potentiation of fas- and trail-mediated apoptosis by ifn-γ in a549 lung epithelial cells:...

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doi:10.1006/cyto.2003.2008 POTENTIATION OF FAS- AND TRAIL-MEDIATED APOPTOSIS BY IFN- IN A549 LUNG EPITHELIAL CELLS: ENHANCEMENT OF CASPASE-8 EXPRESSION THROUGH IFN-RESPONSE ELEMENT Ki-Bae Kim, Yun-Hee Choi, In-Ki Kim, Chul-Woong Chung, Byung Ju Kim, Yang-Mi Park, Yong-Keun Jung* Epithelial cell apoptosis triggered cooperatively by multiple cytokines contributes to the injury induced by inflammatory responses in the lung and elsewhere. Here we show that interferon- (IFN-) sensitizes A549 cells, human lung epithelial cells, to cytokine-mediated apoptosis by upregulating caspase-8 expression. Pretreating the cells with IFN- potentiated Fas- and TNF-related apoptosis inducing ligand (TRAIL)-induced cell death, but other forms of apoptosis, not mediated via receptors, were unaected. Western blotting and inhibitor assays showed that IFN- selectively increased expression of caspases-7 and -8, but not caspases-2, -3, -9, or -10, as a necessary step leading to apoptosis. Assaying promoter activity using a luciferase reporter gene indicated that an IFN- response element was located in the 5-flanking region of the caspase-8 gene, spanning positions -227 to -219. Taken together, these findings suggest that IFN- potentiates Fas- and TRAIL-mediated apoptosis by increasing caspase-8 expression via an IFN- response element in A549 cells. 2003 Published by Elsevier Science Ltd. Apoptosis, or programmed cell death, is a genetically-regulated process that plays an indis- pensable role in development, defense against viral invasion, modulation of immune responses, and homeostasis in multicellular organisms. 13 Apoptosis can be initiated by various cell surface receptors, including Fas (APO-1; CD95), TNF receptors, and TRAIL receptors (DR4 and DR5), which are activated by such cytokines as FasL, TNF and TRAIL. 4 When stimulated, the receptor recruits a set of signaling proteins, leading to the formation of the death- inducing signaling complex (DISC), made up of Fas, FasL, FADD, and Caspase-8. 2,4 Caspase represents a highly conserved family of cysteine proteases that play critical roles in apoptosis. 3,5 Indeed, caspase-3, -9, and -12 (-/-) mutant mice, as well as those administered with caspase inhibitors, show decreased apoptosis in response to a variety of pathological stimuli or to altered nervous system development, 610 which suggests that caspase potentially serves as useful therapeutic targets for controlling inappropriate apoptosis. 10 Thus far, at least 14 caspases having distinct substrate recognition properties and falling into two classes—class I or initiator caspases (e.g., caspases-2, -8, -9, -10) and class II or eector caspases (e.g., caspases-3, -6, -7)—have been identified. 5 Within cells, these enzymes appear to be arranged in complex networks, which produces cascades of activation. 3,5 Although apoptosis is a part of the normal process of epithelial cell renewal, in excess it is pathological. For instance, airway epithelial cells are targets in asthma, viral infection, acute lung injury, and fibrotic lung disease, and apoptosis among the aected cells contributes to the lung injury associated with the resultant inflammatory responses. IFN- may play a role in the pathogenesis of multiple sclerosis, Sjogren’s syndrome, Down syndrome, and acute and chronic graft-versus-host disease. 1113 It has been proposed that in combination with FasL, IFN- induce apopto- sis in several types of cells. 14,15 However, the molecular From the Department of Life Science, Kwangju Institute of Science and Technology, Puk-gu, Kwangju 500-712, Korea Correspondence to: *Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, Korea. Tel: 82-62-970-2492; Fax: 82-62-970-2484; e-mail: [email protected] Received 4 October 2001; received in revised form 27 June 2002; accepted for publication 12 November 2002 1043–4666/03/$-see front matter 2003 Published by Elsevier Science Ltd. KEY WORDS: apoptosis/caspase-8/cytokines/gene regulation/ IFN- CYTOKINE, Vol. 20, No. 6 (21 December), 2002: pp 283–288 283

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doi:10.1006/cyto.2003.2008

POTENTIATION OF FAS- AND TRAIL-MEDIATEDAPOPTOSIS BY IFN-� IN A549 LUNG EPITHELIAL

CELLS: ENHANCEMENT OF CASPASE-8EXPRESSION THROUGH IFN-RESPONSE

ELEMENT

Ki-Bae Kim, Yun-Hee Choi, In-Ki Kim, Chul-Woong Chung, Byung Ju Kim,Yang-Mi Park, Yong-Keun Jung*

Epithelial cell apoptosis triggered cooperatively by multiple cytokines contributes to the injuryinduced by inflammatory responses in the lung and elsewhere. Here we show that interferon-�(IFN-�) sensitizes A549 cells, human lung epithelial cells, to cytokine-mediated apoptosis byupregulating caspase-8 expression. Pretreating the cells with IFN-� potentiated Fas- andTNF-related apoptosis inducing ligand (TRAIL)-induced cell death, but other forms ofapoptosis, not mediated via receptors, were unaffected. Western blotting and inhibitor assaysshowed that IFN-� selectively increased expression of caspases-7 and -8, but not caspases-2, -3,-9, or -10, as a necessary step leading to apoptosis. Assaying promoter activity using a luciferasereporter gene indicated that an IFN-� response element was located in the 5�-flanking region ofthe caspase-8 gene, spanning positions -227 to -219. Taken together, these findings suggest thatIFN-� potentiates Fas- and TRAIL-mediated apoptosis by increasing caspase-8 expression viaan IFN-� response element in A549 cells.

� 2003 Published by Elsevier Science Ltd.

From the Department of Life Science, Kwangju Institute of Scienceand Technology, Puk-gu, Kwangju 500-712, Korea

Correspondence to: *Department of Life Science, Kwangju Instituteof Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju500-712, Korea. Tel: 82-62-970-2492; Fax: 82-62-970-2484;e-mail: [email protected]

Received 4 October 2001; received in revised form 27 June 2002;accepted for publication 12 November 2002

1043–4666/03/$-see front matter � 2003 Published by ElsevierScience Ltd.

KEY WORDS: apoptosis/caspase-8/cytokines/gene regulation/IFN-�

Apoptosis, or programmed cell death, is agenetically-regulated process that plays an indis-pensable role in development, defense against viralinvasion, modulation of immune responses, andhomeostasis in multicellular organisms.1–3 Apoptosiscan be initiated by various cell surface receptors,including Fas (APO-1; CD95), TNF receptors, andTRAIL receptors (DR4 and DR5), which are activatedby such cytokines as FasL, TNF� and TRAIL.4 Whenstimulated, the receptor recruits a set of signalingproteins, leading to the formation of the death-inducing signaling complex (DISC), made up of Fas,FasL, FADD, and Caspase-8.2,4

Caspase represents a highly conserved familyof cysteine proteases that play critical roles in

CYTOKINE, Vol. 20, No. 6 (21 December), 2002: pp 283–288

apoptosis.3,5 Indeed, caspase-3, -9, and -12 (-/-) mutantmice, as well as those administered with caspaseinhibitors, show decreased apoptosis in response to avariety of pathological stimuli or to altered nervoussystem development,6–10 which suggests that caspasepotentially serves as useful therapeutic targets forcontrolling inappropriate apoptosis.10 Thus far, atleast 14 caspases having distinct substrate recognitionproperties and falling into two classes—class I orinitiator caspases (e.g., caspases-2, -8, -9, -10) and classII or effector caspases (e.g., caspases-3, -6, -7)—havebeen identified.5 Within cells, these enzymes appear tobe arranged in complex networks, which producescascades of activation.3,5

Although apoptosis is a part of the normal processof epithelial cell renewal, in excess it is pathological.For instance, airway epithelial cells are targets inasthma, viral infection, acute lung injury, and fibroticlung disease, and apoptosis among the affected cellscontributes to the lung injury associated with theresultant inflammatory responses. IFN-� may play arole in the pathogenesis of multiple sclerosis, Sjogren’ssyndrome, Down syndrome, and acute and chronicgraft-versus-host disease.11–13 It has been proposedthat in combination with FasL, IFN-� induce apopto-sis in several types of cells.14,15 However, the molecular

283

284 / Kim et al. CYTOKINE, Vol. 20, No. 6 (21 December, 2002: 283–288)

mechanism underlying cell death in such cases is notclear yet.

Earlier reports have shown that IFN-� may modu-late apoptosis by regulating expression of caspases-1,-3, and -11 via STAT-1.16,17 Here we show that IFN-�potentiates Fas- and TRAIL-mediated apoptosis inlung epithelial cells and this effect reflects upregulationof caspase-8 expression through IFN-� activatingsequence (GAS) element in the 5�-flanking region.

RESULTS

IFN-� potentiates apoptosis triggered by Fas andTRAIL receptor signals

To examine the effect of IFN-� on Fas-mediatedcell death, A549 cells were exposed to anti-Fas anti-body (Ab) in the presence or absence of IFN-�.Determination of cell viability showed that A549 cellsexposed to either IFN-� or anti-Fas Ab alone exhibitedbasal levels of cell death (10–20%) by day 3 of exposure(Fig. 1A). On the other hand, pre-treatment withIFN-� dramatically sensitized A549 cells to Fas, result-ing in increase of death rate to 75–80% (Fig. 1A).Fluorescence activated cell-sorting (FACS) analysisfollowing staining cells with propidium iodide revealedsubstantial DNA fragmentation (Fig. 1B ) and dyingcells appeared shrunken (data not shown), showingapparent characteristics of apoptosis. Further, whenA549 cells pretreated with IFN-� were exposed tovarious death signals, cell death was synergisticallyincreased by TRAIL (a DR4- and DR5-ligand) (3.6-fold), but not by staurosporine (a kinase inhibitor) oretoposide (a DNA damaging agent) (Fig. 1C). Theseresults indicate that IFN-� selectively sensitizes A549cells to Fas and TRAIL, receptor-mediated deathsignals.

Figure 1. Potentiation of Fas- and TRAIL-induced apoptosis byIFN-�.

(A) A549 cells were pre-incubated for 1 to 3 days with 500 U/mlIFN-� and then with 200 ng/ml anti-Fas Ab. Cell viability (% of celldeath) was determined with trypan blue exclusion; bars depictmeans�SD from three independent experiments. Statistical differ-ence (*P<0.001). (B) FACS analyses demonstrating DNA fragmen-tation after exposure to both IFN-� and anti-Fas Ab for 2 days. (C)After pretreatment with 500 U/ml IFN-� for 36 h, A549 cells wereexposed to 0.2 �M staurosporine (Stau.) for 24 h, 25 �M etoposide(Eto.) for 36 h, or 20 ng/ml TRAIL for 24 h.

Regulation of caspase expression by IFN-�IFN-� has been reported to regulate expression of

caspases-1 and -3.16,17 However, this cannot explainthe selective potentiation of IFN-� on Fas- andTRAIL-induced cell death. Thus, we examinedexpression patterns of other caspases (Fig. 2). Westernblot analysis showed that treatment of A549 cells withIFN-� increased expression of caspases-7 and -8, butnot caspase-2, -3, -9, or -10. Expression level of thepro-caspases-7 and -8 was the highest at day onewithout apparent cell death and returned to basal levelby day 3. Subsequent exposure of A549 cells to anti-Fas Ab following IFN-� treatment decreased levels ofthe pro-caspases-7 and -8 as well as other caspases,which was indicative of proteolytic activation ofcaspases during synergistic cell death. Processed sub-units of caspases-2, -3, -7, and -8 were all detected withWestern blot analysis (data not shown).

Potentiation of caspase 8-mediated apoptosis by IFN-� / 285

Assessment of the enzymatic activities usingfluorogenic substrates showed that IFN-� synergisti-cally induced the activities of caspases-3 and -8 in theanti-Fas Ab-treated cells (Fig. 3A), consistent with theresults of Western blot analysis (Fig. 2). The potentia-tion effects of IFN-� on Fas-mediated cell death wereabolished by treatment with zVAD-fmk, a pan caspaseinhibitor, and IETD-fmk, a caspase-8 inhibitor, indi-cating that caspase-8-like protease plays crucial role intwo cytokine-induced cell death (Fig. 3B).

Figure 2. Western blot analysis showing expression pattern of variouscaspases in A549 cells exposed to IFN-�.

The cells were incubated for the indicated times with IFN-�, anti-FasAb, or both as described in Figure 1, after which cell extracts wereanalyzed with Western blotting using Abs against selected caspasesand FADD.

Figure 3. Contribution of caspase-8 to IFN-�- and Fas-mediated celldeath.

(A) Caspase activity assay. A549 cells were exposed to 500 U/mlIFN-�, 200 ng/ml anti-Fas Ab, or both for 36 h. Cell extracts wereprepared and assayed for caspase-3 and -8 activities using thefluorogenic substrates, DEVD-AMC and IETD-AMC, respectively.Relative caspase activities (fold induction) are represented asmeans�SD. (B) A549 cells were exposed to IFN-�, and anti-Fas Abin the presence or absence of 20 �M zVAD-fmk or 50 �M IETD-fmk. Statistical significance (A, P<0.01; B, P<0.001).

Analysis of the 5�-flanking region of caspase-8gene responsible for IFN-�-response

A genomic DNA fragment composed of 132 bp ofthe 5�-untranslated region and 1200 bp of the5�-flanking sequence of caspase-8 gene was found in theEST data-base and contained three putative GASelements near the transcription start site (Fig. 4A).18

The promoter activity of the caspase-8 gene was exam-ined using the luciferase reporter gene fused to thepromoter of capase-8 containing 370 bp of the5�-flanking region and 102 bp of 5�-untranslatedregion (p472Casp8.Luc) and its deletions (Fig. 4A).Transfection of p472Casp8.Luc induced about 70-foldincrease in the luciferase activity as compared with thecontrol and treatment of the transfectants with IFN-�

further increased the luciferase activity by 3-fold (Fig.4B), demonstrating that the 5�-flanking region ofcapsase-8 gene contains the IFN-�-regulated elements.

Two-fold decrease in luciferase activity wasdetected when the construct was shortened to position-305 (p407Casp8.Luc), indicating that the sequencespanning positions -370 to -305 contains positivelyacting regulatory elements. Further deletion to pos-ition -200 (p302Casp8.Luc) increased the luciferaseactivity 6-fold, indicating the presence of negativeelement within the deleted region. IFN-� treat-ment increased the reporter activity 4.6-fold inthe p407Casp8.Luc construct, but not in thep302Casp8.Luc construct, suggesting that of the three

286 / Kim et al. CYTOKINE, Vol. 20, No. 6 (21 December, 2002: 283–288)

putative GAS elements, one located at position c (-227and -219) functions to control expression of caspase-8gene in response to IFN-�.

DISCUSSION

Fas-mediated apoptosis plays an essential role inthe development of pulmonary fibrosis, asthma, andlymphoproliferation disorder,19–21 and its effects areexacerbated by IFN-�.14 Several lines of evidence indi-cate that IFN-� increase apoptosis through regulationof such apoptosis-related genes as Fas, TRAIL, IAP,TRAIL-R, and caspase.22–24 Similarly, IFN-� and -�also enhanced TRAIL-induced apoptosis.25–27 Here,we present the selective increase of caspase-7 and -8expression by IFN-� in A549 cells, which contributesto the potentiation of caspase-8-dependent apoptosisand may lead to pathology responsible for damage tothe lung associated with the aforementioned ailments.

We observed that IFN-� mainly potentiated apop-tosis mediated by Fas and TRAIL-R, processes in

which caspase-8 played a crucial role.28,29 In addition,we found that by FACS analysis, IFN-� treatment didnot affect expression of Fas or cell cycle process inA549 cells (data not shown). We also observed thatIFN-� increased expression of caspase-8 and potenti-ated Fas-mediated apoptosis in other lung cell linesincluding L132, 738Lu, and MRC5, and liver cells suchas SNU354 (data not shown). Thus, we believe that inthe pulmonary epithelial cells, the potentiating effectsof IFN-� on cell death largely reflect the increasedexpression of caspase-8. Similarly, increase ofcaspase-8 expression was recently reported in breasttumor and erythroid cells.30,31

Still, the molecular mechanism underlying theeffects of IFN-� on the regulation of caspaseexpression is complicated and appears to be celltype-specific. For example, whereas IFN-� increasesexpression of caspases-7 and -8, but not caspase-3, inA549 lung cells, caspases-1, -3 and -8 are upregulatedin erythroid progenitor cells and caspase-11, but notother caspases, is induced in lymphocytes.21 Treatingcells with IFN-� causes activation of JAK1 and JAK2,leading to activation of genes containing GAS elementsthrough STAT1 phosphorylation. In STAT-1-deficientU3A cells, expressions of caspases-1 and -3 are consti-tutively low, and reintroduction of the STAT-1 generestores the expression of those caspases.16 Within thecaspase-8 gene, the GAS consensus (TTNCNNNAA)spanning positions -305 to -200 is a potential bindingsite for IFN-�-activated STAT-1 homodimers. We alsofound the GAS (-590TTACTGAAA-582) in the pro-moter of caspase-7 gene, which remains to be charac-terized. In addition, IFN-�-induced activation of thecaspase-1 promoter is also dependent on the binding ofIRF-1.14,32 On the other hand, there is no IRF-1binding sequence within the �2.5 kb comprisingthe 5�-flanking region of caspase-8 gene, which isindicative of the differential regulation pattern ofcaspases. Identification of the critical element for eachcaspase remains to be clarified to understand diverseregulations of caspase expression in different cell types.

MATERIALS AND METHODS

Figure 4. Promoter analysis of the 5�-flanking region of caspase-8gene.

(A) Schematic diagram showing 5�-flanking region of the caspase-8gene and its deletion constructs fused to a luciferase reporter gene.Numbering is relative to the transcription initiation site and allconstructs include 102 bp of 5�!-untranslated region (5� UTR).Putative GAS elements; a: -256TTGCACAAA-257, b: -227TTCCAAGAA-219, c: -78TTGCTCCAA-70. (B) A549 cells were trans-fected with pGL2-Basic, p472Casp8.Luc, p407Casp8.Luc, orp302Casp8.Luc. pCMV �-gal served as an internal control. One daylater, cells were incubated with 500 U/ml IFN-� for 8 h, after whichluciferase activity was determined and normalized to the�-galactosidase activity. Statistical difference (P<0.05).

ReagentsIFN-�, etoposide, staurosporine, and anti-Fas Ab were

purchased from Sigma (St. Louis, MO, USA) and MBL(Nagoya, Japan), respectively. TRAIL was a kind gift fromGenetech Inc. (San Francisco, CA, USA). Caspase inhibi-tors, acetyl-Ile-Glu-Thr-Asp-fmk (IETD-fmk) and acetyl-z-Val-Ala-Asp-fmk (z-VAD-fmk), and the fluorogeniccaspase substrates, IETD-aminomethylcoumarine (AMC)and acetyl-Asp-Glu-Val-Asp-(DEVD)-AMC, were fromEnzyme System Products (Livermore, CA, USA).

Potentiation of caspase 8-mediated apoptosis by IFN-� / 287

Cell culture and DNA transfectionA549 cells, a human lung epithelial cell line, were grown

in F-12K medium (GibcoBRL) supplemented with 10% fetalbovine serum (FBS, Biofluids). Cells were transfected withthe indicated plasmids using LipofectAMINE according tothe manufacturer’s instructions (GibcoBRL).

Plasmid constructsPcasp8-GFP fusion protein was constructed by sub-

cloning the PCR product into BamHI/HindIII sites ofpEGFP plasmid. p472Casp8.Luc was constructed by ligationinto the KpnI/HindIII sites of pGL2-Basic (Promega) ofthe PCR products; human genomic clone RP11-536I18(AC007283) served as a template. Deletions p407Casp8.Lucand p302Casp8.Luc were constructed into KpnI/HindIII sitesof pGL2-Basic. All PCR products were confirmed by DNAsequencing.

FACS analysisA549 cells (2�106 cells/ml) suspended in 200 �l PBS

were incubated for 30 min with staining solution (0.1%Triton X-100, 2 mM MgCl2, 100 mM NaCl, 10 mM PIPESbuffer (pH 6.8), 10 �g/ml propidium iodide, 20 U/ml DNase-free RNase A) and analyzed on a FACScan flow cytometer(Becton Dickinson).

Antibodies and Western blot analysisAntibodies against caspase-2, -3, -10, and p53 were from

Santa Cruz (Santa Cruz, CA, USA); anti-FADD Ab(F36620) was from Transduction Laboratory (Lexington,KY, USA); anti-caspase-7 Ab and anti-�-tubulin Ab werefrom Pharmingen (San Diego, CA, USA) and Sigma, respect-ively. Western blot analysis and antibody preparation againstcaspase-8 and -9 were described by Kim et al.28

Caspase activity assayCaspase activity was assayed as previously described by

Kim et al.28

Luciferase and �-galactosidase assaysCells were lysed in a lysis buffer [25 mM Tris-phosphate

(pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N�,N�-tetraacetic acid, 10% glycerol, and 1% TritonX-100]. Luciferase activity was measured following theinstruction of manufacturer (Promega Inc.) For �-galactosidase assay, the cell extracts were mixed with�-galactosidase assay buffer [200 mM sodium phosphate(pH 7.3), 2 mM MgCl2, 100 mM �-mercaptoethanol, and1.33 mg/ml ONPG] and incubated for 30 min at 37�C. Theobservance at 420 nm was measured with a microplate reader(Bio-Rad).

Statistical analysisAll results are presented as means�SD of n indepen-

dent experiments and ANOVA using Fisher’s least significantdifference was used.

Acknowledgments

K. Kim, and Y. Choi were supported by the BK21 project. This work was supported in part byNational Research Laboratory program (to Y. Jung)and by a grant from Protein Network Research Centerof MOST in Korea.

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