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• W2057479530 abstract "Glucocorticoids appear to participate in apoptosis of unselected CD4+CD8+thymocytes. Activation of Ca2+-independent novel protein kinase C (nPKC) precedes glucocorticoid-induced thymocyte apoptosis, while proper levels of Ca2+-dependent protein kinase C (cPKC) and calcineurin activities contribute to rescue thymocytes. To clarify the role of nPKC in thymocyte apoptosis, murine thymocytes were stimulated with the diterpene diester, ingenol 3,20-dibenzoate (IDB). IDB induced selective translocation of nPKC-δ, -ε, and -θ and PKC-μ from the cytosolic fraction to the particulate fraction and induced morphologically typical apoptosis through de novo synthesis of macromolecules. The apoptosis was also induced by thymeleatoxin, a diterpene ester, at relatively high concentrations that induced translocation of cPKC, nPKC-θ, and PKC-μ. The IDB- or thymeleatoxin-induced death was inhibited by non-isoform-selective PKC inhibitors, but not by their structural analogs with weak PKC-inhibitory activity or the selective inhibitor of cPKC and PKC-μ, Gö 6976. The death was also inhibited by calcium ionophore ionomycin at concentrations within a narrow range. The range corresponded to the concentration range that contributes to the inhibition of glucocorticoid-induced apoptosis. The antiapoptotic effect was canceled by the immunosuppressant FK506 but not by rapamycin. These results indicate that activation of nPKC, especially nPKC-θ, induces apoptosis in thymocytes and that calcineurin activation regulates the apoptosis. Glucocorticoids appear to participate in apoptosis of unselected CD4+CD8+thymocytes. Activation of Ca2+-independent novel protein kinase C (nPKC) precedes glucocorticoid-induced thymocyte apoptosis, while proper levels of Ca2+-dependent protein kinase C (cPKC) and calcineurin activities contribute to rescue thymocytes. To clarify the role of nPKC in thymocyte apoptosis, murine thymocytes were stimulated with the diterpene diester, ingenol 3,20-dibenzoate (IDB). IDB induced selective translocation of nPKC-δ, -ε, and -θ and PKC-μ from the cytosolic fraction to the particulate fraction and induced morphologically typical apoptosis through de novo synthesis of macromolecules. The apoptosis was also induced by thymeleatoxin, a diterpene ester, at relatively high concentrations that induced translocation of cPKC, nPKC-θ, and PKC-μ. The IDB- or thymeleatoxin-induced death was inhibited by non-isoform-selective PKC inhibitors, but not by their structural analogs with weak PKC-inhibitory activity or the selective inhibitor of cPKC and PKC-μ, Gö 6976. The death was also inhibited by calcium ionophore ionomycin at concentrations within a narrow range. The range corresponded to the concentration range that contributes to the inhibition of glucocorticoid-induced apoptosis. The antiapoptotic effect was canceled by the immunosuppressant FK506 but not by rapamycin. These results indicate that activation of nPKC, especially nPKC-θ, induces apoptosis in thymocytes and that calcineurin activation regulates the apoptosis. T cell receptor protein kinase C atypical protein kinase C classical protein kinase C dexamethasone major histocompatibility complex class I and class II double-knockout ingenol 3,20-dibenzoate nuclear factor of activated T cells novel protein kinase C thymeleatoxin phorbol 12-myristate 13-acetate. Immature T cell clones in the thymus are selected to survive or die at the CD4+CD8+ stage according to the specificity of the T cell receptors (TCRs).1 Useful clones are protected from apoptosis and differentiate into mature CD4+CD8− or CD4−CD8+T cells (positive selection), while self-reactive clones undergo apoptosis (negative selection). Useless clones also appear to undergo apoptosis. The TCR-mediated signals are critical for the fate of CD4+CD8+ thymocytes and involve protein kinase C (PKC) activation. PKC consists of several subfamilies of enzymes including Ca2+-dependent classical PKC (cPKC) and Ca2+-independent novel PKC (nPKC) and atypical PKC (aPKC) (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2368) Google Scholar). Each isoform or subfamily of PKC appears to play its own role in the fate of CD4+CD8+ thymocytes. We have previously indicated that activation of cPKC is involved in positive selection (2Ohoka Y. Kuwata T. Asada A. Zhao Y. Mukai M. Iwata M. J. Immunol. 1997; 158: 5707-5716PubMed Google Scholar). On the other hand, thymocyte apoptosis induced by TCR/CD3- and CD28-mediated stimulation in vitro appears to be accompanied by activation of both cPKC and nPKC, 2A. Asada, Y. Zhao, T. Kuwata, M. Mukai, Y. Tozawa, R. Iseki, K. Fujita, H. Tian, Y. Motegi, R. Suzuki, M. Yokoyama, and M. Iwata, manuscript in preparation. and the death is considered to mimic negative selection (3Punt J.A. Osborne B.A. Takahama Y. Sharrow S.O. Singer A. J. Exp. Med. 1994; 179: 709-713Crossref PubMed Scopus (237) Google Scholar). Glucocorticoid hormones exert pleiotropic effects on thymocyte survival and differentiation (4Willie A.H. Morris R.G. Smith A.L. Dunlop D. J. Pathol. 1984; 142: 67-77Crossref PubMed Scopus (1443) Google Scholar, 5Cohen J.J. Duke R.C. J. Immunol. 1984; 132: 38-42PubMed Google Scholar, 6Gonzalo J.A. González-Garcia A. Martı́nez-A C. Koemer G. J. Exp. Med. 1993; 177: 1239-1246Crossref PubMed Scopus (176) Google Scholar, 7Vacchio M.S. Papadopoulos V. Ashwell J.D. J. Exp. Med. 1994; 179: 1835-1846Crossref PubMed Scopus (317) Google Scholar, 8Gruber J. Sgonc R. Hu Y.H. Beug H. Wick G. Eur. J. Immunol. 1994; 24: 1115-1121Crossref PubMed Scopus (122) Google Scholar, 9Cidlowski J.A. King K.L. Evans-Storms R.B. Montague J.W. Bortner C.D. Hughes Jr., F.M. Recent Prog. Hormone Res. 1996; 51: 457-491PubMed Google Scholar, 10Shortman K. Jackson H. Cell Immunol. 1974; 12: 230-246Crossref PubMed Scopus (140) Google Scholar) and may participate in apoptosis of unselected thymocyte clones. CD4+CD8+thymocytes are highly sensitive to induction of apoptosis by glucocorticoids and appear to undergo apoptosis even at the physiological peak levels at least in mice or rats (5Cohen J.J. Duke R.C. J. Immunol. 1984; 132: 38-42PubMed Google Scholar, 6Gonzalo J.A. González-Garcia A. Martı́nez-A C. Koemer G. J. Exp. Med. 1993; 177: 1239-1246Crossref PubMed Scopus (176) Google Scholar, 10Shortman K. Jackson H. Cell Immunol. 1974; 12: 230-246Crossref PubMed Scopus (140) Google Scholar, 11Iwata M. Hanaoka S. Sato K. Eur. J. Immunol. 1991; 21: 643-648Crossref PubMed Scopus (203) Google Scholar), whereas immature CD4−CD8− thymocytes or mature CD4+CD8− or CD4−CD8+ thymocytes are relatively resistant (12Homo F. Duval D. Hatzfeld J. Evrard C. J. Steroid Biochem. 1980; 13: 135-143Crossref PubMed Scopus (40) Google Scholar, 13Hugo P. Boyd R.L. Waanders G.A. Scollay R. Eur. J. Immunol. 1991; 21: 2655-2660Crossref PubMed Scopus (22) Google Scholar). Glucocorticoid-induced apoptosis in thymocytes is preceded by activation of nPKC including nPKC-ε and is inhibited by non-isoform-selective PKC inhibitors but not by Gö 6976, a specific inhibitor of cPKC isoforms and PKC-μ (11Iwata M. Hanaoka S. Sato K. Eur. J. Immunol. 1991; 21: 643-648Crossref PubMed Scopus (203) Google Scholar, 16Zhao Y. Iwata M. Int. Immunol. 1995; 7: 1387-1396Crossref PubMed Scopus (22) Google Scholar, 17Iwata M. Curr. Topics Microbiol. Immunol. 1995; 200: 81-94PubMed Google Scholar). The apoptosis is also inhibited by proper levels of stimulation through TCR·CD3 complex with co-stimulation through CD4, CD8, or lymphocyte function-associated antigen-1 (11Iwata M. Hanaoka S. Sato K. Eur. J. Immunol. 1991; 21: 643-648Crossref PubMed Scopus (203) Google Scholar, 16Zhao Y. Iwata M. Int. Immunol. 1995; 7: 1387-1396Crossref PubMed Scopus (22) Google Scholar, 17Iwata M. Curr. Topics Microbiol. Immunol. 1995; 200: 81-94PubMed Google Scholar). The antiapoptotic effect is mimicked by moderate stimulation with proper combinations of PMA and the Ca2+ ionophore ionomycin or combinations of thymeleatoxin (TTX) and ionomycin (2Ohoka Y. Kuwata T. Asada A. Zhao Y. Mukai M. Iwata M. J. Immunol. 1997; 158: 5707-5716PubMed Google Scholar, 18Zhao Y. Tozawa Y. Iseki R. Mukai M. Iwata M. J. Immunol. 1995; 154: 6346-6354PubMed Google Scholar, 19Ohoka Y. Kuwata T. Tozawa Y. Zhao Y. Mukai M. Motegi Y. Suzuki R. Yokoyama M. Iwata M. Int. Immunol. 1996; 8: 297-306Crossref PubMed Scopus (54) Google Scholar). PMA activates both cPKC and nPKC (20Ryves W.J. Evans A.T. Olivier A.R. Parker P.J. Evans F.J. FEBS Lett. 1991; 288: 5-9Crossref PubMed Scopus (176) Google Scholar), while TTX at the antiapoptotic doses specifically activates cPKC (2Ohoka Y. Kuwata T. Asada A. Zhao Y. Mukai M. Iwata M. J. Immunol. 1997; 158: 5707-5716PubMed Google Scholar, 20Ryves W.J. Evans A.T. Olivier A.R. Parker P.J. Evans F.J. FEBS Lett. 1991; 288: 5-9Crossref PubMed Scopus (176) Google Scholar, 21Kazanietz M.G. Areces L.B. Bahador A. Mischak H. Goodnight J. Mushinski J.F. Blumberg P.M. Mol. Pharmacol. 1993; 44: 298-307PubMed Google Scholar). On the other hand, the cPKC (and PKC-μ)-specific inhibitor Gö 6976 (22Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marmé D. Schächtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar, 23Gschwendt M. Dieterich S. Rennecke J. Kittstein W. Mueller H.-J. Johannes F.-J. FEBS Lett. 1996; 392: 77-80Crossref PubMed Scopus (566) Google Scholar) cancels the antiapoptotic effect of the antibodies and that of PMA/ionomycin (13Hugo P. Boyd R.L. Waanders G.A. Scollay R. Eur. J. Immunol. 1991; 21: 2655-2660Crossref PubMed Scopus (22) Google Scholar). Gö 6976 also inhibits positive selection in a fetal thymus organ culture system (2Ohoka Y. Kuwata T. Asada A. Zhao Y. Mukai M. Iwata M. J. Immunol. 1997; 158: 5707-5716PubMed Google Scholar). Therefore, proper levels of cPKC activity appear to be involved in both the protection of CD4+CD8+thymocytes from apoptosis and the induction of positive selection. Indeed, transient stimulation of isolated CD4+CD8+ thymocytes with the antiapoptotic combinations of TTX/ionomycin induced differentiation and commitment of the cells to the CD4 or CD8 T cell lineage (2Ohoka Y. Kuwata T. Asada A. Zhao Y. Mukai M. Iwata M. J. Immunol. 1997; 158: 5707-5716PubMed Google Scholar). In the present study, we analyzed a possible relationship between thymocyte apoptosis and activation of nPKC isoforms by using the diterpene esters ingenol 3,20-dibenzoate (IDB) and TTX. We also analyzed the effect of calcineurin activation on nPKC-dependent apoptosis in thymocytes, since activation of calcineurin as well as cPKC was critical for the inhibition of glucocorticoid-induced apoptosis in thymocytes and for thymocyte positive selection (16Zhao Y. Iwata M. Int. Immunol. 1995; 7: 1387-1396Crossref PubMed Scopus (22) Google Scholar, 18Zhao Y. Tozawa Y. Iseki R. Mukai M. Iwata M. J. Immunol. 1995; 154: 6346-6354PubMed Google Scholar). The present results suggest that nPKC activation induces apoptosis in immature CD4+CD8+ thymocytes and that calcineurin activation contributes to protect the cells from the apoptosis. BALB/c mice (4–6 weeks of age) were obtained from Japan SLC (Shizuoka, Japan). BOG8 TCR transgenic mice with RAG-2(−/−), and nonselecting MHC backgrounds were established as described previously (19Ohoka Y. Kuwata T. Tozawa Y. Zhao Y. Mukai M. Motegi Y. Suzuki R. Yokoyama M. Iwata M. Int. Immunol. 1996; 8: 297-306Crossref PubMed Scopus (54) Google Scholar, 24Iwata M. Kuwata T. Mukai M. Tozawa Y. Yokoyama M. Eur. J. Immunol. 1996; 26: 2081-2086Crossref PubMed Scopus (45) Google Scholar). Major histocompatibility complex class I and class II double knockout (DKO) mice (C57BL/6 deficient in Aβb and β2-microglobulin) were obtained from Taconic (Immuno-Biological Laboratories, Gunma, Japan). DEX, PMA, and IDB were obtained from Wako (Osaka, Japan), Sigma, and Biomol (Plymouth Meeting, PA), respectively. Ionomycin, TTX, Gö 6976, Gö 6983, and recombinant human cPKC-α and nPKC-ε were obtained from Calbiochem. H-7 and HA1004 were obtained from Seikagaku Kogyo (Tokyo, Japan). Ro 31–8425 (bisindolylmaleimide X) and Ro 31–6045 (bisindolylmaleimide V) were obtained from LC Laboratories (Läufelfingen, Switzerland). Splenic T cells were obtained from BALB/c mice as described previously (16Zhao Y. Iwata M. Int. Immunol. 1995; 7: 1387-1396Crossref PubMed Scopus (22) Google Scholar). In the splenic T cell preparations, 88–92% of the cells were TCRαβ+ by fluorescence-activated cell sorting analysis. Thymocytes or splenic T cells (3.75–4 × 106) were suspended in 1 ml of Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (JRH Bioscience, Woodland, CA), 3 mml-glutamine, 1 mm sodium pyruvate, 1× minimal essential medium nonessential amino acids, 50 μm 2-mercaptoethanol, 20 mm HEPES (pH 7.2), 20 units of penicillin, and 20 μg of streptomycin (complete Dulbecco's modified Eagle's medium) and were cultured for the indicated times at 37 °C in the presence or absence of the indicated drugs in 24-well tissue culture plates (Corning 25820, Corning, NY). To examine the expression of CD4, CD8, and TCR, the cells were stained with labeled antibodies: R-phycoerythrin-conjugated anti-CD4 monoclonal antibody (RM4–5), fluorescein isothiocyanate-labeled anti-CD8 monoclonal antibody (53–6.7), or fluorescein isothiocyanate-labeled or biotinylated anti-TCRαβ monoclonal antibody (H57–597) (Pharmingen) with or without streptavidin TRI-Color (Caltag Laboratories, San Francisco, CA). Viable cells were gated by using forward and side scatters with a FACScan flow cytometer and FACScan research software (Becton Dickinson, Lincoln Park, NJ) and were analyzed for marker expression. The gate for viable cells was determined by using propidium iodide exclusion and Paint-a-Gate software (Becton Dickinson). DNA fragmentation in thymocytes was determined as described previously (11Iwata M. Hanaoka S. Sato K. Eur. J. Immunol. 1991; 21: 643-648Crossref PubMed Scopus (203) Google Scholar). Briefly, the cells harvested by centrifugation were lysed in 0.5% Triton X-100 containing 5 mm Tris-HCl (pH 7.4) and 1 mm EDTA for 20 min on ice. The lysate and its supernatant after centrifugation at 27,000 × g for 20 min were sonicated for 15 s, and then DNA contents were measured by fluorometry using 4′,6-diamidino-2-phenylindole (Sigma) and Fluoroskan II (Titertek; Flow Laboratories USA, McLean, VA). The percentage of DNA fragmented was calculated as the ratio of DNA content in the supernatant to that in the lysate. Cytoysis was assessed by a trypan blue dye exclusion assay. Immunoblotting analysis of PKC isoforms was performed as described previously (14Iwata M. Iseki R. Sato K. Tozawa Y. Ohoka Y. Int. Immunol. 1994; 6: 431-438Crossref PubMed Scopus (55) Google Scholar) with slight modification. The cultured thymocytes were centrifuged at 450 × gfor 5 min at 4 °C, and each cell pellet (107 cells) was washed with ice-cold phosphate-buffered saline and resuspended in 1 ml of ice-cold buffer A (20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 0.3% mercaptoethanol, 50 μg/ml phenylmethylsulfonyl fluoride, 250 μg/ml leupeptin, and 10 mm benzamidine). After a 5-min incubation, cell lysis was confirmed by trypan blue, and the suspension was centrifuged at 100,000 × g for 70 min. After centrifugation, the supernatant (cytosolic fraction) was removed, and the pellet was resuspended in 1 ml of buffer A supplemented with 1% Triton X-100 and sonicated for 1 min. The homogenate was applied onto DEAE-cellulose DE-52 columns for removing DNA and partial purification. The columns were washed with ice-cold buffer B (20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 0.3% mercaptoethanol, 50 μg/ml phenylmethylsulfonyl fluoride, and 10 mm benzamidine) and eluted with buffer B supplemented with 0.2 m NaCl. The eluate is referred to as the particulate fraction. The proteins in the particulate fractions and those in the cytosolic fractions were precipitated with ethanol (final concentration of 60% (v/v)). Equivalent amounts of proteins were solubilized in sample buffer with 2-mercaptoethanol and separated by SDS-polyacrylamide gel electrophoresis (9% gel) and transferred to nitrocellulose membranes (Micron Separations, Westboro, MA). The membranes were soaked in 5% bovine serum albumin and incubated with monoclonal anti-PKC antibodies (Transduction Laboratories, Lexington, KY). PKC isoforms (α, β, γ, δ, ε, η, θ, μ, λ, and ζ) were detected with horseradish peroxidase-goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and the ECL system (Amersham Pharmacia Biotech, Tokyo, Japan). It should be noted, however, that the anti-cPKC-α cross-reacts with β and that the anti-cPKC-γ cross-reacts with α according to the manufacturer. Cells were prefixed with 2% glutaraldehyde in 0.1 m phosphate buffer (pH 7.4), washed twice with 0.1 m phosphate buffer, postfixed with 2% osmium tetroxide in 0.1 m phosphate buffer, and embedded in 2% agar. The cells were dehydrated in an ethanol series, embedded in Epon 812, and kept at 60 °C for more than 48 h to polymerize the resin. After ultrathin sections were stained with uranyl acetate and lead citrate, they were observed under 1200EX transmission electron microscopy (JEOL, Tokyo, Japan). We have previously shown that glucocorticoids induce an increase in nPKC activity in the particulate fraction of murine thymocytes and translocation of nPKC-ε from the cytosolic fraction to the particulate fraction (14Iwata M. Iseki R. Sato K. Tozawa Y. Ohoka Y. Int. Immunol. 1994; 6: 431-438Crossref PubMed Scopus (55) Google Scholar). Other nPKC isoforms were not significantly detected in these cells by using the antibodies available at that time, but here we could also detect nPKC-δ and -θ by using newly available antibodies. nPKC-η was not detectable. In the normal thymus, 80–85% of thymocytes are immature CD4+CD8+ cells that express low levels of Bcl-2 and are sensitive to induction of apoptosis by glucocorticoids (25Veis D.J. Sentman C.L. Bach E.A. Korsmeyer S.J. J. Immunol. 1993; 151: 2546-2554PubMed Google Scholar). To obtain CD4+CD8+ thymocytes, DKO mice were used, since T cell development is arrested at the CD4+CD8+ stage in these mice, and almost all of the thymocytes are CD4+CD8+ cells (26Grusby M.J. Auchincloss Jr., H. Lee R. Johnson R.S. Spencer J.P. Zijlstra M. Jaenisch R. Papaioannou V.E. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3913-3917Crossref PubMed Scopus (246) Google Scholar). The synthetic glucocorticoid, DEX, induced increases in nPKC-δ and -θ in the particulate fraction of thymocytes from DKO mice after 2–2.5 h of incubation and induced decreases in these isoforms in the cytosolic fraction after 2–3 h of incubation (Fig. 1), suggesting that the nPKC isoforms were translocated. cPKC isoforms in the particulate fraction only slightly increased after 2 h of incubation, while those in the cytosolic fraction did not significantly change (Fig. 1). DEX did not induce translocation of PKC-μ, a PKC distantly related to nPKC (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2368) Google Scholar), or that of aPKC-λ or -ζ (Fig. 1 and data not shown). Similar changes in the intracellular distribution of PKC isoforms were observed in BALB/c mouse thymocytes treated with DEX, and the translocation of nPKC-ε was also confirmed (data not shown). However, the expression of nPKC-ε molecule in C57BL/6 and DKO thymocytes was consistently low, and thus its translocation was hardly detectable. Since DEX-induced DNA fragmentation in thymocytes is inhibited by non-isoform-selective PKC inhibitors but not by the cPKC-specific inhibitor Gö 6976 (14Iwata M. Iseki R. Sato K. Tozawa Y. Ohoka Y. Int. Immunol. 1994; 6: 431-438Crossref PubMed Scopus (55) Google Scholar, 15Iwata M. Ohoka Y. Kuwata T. Asada A. Stem Cells. 1996; 14: 632-641Crossref PubMed Scopus (45) Google Scholar), the results suggest that activation of nPKC isoforms may be involved in the death but that nPKC-ε activation may not be essential. The diterpene diester IDB has once been suggested to be a selective activator of PKC-ε, but it is not specific to this isoform as the manufacturer indicates in the product catalog. IDB at 10 or 50 nm induced translocation of nPKC-δ, -ε, and -θ and PKC-μ from the cytosolic fractions to the particulate fractions of both thymocytes and splenic T cells, whereas it did not induce translocation of cPKC isoforms or aPKC-ζ and -λ (Fig. 2 and data not shown), indicating that IDB selectively activates nPKC isoforms and PKC-μ in these cells in this dose range. On the other hand, as TTX is known as a cPKC-specific activator (20Ryves W.J. Evans A.T. Olivier A.R. Parker P.J. Evans F.J. FEBS Lett. 1991; 288: 5-9Crossref PubMed Scopus (176) Google Scholar, 21Kazanietz M.G. Areces L.B. Bahador A. Mischak H. Goodnight J. Mushinski J.F. Blumberg P.M. Mol. Pharmacol. 1993; 44: 298-307PubMed Google Scholar), incubation of thymocytes with 0.3 ng/ml TTX induced selective translocation of cPKC-α and -β (Fig. 2). However, at higher concentrations (1 ng/ml or higher), TTX is no longer specific for cPKC in thymocytes. Incubation of the cells with 1 ng/ml TTX induced translocation of nPKC-θ and -μ as well as cPKC isoforms (Fig. 2). There was little translocation of nPKC-δ or -ε upon stimulation with 1 ng/ml TTX. The translocational response of each PKC isoform in thymocytes is summarized in Table I.Table ITranslocation of PKC isoforms in thymocytes and splenic T cells upon stimulation with DEX or the diterpene esters, IDB and TTXCellsActivatorcPKCnPKCPKC-μaPKCαβγδεθλζThymocytesDEX−1-aTranslocation of cPKC isoforms in DEX-treated thymocytes was not significantly detected. However, cPKC in the particulate fraction slightly and transiently increased after 2 h of incubation with DEX (Fig. 1).−1-aTranslocation of cPKC isoforms in DEX-treated thymocytes was not significantly detected. However, cPKC in the particulate fraction slightly and transiently increased after 2 h of incubation with DEX (Fig. 1).−1-aTranslocation of cPKC isoforms in DEX-treated thymocytes was not significantly detected. However, cPKC in the particulate fraction slightly and transiently increased after 2 h of incubation with DEX (Fig. 1).++1-bThe DEX-induced translocation of nPKC-ε was confirmed in BALB/c mouse thymocytes.+−−−1-cTranslocation of aPKC-ζ was not detected in BALB/c mouse thymocytes or DKO thymocytes upon stimulation with DEX (data not shown).IDB−−−++++−−TTX1-dThymocytes were incubated with 1 ng/ml TTX.+++−−++−−Splenic T cellsIDB−−−++++−−The results of Figs. 1 and 2 and the data on splenic T cells are summarized.1-a Translocation of cPKC isoforms in DEX-treated thymocytes was not significantly detected. However, cPKC in the particulate fraction slightly and transiently increased after 2 h of incubation with DEX (Fig. 1).1-b The DEX-induced translocation of nPKC-ε was confirmed in BALB/c mouse thymocytes.1-c Translocation of aPKC-ζ was not detected in BALB/c mouse thymocytes or DKO thymocytes upon stimulation with DEX (data not shown).1-d Thymocytes were incubated with 1 ng/ml TTX. Open table in a new tab The results of Figs. 1 and 2 and the data on splenic T cells are summarized. IDB induced DNA fragmentation in thymocytes but not in splenic T cells (Fig. 3, A and B), indicating that IDB induces death in immature T cells but not in mature T cells. IDB as well as DEX induced DNA fragmentation in CD4+CD8+ thymocytes from DKO mice and BOG8 TCR transgenic mice with RAG-2 (−/−) and nonselecting major histocompatibility complex backgrounds (data not shown). In the latter mice, T cell development is also arrested at CD4+CD8+ thymocytes, and almost all of the thymocytes are CD4+CD8+ cells (19Ohoka Y. Kuwata T. Tozawa Y. Zhao Y. Mukai M. Motegi Y. Suzuki R. Yokoyama M. Iwata M. Int. Immunol. 1996; 8: 297-306Crossref PubMed Scopus (54) Google Scholar) as in DKO mice. Incubation of thymocytes with IDB induced morphological changes typical in apoptosis, such as shrinkage of cells, clumping of chromatin into masses, and dilation of endoplasmic vesicles, while keeping mitochondrial structure almost normal (Fig. 4) as observed in glucocorticoid-treated thymocytes (4Willie A.H. Morris R.G. Smith A.L. Dunlop D. J. Pathol. 1984; 142: 67-77Crossref PubMed Scopus (1443) Google Scholar). TTX at 0.3 ng/ml did not induce DNA fragmentation in thymocytes, but TTX at 1 ng/ml did induce it (Fig. 3 C). Thus, there was a close correlation between activation of nPKC, especially the θ-isoform, and induction of apoptosis in thymocytes.Figure 4Morphologically typical apoptosis is induced in thymocytes by IDB stimulation. Thymocytes from BALB/c mice were cultured in the presence (D, E, and F) or the absence (A, B, and C) of 50 nm IDB for 16 h. After the culture, the cells were harvested and fixed, and their morphology was analyzed by electron microscopy.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The IDB-induced DNA fragmentation in thymocytes was inhibited by actinomycin D or cycloheximide (Fig. 5,A and B), suggesting that de novosynthesis of both RNA and proteins is necessary for the apoptosis. Accordingly, there was a time lag of more than 6 h to induce DNA fragmentation after the IDB addition (Fig. 5 B). PKC inhibitors were used to examine if PKC activation is essential for the induction of apoptosis. H-7, an inhibitor of both nPKC and cPKC and some other kinases (27Hidaka H. Inagaki M. Kawamoto S. Sasaki Y. Biochemistry. 1984; 23: 5036-5041Crossref PubMed Scopus (2329) Google Scholar, 28Schaap D. Parker P.J. J. Biol. Chem. 1990; 265: 7301-7307Abstract Full Text PDF PubMed Google Scholar), inhibited IDB-induced DNA fragmentation in thymocytes (Fig. 6 A). Ro 31-8425, a staurosporine-related and highly specific PKC inhibitor (29Muid R.E. Dale M.M. Davis P.D. Elliot L.H. Hill C.H. Kumar H. Lawton G. Twomey B.M. Wadsworth J. Wilkinson S.E. Nixon J.S. FEBS Lett. 1991; 293: 169-172Crossref PubMed Scopus (51) Google Scholar), also inhibited the DNA fragmentation (Fig. 6 A). The inhibition of death was confirmed by a trypan blue dye exclusion assay (data not shown). Ro 31-8425 at 1 μg/ml completely inhibited the activation of recombinant cPKC-α and recombinant nPKC-ε in vitro (data not shown), confirming that Ro 31-8425 is a non-isoform-selective PKC inhibitor. On the other hand, the structural analogs of these inhibitors with weak PKC-inhibitory activity, HA1004 and Ro 31-6045 (27Hidaka H. Inagaki M. Kawamoto S. Sasaki Y. Biochemistry. 1984; 23: 5036-5041Crossref PubMed Scopus (2329) Google Scholar, 30Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar), did not inhibit DNA fragmentation (Fig. 6 A). The selective inhibitor of cPKC and PKC-μ, Gö 6976, also failed to inhibit the IDB-induced DNA fragmentation, whereas its structural analog, Gö 6983 inhibited the death (Fig. 6 B). Gö 6983 has been shown to inhibit various PKC isoforms including cPKC, nPKC-δ, and PKC-ζ, but it does not effectively inhibit PKC-μ (23Gschwendt M. Dieterich S. Rennecke J. Kittstein W. Mueller H.-J. Johannes F.-J. FEBS Lett. 1996; 392: 77-80Crossref PubMed Scopus (566) Google Scholar). Similar effects were observed on TTX-induced DNA fragmentation in thymocytes by using these inhibitors and analogs (Fig. 6 B and data not shown). The results collectively suggest that nPKC activation is essential for the induction of thymocyte apoptosis by IDB. Activation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, contributes to the inhibition of glucocorticoid-induced thymocyte apoptosis (16Zhao Y. Iwata M. Int. Immunol. 1995; 7: 1387-1396Crossref PubMed Scopus (22) Google Scholar, 18Zhao Y. Tozawa Y. Iseki R. Mukai M. Iwata M. J. Immunol. 1995; 154: 6346-6354PubMed Google Scholar) and is essential for positive selection (16Zhao Y. Iwata M. Int. Immunol. 1995; 7: 1387-1396Crossref PubMed Scopus (22) Google Scholar, 31Anderson G. Anderson K.L. Conroy L.A. Hallam T.J. Moore N.C. Owen J.J.T. Jenkinson E.J. J. Immunol. 1995; 154: 3636-3643PubMed Google Scholar,32Wang C.-R. Hashimoto K. Kubo S. Yokochi T. Kubo M. Suzuki M. Suzuki K. Tada T. Nakayama T. J. Exp. Med. 1995; 181: 927-941Crossref PubMed Scopus (93) Google Scholar). Thus, we examined if calcineurin activation also contributes to the inhibition of IDB- or TTX-induced apoptosis in thymocytes. Ionomycin alone induced DNA fragmentation in thymocytes in a dose-dependent fashion but inhibited IDB-induced DNA fragmentation at concentrations within a narrow range (0.2–0.3 μg/ml) (Fig. 7 A). Thus, ionomycin at these concentrations and IDB were mutually antagonistic in the induction of apoptosis. Similar results were obtained with TTX-induced DNA fragmentation (data not shown). The inhibition of apoptosis was canceled by FK506 but not by rapamycin (Fig. 7 B). Although the structurally related immunosuppressive macrolides FK506 and rapamycin commonly bind to FKBP-12, the FK506·FKBP-12 complex but not the rapamycin·FKBP-12 complex inhibits the phosphatase activity of calcineurin (33Lui J. Farmer J.D. Lane W.S. Friedman J. Weissman I. Schreiber S. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3634) Google Scholar). The result thus suggests that calcineurin activation is essential for the antiapoptotic effect. Glucocorticoid-induced DNA fragmentation in thymocytes was not inhibited by ionomycin alone, but it was inhibited by 0.2–0.3 μg/ml ionomycin in the presence of 0.3 ng/ml TTX (Fig. 7 C) (2Ohoka Y. Kuwata T. Asada A. Zhao Y. Mukai M. Iwata M. J. Immunol." @default.
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