Jan A. M. RaaijmakersO, Judith P. Johnson**, and Paul T. van der SaagS Sf. From the ...... Nakabeppu, Y., Ryder, K., and Nathans, D. (1988) Cell 55, 907-925.
Vol. 269, No. 8 , Issue of February 25, pp. 6185-6192, 1994 Printed in U.S.A.
THE JOURNAL of BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc
12-O-Tetradecanoylphorbol-13-acetateand Tumor Necrosis Factor a-mediated Inductionof Intercellular Adhesion Molecule-1Is Inhibited by Dexamethasone FUNCTIONAL ANALYSIS OF THE HUMAN INTERCELLULAR ADHESION MOLECULE-1 PROMOTER* (Received for publication, June 14, 1993, and in revised form, November 9, 1993)
Anja van de StolpeSsnll, Eric CaldenhovenSsII, BarbaraG. Stade**,Leo Koendermans, Jan A. M. RaaijmakersO, Judith P. Johnson**, and Paul T. van der SaagS Sf From the Wubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands, the §Department of Pulmonary Diseases, University Hospital Utrecht, Utrecht, The Netherlands, and the **Znstitute of Immunology, University of Munich, Munich, Germany
(1,4). IncreasedICAM-1 expression may playa role in a variety 'Ikanscription regulation of the human intercellular adhesion molecule-1 gene by the phorbol ester 12-O-tet-of diseases characterized by inflammation; among them is alradecanoylphorbol-13-acetate (TPA), tumor necrosis lergic asthma ( 5 ) . factor a (TNFa),and the glucocorticoid dexamethasone Despite widespread useof glucocorticoids in the treatment of was studied using transient transfections in 293 cells diseases characterizedby inflammation, themolecular mechawith intercellular adhesion molecule-1 promoter-lucif- nism of action of the glucocorticoids remains largely unknown. erase constructs (together with a glucocorticoid recepIn a previous paper we reported that, while the protein kinase tor expression vector). TPA and TNFainduced promoter C-activating phorbol ester TPA induced, the synthetic corticoactivity, which was repressedby dexamethasone. Four steroid dexamethasone repressed ICA"1 protein and mRNA TPA-responsiveDNA regions were identified, each con- expression in both thebronchial epithelial cell line NCI-H292 taining a potentialPA-responsive enhancer sequence: and in themonocytic cell line U937 (6). These results provided 1) -677/-340 an AP3-like sequence; 2) -290/278 a P A a possible mechanism for the anti-inflammatory effect of gluresponse element (TRE); 3) -227/-175 an NFKB-like secocorticoids in general and more specifically in allergic asthma, quence; 4) -105/-38anAP2-like sequence. TNF'a enin which case elevated I C A " 1 levels on bronchial epithelial hanced transcription only through region 3. The TRE in region 2 appeared to be functionally coupled to a distaland inflammatory cells may contribute to destructionof bronTATA box at -313 and differedfrom the consensusTRE chial epithelium, resulting inbronchial hyperreactivity ( 5 ) . Little is known yet about the transcriptional regulation of with respect to binding characteristics for members of the AP1family.The newly identified NFKB enhancer the ICA"1 gene. The 5' region flanking the human ICAM-1 to gene contains many putative regulatory elements in addition (TGGAAATTCC) is boundby a TNFa-induced nuclear two TATA boxes, indicating complex regulation of ICA"1 gene protein and appears to be the key element in rapid transcription induction by TNFa (and TPA), while transac- transcription (7-10). To explore the mechanismby which DEX tivation of this elementis repressed by the ligand-bound repressed ICAM-1 expression, we functionally analyzed the glucocorticoid receptor. We propose a negative crosspromoter region of the human ICA"1 gene. Evidence is proNFKB transcription factor and the glutalk between the vided that severalcis-acting DNA elements in the ICAM-1 prococorticoid receptor. moter region of the human ICAM-1 gene may contribute to TPA-induced transcription. One of these is a newly identified functional NFKB sequence, which also specifically mediates ICAM-1' is constitutively expressedon leukocytes, endothe- rapid inductionof I C A " 1 transcription by TNFa. Remarkably, lial cells, and epithelialcells and can be rapidly up-regulated by the rapid repressive effect of DEX on TPA- and TNFa-induced inflammatory cytokines like TNFa and interleukin 1 and by transcription could also be located to this NFKB element, and lipopolysaccharide (1-3). Interaction between ICA"1 and its we propose the existence of a novel type of negative cross-talk receptor LFA-1 on leukocytes plays a costimulatory role in T- between the NFKB transcription factor family and the glucocell activation and the generation of an inflammatory response corticoid receptor, which may explain the repressive effect of dexamethasone on ICA"1 expression. The induction of ICA"1 transcription by TNFa and repressionby the glucocor* This study was supported by a research grant from Glaxo. Thecosts ticoid receptor may allow the cell to rapidly modify ICA"1 of publication of this article were defrayed in part by the payment of protein expression, which may be essential for its modulatory page charges. This article must therefore be hereby marked"uduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this role in inflammatory processes. fact. 11Present address: Dept. of Hematology, University Hospital NijmeMATERIALS AND METHODS gen (St. Radboud), Nijmegen, The Netherlands. Special Reugents-TPA andDEXwere from Sigma. Recombinant 11 Both authors contributed equally. $$ To whom correspondence should be addressed: Hubrecht Labora- hTNFa (IO4 IU/ml) was obtained from Genzyme (Cambridge,M A ) . Cell Culture-The US37 cell line possesses characteristics of monotory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, cytic cells (11).US37 cells (CRL 1593, American Type Culture Collec3584 CT Utrecht, The Netherlands. The abbreviations used are: ICAM-1, intercellular adhesion mol- tion, Rockville, MD) were cultured in RPMI 1640 medium containing ecule-1; TNFa, tumor necrosis factora; TPA, 12-O-tetradecanoylphor- 10% fetal calf serum. Human 293 embryonal kidney cells (American bol-13-acetate; DEX, dexamethasone; PCR, polymerase chainreaction; Type Culture Collection, CRL 1573) were grown in 1:l mixture of DulEMSA, electrophoretic mobility shift assay; TRE, TPA-responsive ele- becco's modified Eagle's medium and Ham's F-12 medium, buffered ment; bp, base pair(s). with bicarbonate, and containing 7.5% fetal calf serum.
6185
6186
Dunscription Regulation of ICAM-1 B -1352 1
//
N
S
I
I
800
- 1352 1 -1014
600
700
500
A H
S
I
I 200
300
400
S P 1 1 100
N
A 55
1
CAM- 1
0
// //
I
LC-1014
IUC -677
FIG.1.The 5' promoter region ofthe human I C A " 1 gene. Schematic representation of the 5' region of the ICA"1 gene and of the promoter-luciferase constructs used inthis study. This figure shows the restriction sites used for cloning subfragments of the promoter region upstream of a promoterless firefly luciferase cDNA in the PXP2 vector (see "Materials and Methods"). Restriction sites are indicated a s follows: A, AccI; B,BamHI; H, HindIII; N, NheI; P , PstI; S , SmaI; Ss, SstI.
I
4 -339 1
{
IUC
I
LC-677
IUC
1
LC-339
IUC
- 1 7 4 [ -L
-135
I
LC-290-6
IUC
16-277
IUC
I
IUC
PIC-135
-34
I
//
- 1352 I
N
I
N N
-1352
~
1352
LC-34
IUC
I
I
q r l I
I
-277
4 -135
1
4
DIC- 1352
A
DIC-1352
IUC
TRE
PIC- 1352 3. AP2
[yA
d r l
-277
PIC174
TAlA
PIC-277
A
AP2
dC-277
A
W A
471
D C - 135
A AP2
Dansfection Plasmids-Fig. 1 shows relevant restriction sites in the fragment; 3 x NFKB(p1C)tkluccontains three copies of the 16-bp frag5' region of the human ICA"1 gene and the ICA"1 promoter-lucif- ment (as well as 5' one copy of the 38-bp fragment -227/-190). erase constructs,which were used for this study. Nucleotide numbering All constructs were sequenced. Because 293 cells do not contain enstart site TGA, where Gis + l . Constructs dogenous glucocorticoid r e ~ e p t o r , ~ GRGR is relative to the transcription t h e expression vector containPIC-1352, PIC-1014, PIC-677, PIC-339, and PIC-174 were prepared by ing the glucocorticoid receptor was cotransfected (14). To control for polymerase chain reaction (PCR)from the 2032-bp EcoRI genomic frag- differences in transfection efficiency, in all experiments the PDM-P ment described earlier (7). A 3' primer with an internal SstI site was galactosidase (LacZ) vector (15) was cotransfected. Dansient Dansfection and Luciferase Assay-293 Human fetal kidThe 5' primers conused, 5'-ACTCTGAGTAGCAGAGGAGCTC-3'. tained anadditional BamHI siteso that constructs could be cloned with ney cells were grown in 6-well plates (Costar). Supercoiled plasmid BamHIISstI into the promoterless pXP2 luciferase vector, which was a DNA was transfected by the calcium-phosphate precipitation method kind gift of Dr.Steven Nordeen (Universityof Colorado Health Sciences (16). Cells were transfected with a total of 10 pgof DNA, consisting of Center, Denver CO) (12). To create constructsPIC-1352, PIC-1014, PIC- a mixture of 2 pg of luciferase reporter plasmid, 1 pg of GRGR plasmid, 1pg of PDM-LacZ plasmid, adjusted upto 10 pg with pUCl8.Following 677, PIC-339, and PIC-174, the following 5' primers were used, respec16-20 h of exposure t o the calcium-phosphate precipitate, medium was PCR2, 5"cgggatctively: PCR1, 5'-cgggatccGAATTCAGAACTCC-3'; cCTCATAT"AGTGC-3'; PCR3, 5'-cgggatccGAGAGAGGACGCCG-3'; refreshed, and cells were incubated for 8 or 16 h with 16rn TPA, 1p~ PCR4, 5'-cgggatccGTTGGCAGTATTTA-3';PCM, 5'-cgggatccAGCG- DEX, or 250 units/ml TNFa. Transfected cells were subsequently harvested for luciferase assay (17) and LacZ determination (18).Luciferase GCCAGCGAGG-3'.' activity was normalized for variations in transfection efficiency as deConstruct PIC-339 was used to subclone four other regions of the ICA"1 promoter. PIC-290/-6 was prepared by ligating the AccI frag- termined by LacZ measurement. Electrophoretic Mobility Shift Assays-Nuclear extracts were prement into the pXP2 vector. PIC-277, PIC-135, and PIC-34 were prepared by deleting BamHIIHzndIII, BamHYSmaI, and BarnHIINheI frag- pared from U937 cells according to Lee et al. (19). For EMSA with the ments, respectively, and religating the vector. Internal deletion con- TRE oligonucleotide, nuclear extract was dialyzed against Dignam D buffer (20 m~ HEPES pH 8.2, 100 m~ KC1, 0.2 m~ EDTA, 20% (w/v) structs were made as follows. PIC-1352ATRE was prepared by deleting in the BamHYHindIII fragment from PIC-1352 and inserting a BamHII glycerol); in thecase of the NFKB fragment nuclear extract Dignam AccI fragment from PIC-1352, PIC-1352AAP2 by deleting the BamHV C buffer (20 IIIM HEPES pH 8.2, 1.5 m~ MgC12, 420 m~ NaCI,, 0.2 m~ NheI fragment from PIC-1352 and inserting theBamHIIPstI fragment EDTA) was used. Protein concentrations were determined by the BioRad protein assay according to themanufacturer's protocol. To study in from PIC-1352, PIC-1352ATATA by deleting the BamHYHindIII fragment from PIC-1352, PIC-277AAP2 and pIC-135hAP2 by deleting, re- vitro binding of components of the AP1 complex to the TRE-like sespectively, the BamHIIHindIII and BamHIISmaI fragmentsfrom PIC- quence in the ICA"1promoter, we used bacterially expressed purified 1352AAP2,and PIC-277ASma by deleting the SmaI fragment from PIC- JunB, JunD, c-Jun, and c-Fos proteins, containing DNA binding and dimerization domains (a kind gift of Dr. P. J. Coffer and Dr. L. H. K. 277. Defize, Hubrecht Laboratory, Utrecht, The Netherlands). CharacterisThe NFKB-like site in the ICA"1promoter (at -187) was cloned in tics of the JunB andJunD proteins, as well a s the method for bacterial front of the herpes virus thymidine kinase promoter in thepl9Zuc vector expression and purification of the proteins, have been described (20). (13). 1 and 3 x NFKB(p1C)tkluc were prepared by cutting the SmaI The c-Junprotein was madeby ligating the required PCR product (from fragment (-227 t o -135) with AZuI, resulting in a fragment of 16 bp pSelect/c-Jun as a template) into pGEX2T, generating a GST fusion (-189/-174), containing the NFKB-like site, andtwo fragments of 38 bp protein of 43 kDa, comprising amino acids 194-331 (C-terminal do(-227/-190 and -173/136); thefragment mix was cloned intothe pl9tkZuc vector. 1 x NFKB(p1C)tkZuc contains one copyof the 16-bp
J. P. Johnson, unpublished results.
A. van de Stolpe, E. Caldenhoven, L. Koendeman, J.A. M. Raaijmakers, and P. T. van der Saag, unpublished results.
6187
?)-anscription Regulation of ICAM-1 fold induction I
fold induction
25
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a PIC- 1352
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m 1 6 h r PIC- 135
1 6 h DEX PIC- 135
1 6 h TPA
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PIC - 34
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1 6 h TPA/DEX
FIG.2. Promoter activityof the 5' region of the human ICA"1 gene; effectsof 16 h of treatment with TPAandor D M a , TPA- and DEX-responsive DNAregionsare located downstreamof -677. b, region 1 -677/-340; region 2, -290/-278; region 3,-227/-175; region 4, -105/-38 contribute t o the effect of TPA, and regions 3 and 4 to the effect of DEX. Constructs were transiently transfected into 293 cells, together with the GRGR expression vector containingthe glucocorticoid receptor. Cells were incubated withTPA (16 nM) and/or DEX (1 PM) as indicated, 16 hbefore luciferase measurement. Luciferase values were correctedfor transfection efficiency, using a PDM P-galactosidase-containing expression vector. Results are represented as "-fold induction," obtained by dividing (corrected) luciferase activityfrom TPA/DEX-treated cells by control luciferase activity, i.e. from untreated cells transfected with the same construct. Results are expressed as means 2 S.E. of 8-10 experiments. Details are described under "Materials and Methods." main); the c-Fos protein, comprising amino acids 13 to end, was ex- albumin for 30 min at room temperature, and thegel was run at room pressed in HeLa cells using theVaccinia system (21) and purified over temperature. a Ni+ c o l ~ r n n . ~ Sequences of double-stranded DNA used in EMSA were the NCII RESULTS restriction fragment from t h e I C A " 1 promoter (-227/-135), containLocalization of Cis-acting Elementsin the ICAM-1 Promoter, ing theNFKB-like sequence at -187. In addition, the following synthetic Involved in TPA- and Dexamethasone-inducedChanges in oligonucleotides were made (only upper strands are shown), containing single-stranded 5' overhangs (4-5 nucleotides) at both ends after anTbanscription Rate-To localize involved enhancer elements in nealing: 5'-CAGACCGTGATTCA-3', containing the putative TRE a t the promoter region of the human ICA"1gene, we transiently -284 in the ICA"1 promoter (ICAM-TRE); 5"TCATGAGTCAGACGtransfected promoter-luciferase constructs in 293 human em3', containing the consensus TRE from the collagenase promoter (22); bryonal kidney cells, in thepresence of cotransfected glucocor5'-TCTTGGAAA'I"I'CCGGAGC-3', containing the NFxB-like sequence ticoid receptor expression plasmid. Fig. 2a shows that treata t -187 intheICA"1 promoter(ICAM-NFKB); 5"TCAGAGGGGAC'I"I"l'CCGAGAGG-3', containing a consensus NFKB sequence (23); ment for 16hwith TPA resultedinstrong induction of 5'-TCTCAGGAAGATCT-3',containing an ets-1sequence (241, used a s a luciferase activity. Four regions in the ICA"1 promoter connonspecific competitor for the NFKB sequence.Probes were generated tributed to this effect ofTPA,-6771-340,-3391-278,-2771 by labeling the cohesive ends witha-[32P]dATPusing Klenow fragment -175, and -1351-35. DEX repressed TPA-induced reporter acof DNA polymerase I. Labeled DNA fragments were separated from unincorporated oligonucleotides on a polyacrylamide gel. The EMSA is tivity by 50% in thepresence of the promoter region up to-174 based on the procedures described by Fried and Crothers (25) with (PIC-135 and PIC-174) and by 80-90% when the promoter reslight modifications. Binding assays for ICAM-TRE the were performed gion was extended (5') up to -277 (PIC-277, PIC-339, PIC-677, by incubating 2.5-5 pg of nuclear extract or 0.5 pg of each purified PIC-1014, and PIC-1352) (Fig. 2a). TPA had no effect on the protein and 2 pgpoly(d1-dC) (Pharmacia LKE3 Biotechnology Inc.) with construct containing only the proximal TATA box promoter el0.1-1 ng of 32P-labeled 1CA"TRE oligonucleotide, in the presence or deletion of this TATA ement at -30 (PIC-34). On the other hand, absence of a 100-fold excess of unlabeled competitor oligonucleotide, in mM Hepes, 3 mM box (PIC-1352ATATA)completely abolished the effects of TPA a total volume of 20 pl of modified Dignam D buffer (10 Tris, pH 7.9,67mM KC1,33 mM NaCl, 0.2 m~ EDTA, 5 mM MgCL 10% (and DEX) (not shown). These results suggest that, while the (wiv) glycerol) containing 0.5 m~ dithiothreitol and 0.5 pg/pl bovine proximal TATA box is not itself responsive t o TPA, it may be serum albumin, for 30 min at 4 "C. Subsequently, samples wererun on necessary for TPA-induced transcription of the 1CA.M-1 gene. a 5% nondenaturing polyacrylamidegel at 4 "C, and the gel was fEed in Because TPA and DEX had no effect on the empty pXP2 lucif10% fv/v) methanol/lO% (v/v) acetic acid, vacuum-dried, and finally exposed to FujiRX film at -70 "C for 1-3 days. A similar procedure was erase vector, the effects on reporter activity were specifically promoter region. DEX had no followed for binding assays with theNFKB-containing restriction frag- mediated by the inserted ICA"1 ment andoligonucleotide, except that incubation occurred in Dignam D effect when the empty 6R vector was cotransfected, and the buffer(pH 7.5) with 1 mM dithiothreitoland 1 pglpl bovine serum repressive effect of DEX in the presence ofGRGR could be L. H. K. Defize, unpublished results.
blocked by simultaneous administrationof a 1000-foldexcess of the glucocorticoid receptor antagonist RU486 (26) (notshown).
6188
ll-anscription Regulation of ICA"1 TABLE I
dexamethasone, indicating that thisregion is notof major importance for the repressive effect of dexamethasone on reporter activity. The only potential TPA responsive enhancer sequence Sequence Position in region -1051-38 an is AP2-like sequence at -48 Name Human Mouse Human Mouse (CCCCCAGG) (28), which was, however, not present in the mouse ICA"1 promoter (30). Interestingly,deleting region TATA T A T M TATAA -301-26-431-38 TAATAAA -3131-307 -1051-38 (PIC-1352AAP2, PIC-277AAP2, and PIC-35AAP2) SP-1 CCGCCCC CCGCCCC -591-53 -751-69 markedly reduced basal activity (to, respectively, 4% ( n = 6, CCGCCC -2061201 S.E. 1.2%), 18% (n = 13, S.E. 5%), and 5.5% (n = 6, S.E. 1.7)). AP1-like TGACTCC TGATTCA -2951-289 -2841-279 This may be due t o deletion of a SP1 site at -59 (CCGCCCC). AP2-like CCCCCAGG -481-41 AP3-like TGCGAAATGCC TGTGAAATGGA -3911-381 -3751-365 In U937 Cells TPA Induces Specific Binding of Nuclear ProNFKB-like TGGAAATTCC TGGAAATTCC -1911-182 -1871-178 tein to the ICAM-TRE-Because the 1CA"TRE appearedto be an important elementinvolved in TPA-induced promoter actiSee Ref. 7. See Ref. 30. vation, we investigated by EMSA whether TPA treatment of U937 cells would induce specific binding of a nuclear protein to This demonstrates that the repressive effect of DEXon reporter the ICAM-TRE. Nuclear extracts from U937 cells, treated with activity was mediated by the glucocorticoid receptor. Also, the TPA for either 1.5 or 6 h (6 h is not shown) showed a TPAinhibitory effect of DEX appeared to be specific for the gluco- induced increase in specific binding t o the ICAM-TRE, which corticoid receptor, because cotransfecting different members of could be competed away with excess unlabeled ICAM-TRE and the steroidthyroid receptor superfamily, e.g. retinoic acid re- collagenase TRE oligonucleotides (Fig. 3a). No additional ceptor p , estrogen receptoror progesterone receptorin thepres- change ingel mobility was seen with nuclear extracts from cells ence of their proper ligand had no such e f f e ~ t . ~ treated with TPA (1.5 h) in the presence ofDEX (6 h). Our The DNAregions in theICAM-1 promoter, which appeared to finding that the TPA-induced nuclear protein from U937 cells be involved in TPA-mediated induction of luciferase activity, bound to both the 1CA"TRE and the collagenase TRE sugwere further analyzed. TPA is known to activateprotein kinase gested that it mightconsist of a dimer from the AP1 family of C and induce gene transcription through activation of several transcription factors (31). Therefore we subsequently investidifferent transcription factors, e.g. AP1(27), AP2 (28), andAP3 gated whether purified c-Fos, c-Jun, JunB, and JunD proteins (271, and the NFKB transcription complex (291, all binding to a (containing both the DNA-binding and dimerization domains) unique enhancer sequence. Comparison of the human (7) and could bind to the ICAM-TRE. However no binding could be mouse (30) ICAM-1 promoter regions revealed the presence of detected, while c-Jun and JunDhomodimers, as well as c-Jun, several potential enhancer elements for TPA-activated tran- JunB, and JunD heterodimers with c-Fos were observed to bind scription factors with high homology between the human and to the collagenase TRE (Fig. 3, b and e). the mouse promoter, indicating that they probably represent Identification of Regions in the ICAM-1 Promoter, Involved in functional regulatoryelements (Table I). Region -6771-340 Rapid TPA-, TNFa-, and Dexamethasone-induced Changes in only contains a conserved AP3-like site at -375, which may Dunscription Rate-To detectpotential enhancerelements have contributed to the effect of TPA (5'-TGTGAAATGGA-3') specifically involved in rapid induction of ICAM-1 transcrip(27). Region -3391-278 contains both a second TATAbox (at tion, we limited the incubation time of transfected cells with -313) and a TRE-like sequence (ICAM-TRE) (at -284) with the TPA andor DEX to 4 or 8 h before assaying luciferase activity. sequence TGATTCA. Selective deletion of the ICAM-TRE At 4 h no TPA-induced increase in luciferase activity could be measured (not shown). However, TPA clearly induced reporter (-2881-278) from the largest promoter construct (PIC1352ATRE)resulted ina substantial reduction in TPA-induced activity after 8 h (Fig. 4a). As can be seen from Fig. 4a, the reporter activity but did not abolish the repressive effect of promoter region up to -135, containing the putative AP2 seDEX (Fig. 2b). These results indicate that the ICAM-TRE rep- quence, conveyed some TPA-inducibility, while in the presence of region -2771-136 a moreprominent increase in TPA-induced resents a functional TRE within the context of the ICA"1 promoter but is of no importance for the repressive effect of reporter activity was observed, which could be repressed by DEX. As can be seen from Fig. 2b, deleting nucleotides -3391 DEX. In contrast to the situation where we incubated the cells -291, including the TATA box at -313 (pIC-2901-6),resulted in for 16 hwith TPA, increasing the size of the construct to include a marked decrease in TPA-induced luciferase activity, while an the ICAM-TRE and theTATA box at -313 (PIC-339)and region additional 5' deletion up to -277, including the ICAM-TRE -6771-340 (PIC-677) did not increase TPA-induced luciferase (pIC-277), did not reduce TPA-induced reporter activity any activity any further.Deleting the region containing the NFKBfurther. Therefore, the 1CA"TRE is only transactivated in the like site at -187 (PIC-277ASma) resulted in a significant represence of the distalTATA box at -313. A third region confer- duction of the TPA effect and abolishment of most of the rering TPA-induced reporter activity was localized between pressing activity of DEX (Fig. 4a).These experiments indicated nucleotides -277 and -175 (Fig. 2b). Deletion of nucleotides that the NFKB-like site at -187 might be important for medi-2271-136 (PIC-277ASma) reduced the effects of both TPA and ating rapideffects of both TPA and DEX onI C A " 1 expression. The inflammatory cytokine TNFa specifically induces tranDEX, with the residual effect similar to that seen with the PIC-174 construct, thus localizing an importantregulatory el- scription of target genes by activating the NFKB transcription (32). Thereement for both TPA and DEX between nucleotides -227 and factor, which can transactivate an NFKB enhancer -175. This region appeared to harbor NFKB-like an sequence at fore we subsequently investigated the effect of TNFa on the NFKB-like sequence in theI C M - 1 promoter (Fig. 4b). Incubaposition -187, 5'-TGGAAATTCC-3'. Construct PIC-135 demonstrated a small residual effect of tion with TNFa of cells transfected with constructs PIC-677, TPA (10%) that could be partly repressed by DEX (Fig. 2b). In PIC-339, and PIC-277, which all contain the NFKB-like sethis case, deleting region -1051-38 (pIC-135hAp2) completely quence, increased luciferase activity 4-fold, while in the presabolished the residual effects of TPA and DEX (Fig. 2b). Dele- ence of DEX the effect of TNFa was completely inhibited. As expected, the PIC-277ASma and the PIC-135 constructs demtion of this region from the largest promoter construct (PIC1352AAP2) also resulted in a significant reduction of the TPA onstrate no TNFa-induced reporter activity (and no inhibitory effect but had no significant effect on the repressive effect of effect of DEX either) (Fig. 4b). These results localized rapid Feature table of the humana and mousehICAM-1 promotor sequences
Danscription Regulation of ICA"1 COL TRE
CAM
6189
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lRE c-An
a
COL TRE +
I
C A M TRE
1+1
-1 b
C
FIG.3. EMSA using *WlabeledICA"TRJ3 oligonucleotides.a,nuclear proteins fromU937 cells specifically bind to theICAM-TRE and binding increases uponTPA treatment. Binding assays were performed with 5 pg of nuclear extractfrom U937 cells, incubated for 1.5 h with TPA (16 nM), DEX (1 PM), as indicated. The first lune represents free probe. Competition (comp) was performed with 100 x excess of unlabeled ICAM-TRE (icum),collagenase TRE (cons), or unrelated Ets-1( a s p )oligonucleotides. b, cJun and JunD (but not JunB)bind as homodimers to the collagenase (COO TRE but do not bind to the ICAM-TRE. J u n proteins were bacterially expressed and contain DNA-binding and dimerization domains. Binding assay was performed with 0.5 pg of each protein. c, c-Jun, JunB, and JunDbind a s heterodimers with c-Fos to the collagenase TRE but do not bind to the ICAM-TRE. c-Fos protein was expressed in HeLa cells using the Vaccinia system and contains DNA binding and dimerization domains. Binding assay was performed with 0.5 pg of each protein. Details are described under "Materials andMethods."
stimulatory effects of TPA and TNFaon the ICA"1 promoter, NFKB(p1C)tkluc)or 3-fold repeated (3 x NFKB(p1c)tkluc) (see as well as the repressive effect of DEX, to theregion containing "Materials and Methods"). Treatment with TNFa for 8 h induced a 20-fold increase in luciferase activity with the 3 x the NFKB-like sequence. Nuclear Protein from TNFa- and TPA-treated Cells Binds to NFKB(p1C)tkluc construct, and this increasewas partly rethe NFKB-like Sequence (ICAM-NFKB), Which Functions as a pressed by DEX (by 65%) (Fig. 6).After 24 h DEX nearly comTNFa-inducible Enhancer Element-Treatment of U937 cells pletely inhibited the effect of TNFa (by 87%, notshown). With with TNFa for 30 min induced specific binding of nuclear pro- the 1x NFKB(p1C)tkluc construct treatmentwith TNFa for 8 h teins to the NCII restriction fragment (-2271-135) from the induced a 2-fold increase inluciferase activity, which was comICA"1 promoter, containing the ICAM-NFKBsequence (Fig. pletely abolished by DEX. The effect of TPA treatment for 8 h 5b). Competition for binding occurred in the presence of both was small compared with TNFa but increased markedly after consensus- andthe ICAM-NFKB oligonucleotide (Fig. 5a), 24 h of TPA treatment, a 2.2-fold (S.E. 0.25) induction of the 3 strongly suggesting that thebinding protein is an NFKB tran- x NFKB(p1C)tkluc construct after8 h of TPA (inhibited by 55% scription factor. Nuclear extract from cells treated with the in thepresence of dexamethasone) anda 38-fold induction (S.E. combination of TNFa (30 min) and DEX (6 h) demonstrated 16) after24 h (80% inhibition by dexamethasone) (not shown). similar binding characteristics, indicating that (under theexDISCUSSION perimental conditions employed) no binding of glucocorticoid receptor to the NCII fragment occurred nor any durable proTo elucidate the mechanism underlying the repressive effect tein-protein interactions between the glucocorticoid receptor of dexamethasone on (TPA-induced) ICA"1 expression, we and the TNFa-induced NFKB-binding protein. In addition to investigated the promoter region of the human ICA"1gene by TNFa, TPA also induced increased binding of nuclear extract transfecting ICAM-1 promoter-luciferase constructs in 293 from U937 cells (treated for 8 h or longer with TPA) to the cells. Four regions in the ICAM-1 promoter, 1) -6771-340, 2) ICAM-NFKBsequence (Fig. 5c). -290/-278,3) -227/-175,4) -1051-38, appeared tobe involved To demonstrate functionality of the ICAM-NFKBsequence as in TPA-induced enhancement of transcription, of which two a n enhancer element, we performed transient transfection ex- regions (regions3 and 4)mediated rapid effects of both TPA and periments with this NFKBsequence coupled to a heterologous DEX.Region 1 (-677/-340) contains an AP3-like sequence, thymidine kinase promoter, either as a single element (1 x transactivation of which may be induced by TPA (27). Because
6190
Dunscription Regulation of ICAM-1 foldinduction
.
1
I
fold induction
2
3
4
I
I
I
a
dC-339
DIC-277
d C - 2 7 7 A SMA
DlC-277
D
a
b
-
A
SMn
-8" 8 hr DEX PIC- 135
I 8 h r l N F 8 hr TNFlDEX
FIG.4. Promoter activity of the 6' region of the ICA"1 gene; effects of 8 h of treatment with TPA, TNFa,and DEX. a,-227/-175 and -135/-35 are TPA- and DEX-responsive promoterregions. 6, -2271-175 is a TNFa- and DEX-responsive promoter region. Transiently transfected cells were incubated with TPA (16 nM), TNFa (250 unitdml), andor DEX (1 PM) as indicated, 8 h before luciferase measurement. Results are expressed as -fold induction and represent means f S.E. of 4-6 experiments. Further details are described in the legend of Fig. 2 and under "Materials and Methods."
a contribution of this region to TPA-induced reporter gene activity was only detectable after 16 hof TPA, direct activation of the corresponding AF'3 transcription factor by TPA seems unlikely. This is in agreement with the observation that TPAinduced activation of the AP3 transcription factor may involve at leasttwo separate steps(27). Region 2 (-290/-278) contains a TRE-like sequence (ICAM-TRE) at -284, which functioned within thecontext of the ICA"1 promoter as a TPA-responsive element, requiring the presence of the distalTATA box at -313. This finding is in agreement with the observation that TPA may induce the use of an alternative transcription start site at -280 in the ICAM-1 promoter, located very close to the distal TATA box (9). Together, these results suggest that the ICA" TRE may function in concert with the distal TATA box t o enhance I C A " 1 gene transcription. Although in U937 cells TPA induced a nuclear protein that bound to both the ICAM-TRE and thecollagenase TRE, several purified members of the Fos/ J u n family failed to bind as homo- or heterodimers to the ICAM-TRE, indicating that ICAM-TRE and collagenase TRE differ in their binding characteristics toward members of this transcription factor family. Itremainsto be investigated whether the 1CA"TRE specifically binds an unusual dimer combination ofAPl proteins, as described for certain AF'XRElike enhancer sequences (33). Region 4 (-105/-38) contributed t o the rapid transcriptionenhancing effect of TPA and contains only one potential TPAresponsive enhancer sequence, i.e. an AP2-like sequence, which may be rapidly transactivated upon TPA treatment (28). However the fact that the mouse homolog lacks this AP2-like element suggest that may it be of minor physiological importance. On the other hand, the completely conserved SP, site in this region may serve an important function in regulating constitutive ICA"1 transcription (341, because elimination resulted in a significant drop in basal promoter activity. Region 3 (-227/-175) appeared tocontain the key element for rapid TPA-induced transactivation, i.e. an NFKB-like sequence
(5"TGGAAATTCC-3' equals ICAM-NFKB) at -187, which is 100% homologous to the NFKB-like sequence at -191 in the mouse promoter. While the NFKB transcriptionfactor can be activated by TPA through the protein kinase C pathway (29), TNFa is a more specific and powerful inducer of NFKB transactivation (35) and makes use of both a protein kinase C-dependent and a protein kinase C-independent pathway (32).As expected, TNFa-induced luciferase activity was mediated exclusively by region 3. This is in agreementwith the finding of Voraberger and co-workers (91,who located TNFa-induced transcription of the ICA"1 gene in the promoter region between -280 and -137 bp. Subsequently, we demonstrated that the NFKB-like sequence in region 3 functions, independent of promoter context, as a TNFa- (and TPA-) inducible enhancer element when coupled to a heterologous (thymidine kinase) promoter. Furthermore, the finding that TNFa (and TPA) induced in U937 cells a nuclear protein (complex), which bound not only the ICA" but also the consensus-NFKB sequence, suggests a role for the NFKB transcriptionfactor in transactivation of the ICAM-NFKBenhancer. The transcription factor NFKB has originally been described to consist of two protein subunits, p50 and p65; more recently several other members of the NFKB transcription factor familyhave been described (for a review, see Ref. 361, varying in their bindingaffinities for different NFKB enhancer sequences (37).Based on the sequence of the ICAM-NFKBenhancer we would expect strong binding of the p65 subunit of the NFKBcomplex and, accordingly, in preliminary experimentswe were able t o show that cotransfection of p65 expression plasmid strongly transactivated the I C A " NFKBsequence, while p50 was i n a ~ t i v eThe . ~ ICAM-NFKBenhancer sequence has not been described before and deviates from the consensus sequence in thehighly conserved 5' G-rich part of the enhancer. Interestingly, compared with the protein kinase C-activating agent TPA, TNFa induced more rapid binding of a nuclear protein to the ICAM-NFKBsequence, and, in agreement with this, induced rapid, andvery specific, transac-
Danscription Regulation of ICAM-1 T
N
C O ~ D
-
-
+
-
+ + + cons cam asp
6191
1 8 h r 8 hr DEX 1 8 h r T N F
D8
hr TNFlDEX
-I d TKLUC
lxNkB(IC) T K L K
3XNkB(ICl T K L W
FIG.6. TNFa induces transactivation of the ICAM-NFKBenhancer, whichis repressed by DEX One copy (1x NFKB(p1C)tkluc) or threecopies (3 x NFKB(p1C)tkluc)of the ICM-NFKB sequence were cloned upstream of the thymidine kinase promoter in the pl9luc vector and transiently transfected into 293 cells, together with the 6RGR expression vector containing theglucocorticoid receptor. Cells were incubated with TNFa(250 units/ml) and/or DEX (1 p ~ a)s indicated, 8 h before luciferase measurement. Results are expressed as -fold induction and represent means 2 S.E. of 4 experiments. Further details are described in thelegend of Fig. 2 and under "Materials and Methods."
"
a
b
tivation of the ICAM-NFKBenhancer element,suggesting that TNFa predominantly uses a protein kinase C-independent messenger pathway to activate the NFKB transcription factor. Inagreement with this,TNFahas been shown to induce ICA"1 protein and mRNA expression independent of the protein kinase C pathway (4). Interestingly, two other physiological stimulators of ICAM-1 expression, interleukin 1 and lipopolysaccharide, also activate the NFKB transcription factor ( 3 Q as well as induce ICA"1 expression, by a protein kinase C-independent mechanism (4). Taken together, this suggests that protein kinase C-independent transactivation of the ICAM-NFKBenhancer element may be the major mechanism to rapidlyinduce ICA"1 gene transcription in vivo, while the protein kinase C signal transduction pathway may mediate a slower increase in ICA"1 expression. Extending our earlierfindings that DEX could repress TPAinduced ICA"1 protein and mRNA expression, we now report that the activated glucocorticoid receptor represses not only TPA-induced but alsoTNFa-induced transcription of the ICA"1 gene. This locates the repressive effect of DEX to region 3, containing the ICAM-NFKBelement. In addition, DEX also partly repressed the effect of TPAon region 4, an effect that a t present we did not investigate any further. Subsequent analysis of the inhibitory effect of DEX on transactivation through region 3 revealed that the glucocorticoid receptor represses transactivation of the ICAM-NFKBenhancer, independent of
teins from TNFa-treated U937 cells specifically bind to the NCII fragment from the ICA"1 promoter, containing the ICM-NFKB sequence. Binding assay wasperformed with 10 pg of nuclear extractfrom U937 cells and incubatedfor 30 min with TNFa (250 units/ml). Thefirst lane represents free probe. Competition (cornp)was performed with 100 x excess of unlabeled ICAM-NFKB (icarn),consensus-NFKB (cons),or unrelated COL-TRE (asp) oligonucleotides. b, in U937 cells TNFa induces increased binding of nuclear proteins to the NCII fragment. Binding assay wasperformed with 10 pg of nuclear extract from untreated U937 cells or cells treated with TNFa (250 units/ml), with or without DEX (1 p ~ ) as , indicated. c, in U937 cells TPA induces increased binding of nuclear proteins to the ICAM-NFKBoligonucleotide. Binding assay was performed with 10 pg of nuclear extract from U937 cells treated FIG.5. EMSA, demonstratingTNFa-and "PA-induced binding with TPA (16 nM) for the indicated time periods. Details are described of nuclear protein to the ICA"NF& sequence. a, nuclear pro- under "Materials andMethods."
Dunscription Regulation of ICAM-1
6192
promoter context (ICA"1 or thymidine kinase promoter) or the vector used. Interestingly, these resultspoint to a new type of negative cross-talk between the NFKB transcriptionfactor and theglucocorticoid receptor. Supporting our findings, inhibitory effects of DEX on TNFa-mediated induction of the TNFa gene (39) and of the acute phaseprotein C4BP (40) have been described, indicating that the interaction between the glucocorticoid receptor and NFKB is probably not restricted to the t o our results,no repressive ICAM-1 gene. In apparent contrast effect of DEX on ICAM-1 transcription in A549 cells was observed by Voraberger and co-workers (91, which may, however, be due to lack of (endogenous) glucocorticoid receptors in the A549 cells. Negative interaction between transcription factors can be based on several mechanisms (41). Direct bindingof the glucocorticoid receptor to the NFKB sequence, resulting in displacement of the NFKB transcriptionfactor, is unlikely for two reasons. First, the promoter element involved in the repressive effect of DEX does not contain a glucocorticoid receptor binding element (eithera GRE or a negative GRE)(for a review, see Ref. 421, and second, the effect of DEX was inhibited by the glucocorticoid antagonist RU486, a receptor ligand that does not prevent DNA-binding of the glucocorticoid receptor (43). More likely, the glucocorticoid receptor directly or indirectly binds and inactivates the NFKB transcription factor, analogous to the mechanism responsible for the negative interaction between the glucocorticoid receptor and the AP1 transcription complex (44-46). In our experimental settingwe could not demonstrate this type of protein-protein interaction in an EMSA, but this may be due to relative labile binding between the NFKBprotein complex and theglucocorticoid receptor. An intriguing alternative mechanism may bespecific modification by the glucocorticoid receptor of the IKB protein (which is released from the IKB-NFKBcomplex upon activation of NFKB) in such a way that it can specifically block the NFKB-induced transcription initiation complex, as has been described for IKB ina cell-free transcription assay (47). Finally, it is possible that activated glucocorticoid receptor specifically binds to and squelches certain (co)factors, necessary for the NFKB transcriptionfactor to enhance transcription. Theexistence of a negative interaction between NFKB and the glucocorticoid receptor may have far reaching implications, because many genes thatplay a role in in host defense mechanisms containa NFKB enhancer element their promoter region and thereforecould explain the profound, though largely unexplained, repressive effects of glucocorticoids on the immune system. Acknowledgments-We thank Dr. Siegfried W. de Laat and Dr. JanWillem J . Lammers for continuous support and interest. REFERENCES
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