SHIMBARA Satoko Showa University, School of Pharmaceutical Sciences, Research Assistant, 薬学部, 助手 (60266161)
NAKATANI Yoshihito Showa University, School of Pharmaceutical Sciences, Research Assistant, 薬学部, 助手 (80266163)
ATSUMI Gen-ichi Showa University, School of Pharmaceutical Sciences, Research Assistant, 薬学部, 助手 (70276608)
長岡 寛明 昭和大学, 薬学部, 助手 (90255860)
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¥7,100,000 (Direct Cost : ¥7,100,000)
Fiscal Year 1996 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1995 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1994 : ¥3,800,000 (Direct Cost : ¥3,800,000)
We have examined the expression and function of 85-kDa cytosolic phospholipase A_2 (cPLA_2) in mammalian cells.
1. Whereas cPLA_2 is ubiqitously and constitutively expressed in most mammalian cells, the regulation of its expression after cell activation was found to differ among cell types : (1) in fibroblasts, cPLA_2 expression was almost constant even after cell activation by proinflammatory cytokines ; (2) in osteoblasts and mast cells, cPLA_2 protein expression increased markedly after cytokine stimulation, without accompanied by concomitant increase in its mRNA level, revealing significant post-translational regulation of cPLA_2 expression ; and (3) in macrophages, both cPLA_2 mRNA and protein increased after lipopolysaccharide stimulation. Of note, we found for the first time that increase in cPLA_2 expression underwent positive-feedback augmentation by PGA_2, which is an endoproduct of the prostanoid biosynthetic pathway, in mouse osteoblastic cells.
2. The functional coupling of
cPLA_2 with downstream cyclooxygenase (COX) enzymes in the different phases of the prostanoid biosynthetic pathway was investigated. (1) Fibroblasts, mast cells and macrophages produced PGE_2, PGD_2 and TXA_2, respectively, in the immediate phase of cell activation through functional coupling of cPLA_2 with constitutive COX-1. In contrast, cytokine-primed macrophages or osteoblasts, in which COX-2 had been already induced, produced PGE_2 in preference to other prostanoids in the immediate response where cPLA_2 and inducible COX-2 were functionally linked. (2) In the delayd phase of cell activation, most cells produced PGE_2 as a consequence of the functional coupling of cPLA_2 and COX-2. In macrophages, mast cells and fibroblasts, secretory type II PLA_2, often inducible, was also required for the optimal delayd prostanoid generation, revealing an unexplored crosstalk between the two distinct PLA_2 enzymes. Nevertheless, these studies imply that cPLA_2 is prerequisite for both immediate and delayd prostanoid biosynthesis, irespective of the differential regulation of its expression in various cells.
3. Finally, in search for the protein that specifically interacts with cPLA_2, we identified a 60-kDa intracellular protein in several cell types. We aim to clarify the structure and function of this 60-kDs cPLA_2-interacting protein, which is assumed to act as a regulator of cPLA_2 function in cells. Less