Prostaglandin E2 induction of monoblastic differentiation utilizes both cAMP and non-cAMP dependent signalling...

U937 cells can be induced to express receptor for complement 5a (C5aR) by sequential 2 day treatments of cells with dihydroxyvitamin D-3 (1,25(OH)2D3) followed by prostaglandin E2. We asked whether the action of prostaglandin E2 to cause maximal C5aR expression required only activation of the cAMP-dependent protein kinase (PKA). Prostaglandin E2 dose dependently activated PKA in control and 1,25(OH)2D3 treated cells; by 4 h the PKA did not respond to further prostaglandin E2 challenge. We hypothesized that prostaglandin E2 actions transduced via PKA should be complete by 4 h; i.e., C5aR induction should be equivalent in cells treated with prostaglandin E2 for 4 h and for 2 days. All cells were treated for the first 2 days with 1,25(OH)2D3 and the second 2 days with prostaglandin E2 or cAMP analogs. C5aR number was measured after 4 days total culture. 4 h pulse treatments with agents were given at the end of the 1,25(OH)2D3 treatment. Cells exposed to a 4 h pulse of prostaglandin E2 had only 68.2 +/- 4.4% the amount of C5aR seen in cells continuously exposed to prostaglandin E2. Continuous culture with a cAMP analog pair (50 microM each of 8-thiomethyl-cAMP + N6-benzoyl-cAMP), which caused a 41.7% +/- 10.8% increase PKA activation above basal, resulted in only 51% +/- 16% of the C5aR numbers seen in cells cultured for 2 days with prostaglandin E2, where PKA remained at basal activity. We therefore concluded that C5aR expression caused by prostaglandin E2 could not be ascribed entirely to duration or degree of activation of cAMP-dependent signalling pathways. We investigated the possibility that the calcium sensitive protein kinase C was involved. Cytoplasmic protein kinase C was increased 154% +/- 14% above control in cells treated with sequential 2 days treatments of 1,25(OH)2D3 and prostaglandin E2. A 147% +/- 2% increase in membrane associated protein kinase C was also seen 10 min after phorbol myristate acetate stimulation in the above treatment group. Finally, phorbol myristate acetate augmented the C5aR induction caused by cAMP analog. We propose that the mechanism of prostaglandin E2 synergism with 1,25(OH)2D3 in causing C5aR induction in U937 cells includes signal transduction not only by the cAMP cascade, but also via protein kinase C modulated pathways.