HL60 and EL4 cells incubated with tumor necrosis factor-a (TNF-a) plus staurosporin, a potent inhibitor of protein kinases, showed at least S-fold increased.
THE JOURNAL. OP BIOLOGICAL. CHEMISTRY 0 1992 by The American Society for Biochemistry
Vol. 267, No. 3, Issue of January 25, pp. 2065-2072,1992 Printed in U.S.A.
and Molecular Biology, Inc.
Protein Kinases Negatively Affect Nuclear Factor-& Activation by Tumor Necrosis Factor-a! at Two Different Stages in Promyelocytic HL60 Cells* (Received
Hans-Peter Hohmann@, Roland Remy$$, Ludwig Adolphus P. G. M. van Loon@ll From the $Pharmaceutical F. Hoffmann-La Roche
Ltd.,
Research Department, New CH-4002 Base& Switzerland
Aignere, Manfred
Technologies,
HL60 and EL4 cells incubated with tumor necrosis factor-a (TNF-a) plus staurosporin, a potent inhibitor of protein kinases, showed at least S-fold increased levels of nuclear factor-rB (NF-KB) activity compared with TNF-a alone both during rapid NF-KB activation from the cytosolic pool and protein synthesis-dependent NF-KB activation. NF-KB activation by phorbol 12myristate 13-acetate (PMA) and interleukin- 1 was inhibited by staurosporin. Staurosporin treatment hardly affected the TNF-a-induced increase in mRNA for the ~61 subunit of NF-KB but interfered with any phorbol ester (PMA)-induced increase in ~61 mRNA. Thus, induction of NF-KB and p51 mRNA by TNF-a was not mediated by a staurosporin-sensitive factor, but NF-KB activation by TNF-a was even reduced by action of a staurosporin-sensitive factor. Decreased levels of phosphorylation of TNF-Ra (TNF receptor type a) after staurosporin-treatment correlated with increased induction of NF-KB by TNF-a. Staurosporintreatment did not affect TNF-R levels. Although protein kinase C stimulation by PMA inhibited NF-KB activation by TNF-a, its action mechanism may be different from that of the staurosporin-sensitive factor. PMA induced disappearance of TNF-Ra by shedding into the surrounding medium, with kinetics similar to those of its inhibition of NF-KB activation by TNF-a. Phosphorylation may not mediate receptor shedding, since PMA treatment did not detectably affect TNF-Ra phosphorylation.
Transcription factor NF-KB~ (for reviews, see Refs. l-3) is present in the cytosol of most cell types in an inactive form bound to an inhibitor protein IKB (4, 5). However, in some cell types, such as mature B-lymphocytes (6), monocytes, and macrophages (7), NF-KB is constitutively active. Active NFKB consists of two different subunits: a DNA-binding protein of 51 kDa (~51) associated with a 65-kDa protein (~65) (8* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: Bldg. 64-43, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland. Tel.: 41-61688-1027; Fax: 41-61-688-1645. 1 The abbreviations used are: NF-KB, nuclear factor-KB; PMA, phorbol 12-myristate 13-acetate; TNF, tumor necrosis factor; TNFR, TNF receptor; IL-l, interleukin-1; EMSA, electrophoretic mobility shift assays; PBS, phosphate-buffered saline; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.
and 3 Vitamin
for publication,
BrockhausS,
Research,
June
28, 1991)
and
Biotechnology,
10). The gene encoding p51 consists of an open reading frame for a 105- to 107-kDa protein of which the amino-terminal half represents ~51. In this part, regions with high homology to the v-rel oncogene, its cellular homolog c-rel, and the Drosophila maternal morphogen dorsal were identified. P51 was identical with the constitutive transcription factor KBFl, which binds to DNA sequences similar to those that NF-KB binds to, although with different affinity. The exact function of the carboxyl-terminal part of the open reading frame is as yet unknown. Interestingly, the full-sized 105- to 107-kDa protein is inactive in DNA binding, suggesting that the carboxyl-terminal half inhibits the DNA binding by p51 (10-13). Recently, another protein with characteristics very similar to the p105/p51 protein was described. This protein is synthesized as a lOO-kDa precursor, is cleaved to a 50- or 49-kDa protein, and can also associate with p65 (14). The gene for p65 was also isolated and its DNA sequence showed p65 to belong to the rel family. P65 has weak DNA binding activity and interacts with IKB (9, 15, 16). In addition, several differently sized proteins were found as components of active NFKB (17), all of which may belong to the rel family of proteins. Inactive NF-KB can be activated posttranslationally in uivo via at least two different pathways (18-20). Activation requires dissociation from IKB and nuclear translocation of the active transcription factor (5). One pathway involves protein kinase C, since stimulation of cells with bacterial lipopolysaccharide or phorbol esters, such as phorbol 12-myristate 13acetate (PMA), all of which activate protein kinase C (2123), leads to NF-KB activation. TNF-(U and TNF-/3 activate NF-KB via a protein kinase C-independent pathway (19, 20). Activation of NF-KB by interleukin-1 (IL-l) may involve CAMP and, thus, protein kinase A at least in some (24-27), but not in all, cell types (28,29). NF-KB activity is also induced by double-stranded RNA (30), but at present, it is unclear whether or not this represents another, independent mechanism of NF-KB activation. Zabel and Baeuerle (31) characterized two different IKB molecules and suggested that the occurrence of distinct forms of IKB could explain NF-KB activation by different intracellular messenger systems. A third inhibitor protein, pp40, was described very recently (32). We analyzed NF-KB activation by TNF-a. Two different TNF receptors (TNF-Ra and TNF-R/I, which are also referred to as type A and type B TNF receptors) exist, which have slightly different affinities for TNF-a (33, 34) or TNF/3 (35). Cells contain either TNF-Ra! and TNF-R/3 in various ratios (HL60 or SW480T cells) or only TNF-Rfl (HEp2 or human umbilical vein endothelial cells) (33, 36). The degree of binding of TNF-a! and TNF-@ to both TNF receptors correlated well with the amount of activation of NF-KB.
2065
2066
Protein Kinases Negatively
Affect NF-KB Activation
Maximal levels of active NF-KBwere seen upon binding of TNF-a toless than a few hundred TNF receptors, representing maximally 20-25% of all TNF receptors on HL60 and HEp2 cells. Activation of NF-KBby TNF-a was a very rapid process and was maximal 2-4 min after addition of TNF-a. Both receptors mediated activation of NF-KBby TNF-a and TNF-P (35, 36). Two stages of NF-KB activationby TNF-a could be distinguished. The first stage leads to rapid NF-KB activation, is protein synthesis-independent, and thus, may involve activation of NF-KBfrom the inactive complex with IKB.Active NF-KBdisappears rapidly. Maintenance of NFKBactivity required continuous stimulation with TNF-a and de n o m protein synthesis, possibly since the pool of active NF-KBwas rapidly exhausted (20). In addition, the level of the mRNA encoding the DNA-binding subunit of NF-KBwas also increased after cell stimulation with TNF-a (10). Cell-specific differences in NF-KBinduction by the protein kinase C activator PMA were observed. In 70Z/3 cells (a mouse pre-B-lymphocytic cell line), PMA rapidly induced NFKB activity in a protein synthesis-independent fashion. In contrast, in HL60 cells (a human promyeloid cell line) activation of NF-KBby PMA was slowand completely dependent on ongoing protein synthesis, resembling the second stage of TNF-a-induced NF-KBactivation (20). Thus, it seems that the PMA-induced protein kinase C can mobilize the preformed NF-KBprecursor pool in 702/3 cells, but not inHL60 cells. Although TNF-a activates NF-KBin a protein kinase Cindependent fashion (19), TNF-a also induces protein kinase C activity in certain cell types (37). Several groups showed that activation of protein kinase C by phorbol estersor lipopolysaccharide led to down-modulation and total disappearance of all TNF receptors in many but not all cells (3843). Receptor down-modulation can result from shedding of part of the receptor protein into the surrounding medium (44). Protein kinase C, therefore, plays a role in TNF-ainduced signal transduction. In this paper, we show that protein kinase C and another staurosporin-sensitive factor, possibly another kinase, affect NF-KBactivation by TNF-a attwo different stages. Inhibition of protein kinases enhanced NF-KB activation by TNF-a. This effect correlated with reduced TNF-Ra phosphorylation. Protein kinase C affected NF-KBactivation by receptor downmodulation resulting from receptor shedding by a mechanism not involving a change in theamount of TNF-Ra phosphorylation. MATERIALS ANDMETHODS
Reagents-The stock solutions usedwere staurosporin(Fluka Chemical Corp., 2.5 mM in dimethyl sulfoxide) and PMA (Fluka, 2 mM in dimethyl sulfoxide). Recombinant human TNF-a andhuman IL-lP were obtained from Dr. E. Hochuli (F. Hoffmann-La Roche Ltd., Basel) and Dr. P. Lomedico (F. Hoffmann-La Roche Ltd., Nutley), respectively. Monoclonal antibodies against human TNFR a are described elsewhere (34). Cell Culture-The human promyelocytic cell line HL60 and the mouse pre-B and lymphoma cell lines 702/3 and EL4 were obtained from the ATCC and maintained over many passages in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum or 10% (v/v) horse serum. 70Z/3 cells were cultivated in the presence of 50 nM mercaptoethanol (Fluka). SW480T cells, which are from an epithelial cell line SW480 and express about 10 times more TNF-R on their cell surfaces, as compared with HL60 cells, were kindly provided by Dr. T. Espevik, University of Trondheim. These cells were grown as described above. Allcells were used after reaching near confluence or a density of 1-1.5 X lo6 cells/ml of culture medium. Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSA)-Nuclear extracts were prepared as described before (35, 52). EMSA were performed as described by Sen and Baltimore
by TNF-a
(53). 5,000 cpm of the 32Pend-labeled DdeIIHaeIII fragment of the K-light chain enhancer containing the NF-KB-binding site were used per assay. A restriction fragment mutated in the NF-KB-binding site, but otherwise identical with the wild-type fragment (54), was used as a negative control. In all cases, the DNA/NF-KBcomplexes wereonly observed using the wild-type, but not the mutant, oligonucleotide. RNA (Northern) Blot Analysis-Northern blot analysis was performed essentially as described (10). The 1-kilobase EcoRI fragment of clone 3, which encodes a part of the DNA-binding domain of the 105-kDa protein, was used as a probe. The filter was rehybridized with a mouse /3-actin DNA probe under identical hybridization conditions afterremoval of the first probe by heating in a low salt buffer. Detection of TNF-R on Whole Cells and in Culture Supernatants UsingMonoclonalAntibodies and Radiolobeled TNF-a"SW480T cells were grown to near confluence in 24-well Petri dishes (1.6-cm diameter). In individual wells, culture media werereplaced at various times by 0.3 ml of fresh prewarmed medium supplemented with 40 nM PMA/well, incubation was continued at 37 "C, and the entire Petri dish was then rapidly cooled. Culture media were collected and centrifuged for 10 min at 70,000 X rpm in a Beckman TLlOO tabletop ultracentrifuge. 45-pl aliquots of culture media were analyzed for the presence of soluble TNF receptors using a sandwich assay. Purified monoclonal antibody Ut& which is specific for TNF-Ra, was coated to thewells of a microtiter plate, incubation with culture supernatants was performed, and radiolabeled TNF-a was then used to detect any bound TNF receptor molecules. Experimental details are described elsewhere.' For determination of the numbers of TNF receptors present at the cell surface, PMA-treated SW480T and HL60 cells were either incubated for 2 h at 0 "C with 3 nM radiolabeled TNFa as described previously (33, 35) or with 5 pg of purified monoclonal antibodies specific for TNF-Ra (utrl andutr4, both of which recognize structural epitopes of TNF-Ra (34)) per ml of phosphate-buffered saline (PBS). Unbound antibodies were removed together with the incubation medium and cells were washed and then incubated with 1 X lo6 cpm of radiolabeled sheep anti-mouse IgG (Amersham Corp.)/ml of PBS for 2 h and washed again. The amount of radiolabeled anti-mouse IgG bound to cells was analyzed in a y-counter. The amount of specific binding was calculated from the difference of radiolabeled second antibody bound to cells, which were preincubated with or without the mixture of utrl and utr4antibodies. Receptor Phosphorylation.-After growing nearly to confluence in six-well Petri dishes (3.5-cm diameter, about 2.5 X lo6 cells/well), SW480T cells were washed once with a buffer containing 20mM HEPES-NaOH (pH7.4), 1mM MgCl,, 5 mM KC1,120 mM NaC1, and 5 mM glucose. Cells werethen incubated for 1 h at 37 "C in the same buffer containing 0.5 mCi of 32P-labeledinorganic phosphate (Amersham Corp.)/ml. Where indicated, PMA was added to 40nM, and incubation was continued for 15 min at 37 "C. The medium was removed, and 1 ml of ice-cold PBS containing 10 pgof purified monoclonal antibodies utrl and utr4was added to each well. After 2 h on ice, the antibody solution was removed. Cells were lysed and extracted with 1 ml of RIPA buffer (10 mM Tris-HC1 (pH 7.5), 150 mM NaCl, 1%Nonidet P-40 (v/v), 1%deoxycholate, 0.1% SDS, supplemented with 10 mM benzamidine HCl (Fluka) and 1 mM phenylmethylsulfonyl fluoride (Fluka)), undissolved material was removed by centrifugation for 10 min at 70,000 X rpm in a Beckman TLlOO tabletop ultracentrifuge, and immunocomplexeswere collected from the detergent extracts using 50 p1 of Sepharose beads with coupled protein G (Pharmacia LKB Biotechnology Inc.). Beads were extensively washed with RIPA buffer, followed bywashing with PBS. Pellets were dissolved in 100 pl of Laemmli sample buffer. Similarly, immunoprecipitates were prepared from 1 X 10' unlabeled SW480T cells. Immunoprecipitates from labeled cells were subjected to SDSPAGE and analyzed by autoradiography. Immunoprecipitates from unlabeled cells were also subjected to SDS-PAGE (1 X 108 cell equivalents/lane) and transferred toan Immobilon-P membrane (Millipore Corp.). The membranes were incubated in 1%milk powder in 50 mM Tris-HC1 (pH 7.4), 140 mM NaC1,5 mM EDTA, and 0.02% sodium azide (34), followed by 2 h of incubation with 5 pg/ml utrl plus 5 pg/ml Ut14. After washing, membranes were incubated for 2 h with 1 X IO6 cpm of radiolabeled sheep anti-mouse IgG (Amersham Corp.)/ml and were washed again using milk powder-containing buffer.
' W. Digel, F.Porzsolt, W. Lesslauer, and M. Brockhaus, submitted for publication.
Protein Kinases Negatively Affect NF-KBActivation by TNF-a RESULTS
1
@
The Protein Kinase Inhibitor Staurosporin Did Not Interfere with Protein Synthesis-dependent Activation of NF-KBAfter a transient phaseof protein synthesis-independent NFK Bactivation, which only lasts for 1-2 h when excess TNF-a is present, NF-KB activation HL60 in cells by TNF-a depends on de novo protein synthesis (20). PMA-induced NF-KB activation in HL60 cells showed characteristics similar only to this second stage of NF-KB activation (20). T o determine whether protein kinase C is involved in the second stage of NF-KB activationby TNF-a, staurosporin, a potent protein kinase inhibitor (45), was added to H L 6 0 cells 1 h prior to a 4-h incubation with TNF-a or PMA (Fig. 1).NF-KB activity was measured in nuclear extractsby EMSA. After incubation with TNF-a, high NF-KB activity was found in the staurosporin-treated cells (Fig. 1,lane 5 ) ,whereas PMA-induced NFKB activationwas totally abolished (Fig. 1, lane 6 ) .
2
none
3
2067 4
TNFa
5
6
PMA
P51
p actin
- +Staurosporin- + - + -
Preincubation with Staurosporin HardlyAffected the TNFa-induced Increase in the Level of mRNA Encoding the p51 Subunit of NF-KB(p51) but Abolished the Increase Inducedby
PMA-The level of mRNA encoding p51 increased 4-fold, compared with the level of actin mRNA, after treatment of H L 6 0 cells with TNF-a for 3 h and 3.5-fold afterPMA treatment (Fig. 2, A, lanes 1,3,5, and B ) . The PMA-induced increase in mRNA levels was completely abolished by staurosporin, as expected, whereas the TNF-a-induced increase was hardlyaffected (Fig. 2A, lanes 2, 4, 6, and B ) . These results, together with the results from the gel shift assays documented in Fig. 1, indicated that maintenance of NF-KB activity inHL60 cells after TNF-a-stimulation did not depend + + + on staurosporin-sensitive protein kinases. Furthermore, inStimulationwith FIG.2. Staurosporin treatment hardly affected theTNF-a- Wildtype - - Mutant induced increase in levels of p51 mRNA but abolished the 1 2 3 4 5 6 7 8 9 101112 increase induced by PMA treatment. HL60 cells were preincuSTAUR. - - - + + + --- +++ bated for 1 h with 250 nM staurosporin (stauro),and 10 nM TNF-n
NF-KE
or 40 nM PMA were added where indicated. After 3 h, cells were collected, poly(A+) RNA was isolated, and the mRNAs encoding the p51 subunit of NF-KBor @-actinwere visualized by Northern analysis. Hybridization results shown in panel A were quantitated using a Phospho-imager (Applied Biosystems) (panel E).The amount of p51 mRNA in each lane was expressed as -fold induction compared with determined levels of @-actin mRNA, whichwere assumed to be unaffected by the different stimuli applied to the cells.
creased levels of mRNA encoding the p51 NF-KB subunit after incubation with TNF-a or PMA could be obtained by different mechanisms, which were distinguished by their requirement for proteinkinases. Inhibition of Protein Kinase Activity by Staurosporin Increased the Amount of NF-KBActivated by TNF-a but Completely Inhibited NF-KBActivation by IL-1 or PMA-HL6O
cells were incubated with staurosporin for 1 h followed by 1 h of incubation with TNF-a. During thatperiod, active NFKB is mainly derived from thecytosolic NF-KBprecursor pool (20). Staurosporin pretreatment of the cells increased the efficacy of NF-KB activation by TNF-a (Fig. 3A; compare lanes 1-4 with lanes 6-9 and Fig. 3B),andas expected, FIG. 1. The protein kinase inhibitor staurosporin did not interfere with protein synthesis-dependentNF-KBactivation. inhibited activation of NF-KBby PMA (Fig. 3A, lanes 5 and Human promyelocytic HL60 cells were preincubated for 60 min a t 10). Two-fold increased amountsof active NF-KBwere found 37 “C in the absence or presence of250nM staurosporin (STAUR.). with saturating concentrationsof TNF-a and concentrations Then 10 nM TNF-n or 40 nM PMA were added, and incubation was below 4 PM (Fig. 3 B ) when the TNF-a concentrationis ratecontinued for 4 h. NF-KBactivity was measured in nuclear extracts limiting for NF-KB activation (35). In the mouse lymphoma by EMSA using an oligonucleotide containing the wild-type binding cell line EL4, similar amounts of NF-KBwere activated after site for NF-KBfrom the K light chain (Wildtype) or, as control, a mutated binding site that does not bind NF-KB(Mutant).Nuclear incubation with TNF-a or staurosporin. When both stimuli extracts obtained from equal aliquots of cells were used in each lane. were applied together, a synergistic effecton NF-KB activation The position of the complex between DNA and NF-KB (NF-KB)is was observed (Fig. 3C). In the mouse pre-B cell line 702/3, indicated. NF-KB activationby PMA and IL-lPwas inhibited by stau-
Protein Kinases Negatively
2068 69
HL60
-
Wildtype 1 2 3 4 5 6 7 STAUR.-----
8 910
+++++
- Mulant
Affect NF-KBActivation by TNF-CY
-
-1 000
11131124
- +- +
h
!!?
-800 a) a NF-
-600
a .c
*-
E, 0
v
staurosporin
FIG. 4. Staurosporin treatment did not increase the number of TNF receptorsat the cell surface. HL60 cells were incubated for 60 min at 37 "C in the absence (-) or presence (+) of250 nM staurosporin. Then each culture was divided into two parts. One part was used to determine the number of TNF-binding sites using radiolabeled TNF-a, as described previously (33). Solid bars indicate specific TNF-a binding after a 2-h incubation with or without staurosporin treatment. Unspecific binding, determined by binding of radiolabeled TNF-a in the presence of excess unlabeled TNF-a, was 150 cpm and was subtracted from total binding. To the second part, 10 nM unlabeled TNF-a was added, incubation was continued for one additional hour, nuclear extracts were prepared, and activated NFKBwas visualized by EMSA. Radioactivity present in the specifically retarded NF-KB/DNAcomplex was quantified by Cerenkov counting (stippled bars).
Q - P 2
0
@ -
6 8 10 TNFa-concentration (pM)
EL4
- Wildtype 1
STAUR. VNFa]
4
2
3
- - + - f -
100
Mulant 5 6
4
+
+
-
+
- +
-702/3 -Wildtype - Mutant 1 2 34 5 6 7 89 1 0 -STAUR.+-+-+-+-
@
NF-rB-
FIG. 3. Inhibition of protein kinases by staurosporin enhanced TNF-a-induced NF-KB activation, but inhibited NFKB activation by PMA a n d IL-1. Human promyelocytic HL60 (panels A and B ) , mouse lymphoma EL4 (panel C), or mouse pre-B-
rosporin pretreatment (Fig. 30, lunes 3-6). TNF-a did not activate NF-KBin 70Z/3 cells (Fig. 30, lanes 7 and 8), which lack any detectable TNF receptors (not shown). Thus, NFKBactivation from the pre-existing precursor pool by TNF-a in HL60 or EL4 cells, in contrast to activation by PMA in HL60 cells or PMA or IL-lPin 70Z/3 cells, was not mediated by a staurosporin-sensitive protein kinase. In contrast, upon action of a staurosporin-sensitive factor, most likely a protein kinase, the efficacy of TNF-a-induced NF-KBactivation was reduced in HL60 and EL4cells. Pretreatment of CelLs with Staurosporin Did Not Affect the Number of TNF-a Receptors but Drastically Reduced the Degree of Constitutive Phosphorylation of TNF-Ra-To gain more insight into the mechanism of action of the staurosporin-sensitive factor (perhaps a protein kinase), that interferes with TNF-a-induced NF-KBactivation, the number of TNF-R on the surface of staurosporin-treated HL60 cells was compared with that of untreated cells. No difference in TNFR number was found, whereas the efficacy of TNF-a-induced NF-KBactivation was 2-fold increased in staurosporin-treated cells (Fig. 4). Phosphorylation of TNF-Ra was analyzed. For practical reasons, SW480T cells were used, which contain lymphocytic 70Z/3 cells (panel D )were preincubated for 60 min at 37 "C in the absence or presence of 250 nM staurosporin (STAUR.). Then TNF-a or PMA were added to the indicated concentrations (panels A and B ) or to 10 nM TNF-a, 40 nM PMA, or 5 ng of &la/ ml (panels C and D),and incubation was continued for 60min (panels A, C, and D) or 10 min (panel B ) . Nuclear extracts from equal aliquots of each cell sample were prepared, and NF-KBactivity was measured by EMSA. The position of the complex between DNA and NF-KB( N F - K B )is indicated. For quantitation, parts of the gel containing the NF-KB/DNAcomplex were cut out, and radioactivity was measured by Cerenkov counting (panel B ) . In panel C, radioactivity in the position of the specifically retarded complex was determined by densitometry using properly exposed films. arbi.units, arbitrary units.
Protein K i m e s Negatively Affect NF-KBActivation by TNF-cu about 10 times more TNF receptors, mainly TNF-Ra, than HL60 cells (36). TNF-a also activates NF-KB inSW480T cells (36). SW480T cells were pretreated for 1 hwith or without staurosporin followed by incubation with "P-labeled inorganic phosphate. Intact cells were then incubated at 0 "C with the monoclonal antibodies utrl plus utr4, which are specific for the TNF-Ra, excess unbound antibodies were removed, and immunocomplexes were collected from detergent extracts. This procedure, which specifically immunoprecipitated TNF-Ra (see Fig. 8), should allow immunoprecipitation only of those receptor molecules that are exposed to the outside of the cell. Staurosporin pretreatment drastically reduced the amount of phosphorylation of TNF-Ra (Fig. 5), whereas staurosporin treatment also did not affect the number of TNF receptors at thecell surface of SW480T cells (Fig. 5), as shown above for HL60 cells. We assume that constitutive phosphorylation of TNF-Ra by staurosporin-sensitive kinases might also occur in HL60 cells, but we were unable to directly confirm this, possibly due to the much lower level of receptor expression in HL60 cells. Thus, enhanced efficacy of TNF-a-induced NF-KBactivation in HL60 cells correlated with inhibitionof constitutive TNF-Ra phosphorylation, suggesting, but not proving, that TNF-a signal transduction is negatively regulated through phosphorylation of TNF receptors. The Kinetics of Inhibition by PMA of TNF-a-induced NFKBActivation Is Similar to the Kinetics of Down-modulation of TNF Receptors-Which protein kinase could be involved as a negative factor in NF-KBactivation by TNF-a? Activation of protein kinase C by PMA leads to down-modulation of TNF receptors (see the Introduction) but could theoretically also affect other stages of signal transduction. PMA-
'
-200 -1 16
- 97 TNF-Rad
I)
induced effects on binding of TNF-a to intact cells were directly compared with effects on NF-KBactivation by TNFa. Although both PMA and TNF-a are activators of NF-KB, analysis of NF-KBactivity induced by TNF-a is possible in PMA-treated HL60 cells, because NF-KB activationby PMA is significantly slower and leads to lower levels of NF-KB activity (20). Cells were incubated for various times with PMA, and TNF-a was then added during the last 10 min of incubation. A 50% reduction in the number of TNF-R was found within 15 min of incubation with PMA, whereas no detectable TNF-R were found after 1 h of PMA incubation (Fig. 6A). The highest levels of active NF-KBwere found in cells stimulated with TNF-a (Fig. 6B). Preincubation of cells with PMA for longer than about 30 min reduced the amount of active NF-KBinduced by TNF-a. The final levels of active NF-KB after more than 1h of treatment with PMA plus TNFa was similar to thatobserved with PMA only (Fig. 623). The amount of NF-KBactivated by TNF-a (Fig. 6B, dotted line) during incubations with PMAplus TNF-a may be calculated by subtracting the amount activated by PMA from the amount observed with PMA plus TNF-a, assuming both systems are independent. PMA-induced reduction of NF-KBactivation by TNF-a showed kinetics similar to those of receptor downmodulation, although a time delay for effects on NF-KBactivation was observed. Down-modulation of TNF-Ra by PMA Resulted in Receptor Shedding into the Culture Medium-TNF-R levels at thecell surface of SW480T cells were measured after protein kinase C activation by PMA,using TNF-a (Fig. 7A, squares) or TNF-Ra-specific monoclonal antibodies utrl plus utr4 (Fig. 7A, triangles). Very similar resultswere obtained, since TNFRP, which reacts with TNF-a but not with the antibodies, forms only a very minor fraction of all TNF-R of SW480T cells (36). TNF receptors disappeared from the cell surface of SW480T cells upon PMA stimulation with kinetics similar to those observed with HL60 cells (compare Fig. 7A with Fig. 6A). Disappearance of TNF-Ra from the cell surface after PMA treatment (Fig. 7B, closed squares) was related to the simultaneous appearanceof TNF-Ra protein in thesurround-
- 66
I I ,
"1
@ 1200
1
100
- 42
5 - 31 kDa
spec. TNF-a bdg. (CPW % f,
2069
,f0
-
,,f +
staurosporin FIG.5. The degree of phosphorylation of TNF-Ra in SW480T cells is drastically decreased upon incubation with staurosporin. SW480T cells were incubated a t 37 "C for 1 h without (-) or with (+) 250 nM staurosporin followed by a 1-h incubation with 'jP-labeled inorganic phosphate. Cells were incubated with the monoclonal antibodies utrl plus utr4specific for TNF-Ra. Detergent extracts were preparedafter removal of unbound antibodies and immunocomplexes were collected, subjected to SDS-PAGE, and analyzed by autoradiography.2-Mercaptoethanol was presentduring sample preparation. The position of TNF-Ra is indicated on theleft; positions of molecular mass markers (in kDa) are indicated on the right. Theamount of radiolabeled TNF-a specifically boundto SW480T cells (spec. TNF-a bdg.), indicated as cpm at thebottom of the figure, was determined using parallel cultures, which were incubated with 250 nM staurosporin for 2 h or left untreated. Unspecific binding was less than 5% of total binding.
$
1000
60
40
d 20
0
30 60 90 Treatment PMA
120 150 180 (m~n)
70 100 130 160 190 Total incubam hmo (man)
10 40
FIG. 6. PMA inhibited NF-KB activation by TNF-a with kinetics similar to the PMA-induced disappearance of TNFR from the cellsurface. Panel A, HL60 cells were incubated in the presence of 40 nM PMA, samples were taken after the indicated times, and the numberof TNF-R was determined by incubation with radioiodinated TNF-a. 100% binding corresponded to 2650 cpm of '2sI-TNF-a bound to 1 X loficells. Unspecific binding was less than 7% of total binding. Panel B, cells were incubated without orwith 40 nM PMA followed, where indicated, by 10 min of incubation with TNF-a and rapidly cooled in ice water, and nuclear extracts from identical cell aliquots were prepared. NF-KBactivity was visualized by EMSA and quantitated using a Phospho-imager (solid lines).To determine theeffect of PMA treatment on NF-KB activation by TNFa, NF-KB activity in cells treated with PMA alone was subtracted from the NF-KB activity of cells,which were treated with PMA followed by T N F a (dashed line).
Protein Kinases Negatively Affect
2070
NF-KBActivation by TNF-CY 1
2
3
4
J1
5
6
.
7
.=
8.?00
1I16
97
+
66
42
0
Incubation time (min)
