Rubin J E, Titus L, Nanes M S
Research Service, Veterans Administration Medical Center, Clairmont, GA.
Biochim Biophys Acta. 1991 Jan 10;1091(1):87-95. doi: 10.1016/0167-4889(91)90226-n.
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.
通过用二羟基维生素D - 3（1,25(OH)2D3）连续处理细胞2天，随后用前列腺素E2处理，U937细胞可被诱导表达补体5a受体（C5aR）。我们询问前列腺素E2引起最大C5aR表达的作用是否仅需要激活环磷酸腺苷依赖性蛋白激酶（PKA）。前列腺素E2在对照细胞和1,25(OH)2D3处理的细胞中均呈剂量依赖性激活PKA；到4小时时，PKA对进一步的前列腺素E2刺激不再有反应。我们推测通过PKA转导的前列腺素E2作用在4小时时应该完成；即，在用前列腺素E2处理4小时和2天的细胞中，C5aR诱导应该是等效的。所有细胞在最初2天用1,25(OH)2D3处理，在接下来的2天用前列腺素E2或环磷酸腺苷类似物处理。在总共培养4天后测量C5aR数量。在1,25(OH)2D3处理结束时用试剂进行4小时脉冲处理。暴露于前列腺素E2 4小时脉冲的细胞中C5aR的量仅为持续暴露于前列腺素E2的细胞中的68.2±4.4%。用一对环磷酸腺苷类似物（每种8 - 硫代甲基 - 环磷酸腺苷+ N6 - 苯甲酰 - 环磷酸腺苷各50 microM）连续培养，其导致PKA激活比基础水平增加41.7%±10.8%，在这些细胞中C5aR数量仅为用前列腺素E2培养2天的细胞中的51%±16%，而此时PKA保持在基础活性水平。因此，我们得出结论，前列腺素E2引起的C5aR表达不能完全归因于环磷酸腺苷依赖性信号通路激活的持续时间或程度。我们研究了钙敏感蛋白激酶C参与的可能性。在用1,25(OH)2D3和前列腺素E2连续处理2天的细胞中，细胞质蛋白激酶C比对照增加了154%±14%。在上述处理组中，佛波酯肉豆蔻酸酯刺激10分钟后，膜相关蛋白激酶C也增加了147%±2%。最后，佛波酯肉豆蔻酸酯增强了环磷酸腺苷类似物引起的C5aR诱导。我们提出，前列腺素E2与1,25(OH)2D3协同作用在U937细胞中引起C5aR诱导的机制不仅包括通过环磷酸腺苷级联的信号转导，还包括通过蛋白激酶C调节的途径。
