Microtubule depolymerization selectively down-regulates the synthesis of proinflammatory secretory nonpancreatic phospholipase A2.
Microtubule depolymerizing agents (MTD) diminish the expression of cell surface receptors for TNF-alpha. Because TNF-alpha along with IL-1 beta markedly enhance the gene expression and extracellular release of proinflammatory secretory nonpancreatic phospholipase A2 (sPLA2), we tested the impact of MTD on the expression of sPLA2. We report that MTD markedly inhibit the expression and release of sPLA2 by fetal rat calvarial osteoblasts (FRCO), which synthesize and release sPLA2. When FRCO were pretreated with colchicine and then stimulated with IL-1 beta 0.2 ng/ml and TNF-alpha 25 ng/ml (IL-1/TNF), minute quantities of colchicine (1.25 nM) reduced the released sPLA2 activity to 11% of that in controls. IC50 was 0.75 nM. When IL-1/TNF and colchicine were added simultaneously, similar inhibition (8% of that in controls) required higher concentrations of colchicine (0.125 microM). IC50 was 68.75 nM. When FRCO were prestimulated by IL-1/TNF, much higher concentrations of colchicine were required to reduce sPLA2 activity. MTD inhibited the expression of sPLA2 by a mechanism(s) different from the way in which they impact TNF surface receptors, because they inhibited sPLA2 expression in FRCO stimulated by IL-1 beta or by cell-permeable cAMP analogs. Colchicine (1 microM) reduced the expression of sPLA2 induced by dibutyryl cAMP (2 mM) and 8-bromo-cAMP (4 mM) to 38% and 58% of that n controls, respectively. Photoinactivated lumicolchicines beta and gamma were noninhibitory. Microtubular stabilizer taxol (5 microM) abolished inhibitory activity of colchicine, increasing the expression of sPLA2 3.2-fold compared with that in control cells cultured without taxol. Other MTD, such as vinblastine (0.01 microM), inhibited sPLA2 release to 27% of the controls, whereas nocodazole (10 microM) was less inhibitory. Northern blot analysis of FRCO showed that sPLA2 mRNA was greatly induced by IL-1/TNF. The induction of sPLA2 mRNA by IL-1/TNF was nearly completely abolished by colchicine in a dose-related manner. Western blot analysis of intra- and extracellular sPLA2 protein showed complete inhibition of the synthesis by MTD. To determine whether the inhibition of sPLA2 is selective, mRNA levels of cytosolic PLA2 and of inducible cyclooxygenase-2 were investigated. Colchicine had no effect on the mRNA levels of these two enzymes, which suggests that the inhibitory effect of MTD on sPLA2 expression is selective and occurs at the transcriptional level. Thus, the microtubular system plays a significant role in the synthesis of proinflammatory sPLA2, a fact that may explain in part the anti-inflammatory activity of microtubular disrupters.
Pruzanski
W
W
Wellesley Hospital Research Institute, University of Toronto, Ontario, Canada.
Kennedy
B P
BP
van den Bosch
H
H
Stefanski
E
E
Vadas
P
P
D000818:Animals; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D015536:Down-Regulation; D007375:Interleukin-1 / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects* / Q000502:physiology; D015739:Nocodazole / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D010741:Phospholipases A / Q000096:biosynthesis*; D054467:Phospholipases A2; D051381:Rats; D014409:Tumor Necrosis Factor-alpha; D014747:Vinblastine / Q000494:pharmacology
D007375:Interleukin-1; D014409:Tumor Necrosis Factor-alpha; D014747:Vinblastine; D010741:Phospholipases A; D054467:Phospholipases A2; D017239:Paclitaxel; D015739:Nocodazole; D003078:Colchicine