Identification of genomic targets downstream of p38 mitogen-activated protein kinase pathway mediating tumor necrosis factor-alpha signaling

Abstract

Inhibition of p38 MAPK suppresses the expression of proinflammatory cytokines such as TNF-alpha and IL-1 beta in macrophages and fibroblast-like synoviocytes (FLS). However, there have been no genomewide studies on the gene targets of p38 MAPK signaling in synoviocytes. Microarray technology was applied to generate a comprehensive analysis of all genes regulated by the p38 MAPK signaling pathway in FLS. Gene expression levels were measured with Agilent oligonucleotide microarrays. Four independent sets of mRNA modulated by TNF-alpha and vehicle were used to measure the change of gene expression due to TNF-alpha, and three experiments were done to ascertain the effect of SB-203580, a p38 MAPK inhibitor, on TNF-alpha-induced genes. Microarray data were validated by RT-quantitative polymerase chain reaction. One hundred forty-one significantly expressed genes were more than twofold upregulated by TNF-alpha. Thirty percent of these genes were downregulated by the p38 inhibitor SB-203580, whereas 67% of these genes were not significantly changed. The SB-203580-inhibited genes include proinflammatory cytokines such as interleukins and chemokines, proteases including matrix metallopeptidases, metabolism-related genes such as cyclooxygenases and phosphodiesterase, genes involved in signal transduction, and genes encoding for transcription factors, receptors, and transporters. Approximately one-third of the TNF-alpha-induced genes in FLS are regulated by the p38 MAPK signal pathway, showing that p38 MAPK is a possible target for suppressing proinflammatory gene expressions in rheumatoid arthritis.

Fig. 1

A: inhibition of rat fibroblast-like synoviocyte (FLS) p38 mitogen-activated protein kinase (MAPK) by SB-203580. The effect of SB-203580 on the activity of p38 MAPK was measured by an in vitro kinase assay. Phospho-p38 was immunoprecipitated from tumor necrosis factor (TNF)-α-stimulated rat FLS cell lysate and was used to phosphorylate ATF-2 fusion protein in the presence of 0, 0.1, 0.3, 1.0, or 10.0 μM SB-203580. B: SB-203580 effect on rat FLS c-Jun NH₂-terminal kinase (JNK). The effect of SB-203580 on the activity of JNK was measured by an in vitro kinase assay. JNK was immunoprecipitated with immobilized c-Jun fusion protein from either TNF-α- or control-treated rat FLS cell lysates and was used to phosphorylate c-Jun in the presence of 0, 0.01, 0.1, or 1.0 μM SB-203580. C: p38 MAPK kinase assay. The effect of SB-203580 on p38 MAPK in rat FLS in vivo was measured at 15 min, 1 h, 6 h, and 24 h after TNF-α stimulation. The cells were first preincubated with 0.3 μM SB-203580 or vehicle for 2 h, and then 10 ng/ml TNF-α was added. A negative control without TNF-α stimulation was also included. The cells were harvested at the time points indicated, and the activity of p38 MAPK was measured. SB80, SB-203580.

Fig. 2

Induced gene expression at 24 h after TNF-α stimulation and SB-203580 inhibition. A: 141 genes with signal intensity >1,000 were upregulated >2-fold by TNF-α. Pie chart represents the subsets of functionally assigned genes with the numbers found in each group. B: FLS were preincubated with 0.3 μM SB-203580 for 2 h, followed by 24-h TNF-α stimulation. c upregulated 3% and downregulated 30% of the 141 genes upregulated >2-fold by TNF-α shown in A.

Fig. 3

Gene targets of p38 MAPK inhibition. Functional categories of TNF-α-induced genes found to be inhibited by SB-203580 in the microarray experiments are shown. They can be categorized into genes encoding for proteases and cytokines, metabolism-related genes, and genes involved in signal transduction, proliferation, and apoptosis (see Supplemental Data 1). Many of these genes, shown in bold, have been associated with rheumatoid arthritis.