The Role of Protein Kinase C and Protein Tyrosine Kinase in the Signal Transduction Pathway of stimulus Induced by Endotoxin in Peripheral Blood Monocyte

Kim, Jae Yeol; Park, Jae Suk; Lee, Gwi Lae; Yoo, Chul Gyu; Kim, Young Whan; Han, Sung Koo; Shim, Young Soo

Abstract

BACKGROUND: Endotoxin, the component of outermembrane of gram negative organism, plays an important role in the initiation and amplification of inflammatory reaction by its effects on inflammatory cells. Until recently, there have been continuing efforts to delinate the mechanisms of the signal trasduction pathway of endotoxin stimuli on inflammatory cells. By uncovering the mechanisms of signal transduction pathway of endotoxin stimuli, we can expect to have tools to control the excessive inflammatory responses which sometimes may be fatal to the involved host. It was generally accepted that endotoxin exerts its inflammatory effects through inflammatory cytokines that are produced by endotoxin-stimulated inflammatory cells and there were some reports on the importance of protein kinase C and protein tyrosine kinase activation in the production of inflammatory cytokines by endotoxin. So we evaluated the effect of pretreatment of protein kinase C inhibitors (H7, Staurosporin) and protein tyrosine kinase inhibitors(Herbimycin, Genistein) on the endotoxin-stimulated cytokines(IL-8 & TNF-alpha) mRNA expression. METHOD: Peripheral blood monocytes were isolated from healthy volunteers by Ficoll-Hypaque density gradient method and purified by adhesion to 60mm Petri dishes. Endotoxin(LPS 100ng/ml) was added to each dishes except one control dish, and each endotoxin-stimulated dishes was preincubated with H7, Staurosporin(protein kinase C inhibitor), Herbimycin or Genistein(protein tyrosine kinase inhibitor) respectively except one dish. Four hours later the endotoxin stimulation, total RNA was extracted and Northern blot analysis for IL-8 mRNA and TNF-alpha mRNA was done. RESULT: Endotoxin stimulation increased the expression of IL-8 mRNA and TNF-alpha mRNA expression in human peripheral blood monocyte as expected and the stimulatory effect of endotoxin on TNF-alpha mRNA expression was inhibited by protein kinase C inhibitors(H7, Staurosporin) and protein tyrosine kinase inhibitors (Herbimycin, Genistein). The inhibitory effect of each drugs was increased with increasing concentration. The stimulatory effect of endotoxin on IL-8 mRNA was also inhibited by H7 and protein tyrosine kinase inhibitors (Herbimycin, Genistein) dose-dependently but not by Staurosporin.
CONCLUSION
Protein kinase C and protein tyrosine kinase are involved in the endotoxin induced signal transduction pathway in human peripheral blood monocyte.