PURPOSE: In order to understand the intracellular signaling pathway involving the c-fos gene expression that is caused by Titanium-particles, we analyzed the involvement of Rac, cytosolic phospholipase A2, and eicosanoids (e.g. leukotriene B4 and prostaglandin E2) as well as c-fos. MATERIALS AND METHODS: We tested whether or not Titanium-particles activate a c-fos serum response element in Rat-2 fibroblasts. To measure the activity of the c-fos serum response element, we analyzed the serum response element using a luciferase reporter system. The luciferase activity was measured using a scintillation spectrophotometer. Next, we analyzed the involvement of Rac and the eicosanoid synthesis mechanisms which are downstream mediators of Rac in the c-fos serum response element activation cascade. RESULTS: Titanium-particles cause an activation of the c-fos serum response element and this activation was selectively repressed by RacN17 and by pretreatment of the inhibitors of cytosolic phospholipase A2, cyclooxygenase or 5-lipoxygenase. Eicosanoid synthesis was increased in a Rac-dependent manner in response to the presence of Titanium- particles. CONCLUSION: 'Rac, a member of G-protein, which is involved in the eicosanoid synthesis' may play a critical role in the Titanium-induced signaling cascade. Thus, we speculated that the 'Rac-cytosolic phospholipase A2-eicosanoids-c-fos cascade' may be a possible mechanism that produces eicosanoid synthesis caused by Titanium-particles in the periprosthetic osteolytic process.
Arachidonate 5-Lipoxygenase ; Cytosol ; Eicosanoids ; Fibroblasts ; Genes, fos ; GTP-Binding Proteins* ; Leukotriene B4 ; Luciferases ; Phospholipases ; Phospholipases A2 ; Prostaglandin-Endoperoxide Synthases ; Serum Response Element ; Titanium*

The early growth response-1 gene (egr-1) encodes a zinc-finger transcription factor Egr-1 and is rapidly inducible by a variety of extracellular stimuli. Anisomycin (ANX), a protein synthesis inhibitor, stimulates mitogen-activated protein kinase (MAPK) pathways and thereby causes a rapid induction of immediate-early response genes. We found that anisomycin treatment of U87MG glioma cells resulted in a marked, time-dependent increase in levels of Egr-1 protein. The results of Northern blot analysis and reporter gene assay of egr-1 gene promoter (Pegr-1) activity indicate that the ANX- induced increase in Egr-1 occurs at the transcriptional level. Deletion of the serum response element (SRE) in the 5'-flanking region of egr-1 gene abolished ANX-induced Pegr-1 activity. ANX induced the phosphorylation of the ERK1/2, JNK, and p38 MAPKs in a time-dependent manner and also induced transactivation of Gal4-Elk-1, suggesting that Elk-1 is involved in SRE-mediated egr-1 transcription. Transient transfection of dominant-negative constructs of MAPK pathways blocked ANX-induced Pegr-1 activity. Furthermore, pretreatment with specific MAPK pathway inhibitors, including the MEK inhibitor U0126, the JNK inhibitor SP600125, and the p38 kinase inhibitor SB202190, completely inhibited ANX-inducible expression of Egr-1. Taken together, these results suggest that all three MAPK pathways play a crucial role in ANX-induced transcriptional activation of Pegr-1 through SRE-mediated transactivation of Elk
p38 Mitogen-Activated Protein Kinases/genetics/metabolism ; ets-Domain Protein Elk-1/genetics/*metabolism ; Trans-Activation (Genetics)/*drug effects ; Serum Response Element ; Protein Kinase Inhibitors/pharmacology ; Protein Biosynthesis/*drug effects ; Promoter Regions (Genetics)/genetics ; *MAP Kinase Signaling System/drug effects ; JNK Mitogen-Activated Protein Kinases/genetics/metabolism ; Humans ; Extracellular Signal-Regulated MAP Kinases/genetics/metabolism ; Early Growth Response Protein 1/genetics/*metabolism ; Cell Line, Tumor ; Anisomycin/*pharmacology