 0.01 difference from time point 30 min. ## p < 0.01 compared with control. * p < 0.01 reduction of the 0–30 min difference obtained when stimulating with DEPs. ### p < 0.01 difference between DEP-stimulated and control-stimulated. † p < 0.001 down-regulation.

Article Snippet: We chose the concentrations based on recommendations of the supplier (Tocris) or those commonly used in previous studies ( ; ), usually 5–10 μM, except for LBT613, which we used at 1 μM ( ).

Techniques: Immunofluorescence, Western Blot, Inhibition, Knockdown, Quantitation Assay, Control