MICROTUBULE DEPOLYMERIZATION SELECTIVELY DOWN-REGULATES THE SYNTHESISOF PROINFLAMMATORY SECRETORY NONPANCREATIC PHOSPHOLIPASE A(2)

Microtubule depolymerizing agents (MTD) diminish the expression of cel l surface receptors for TNF-alpha. Because TNF-alpha along with IL-1 b eta markedly enhance the gene expression and extracellular release of proinflammatory secretory nonpancreatic phospholipase A(2) (sPLA(2)), we tested the impact of MTD on the expression of sPLA(2). We report th at MTD markedly inhibit the expression and release of sPLA(2) by fetal rat calvarial osteoblasts (FRCO), which synthesize and release sPLA(2 ). 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 quantiti es of colchicine (1.25 nM) reduced the released sPLA(2) 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 mu M). IC50 was 6 8.75 nM. When FRCO were prestimulated by IL-1/TNF, much higher concent rations of colchicine were required to reduce sPLA(2) activity. MTD in hibited the expression of sPLA(2) by a mechanism(s) different from the way in which they impact TNF surface receptors, because they inhibite d sPLA(2) expression in FRCO stimulated by IL-1 beta or by cell-permea ble cAMP analogs. Colchicine (1 mu M) reduced the expression of sPLA(2 ) induced by dibutyryl cAMP (2 mM) and 8-bromo-cAMP (4 mM) to 38% and 58% of that in controls, respectively. Photoinactivated lumicolchicine s beta and gamma were noninhibitory. Microtubular stabilizer taxol (5 mu M) abolished inhibitory activity of colchicine, increasing the expr ession of sPLA(2) 3.2-fold compared with that in control cells culture d without taxol. Other MTD, such as vinblastine (0.01 mu M), inhibited sPLA(2) release to 27% of the controls, whereas nocodazole (10 mu M) was less inhibitory. Northern blot analysis of FRCO showed that sPLA(2 ) mRNA was greatly induced by IL-1/TNF. The induction of sPLA(2) mRNA by IL-1/TNF was nearly completely abolished by colchicine in a dose-re lated manner. Western blot analysis of intra- and extracellular sPLA(2 ) protein showed complete inhibition of the synthesis by MTD. To deter mine whether the inhibition of sPLA(2) is selective, mRNA levels of cy tosolic PLA(2) and of inducible cyclooxygenase-2 were investigated. Co lchicine had no effect on the mRNA levels of these two enzymes, which suggests that the inhibitory effect of MTD on sPLA(2) expression is se lective and occurs at the transcriptional level. Thus, the microtubula r system plays a significant role in the synthesis of proinflammatory sPLA(2), a fact that may explain in part the anti-inflammatory activit y of microtubular disrupters.