Downloaded from
`
`http://science.sciencemag.org/
`
`
`
`on March 20, 2019
`
`stantially decrease. HeLa cultures were met-
`4. T. K. Kerppola, D. Luk, T. Curran, Mol. Cell. Biol. 13,
`~~~~~~~~~~~Hertlich,EMBO J. 11, 2241 (1992); R. Schule et a!.,
`3782(1993); H. Konig, H. Ponta, H. J. Rahmsdorf, P.
`abolically labeled in the absence or pres-
`abolically
`ence of DEX for several hours. The labeling
`Cell 62, 1217 (1990); H. F. Yang Yen et a!., ibid., p.
`medium was then replaced with chase me-
`1205; M. I. Diamond, J. N. Miner, S. K. Yoshinaga, K.
`dium containing DEX and TNF-ot. Cultures
`R. Yamamoto, Science 249,1266 (1990); F. C. Lu-
`cibello, E. P. Slater, K. U. Jooss, M. Beato, R. Muller,
`were harvested and extracts were immuno-
`EMBO J. 9, 2827 (1990); C. Jonat et al., Cell 62,
`precipitated with the p65-specific antibody.
`1189 (1990).
`5. A. Ray and K. E. Prefontaine, Proc. Natl. Acad. Sci.
`Under these conditions, the IKBot signal
`U.S.A. 91, 752 (1994); N. Mukaida et al., J. Biol.
`rapidly disappeared (Fig. 4B). Thnus, DEX
`rapidly disappeared (Fig. 4B). Thus, DEX
`Chem. 269, 13289 (1994); E.. Caldenhoven et a!.,
`does not appear to alter the signal transduc-
`Mol. Endocrinol. 9, 401 (1995).
`tion pathway leading to the induced disso-
`6. R. I. Scheinman, A. Gualberto, C. M. Jewell, J. A.
`nCidlowski, A. S. Baldwin Jr., Mol. Cell. Biol. 15, 943
`NF-KB andIKBot.
`ciationof
`(1995).
`Together, these data indicate that DEX
`7. M. Grilli, J. J.-S. Chiu, M. J. Lenardo, Int. Rev. Cytol.
`treatment induces the transcription of the
`143, 1 (1993); P. A. Baeuerle and T. Henkel, Annu.
`Rev. Immunol. 12,141 (1994); U. Siebenlist, G. Fran-
`IKBax gene. Thils induction results In the
`IKBot gene.This induction results in the
`zoso, K. Brown, Annu. Rev. Cell Biol. 10,405 (1994).
`increased synthesis of IKBot protein. This
`8. s. Haskill et a!., Cell 65,1281 (1991).
`increase in protein synthesis leads to the
`9. N. R. Rice, M. L. MacKichan, A. Israel, ibid. 71, 243
`(1992); R. I. Scheinman, A. A. Beg, A. S. Baldwin Jr.,
`rapid turover Of IKB
`protein associated
`Mo!. Cell. Biol. 13, 6089 (1993); F. Mercurio, J. A.
`with preexisting NF-KB complexes. In the
`DiDonato, C. Rosette, M. Karin, Genes Dev. 7, 705
`presence of an activator such as TNF-x,
`(1993).
`newly released NF-KB reassociates with the
`10. R. I. Scheinman, P. C. Cogswell, A. K. Lofquist, A. S.
`Baldwin, unpublished observations.
`DEX-induced IKBot and thus reduces the
`11. N. Auphan, J. A. DiDonato, C. Rosette, A. Helmberg,
`amount of NF-KB translocating to the nu-
`M. Karin, Science 270, 286 (1995).
`cleus. Additionally, newly synthesized IKBox
`12. K. Brown, S. Park, T. Kanno, G. Franzoso, U.
`Siebenlist, Proc. Natl. Acad. Sci. U.S.A. 90, 2532
`(1993); S.-C. Sun, P. A. Ganchi, D. W. Ballard, W. C.
`may enter the nucleus and inhibit NFU-KB
`enterthe nucleus and inhibit NF-KB
`DNA binding (15). A model of this process
`Greene, Science 259, 1912 (1993); A. A.Beg, T. S.
`Finco, P. V. Nantermet, A. S. Baldwin Jr., Mol. Cell.
`is shown in Fig. 5. Consistent with this
`BioL 13, 3301 (1993).
`mode,iwe sow thatBinuthe nCC-medated in-
`model, we show that the GC-mediated in-
`13. J. E. Thompson, R. J. Phillips, H. Erdjument-Bro-
`hibition of NF-KB induction by means of
`TNF-ot is blocked by CHX (Fig. 1). Previ-
`ously we and others showed that activated
`GR could physically associate with NF-KB
`subunits and that DEX represses the DNA
`binding activity of nuclear NF-KB (5, 6).
`Here we demonstrate a second independent
`mechanism through which the NF-KB and
`GC signal transduction systems interact. As
`NF-KB is a critical regulator of cytokine
`genes, the inhibition of the activity of this
`transcription factor would effectively block
`cytokine secretion, thus explaining an im-
`munosuppressive function of GCs. It has
`also been reported recently that salicylates,
`at concentrations corresponding to doses
`prescribed for arthritis patients, also block
`NF-KB activity (16). Thus, NF-KB activa-
`tion serves as a target for two distinct im-
`munosuppressive therapies. The presence of
`multiple levels of interaction between the
`NF-KB and GC systems suggests that these
`interactions may have evolved to serve a
`physiological role in the development of
`the immune system and in modulation of
`the immune response.
`
`mage, P. Tempst, S. Ghosh, Cell 80, 573 (1995).
`14. N. R. Rice and M. K. Enst, EMBO J. 12, 4685
`(1993).
`15. F. Arenzana-Seisdedos et al., Mol. Cell. Biol. 15,
`2689 (1995).
`16. E. Kopp and S. Ghosh, Science 265, 956 (1994).
`17. J.
`J.Chirgwin, A. E. Przbyla, R. J. MacDonald, W. J.
`Rutter, Biochemistry 18, 5294 (1979).
`18. A. K. Lofquist, K. Mondal, J. S. Morris, J. S. Haskill,
`Mol. Cell. Biol. 15, 1737 (1995).
`19. HeLa cultures were metabolically labeled as follows:
`Cell culture plates were washed once with phos-
`phate-buffered saline (PBS) and incubated with
`starve medium [Eagle's minimum essential medium
`(MEM) without methionine or cysteine, plus 15 mM
`Hepes] for 1 hour. Starve medium was replaced with
`fresh starve medium to which was added Express
`Label (NEN) to a final concentration of 200 ,uCi/ml.
`Cultures remained in a tissue culture incubator until
`harvest or addition of chase medium (Eagle's MEM
`containing 25 mM methionine and 25 mM cysteine).
`Cells were harvested by being scraped into ice cold
`PBS and lysed for 5 min on ice in RIP buffer [25 mM
`tris (pH 7.6),150mM NaCI, 2mM EDTA, 0.5% Non-
`idet P-40, and 2 mM phenylmethylsulfonyl fluoride].
`Membranes were removed by spinning of cells for 10
`min in a microfuge at 40C.
`20. We thank members of the Baldwin lab for helpful
`discussion, M. Karin for generously sharing data be-
`fore publication, P. Cohen for 2B4.1 1 cultures, and
`S. Ghosh for providing IKB3 antibody. R.I.S. is sup-
`ported by a fellowship from the Arthritis Foundation
`of America. This research was supported by grants
`to A.S.B. from the Arthritis Foundation of America
`and from NIH (grants A135098 and CA52515).
`27 April 1995; accepted 9 August 1995
`
`Immunosuppression by Glucocorticoids:
`Inhibition of NF-KB Activity Through
`Induction of IKB Synthesis
`Nathalie Auphan,*t Joseph A. DiDonato,t Caridad Rosette,
`Arno Helmberg,t Michael Karin§
`
`Glucocorticoids are among the most potent anti-inflammatory and immunosuppressive
`agents. They inhibit synthesis of almost all known cytokines and of several cell surface
`molecules required for immune function, but the mechanism underlying this activity has
`been unclear. Here it is shown that glucocorticoids are potent inhibitors of nuclear factor
`kappa B (NF-KB) activation in mice and cultured cells. This inhibition is mediated by
`induction of the IKBa inhibitory protein, which traps activated NF-KB in inactive cyto-
`plasmic complexes. Because NF-KB activates many immunoregulatory genes in response
`to pro-inflammatory stimuli, the inhibition of its activity can be a major component of the
`anti-inflammatory activity of glucocorticoids.
`
`REFERENCES AND NOTES
`1. T. R. Cupps and A. S. Fauci,/!mmuno!. Rev. 65,133
`(1982); R. C. Haynes Jr., in Goodman andGilman's
`the Pharmaco!ogica! Basis of Therapeutics, A. G.
`Gilman, T. W. Roll, A. S. Nies, P. Taylor, Eds. (Per-
`gamon, New York, ed. 8,1990), pp. 1431-1462.
`2. R. M. Evans, Science 240, 889 (1988); M. J. Tsai and
`B. W. O'Malley,Annu. Rev. Biochem. 63,451 (1994);
`M. Truss and M. Beato, Endocr. Rev. 14,459(1993).
`3. K. A. Smith, Immuno!. Rev. 51, 337 (1980); P. J.
`Knudsen, C. A. Dinarello, T. B. Strom, J. !mmuno!.
`139,4129(1987); B. Beutler, N. Krochin,. W.Milsark,
`C. Luedke, A. Cerami, Science 232, 977 (1986).
`
`Glucocorticoids (GCs) are physiological
`anti-inflammatory agents (1). Interference
`inhibitors of inflammatory responses and
`with GC action or synthesis increases ani-
`are widely used as immunosuppressive and
`mal mortality after challenge with bacterial
`superantigens (2). GCs induce lymphocyte
`Department of Pharmacology, Program in Biomedical
`supetois (2). CCs induce lymphocyte
`Sciences, Center for Molecular Genetics, School of Med-
`apoptosis (1, 3) and inhibit synthesis of
`icine, University of California, San Diego, La Jolla, CA
`lymphokines (4) and cell surface molecules
`92093-0636, USA.
`required for immune functions (5). In spite
`wPresent address: Centre d'lmmunologie, Institut Nation-
`of the widespread use of CCs, the molecular
`al de Ia Sante et de Ia Recherche Medicale-Centre Na-
`mechanisms that underlie their therapeutic
`tional de Ia Recherche Scientifique, Marseille Laminy
`r
`X>w
`Case 906,13288 Marseille, Cedex 9, France.
`effects are poorly understood (1)J. GCs in-
`tThese authors contributed equally to this work.
`duce target genes through the glucocorti-
`tPresent address: Institute for General and Experimental
`coid receptor(CR), a ligand-activated tran-
`of Innsbruck Medical School, ..
`Pathology,
`University
`A-6020Innsbruck,Austria.
`scription factor (6). CCs repress gene ex-
`§To whom correspondence should be addressed.
`pression through transcriptional
`interfer-
`
`286
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`A
`
`E
`
`C
`
`505
`E 120,000o
`100,000
`4
`X
`°80,000
`600020
`40,000
`820,000
`0
`
`----L0--I
`l+T I+T+D D
`
`UN
`
`0
`
`c
`
`Y
`
`Fig. 1. Inhibition of IL-2 promoter activa-
`tion by DEX correlates with decreased I
`DNA binding of both AP-1 and NF-KB. (A)
`FJ8.1 cells (16) were seeded into microtiter
`plates (2 x 104 cells per well) and incubat-
`ed for 24 hours with either medium alone
`(UN) or with 4 pM ionomycin (I) and TPA (T)
`(100 ng/ml) in the absence or presence of *
`1 ,uM DEX (D). Culture supernatants were
`harvested, and their IL-2 content was
`measured through its mitogenic effect on
`theTcell line HT-2 (16). The results shown are averages of triplicates. (B) FJ8.1 cells were electroporated with the
`18). After 12 hours, cells were treated as indicated above, and luciferase activity was
`IL-2-LUC plasmid (17,
`determined 7 hours later. The results shown are averages of three separate experiments and are presented as
`fold of induction above basal IL-2-LUC expression. (C) FJ8.1 cells were treated as indicated, and after 1 hour
`nuclear extracts were prepared. Five micrograms of each extract were incubated with 20,000 cpm of 32P-labeled
`KB, Oct-1, or AP-1 (TRE) binding site probes, and DNA binding activity was measured by EMSAs. The increase
`in AP-1 binding activity in response to DEX was not reproducibly seen and was probably the result of loading
`more protein in that lane. (D) Inhibition of NF-KB induction in T lymphocytes in vivo. BIO.BR mice (10 weeks old)
`were injected with (i) phosphate-buffered saline (PBS), (ii) 100 Kg of CD3 monoclonal antibody (1 45.2c.1 1) in
`PBS, (iii) DEX-acetate (1 mg per kilogram of body weight) together with 100 ,ug of anti-CD3 in PBS, or (iv)
`DEX-acetate (1 mg/kg). In the experiment shown here, DEX (D) was administered 2 hours before anti-CD3. Two
`hours after anti-CD3 injection, thymuses and lymph nodes were isolated, and their cells were retrieved by being
`squeezed through a nylon mesh. After being washed in PBS, whole-cell extracts were prepared (36). Fifteen-
`microgram samples were incubated with the KB probe, and NF-KB binding activity was analyzed as described
`above.
`
`B
`
`30
`
`0
`
`l+T I+T+D
`
`D
`
`C
`
`KB
`
`Oct-1
`
`TRE
`
`I+TI+T
`+
`UN D I+T +D UN D I+T+D UN D I+T +D
`
`Lymph node
`Thymus
`ymp no eIThymus
`++
`+
`-
`-
`+
`_+
`+
`
`+
`
`+
`
`D
`
`D
`Anti-
`CD3
`l
`
`tion (Fig. 1A) and transcription, as mea-
`sured by an IL-2-luciferase reporter plasmid
`(19) (Fig. IB). Electrophoretic mobility-
`shift assays (EMSAs) (20) revealed that
`both AP-l and NF-KB DNA binding activ-
`ities were elevated in nuclear extracts of
`activated cells (Fig. IC). DEX inhibited
`induction of NF-KB binding activity and
`
`reduced the amount of AP-1 binding activ-
`ity, whereas OBP binding activity was un-
`affected. DEX also inhibited induction of
`NF-KB in mouse T lymphocytes in vivo
`(Fig. ID). Administered either 2 hours be-
`fore or simultaneously with anti-CD3, a
`potent T cell-activating monoclonal anti-
`body (21), DEX inhibited induction of NF-
`
`+GR
`T
`D D UN T
`
`Parental
`T
`D
`
`D
`
`UN T
`
`B
`
`30 min
`T
`D
`
`UN T
`
`45 min 60 min
`T
`T
`D
`D
`
`T
`
`T
`
`60 min
`+ CHX
`T
`D
`
`UN T
`
`D
`
`D
`
`ence between the activated GR and other
`transcription factors, most notably AP-1, by
`a mechanism likely to involve protein-pro-
`tein interactions (7, 8). Although AP-1 is
`involved in lymphokine gene induction (9),
`interference with AP-1 activity cannot ac-
`count for the full spectrum of immunoregu-
`latory genes affected by GCs. Another tran-
`scription factor that activates immunoregu-
`latory genes is NF-KB, whose predominant
`form is a heterodimer composed of p5O and
`Fig. 2. Inhibition of NF- A
`p65 subunits (10). In unstimulated cells, the
`KB activation requires in-
`NF-KB heterodimer is kept as an inactive
`duction of a short-lived
`inhibitory protein. (A) A
`cytoplasmic complex by inhibitory proteins,
`such as IKBot andlIKB I3(1). After cell stim-
`ubonofJra-
`GR
`K ( ) ~~~~~cells (parental) or Jurkat _,
`ulation, the IKBs are rapidly degraded (12,
`cells (tal)
`Jurkat
`~~~~~~~~~~~~cells
`stably transfected
`3
`13 ) and free NF-KB dimers translocate to the
`with an expression vec-
`nucleus and activate target genes (10, 14).
`orencodingratGRand
`This process is transient and is terminated
`expressing 20,000 re-
`through delayed NF-KB-mediated IKBot in-
`ceptors per cell (+GR)
`duction (12, 14). Because of its role in the
`were incubated with TPA
`activation of lymphokine genes and other
`(T) (100 ng/ml), DEX (D)
`pM), aDcombination of
`immunoregulatory
`examined
`(1
`genes,
`we
`whether NF-KB activity is inhibited by GCs.
`TPA plus DEX, or no fur-
`the7r addition (UN) After
`Here we show that GCs are potent inhibitors
`, \ ^ . . ~~~~~~~~1 h...........1our,
`nuclear extracts S
`of NE-KB activation. This inhibition is the
`1hu,nceretat
`were prepared and 5-pg
`result of induction of IKBot expression, fol-
`samples were examined
`lowed by sequestration of NF-KB dimers in
`for NF-KB binding activity as described above. The arrowhead indicates the NF-KB-DNA complex. (B)
`the cytoplasm.
`GR+ Jurkat cells were treated with TPA (T), TPA plus DEX, or DEX (D) for the indicated times. Untreated
`The interleukin-2 (IL-2) promoter con-
`(UN) and DEX-treated cells were harvested after 60 min. When indicated, cells were pretreated with CHX
`(10 ,ug/ml) for 30 min and then incubated with TPA, TPA plus DEX, or DEX in the presence of CHX. NF-KB
`tains cis elements required for maximal in-
`DNA binding activity in nuclear extracts was assayed as described above. (C) GR+ Jurkat cells were
`duction during T cell activation, which are
`recognized by AP-1, NF-KB, NF of activat-
`either untreated (UN) or incubated in the presence of TPA (T) or TPA plus DEX (D) as described above.
`After 60 min, nuclear and cytoplasmic extracts were prepared. Samples of nuclear (Nuc.) (5 mcg) and
`ed T cells, and ocamer-binding prteins
`(OBP) (9, 15). WVe investigated which Of
`cytosolic (Cyt.) (10 p4) extracts were treated with 0.8% Na-deoxycholate (DOG) and 1.2% NP-40 and
`ths fatrs medat th iniitr efec
`analyzed for NF-KB binding activity by EMSA. (D) GR + Jurkat cells were incubated in medium alone (lane
`1), in medium plus TPA (T) (lane 2), or in TPA plus DEX (D) (lanes 3 to 7) for 90 min. After two washes, cells
`tneseractrsmelatethe Ihlblory efect
`(of the synthetic CC dexamethasone (DEX)
`treated withTPA plus DEX were divided into aliquots and incubated for the indicated times with either TPA
`(lanes 4 to 7) or TPA plus DEX( (lane 3) for 120 min. In lanes 1 and 2, cells were maintained under the same
`on IL-2 production by a murine T cell
`hybridoma (16). As shown previously (17,
`conditions (lane 1, medium; lane 2, TPA) for another 120 min. Nuclear extracts were prepared and
`18), DEX inhibited inductiohn of IL-2 secre-
`analyzed for NF-KB binding activity.
`
`C
`
`DCUN
`
`T
`
`+
`D
`
`D
`
`Nuc.
`
`T
`D
`
`,
`
`+
`
`3n 60iffin90
`
`rn 120flin
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`SCIENCE *
`
`VOL. 270
`
`*
`
`13 OCTOBER 1995
`
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`
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`
`1
`
`Antl-GR
`
`D_
`
`T
`UN 102030
`
`T+D
`102030
`
`3C). Treatment of TNF-ox-stimulated cells
`with DEX resulted in retention of p65 in
`the cytoplasm. Staining with antibody to
`GR indicated that DEX induced nuclear
`translocation of GR, regardless of whether
`the cells were stimulated with TNF-ot or
`not. Recently a second form of IKB, IKB3,
`was molecularly cloned (27). Immunoblot
`analysis of HeLa cell extracts with antibod-
`ies to IKBot did not reveal any effect of DEX
`on IKBox expression (28).
`Because DEX did not prevent IKBoL deg-
`radation at early time points, the increase in
`IKBot abundance in DEX-treated cells
`is
`probably the result of increased IKBox syn-
`thesis. Northem (RNA) blot analysis veri-
`fied this assumption and indicated that DEX
`treatment of GR+ Jurkat cells, but not the
`GR- parental cell line, led to sevenfold in-
`
`s
`
`TNF-a
`
`chemoluminescence-sensitive screen and a Phos-
`
`pholmager and normalized to the level of the non-
`specific protein. (B) BIO.BR mice were injected with
`PBS, anti-CD3, DEX (D), or anti-CD3 plus DEX, as
`described in the legend to Fig. 1 D. After 2 hours, the thymuses were isolated and whole-cell extracts were
`prepared. Fifty micrograms of each extract were separated by SDS-PAGE, transferred to lmmbilon P mem-
`brane, and probed with antibodies to IKBQ and p65. (C) HeLa cells were plated on glass cover slips and were
`~~~~~~~~~~~~left
`untreated (UN) or were treated with DEX (D) for 30 min TNF-a for 10min or DEX for 30 min and then TNF-cx
`for 10 mi. After treatment, the cells were fixed and the intracellular locations of p65 and GR were determined
`by indirect immunofluorescence with the use of anti-p65 (Pharmingen, La Jolla, Califomnia) and anti-GR, as
`described (37). Shown are typical fields of stained HeLa cells showing the exclusive cytoplasmic location
`of p65 in untreated, DEX-treated, or TNF- x-plus-DEX-treated cells and its nuclear translocation in
`TNF-a-treated cells.
`
`288
`
`SCIENCE *
`
`VOL. 270
`
`*
`
`13 OCTOBER 1995
`
`.r.rr..~~~PBSCD3
`
`cells. The kinetics of DEX-induced IKBoX
`accumulation paralleled the kinetics of NF-
`KB inhibition. Sensitivity to CHX indicat-
`ed that increased IKBot abundance in cells
`treated with TPA plus DEX required new
`protein synthesis, whereas the TPA-stimu-
`lated degradation of IKBo did not. None of
`the treatments affected p65 abundance.
`Similar results were obtained in vivo: Injec-
`tion of mice with anti-CD3 caused degra-
`dation of thymic IKBa, but simultaneous
`administration of DEX resulted in IKBao
`levels similar to those in untreated animals
`(Fig. 3B). Examination of GR+ Jurkat cells
`(26) or HeLa cells stained with an antibody
`to p65 by indirect immunofluorescence in-
`dicated that p65 was cytoplasmic in un-
`stimulated cells, but after 10 min of TNF-a
`stimulation, most of it was nuclear (Fig.
`
`A
`
`+CHX
`30 mm45min 60minm
`
`a
`
`0
`
`a
`
`-
`
`o F
`
`a
`
`o
`a
`
`.
`
`Z F
`
`___
`MAD4-0-_
`NS-_*.
`
`B
`
`P65*
`
`Anti-
`C
`
`PB
`
`I
`
`I
`
`I
`
`C
`
`ACnD3
`+
`D
`
`D
`
`Atip65
`
`UN.
`
`KB activity in both thymocytes and lymph
`nodes. In the latter, DEX also inhibited
`basal NF-KB binding activity. In addition,
`DEX inhibited induction of NF-KB activity
`in splenocytes of mice injected with lipo-
`polysaccharide (22). Inhibition of NF-KB
`activation by DEX, with a median inhibi-
`tory dose (IC50) of 5 nM (22), was also
`observed in a Jurkat human T cell leukemia
`line stably transfected with a GR expression
`vector (Fig. 2A). No inhibition was ob-
`served in the parental GR Jurkat subclone.
`Therefore, the inhibitory effect of DEX is
`mediated through the GR. Inhibition of
`NF-KB activity was also observed in cells
`stimulated by either 12-0-tetradecanoyl-
`phorbol 13-acetate (TPA) alone or by tu-
`mor necrosis factor (TNF-a) (23). The NF-
`KB binding activity inhibited by DEX was
`composed mostly of the p5O and p65
`(ReIA) subunits (23).
`Inhibition of NF-KB activity required 45
`to 60 min of DEX treatment (Fig. 2B).
`Because initial induction of NF-KB binding
`activity was unaffected, DEX is unlikely to
`interfere with the signaling cascade that
`leads to NF-KB activation. Inhibition of
`NF-KB activity required new protein syn-
`thesis; it was prevented by cycloheximide
`(CHX) (Fig. 2B). The inactive, cytoplasmic
`NF-KB-IKB complex dissociates after de-
`oxycholate (DOC) treatment (24). DOC
`treatment of cytoplasmic extracts of DEX-
`treated cells revealed the same amount of
`NF-KB binding activity as in untreated cells
`(Fig. 2C). Thus, inhibition of NF-KB bind-
`ing activity is not caused by degradation or
`covalent modification of any of its constit-
`uents. Upon DEX removal, but in the con-
`MAD-3-
`tinued presence of TPA, NF-KB binding
`-3
`acftivityereappare after 30mmrs
`and pekdl5 1 1
`at 60 min (Fig. 2D). However, if DEX was
`Fig. 3. DEX treatment increases the abundance of
`left on the cells for 5 hours, NF-KB activity
`IKBa (MAD-3) and traps p65 in the cytoplasm. (A)
`remained repressed. These results suggest
`GR+Jurkatcells(5x106perlane)weretreatedas
`that inhibition of NF-KB IS mediated by a
`indicated for different lengths of time (in minutes).
`short-lived DEX-induced protein.
`Cells were lysed and 40 ,ug of each lysate was sep-
`A possible mediator of the inhibitory
`arated by SDS-polyacrylamide gel electrophoresis
`action of DEX is IKBot, encoded by the
`(PAGE), transferred to a polyvinylidene difluoride
`MAD-3 gene (25), as it
`is a short-lived
`membrane (Millipore), and immunoblotted with either
`anti-IKBa or anti-p65 and donkey anti-rabbit immu- TNF-
`protein whose degradation is further en-
`hanced by various NF-KB inducers (12, 13).
`noglobulin conjugated to peroxidase as described
`(20). The migration positions of both MAD-3 (IKBQx)
`These inducers also cause IkBot resynthesis
`and p65 are indicated. UN, untreated; T, TPA; D,
`by stimulating MAD-3 transcription, a feed-
`DEX; NS, a nonspecific cross-reacting protein. The
`back response that terminates NF-KB acti-
`levels of IKBa were quantitated with the use of a
`vation (12). The effect of DEX on IKBaL
`metabolism was examined by immunoblot-
`ting with an antibody to IKBa. Treatment
`with TPA led to the partial disappearance
`of IKBa, which reached 20% of its basal
`level after 30 min (Fig. 3A). At 10 and 20
`min DEX had an insignificant effect on the
`'
`amount of IKBat in TPA-treated cells, but
`after 30 min the amount of IKBat in cells
`treated with TPA plus DEX was similar to
`that in unstimulared cells, and after 45 min
`it was 50% higher than in unstimulated
`
`SAN EX 1022, Page 3
`
`

`

`duction of MAD-3 (IKBox) mRNA (Fig. 4A).
`Stimulation of either GR+ or GR- cells with
`TPA resulted in sixfold induction of MAD-3
`mRNA after 1 hour. Treatment with both
`TPA and DEX resulted in a synergistic in-
`duction (23-fold), but only in the GR+ sub-
`clone. None of the treatments had a substan-
`tial
`effect on expression of p65 (RelA)
`mRNA. Induction of IKBot expression by
`both TPA and DEX is most likely the result
`of increased transcription because it was in-
`hibited by actinomycin D (Fig. 4B). In an
`accompanying report (29), Scheinman et al.,
`who obtained similar results, demonstrate
`that DEX increases the transcription rate of
`the MAD-3 gene. Actinomycin D also pre-
`vented DEX-induced inhibition of NF-KB
`binding activity (Fig. 4C).
`Collectively, these results define a sim-
`ple mechanism through which GCs repress
`NF-KB activity. Unlike previously described
`transcriptional interference (8), inhibition
`of NF-KB activity does not rely on direct
`interaction between the activated GR and
`any NF-KB constituent. Rather, the inhibi-
`tion is based on induction of IKBot expres-
`sion. Induction of MAD-3 mRNA results in
`faster reappearance of IKBot in activated
`cells treated with DEX than in activated
`cells not exposed to DEX. Reappearance of
`
`IKBot correlates with termination of NF-KB
`activation. Even a modest increase in the
`amount of IKBot, not greatly exceeding its
`basal level in unstimulated cells, is suffi-
`cient to cause redistribution of active p65
`from the nucleus to the cytoplasm, where it
`is sequestered as an inactive complex.
`It was previously suggested that inhibi-
`tion of NF-KB activation by DEX is medi-
`ated by physical interactions between the
`activated GR and the p65 component of
`NF-KB (30). However, those results were
`obtained by transient cotransfection exper-
`iments, and although it is feasible that once
`overexpressed the GR and p65 can directly
`interact, this mechanism is not necessary to
`explain the results described above that
`were obtained under physiological condi-
`tions. Furthermore, DEX treatment causes
`translocation of p65 from the nuclei of
`stimulated cells to the cytoplasm, whereas
`the activated GR remains nuclear. It
`is
`therefore unlikely that direct binding of GR
`to p65 mediates cytoplasmic sequestration
`of p65. Because DEX induces MAD-3 ex-
`pression in nonactivated T cells, the induc-
`tion response itself does not appear to rely
`on direct interaction between the activated
`GR and NF-KB. As is consistent with other
`work (31), the simplest explanation of the
`
`A
`Parental
`
`MAD-3
`
`MAD.3
`
`Kl
`
`D
`T+D
`T
`UN
`60 30 45 60 30 45 60
`60
`i| | ffi S S S 2 ftXh>A+>ilfvv
`|1SS
`
`B
`
`-ActD
`1
`
`I
`
`l
`
`lll
`
`*l fz zi 0ll
`
`+ActD
`4
`1
`
`I
`
`1
`
`T
`
`p65
`
`2
`
`current results is that newly synthesized
`IKBot translocates to the nucleus, where, as
`shown in vitro (32), it can sequester free
`NF-KB and thereby promote net dissocia-
`tion of DNA-bound NF-KB. This is fol-
`lowed by translocation of IKBo- and p65-
`containing complexes to the cytoplasm.
`This mechanism also differs from the one
`proposed to explain inhibition of NF-KB
`activation by the relatively weak anti-in-
`flammatory agent aspirin (33).
`Inhibition of NF-KB activation can ac-
`count for many of the immunosuppressive
`and anti-inflammatory activities of GCs,
`which are amongst the most potent anti-
`inflammatory agents known. NF-KB plays a
`central role in induction of a large number of
`important immunoregulatory genes, includ-
`ing those encoding IL-1, IL-2, IL-3, IL-6,
`IL-8, TNF-ox, interferon y (IFN--y), granulo-
`cyte-macrophage colony-stimulating factor,
`class I and class II major histocompatibility
`complexes, the K light chain, and endothe-
`lial leukocyte adhesion molecule 1 and in-
`tercellular adhesion molecule 1 (10). Several
`of these genes are also regulated by AP-1 (9),
`which synergizes with NF-KB (34). Because
`GCs inhibit both NF-KB and AP-1 activi-
`ties, albeit through different mechanisms, it
`is no longer a surprise that they repress ex-
`pression of a very wide spectrum of immu-
`noregulatory genes. Indeed, administration
`of GC prevents systemic release of IL-2, IL-6,
`IFN--y, and TNF-cx in response to anti-CD3
`or to superantigen (2, 35). It is anticipated
`that other effective inhibitors of NF-KB and
`1;0ql|!|>w00000*0*ijlAP-I
`_! | -w w1 aXitUf0L
`also turn out to be useful immu-
`may
`nosuppressive and anti-inflammatory agents.
`
`G+
`
`UN
`T
`T+D
`D
`60 30 45 60 30 45 60 30 45 60
`
`MAD-3
`
`p65
`
`T+D
`
`UD F
`F
`D |w 11* +***
`[ .i1!.|
`L
`3
`
`6
`
`F
`
`9
`
`60 go
`
`30
`
`45
`
`60
`
`75
`
`90
`
`60
`
`+
`+
`
`Fig. 4. DEX induces expression of the gene encoding
`IBx(MAD-3), which is required for inhibition of NF-KB
`IKBLS
`activation. (A) GR- (parental) and GRt Jurkat cells were
`treated for different lengths of time (in minutes), as indi-
`cated. Total cytoplasmic RNA was extracted and 20-
`g samples were separated on a 1% agarose gel,
`transferred to a nylon membrane, and analyzed by hy-
`bridization to MAD-3 (IKBa) and p65 (Re/A) comple-
`mentary DNA probes. UN, untreated; T, TPA; D, DEX.
`(B) GR+ Jurkat cells were treated with TPA (T) or DEX
`(D) or both for different times (in minutes) as indicated.
`3
`2
`1
`6
`5
`4
`Boxes 1 to 3 show no further additions. In boxes 4
`and 5, actinomycin D (ActD; 3 ,ug/ml), was added 15 min after the initial exposure to TPA or DEX, then
`cells were harvested every 15 min as indicated. In box 6, cells were pretreated with DEX for 15 min,
`followed by ActD, and after 15 min TPA was added. In boxes 7 to 9, ActD was added 15 min before
`incubation with TPA or DEX or both. Total cellular RNA was prepared and 30-,ug samples were
`separated on an agarose gel, transferred to nylon membrane, and analyzed for the presence of MAD-3
`(lKBcL) mRNA. (C) GR+ Jurkat cells were incubated with DEX (D) for 15 min, then ActD (3 IJg/ml) was
`either added (lane 3) or not (lane 2), and after 15 min TPA (T) was added. As a control, ActD was also added
`15 min before incubation with TPA plus DEX (lane 4), TPA (lane 5), or DEX (lane 6). In addition, TPA was
`added alone (lane 1). Nuclear extracts were prepared, and NF-KB binding activity was examined.
`
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`kA *
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