Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 Sep;18(9):5414-24.
doi: 10.1128/MCB.18.9.5414.

Stress-induced Fas ligand expression in T cells is mediated through a MEK kinase 1-regulated response element in the Fas ligand promoter

M Faris  1 , K M Latinis, S J Kempiak, G A Koretzky, A Nel
Affiliations

Stress-induced Fas ligand expression in T cells is mediated through a MEK kinase 1-regulated response element in the Fas ligand promoter

M Faris et al. Mol Cell Biol. 1998 Sep.

Abstract

T lymphocytes undergo apoptosis in response to a variety of stimuli, including exposure to UV radiation and gamma-irradiation. While the mechanism by which stress stimuli induce apoptosis is not well understood, we have previously shown that the induction of Fas ligand (FasL) gene expression by environmental stress stimuli is dependent on c-Jun N-terminal kinase (JNK) activation. Using inducible dominant-active (DA) JNK kinase kinase (MEKK1) expression in Jurkat cells, we map a specific MEKK1-regulated response element to positions -338 to -316 of the Fas ligand (FasL) promoter. Mutation of that response element abrogated MEKK1-mediated FasL promoter activation and interfered in stress-induced activation of that promoter. Using electrophoretic mobility shift assays, we demonstrate that activator protein 1 (AP-1) binding proteins, namely, activating transcription factor 2 (ATF2) and c-Jun, bind to the MEKK1 response element. Transient transfection of interfering c-Jun and ATF2 mutants, which lack the consensus JNK phosphorylation sites, abrogated the transcriptional activation of the FasL promoter, demonstrating the involvement of these transcription factors in the regulation of the FasL promoter. Taken together, our data indicate that MEKK1 and transcription factors regulated by the JNK pathway play a role in committing lymphocytes to undergo apoptosis by inducing FasL expression via a novel response element in the promoter of that gene.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Inducible expression of DA-MEKK1 in Jurkat cells leads to constitutive JNK activation and induction of apoptosis. (A) Western blot showing the inducible expression of DA-MEKK1 in stably transfected Jurkat-tTA cells. Jurkat-tTA cells were transfected with 30 μg of cDNA encoding DA-MEKK1 in the pUHD10.3 vector (lanes 1 and 2). Cells in lane 3 were untransfected Jurkat-tTA cells. Following selection in 270 μg of hygromycin/ml for 4 weeks, the cells were grown in the presence (+) or absence (−) of 0.1 μg of tetracycline/ml for 24 h. Total cell lysates from 5 × 106 cells were separated by SDS–10% PAGE and transferred to an Immobilon-P membrane. The membrane was overlaid with 0.1 μg of anti-MEKK1 antibody/ml, followed by HRP-conjugated protein A, and was developed by ECL. (B) In vitro kinase assay showing the constitutive activation of JNK by DA-MEKK1. The transfected cells described above were either left untreated (lanes 1 and 3) or stimulated for 10 min with 100 nM PMA and 1 μg of ionomycin/ml (P+I) at 37°C (lanes 2 and 4), and JNK activity was measured as previously described (14). (C) Cell viability assay in DA-MEKK1-expressing cells and the effect of Fas-Fc fusion protein. DA-MEKK1 Jurkat cells were incubated in the presence or absence of 30 μg of Fas-Fc/ml and grown under tet+ or tet− conditions for 72 h. Cell viability was measured by trypan blue exclusion. Duplicate counts were performed by two independent observers. We have previously shown that in these cells, trypan blue uptake is accompanied by 7AAD uptake and DNA laddering (14). Similar results were obtained in three separate experiments.
FIG. 2
FIG. 2
Immunostaining showing enhanced FasL expression in cells expressing DA-MEKK1. DA-MEKK1 cells, grown under tet+ or tet− conditions for 36 h (A), were stained with anti-FasL (NOK1) MAb, followed by FITC-coupled anti-mouse immunoglobulin, and were analyzed by flow cytometry by using the Cell Quest program (Becton Dickinson). For comparison, Jurkat-tTA cells were either left untreated or stimulated with 100 nM PMA plus 1 μg of ionomycin (Iono)/ml for 12 h (B) and analyzed as above.
FIG. 3
FIG. 3
Regulation of the transcriptional activation of the FasL promoter by DA- and DN-MEKK1. (A) Luciferase assay showing the regulation of the FasL promoter by DA-MEKK1. DA-MEKK1 cells, transiently transfected with 30 μg of FasL-486 luciferase construct, were grown in the presence [Tet(+)] or absence [Tet(−)] of 0.1 μg of tetracycline/ml for 24 h. Cells were left unstimulated or were treated with 100 nM PMA plus 1 μg of ionomycin (Iono)/ml for 8 h. The cells were lysed, and 100 μg of cell lysates was analyzed for luciferase activity. The fold increase in luciferase activity was calculated over the value for unstimulated tet+ cells, which amounted to 6,576 relative light units. These data are representative of four experiments. (B) Luciferase activity showing the effect of DN-MEKK1 on the activation of the FasL promoter by stress stimuli. Jurkat tTA cells were transiently cotransfected with 30 μg of FasL-486 luciferase and 20 μg of DN-MEKK1 subcloned into the pUHD10.3 vector. The cells were grown in the presence or absence of 0.1 μg of tetracycline/ml for 24 h and were stimulated with 200 J of UVR/m2 or 3,300 rads of γ-irradiation in the presence of 30 μM Z-Val-Ala-Asp(OMe)-CH2F (Z-VAD). The cells were lysed 8 h later and analyzed for luciferase activity. Fold increase in luciferase activity was calculated over the value for unstimulated tet+ cells, which amounted to 7,380 relative light units. These data are representative of three experiments.
FIG. 4
FIG. 4
The MEKK1 response element maps downstream of position −335 in the FasL promoter. (A) Schematic representation of the FasL promoter-reporter constructs showing the MEKK1- and NF-AT-responsive elements (RE) in the 486-bp promoter. Serial 5′ deletion mutants were generated as described in Materials and Methods. (B) Comparison of luciferase activity in the FasL promoter deletion mutants. DA-MEKK1 cells were transiently transfected with 30 μg of the 486-bp FasL luciferase construct or its 5′ deletion mutants. Luciferase activity was determined as described above. The fold increase in luciferase activity was calculated over the value of tet+ cells, which amounted to 10,142 relative light units. These data are representative of three experiments.
FIG. 4
FIG. 4
The MEKK1 response element maps downstream of position −335 in the FasL promoter. (A) Schematic representation of the FasL promoter-reporter constructs showing the MEKK1- and NF-AT-responsive elements (RE) in the 486-bp promoter. Serial 5′ deletion mutants were generated as described in Materials and Methods. (B) Comparison of luciferase activity in the FasL promoter deletion mutants. DA-MEKK1 cells were transiently transfected with 30 μg of the 486-bp FasL luciferase construct or its 5′ deletion mutants. Luciferase activity was determined as described above. The fold increase in luciferase activity was calculated over the value of tet+ cells, which amounted to 10,142 relative light units. These data are representative of three experiments.
FIG. 5
FIG. 5
MEKK1 activation induces a mobility shift complex with an oligonucleotide corresponding to positions −336 to −318 of the FasL promoter. (A) EMSA showing the association of c-Jun and ATF2 with the MEKK1-responsive site of the FasL promoter. A total of 5 × 106 Jurkat-tTA cells were either left untreated or stimulated with 100 nM PMA plus 1 μg of ionomycin (Iono)/ml for 3 h. Nuclear extracts (10 μg) were incubated in buffer or pretreated with 0.5 μg of anti-pan-Jun (lane 3), anti-c-Jun (lanes 4 and 11), anti-pan-Fos (lanes 5 and 12), or anti-ATF2 (lane 13) polyclonal antibodies or with nonimmune serum (NIS) (lanes 6 and 10) for 40 min. EMSA was performed by using a 32P-labeled AP-1 oligonucleotide (lanes 1 through 7) or the oligonucleotide corresponding to positions −336 to −318 from the start site of the FasL promoter (lanes 8 through 14). The DNA-binding complexes were separated by 4.5% acrylamide gel electrophoresis. The gel was dried and visualized by autoradiography. (B) EMSA showing that DA-MEKK1 induces DNA shift complexes with the MEKK1-responsive site of the FasL promoter. A total of 5 × 106 Jurkat-tTA cells, stably transfected with DA-MEKK1, were grown in the presence (+) or absence (−) of tetracycline for 24 h. The cells were either left untreated or stimulated with 100 nM PMA plus 1 μg of ionomycin (Iono)/ml for 3 h. Nuclear extracts (10 μg) were incubated with 2 ng of 32P-labeled AP-1 oligonucleotide (lanes 1 through 5) or the oligonucleotide corresponding to the JNK response element (lanes 6 through 10). The DNA-binding complexes were analyzed as described above. (C) EMSA showing the effects of anti-c-Jun and anti-ATF2 on DA-MEKK1-induced shift complexes. EMSA was conducted by using the MEKK1-responsive oligonucleotide together with nuclear extracts from DA-MEKK1-expressing Jurkat cells as described for panel B. Anti-c-Jun, anti-ATF2, and anti-Fos antibodies were incubated together with the nuclear extracts as described above for panel A.
FIG. 5
FIG. 5
MEKK1 activation induces a mobility shift complex with an oligonucleotide corresponding to positions −336 to −318 of the FasL promoter. (A) EMSA showing the association of c-Jun and ATF2 with the MEKK1-responsive site of the FasL promoter. A total of 5 × 106 Jurkat-tTA cells were either left untreated or stimulated with 100 nM PMA plus 1 μg of ionomycin (Iono)/ml for 3 h. Nuclear extracts (10 μg) were incubated in buffer or pretreated with 0.5 μg of anti-pan-Jun (lane 3), anti-c-Jun (lanes 4 and 11), anti-pan-Fos (lanes 5 and 12), or anti-ATF2 (lane 13) polyclonal antibodies or with nonimmune serum (NIS) (lanes 6 and 10) for 40 min. EMSA was performed by using a 32P-labeled AP-1 oligonucleotide (lanes 1 through 7) or the oligonucleotide corresponding to positions −336 to −318 from the start site of the FasL promoter (lanes 8 through 14). The DNA-binding complexes were separated by 4.5% acrylamide gel electrophoresis. The gel was dried and visualized by autoradiography. (B) EMSA showing that DA-MEKK1 induces DNA shift complexes with the MEKK1-responsive site of the FasL promoter. A total of 5 × 106 Jurkat-tTA cells, stably transfected with DA-MEKK1, were grown in the presence (+) or absence (−) of tetracycline for 24 h. The cells were either left untreated or stimulated with 100 nM PMA plus 1 μg of ionomycin (Iono)/ml for 3 h. Nuclear extracts (10 μg) were incubated with 2 ng of 32P-labeled AP-1 oligonucleotide (lanes 1 through 5) or the oligonucleotide corresponding to the JNK response element (lanes 6 through 10). The DNA-binding complexes were analyzed as described above. (C) EMSA showing the effects of anti-c-Jun and anti-ATF2 on DA-MEKK1-induced shift complexes. EMSA was conducted by using the MEKK1-responsive oligonucleotide together with nuclear extracts from DA-MEKK1-expressing Jurkat cells as described for panel B. Anti-c-Jun, anti-ATF2, and anti-Fos antibodies were incubated together with the nuclear extracts as described above for panel A.
FIG. 5
FIG. 5
MEKK1 activation induces a mobility shift complex with an oligonucleotide corresponding to positions −336 to −318 of the FasL promoter. (A) EMSA showing the association of c-Jun and ATF2 with the MEKK1-responsive site of the FasL promoter. A total of 5 × 106 Jurkat-tTA cells were either left untreated or stimulated with 100 nM PMA plus 1 μg of ionomycin (Iono)/ml for 3 h. Nuclear extracts (10 μg) were incubated in buffer or pretreated with 0.5 μg of anti-pan-Jun (lane 3), anti-c-Jun (lanes 4 and 11), anti-pan-Fos (lanes 5 and 12), or anti-ATF2 (lane 13) polyclonal antibodies or with nonimmune serum (NIS) (lanes 6 and 10) for 40 min. EMSA was performed by using a 32P-labeled AP-1 oligonucleotide (lanes 1 through 7) or the oligonucleotide corresponding to positions −336 to −318 from the start site of the FasL promoter (lanes 8 through 14). The DNA-binding complexes were separated by 4.5% acrylamide gel electrophoresis. The gel was dried and visualized by autoradiography. (B) EMSA showing that DA-MEKK1 induces DNA shift complexes with the MEKK1-responsive site of the FasL promoter. A total of 5 × 106 Jurkat-tTA cells, stably transfected with DA-MEKK1, were grown in the presence (+) or absence (−) of tetracycline for 24 h. The cells were either left untreated or stimulated with 100 nM PMA plus 1 μg of ionomycin (Iono)/ml for 3 h. Nuclear extracts (10 μg) were incubated with 2 ng of 32P-labeled AP-1 oligonucleotide (lanes 1 through 5) or the oligonucleotide corresponding to the JNK response element (lanes 6 through 10). The DNA-binding complexes were analyzed as described above. (C) EMSA showing the effects of anti-c-Jun and anti-ATF2 on DA-MEKK1-induced shift complexes. EMSA was conducted by using the MEKK1-responsive oligonucleotide together with nuclear extracts from DA-MEKK1-expressing Jurkat cells as described for panel B. Anti-c-Jun, anti-ATF2, and anti-Fos antibodies were incubated together with the nuclear extracts as described above for panel A.
FIG. 6
FIG. 6
The response element between positions −336 and −318 is critical for MEKK1-mediated FasL promoter activity. (A) Luciferase assay showing the effect of the DA-MEKK1 on the transcriptional activation of the MEKK1-responsive element. DA-MEKK1 cells were transiently transfected with 30 μg of luciferase constructs representing the 486-bp FasL promoter or the triplicated −336-to-−318 site. The cells were grown in the presence or absence of 0.1 μg of tetracycline/ml for 24 h. Cells were either left unstimulated or treated with 100 nM PMA plus 1 μg of ionomycin (Iono)/ml for 8 h. The cells were lysed and analyzed for luciferase activity as for Fig. 3. The fold increase in luciferase activity was calculated based on the value for tet+ cells. Transfection efficiency was monitored by cotransfection of a β-galactosidase-encoding plasmid (CMV-β-Gal). These data are representative of three experiments. (B) Luciferase assay showing the regulation of the FasL promoter by the MEKK1 response element. DA-MEKK1 cells were transiently transfected with 30 μg of the wild-type FasL-486 reporter or the FasLδ−336/−318 construct carrying a mutant MEKK1-responsive element. The cells were grown in the presence or absence of 0.1 μg of tetracycline/ml for 24 h, lysed, and analyzed for luciferase activity. The fold increase in luciferase activity was calculated based on the value for tet+ cells.
FIG. 7
FIG. 7
Distinct response elements mediate the activation of the FasL promoter by the TCR and DA-MEKK1. DA-MEKK1 cells, transiently transfected with 30 μg of FasL−486, FasLδ−336/−318, or FasLδNFAT luciferase constructs, were grown in the presence or absence of 0.1 μg of tetracycline/ml for 24 h. The cells were either left unstimulated or treated with 10 μg of anti-CD3 MAb/ml for 8 h. The cells were lysed and analyzed for luciferase activity. The fold increase in luciferase activity was calculated based on the value for tet+ cells. Transfection efficiency was monitored by cotransfection of a β-galactosidase-encoding plasmid. Similar results were obtained in two experiments.
FIG. 8
FIG. 8
Involvement of c-Jun and ATF2 in the MEKK1-mediated activation of the FasL promoter. (A and C) Luciferase assay showing the effect of mutant c-Jun on the transcriptional activation of the FasL promoter (A) and the triplicated MEKK1-responsive element (C). DA-MEKK1 cells were transiently transfected with 30 μg of FasL−486 or triplicated JNK response element reporter constructs in the presence of 20 μg of either wild-type (WT) or mutant (63/73) c-Jun. The experiment was performed as described for Fig. 3. The intersample variation in transfection efficiency was adjusted by using CMV-β-Gal cotransfection. (B and D) Luciferase assay showing the effect of mutant ATF2 on the transcriptional activation of the FasL promoter (B) and the triplicated MEKK1-responsive element (D). DA-MEKK1 cells were transiently transfected with 30 μg of FasL−486 or triplicated JNK response element reporter constructs in the presence of 20 μg of either wild-type (WT) or mutant (69/71) ATF2. The experiment was performed as described above.
See this image and copyright information in PMC

References

    1. Boldin M P, Goncharov T M, Goltsev Y Y, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell. 1996;85:803–815. - PubMed
    1. Brenner C, Hirsch T, Petit P X, Geuskens M, Kroemer G. A cytofluorometric assay of nuclear apoptosis induced in a cell-free system: application to ceramide-induced apoptosis. Exp Cell Res. 1997;236:397–403. - PubMed
    1. Butterfield L, Storey B, Maas L, Heasley L E. c-Jun NH2-terminal kinase regulation of the apoptotic response of small cell lung cancer cells to ultraviolet radiation. J Biol Chem. 1997;272:10110–10116. - PubMed
    1. Cahill M A, Peter M E, Kischkel F C, Chinnaiyan A M, Dixit V M, Krammer P H, Nordheim A. CD95 (APO-1/Fas) induces activation of SAP kinases downstream of ICE-like proteases. Oncogene. 1996;13:2087–2096. - PubMed
    1. Calnan B J, Szychowski S, Chan F K, Cado D, Winoto A. A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection. Immunity. 1995;3:273–282. - PubMed

Publication types

MeSH terms