Figure 5.

Differential MAP kinase activation is necessary for optimal TNF-α gene expression. (A) Blood monocytes were cultured in the presence or absence of SB203580 (SB) for 30 minutes before exposing to crocidolite asbestos for 3 hours. Total RNA was reverse transcribed to cDNA. Real-time PCR amplification was performed as described in Materials and Methods. Data are expressed as fold induction of TNF-α mRNA expression from control. For statistical analysis, * denotes a comparison of crocidolite asbestos to control (P < 0.0037), and ** denotes a comparison of crocidolite to crocidolite + SB203580 (P < 0.0046). (B) THP-1 cells were transiently transfected with the -600 TNF-α-CAT reporter plasmid and either an empty vector (shaded bars) or pcDNA-HA-ERK2 (K/A) (solid bars) expression vector. After 24 hours, cells were exposed to crocidolite asbestos for 24 hours. Whole cell lysates, which were normalized to protein, were incubated with 0.1 μCi of [¹⁴C] chloramphenicol and 1.0 mM acetyl coenzyme A for 2 hours at 37°C. Acetylated derivatives (CM3-AC and CM1-AC) were separated from nonacetylated chloramphenicol (CM) by ascending thin layer chromatography in chloroform/methanol (95:5) solvent. *P < 0.0175, **P < 0.0050. (C) THP-1 cells were transiently transfected with the −600 TNF-α-CAT reporter plasmid and either an empty vector (shaded bars) or the constitutive active pCMV-MEK1 (solid bars) expression vector. After 24 hours, cells were exposed to crocidolite asbestos for 24 hours, and the CAT assay was performed as described in B. *P < 0.012, **P < 0.038. In B and C, densitometry of three separate experiments was performed and is expressed graphically in arbitrary units. For statistical analysis, * denotes a comparison of control to crocidolite (vector); and ** denotes a comparison of crocidolite (vector) to (B) crocidolite (pcDNA-HA-ERK2 [K/A]) and (C) crocidolite (pCMV-MEK1). (D) THP-1 cells were transiently transfected with pTRE-luc and pTET-ATF and either an empty vector (solid bars) or the constitutive active MEK1 (shaded bars). After 24 hours, cells were exposed to crocidolite asbestos for 6 hours. Luciferase activity, which was normalized to Renilla luciferase, is expressed in real light units. For statistical comparisons, * denotes a comparison of vector to pCMV-MEK1 (P < 0.0031).