ENDOTOXIN ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASE IN THP-1 CELLS - DIMINISHED ACTIVATION FOLLOWING ENDOTOXIN DESENSITIZATION

The signal transduction events occurring in monocytes in response to e ndotoxin (LPS) stimulation are incompletely delineated, although pertu ssis toxin (PT)-sensitive G proteins and the mitogen-activated protein kinase (MAPK) cascade have been implicated. Cellular desensitization in response to 18-h pre-exposure to 1 mu g/mL LPS alters signal transd uction pathways of cellular activation and decreases production of cer tain inflammatory mediators such as thromboxane (Tx)B-2, the stable me tabolite of TxA(2). We hypothesized that LPS stimulation of the human monocyte cell hue THP-I occurs ria MAPK activation, and that LPS desen sitization, induced by pre-exposure to LPS, is associated with altered signaling through the MAPK cascade, involvement of a specific MAPK, E RK, in LPS-stimulated TxB(2) production was further tested using a spe cific MAPK cascade inhibitor, PD98059 (PD), PD inhibited LPS and phorb ol myristate acetate (PMA)-stimulated ERK activation as demonstrated b y immunoblots using anti-activated ERK antibodies, PD significantly in hibited LPS and PMA-stimulated TxB(2) synthesis to non-detectable leve ls, suggesting an involvement of MAPK in LPS-stimulated activation. Be cause PT-sensitive G proteins mediate LPS-stimulated signal transducti on, their role in MAPK activation was tested. Pretreatment with PT inh ibited basal and LPS-stimulated, but not PMA-stimulated ERK activation . Activation of ERK after LPS desensitization was also assessed. LPS p re-exposure resulted in a profound decrease in LPS-stimulated activati on of ERK, but did not affect PMA activation of ERK. These data implic ate the involvement of the MAPK cascade in LPS-stimulated activation o f THP-1 cells and suggest coupling of Gi proteins and MAPKs in LPS-sti mulated events, LPS desensitization is associated with decreased MAPK activation, but does not impair MAPK activation by PMA, Thus, LPS dese nsitization appears to selectively alter signal transduction upstream of ERK.