Regulation of thymocyte lineage commitment by the level of classical protein kinase C activity.
Thymocyte-positive selection involves signaling through TCR and accessory molecules, and the signaling intensity appears to be critical for this event. The specific inhibitor of classical Ca2+-dependent protein kinase C (cPKC), Gö 6976, inhibited positive selection in fetal thymus organ culture, indicating that cPKC activation is essential for positive selection. The major protein kinase C isoforms in CD4+ CD8+ thymocytes are cPKC-alpha, cPKC-beta, and the novel Ca2+-independent protein kinase C, nPKC-epsilon. To analyze the effect of cPKC activation level on positive selection, we used thymocytes from TCR transgenic mice with nonselecting and RAG-2 -/- backgrounds as they were developmentally arrested at the CD4+ CD8+ stage without positive selection signals. These thymocytes survived and acquired CD4/CD8 lineage commitment in suspension culture upon transient stimulation with limited concentrations of the selective activator of cPKC-alpha and -beta, thymeleatoxin, and the calcium ionophore, ionomycin. However, neither 12-deoxyphorbol 13-phenylacetate 20-acetate, which selectively activates cPKC-beta, nor ingenol 3,20-dibenzoate, which selectively activates nPKC-epsilon, exerted such an effect. The thymeleatoxin/ionomycin concentrations corresponded to those that inhibit glucocorticoid-induced apoptosis in thymocytes and were lower than those that induce proliferation of mature T cells. The CD4 lineage commitment required a higher level of cPKC activity than the CD8 lineage commitment. CD8alpha or CD4 mRNA expression was down-regulated. Functional helper and killer T cells were induced from the CD4 and CD8 lineage-committed cells, respectively, by additional stimulation. These results suggest that thymocyte lineage commitment in positive selection is regulated by the level of cPKC-alpha activity or by the levels of cPKC-alpha and -beta activities.
Ohoka
Y
Y
Project Research Center, Mitsubishi Kasei Institute of Life Sciences, Machida-shi, Tokyo, Japan.
Kuwata
T
T
Asada
A
A
Zhao
Y
Y
Mukai
M
M
Iwata
M
M
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002118:Calcium / Q000502:physiology; D019070:Cell Lineage; D002478:Cells, Cultured; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009924:Organ Culture Techniques; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D013601:T-Lymphocytes / Q000502:physiology*; D015854:Up-Regulation
D007527:Isoenzymes; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D002118:Calcium