AN ANTIINFLAMMATORY BENZAMIDE DERIVATIVE INHIBITS THE PROTEIN-KINASE-C (PKC)-DEPENDENT PATHWAY OF ERK2 PHOSPHORYLATION IN MURINE MACROPHAGES

We have previously described benzamide derivatives that inhibited tumo r necrosis factor (TNF) production from activated macrophages (M phi) probably by interacting with a protein kinase C (PKC)-dependent pathwa y. To investigate their mode of action further, we first tested their effect on isolated PKC in vitro, using the selective inhibitor bisindo lylmaleimide (BIM) as a positive control. We found that our representa tive compound JM34 did not inhibit PKC activity in vitro. We then inve stigated pathways located downstream of PKC and focused on the RaF1/ME K1,2/Erk1,2 cascade known to be preferentially activated by PKC activa tors such as phorbol esters. We found that JM34 dose-dependently inhib ited Erk2 phosphorylation in Md, stimulated by phorbol dibutyrate and calcium ionophore (maximal inhibition of 85% at 300 mu M). BIM at 3 mu M totally abrogated Erk2 phosphorylation. After stimulation with endo toxin or zymosan, Erk2 phosphorylation was only partially inhibited (2 5-30%) by JM34 or BIM, which confirmed that PKC-independent events wer e also involved in Erk2 phosphorylation. Because activated Erk2 has be en shown to activate phospholipase A(2), we tested the effect of JM34 and BIM on the release of arachidonate metabolites from activated M ph i. We found that both products partially inhibited the release of arac hidonate metabolites from zymosan-activated M phi at levels comparable to their inhibition of Erk2 phosphorylation. In contrast, JM34 and BI M markedly differed in their ability to inhibit TNF production. Taken together, our results suggest that JM34 inhibited the PKC-dependent pa thway of Erk2 phosphorylation, which may fully account for its inhibit ory effect on phospholipase A(2) activation. However, the inhibition o f TNF release by JM34 probably involved inhibition of an additional pa thway, distinct from the Erk1/Erk2 cascade.