巨噬细胞免疫调变信号:Raf-1,MAPK p44,MAPK p42和p38 MAPK的研究

STUDIES ON CELL SIGNALING IMMUNOMODULATED MURINE PERITONEAL SUPPRESSOR MACRO-PHAGES: LPS ANDPMA MEDIATE THE ACTIVATION OF RAF-1, MAPK p44 AND MAPK p42 AND p38 MAPK

为了了解巨噬细胞免疫调变机理,我们应用LPS和PMA处理小鼠抑制性巨噬细胞,观察到Ras下游信号分子Raf-1,分裂原激活蛋白激酶MAPK p44,MAPK p42和p38 MAPK均被活化,发现forskolin能增强p38 MAPK的活性,进一步提示PKC和PKA途径增强了p38 MAPK的磷酸化效应,为我们了解LPS如何激活p38 MAPK信号通路提供了一个新的机会。

Monocytes-macrophages which serve as host immune cells to kill pathogens can often be 'activated' after exposing to viruses, bacteria, cytokines as well as chemical substances, Ho- wever, it is paradoxical that highly activated macrophages can be induced to become the suppressor ones by live microbes, microbial products, tumor, and autoimmune disease, althou- gh the mechanism remains unknown. Our previous experimental studies have shown that immuno-suppressor activities of suppressor ma-crophages on T, B and NK cells can be prevented by the treatment with LPS or supernatant in vitro from mitogen-stimulated lymphocytes, while, at the same time, the tumo-ricidal activities of those macrophages can be kept or even enhanced following the same treatment. This phenomenon was then termed as 'immune modulation' For the understanding of its mechanism, we are now undertaking signal transduction in modulated macrophages. Since mitogen-activated protein kinase (MAPK) is an integration point of different signal transduction pathways, its cascade and regulation of activation are being investigated extensively by the assay of electrophoresis mobility shift. Recent results suggested that interaction of ligand-receptor triggers protein tyrosine kinase(PTK) activation leading to Ras-GTP binding with Raf-1 to phosphorylate MAPK kinase (MAPKK), the specific activator of MAPK. It is reported that PKC-α can directly phosphorylate or activate Raf-1 in NIH3 T3 cells. Raf-1 (74KDa), with an intrinsic serine (Ser)-threonine (The) kinase activity, becomes hyperphosphorylated after activation which can be followed by gel mobility shift test. It has also been shown that a variety of extracellular factors stimulate a pair of MAPK p44 and MAPK p42 of MAPK family members. A significant property of activation of ERK 1 and ERK 2 is the requirement for the phosphorylation of both Thr-183 and Tyr-185 (at TEY motif) within in its protein kinase subdomain VIII. More recently, two other MAPK subtypes, p38 MAPK (mammalian equivalents of HOG1 in yeast) and JNK MAPK have been discovered. The requirement for activation of p38 MAPK for both Thr-180 and Tyr-182 (at TGY motif) has been shown. p38 MAPK is important in certain trans-criptional regulatory pathways, since it can phosphorylate the following transcriptional factors:1) Elk at Ser 383/389 for binding with SRE motif; 2). ATF 2 at Ser 69/71, forming a complex with Myc for DNA binding at CRE motif; 3) Max at Ser-62 to combine DNA of E-Box motif. p38 MAPK can be ac- tivated by LPS, inflammatory cytokines, such as TNF and IL-1, osmolarity. To examine the possibility that whether activation of Raf-1 and ERK 1, ERK2 and p38 MAPK can be regulated directly or/and differently by PKC and PKA pathways, her-bimycin A (Ki=0.9μmol/L), a potent PTK inhibitor (J. Immunol. 155:3944-4003, 1995) at 2μmol/L concentration was utilized to block Ras/Raf-1/MAPK cascade. After pre-incu-bation of macrophages with herbimycin A for 30 min or 90 min, cells were treated with LPS (10μg/ml) and PMA (100 nmol/L) for 15 min. No inhibition of phosphorylation of Raf-1, MAPK p44 and MAPK p42 in response to LPS and PMA was observed (Fig. 1 and 3). However, forskolin, a cAMP inducer for protein kinase A (PKA) activation, inhibited the phosphorylation of LPS - and PMA-stimu-lated Raf-l,MAPK p44 and MAPK p42 (Fig.2 and 4). Similarly, in agreement with a very recent report from David, M et al in NIH, in which they indicated that forskolin (30 μmol/ L) inhibited IFN-β-stimulated ERK activity by U 266 cells (J. Biol. Chem. 271:4585-4588 1996), we found that the levels of phosphory-lations of Raf-1 and ERK1 and ERK2 were declined when forskolin (30μmol/L) was added to macrophages for 20 min at 37℃ prior to the stimulation by LPS and PMA. Interestingly, under the same condition, forskolin (30μmol/L) stimulated the phosphorylation of LPS- and PMA-triggered p38 MAPK of murine peritoneal suppressor macrophages, suggesting that activation of p38 MAPK is regulated positively by both PKC and PKA (Fig. 5 and Fig. 6) Our finding also indicates that regulation of activity of p 38 MAPK is different from that of ERK1 and ERK2 in PKA pathway It seems also to implicate that the motifs of TEY (ERK) and TGY( p38 MAPK) are targeted by different dual-specific kinases through PKC and PKA. In contrast to the data by Han, J. et al ( Science 265:808-811, 1994), in which they indicated that p38 MAPK was not activated by TPA, the discrepancy between two groups may be due to their differences in cells and method used. Maybe a sensitive and reliable method is very important for the assay of phosphorylation of various kinases, and to our knowledge, an anti-phospho Plus p38 MAPK (Try 182) antibody kit is the choice for the rapid analysis of p38 MAPK of phosphoryla-tion. Taken together, two conclusions can be emerged from our findings: 1. LPS- and PMA(PKC activator) -responsive pathways are capable of inducing activation of Raf-1, ERK 1. ERK 2 and p38 MAPK However, only forskolin can enhance phosphorylation of p38 MAPK in response to LPS, or even to PMA. 2. PMA can activate p38 MAPK of macro-phages in our system, but further investigation of the role of p38 MAPK in immune modulation, including IL-1 and IL-12 secretion and adhesive molecule production is needed.