An anti-inflammatory benzamide derivative inhibits the protein kinase C (PKC)-dependent pathway of ERK2 phosphorylation in murine macrophages.
We have previously described benzamide derivatives that inhibited tumor necrosis factor (TNF) production from activated macrophages (Mphi) probably by interacting with a protein kinase C (PKC)-dependent pathway. To investigate their mode of action further, we first tested their effect on isolated PKC in vitro, using the selective inhibitor bisindolylmaleimide (BIM) as a positive control. We found that our representative compound JM34 did not inhibit PKC activity in vitro. We then investigated pathways located downstream of PKC and focused on the Raf1/MEK1,2/Erk1,2 cascade known to be preferentially activated by PKC activators such as phorbol esters. We found that JM34 dose-dependently inhibited Erk2 phosphorylation in Mphi stimulated by phorbol dibutyrate and calcium ionophore (maximal inhibition of 85% at 300 microM). BIM at 3 microM totally abrogated Erk2 phosphorylation. After stimulation with endotoxin or zymosan, Erk2 phosphorylation was only partially inhibited (25-30%) by JM34 or BIM, which confirmed that PKC-independent events were also involved in Erk2 phosphorylation. Because activated Erk2 has been shown to activate phospholipase A2, we tested the effect of JM34 and BIM on the release of arachidonate metabolites from activated Mphi. We found that both products partially inhibited the release of arachidonate metabolites from zymosan-activated Mphi at levels comparable to their inhibition of Erk2 phosphorylation. In contrast, JM34 and BIM markedly differed in their ability to inhibit TNF production. Taken together, our results suggest that JM34 inhibited the PKC-dependent pathway of Erk2 phosphorylation, which may fully account for its inhibitory effect on phospholipase A2 activation. However, the inhibition of TNF release by JM34 probably involved inhibition of an additional pathway, distinct from the Erk1/Erk2 cascade.
Vernhet
L
L
Department of Pharmacology and Pharmacokinetics, College of Pharmacy, University of Nantes, France.
Petit
J Y
JY
Lang
F
F
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D016718:Arachidonic Acid / Q000378:metabolism; D001549:Benzamides / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004075:Diglycerides / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D007211:Indoles / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D000894:Anti-Inflammatory Agents, Non-Steroidal; D001549:Benzamides; D004075:Diglycerides; D007211:Indoles; C096171:JM 34; D008070:Lipopolysaccharides; D008301:Maleimides; D014409:Tumor Necrosis Factor-alpha; D016718:Arachidonic Acid; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; C088060:bisindolylmaleimide