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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 1 250-257
Copyright © 2004 by The Endocrine Society

Interleukin-1ß and Tumor Necrosis Factor (TNF)-{alpha} Sensitize Human Thyroid Epithelial Cells to TNF-Related Apoptosis-Inducing Ligand-Induced Apoptosis through Increases in Procaspase-7 and Bid, and the Down-Regulation of p44/42 Mitogen-Activated Protein Kinase Activity

Emese Mezosi, Su He Wang, Saho Utsugi, Laszlo Bajnok, James D. Bretz, Paul G. Gauger, Norman W. Thompson and James R. Baker, Jr.

Center for Biologic Nanotechnology (J.R.B.) and the Departments of Medicine (E.M., S.H.W., S.U., J.D.B., J.R.B.), Physiology (L.B.), and Surgery (P.G.G., N.W.T.), University of Michigan Medical Center, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: James R. Baker, Jr., M.D., University of Michigan Medical Center, 9220 MSRB III, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0648. E-mail: jbakerjr{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Primary thyroid cells are resistant to TNF-related apoptosis-inducing ligand (TRAIL). Previously we showed that the combination of IL-1ß and TNF{alpha} facilitated TRAIL-mediated apoptosis in these cells and enhanced cell surface expression of TRAIL receptors. The aim of this study was to further characterize the mechanism by which these cytokines sensitized primary thyroid cells to TRAIL-mediated apoptosis. IL-1ß and TNF{alpha} increased the concentrations of procaspase-7 and Bid. In contrast, the p44/42 MAPK (Erk) pathway was active in thyroid cells and this activity was significantly decreased after exposure to IL-1ß/TNF{alpha}. A MAPK kinase inhibitor (U0126) could enhance the cytokine-induced sensitization of thyroid cells to TRAIL, reinforcing the inhibitory role of Erk on TRAIL signaling. In conclusion, IL-1ß/TNF{alpha} treatment sensitizes human thyroid cells to TRAIL-mediated apoptosis through increased surface expression of TRAIL receptors, increased expression of procaspase-7 and Bid, and the inhibition of p44/42 MAPK (Erk) pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A RECENTLY CHARACTERIZED member of the tumor necrosis factor family, TNF-related apoptosis-inducing ligand (TRAIL) (1), has importance as it induces apoptosis in many transformed cell lines but not in normal cells (2, 3, 4, 5). Recent studies suggested that a physiologic role of TRAIL is to remove virus-infected and neoplastic cells (6, 7, 8). TRAIL, however, is expressed in a wide variety of normal tissues, suggesting that this pathway is regulated and protective mechanisms exist in normal cells (9, 10, 11, 12). TRAIL induces apoptosis by signaling through either of two membrane death receptors (DRs), DR4 (TRAIL-R1), and DR5 (TRAIL-R2) (13, 14). Three additional TRAIL decoy receptors (DcR) have also been identified: DcR1 (TRID, TRAIL-R3), DcR2 (TRUNDD, TRAIL-R4), and osteoprotegerin (15, 16, 17, 18). These receptors cannot transduce an apoptotic signal but can competitively block signal transduction. This information suggests that TRAIL signaling is complex.

During DR signaling, the intracellular death domains of these receptors bind to an adapter protein that activates an apical caspase (19), forming a complex known as the death-inducing signaling complex. It is generally accepted that TRAIL DRs recruit Fas-associated death domain and caspase-8 (20, 21, 22), activating the caspase signaling cascade. Apoptotic signaling by TRAIL has been mainly investigated in cancer cell lines, in which the cleavage of Bid by caspase-8 and the loss of mitochondrial membrane potential are seen after TRAIL administration (23, 24, 25). Besides initiating a caspase-signaling cascade, TRAIL receptors also activate nuclear factor-{kappa}B (NF-{kappa}B) (26, 27).

Signaling in the TNF family of DRs is reported to be controlled at several levels including the expression of DRs and DcR, by inhibitors of death-inducing signaling complex formation and apical caspase activation, and through caspase inhibitors including inhibitors of apoptosis proteins (IAPs) and bcl-2 types of proteins (28, 29). Most recently the rapid activation of p44/p42 MAPK (also called as Erk1/2) was reported to occur in response to death ligands, which protects the cell from apoptosis by inhibiting the caspase effector machinery (30). In various cell types, resistance to TRAIL appears to be mediated by distinct mechanisms. These include an absence of cell surface expression of DRs, the expression of DcR, or intracellular inhibitors (31).

Normal primary thyroid epithelial cells (TECs) are also resistant to apoptosis induction by TRAIL (32). However, we identified physiologically relevant conditions in which TRAIL can induce apoptosis in TECs in vitro (33). IL-1ß, alone and in combination with TNF{alpha}, sensitized TECs to TRAIL-induced apoptosis, whereas the addition of interferon (IFN){gamma} made these cells resistant to TRAIL (33). This pattern of TRAIL sensitivity was correlated to a degree with enhanced cell surface expression of DR5 and DcR1, and the death signal was mediated by DR5 (32). In the present study, we further investigated the mechanism of TEC sensitization to TRAIL-induced apoptosis by a specific combination of inflammatory cytokines. Our studies indicate TNF{alpha}/IL-1ß sensitizes TECs to TRAIL through the up-regulation of Bid associated with down-regulation of the p44/42 MAPK pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

This study and tissue procurement was approved by the University of Michigan Institutional Review Board. Normal thyroid tissue was obtained from patients at thyroidectomy from the uninvolved, contralateral lobes of thyroids resected for tumors. Seventeen normal thyroid samples were used in the study. Histological examination of adjacent paraffin-embedded tissue was made in every case to confirm the normal structure of thyroid samples. All excised tissues were prepared for cell culture as previously described (34). The primary cultures were passed in CellGro Complete media (Mediatech, Herndon, VA) supplemented with 20% NuSerum IV (Collaborative Biomedical Products, Bedford, MA), 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mIU/ml bovine TSH (Sigma, St. Louis, MO). Nuserum IV is a partly artificial serum, which contains 25% fetal calf serum, so the final concentration of fetal calf serum in the culture medium was 5%. The purity of thyroid cell population was verified by staining with anticytokeratin 18 antibody (a marker for epithelial cells), quantitated by flow cytometry, and only cultures that were more than 90% cytokeratin positive were used for experiments.

Cytokines, TRAIL, and MAPK kinase (MEK) inhibitor treatment

Primary thyroid cells were treated for four days with 50 ng/ml TNF{alpha} (Collaborative Biomedical Products), 50 U/ml IL-1ß (Sigma) or 100 U/ml IFN{gamma} (Roche Molecular Biochemicals, Indianapolis, IN). Cells were then treated overnight with 800 ng/ml TRAIL. The concentration dependence and time course of the TRAIL activity were published in our previous paper (33), with a TRAIL concentration that resulting in the maximum cell death being used in this study. TRAIL (a kind gift of A. Chinnaiyan, Department of Pathology, University of Michigan, Ann Arbor, MI) was affinity purified as described (35) from lysates of bacteria transformed with the plasmid pET15b-His-FLAG-TRAIL. The purity of the TRAIL preparation was confirmed by silver stained SDS-PAGE and limulus amoebocyte lysate assay was performed to assure there was no contamination with lipopolysaccharide (35). The p44/p42 MAPK was inhibited by the MEK inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene) (Promega, Madison, WI) added 30 min before TRAIL administration.

Determination of cell viability

Cell viability was measured 20 h after TRAIL administration by staining the cells with fluorescein diacetate and propidium iodide, followed by flow cytometric analysis as described by Killinger (36).

Flow cytometric determination of cytokeratin 18

Total cytokeratin 18 expression was determined as described by the vendor (Chemicon, Temecula, CA) of the antibody. Briefly, trypsinized cells were washed and fixed in ice-cold methanol for 30 min and incubated for 15 min in blocking buffer (2% fetal calf serum, 0.1% Tween 20 in PBS). Blocking buffer was replaced with anticytokeratin 18 antibody diluted to 2.0 µg/ml and incubated for 1 h. Cells were then re-suspended in antimouse fluorescein isothiocyanate-conjugated antibody (Jackson ImmunoResearch, West Grove, PA) diluted to 1:100 in blocking buffer for 30 min. A mouse IgG1 (MOPC21, Sigma) was used as an isotype-matched control antibody.

Flow cytometric determination of DR5 surface expression

Cytokine-treated thyroid cells were made nonadherent by incubation with 0.265 mM EDTA in PBS and incubated for 15 min in blocking solution (2% normal horse serum in PBS) at 4 C. Goat polyclonal antihuman DR5 antibody (R&D Systems, Minneapolis, MN) was diluted in blocking solution to 5 µg/ml, and cells were incubated with the antibody for 20 min at 4 C. Then cells were incubated with fluorescein isothiocyanate-conjugated antigoat F(ab')2 fragment (Jackson ImmunoResearch) at 1:100 dilution for 20 min, at 4 C. Purified goat IgG (R&D Systems) served as control. Cells (2 x 104 total) were acquired for each sample and quantitated on a FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

RNase protection assay

RNA was isolated from cells using Trizol Reagent according to the manufacturer’s protocol (Gibco BRL, Grand Island, NY). RiboQuant MultiProbe RNase protection assay system (PharMingen, San Diego, CA) was used for the detection and quantitation of bcl-2 family mRNA expression (hAPO-2b template set). 32P-labeled antisense RNA probes were prepared and hybridized with 5 µg total RNA from primary cultures of thyrocytes. After hybridization, the samples were subjected to RNase treatment followed by purification of RNase-protected probes. The protected probes were resolved on a 5% denaturing polyacrylamide gel, and transcripts were quantified by autoradiography followed by densitometry (Quantity One, Bio-Rad Laboratories, Hercules, CA). The relative signal intensity was corrected for RNA loading by comparison with the glyceraldehyde-3 phosphate dehydrogenase band intensity for each sample.

Immunoblot analysis

Cytokine pretreated and untreated primary thyroid cells were lysed in Triton X lysis buffer (150 mM NaCl; 10 mM Tris, pH 7.4; 5 mM EDTA; 1% Triton X-100), radioimmunoprecipitation assay lysis buffer [1% Nonidet-P40 (NP-40), 0.5% sodium deoxycholate, 0.1% sodium-dodecyl sulfate in PBS] or Chaps buffer [50 mM 1,4-piperazinediethanesulfonic acid/HCl (pH 6.5), 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 5 mM dithiothreitol] with protease inhibitors (Complete, Roche Molecular Biochemicals), depending on the primary antibodies. Insoluble material was removed by centrifugation and supernatants were stored frozen at -20 C until used for immunoblot analysis. Total protein concentration was determined by BCA protein assay kit (Pierce Chemical Co., Rockford, IL) and equivalent amounts of each sample were electrophoretically separated on a 12.5% polyacrylamide gel and transferred to polyvinyl difluoride membrane. Goat polyclonal anti-DR5 (R&D Systems), rabbit polyclonal anti-bcl-X, anti-bax, mouse monoclonal anti-Bid, anti-bak and hamster monoclonal anti-bcl-2 antibodies (BD PharMingen), rabbit polyclonal anti-caspase-9, anti-caspase-7, anti-caspase-3 (cleaved), mouse monoclonal anti-caspase-8 (clone 1C12) (Cell Signaling Technology, Beverly, MA) and anti-caspase-10 (MBL, Naka-ku, Nagoya, Japan) were used according to the manufacturer’s protocol. The vendor of the rabbit polyclonal anti-p44/p42 MAPK, phospho-p44/p42 MAPK, phospho-Raf, phospho-MEK1/2, and phospho-ribosomal S6 kinase (p90RSK) antibodies was the Cell Signaling Technology. Mouse monoclonal anti-actin (Ab-1) antibody was provided by Oncogene Research Products (San Diego, CA). The results were visualized by ECL (Amersham, Arlington Heights, IL) followed by autoradiography.

Detection of mitochondrial activation

The mitochondrial activation in response to TRAIL was examined by ApoAlert mitochondrial membrane sensor kit (Clontech Laboratories, Palo Alto, CA), according to the manufacturer’s protocol and was analyzed by flow cytometry.

Nuclear extract preparation

Thyroid cells were treated for 30 min with 50 ng/ml TNF{alpha}, 50 U/ml IL-1ß, or the combination of these cytokines; washed in PBS; scraped in hypotonic lysis buffer [20 mM Tris (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 1 mM dithiothreitol, 1 mM Na-ortho-vanadate, 30 mM b-glycerol-phosphate, protease inhibitors (Complete, Roche Molecular Biochemicals)], and transferred on ice to a Dounce homogenizer. NP-40 was added in 0.1% final concentration, and cell membranes were broken with 20 strokes. The crude nuclear pellet was resuspended in hypotonic lysis buffer, spun down, and resuspended in nuclear storage buffer (40% glycerol; 50 mM Tris, pH 8.0; 3 mM MgCl2; 1 mM dithiothreitol; 1 mM Na-ortho-vanadate; 30 mM b-glycerol-phosphate; and protease inhibitors). After a spin for 20 sec at 8000 rpm, nuclei were resuspended in 10 volumes of NUN buffer (1.1 M urea, 0.33 M NaCl, 1.1% NP-40, 17.5 mM HEPES, pH 7.6), 1 mM dithiothreitol, 1 mM Na-ortho-vanadate, 30 mM b-glycerol-phosphate, protease inhibitors) and incubated on ice for 30 min. After spin at 14,000 rpm for 10 min, the supernatant was transferred to a fresh microfuge tube, and glycerol was added to 10% final concentration. Total protein concentration was quantitated by BCA protein assay kit (Pierce Chemical Co., Rockford, IL), and the nuclear extract was stored at -70 C.

EMSA

The activation of NF-{kappa}B in response to TNF{alpha} and IL-1ß was evaluated by NF-{kappa}B/Rel family Gelshift kit (Active Motif Inc., Carlsbad, CA), according to the manufacturer’s protocol. Briefly, the NF-{kappa}B consensus oligonucleotid probe (5'-GGGACTTTCC-3') was radiolabeled by 32P-ATP and purified. The extract premix (using 5 µg nuclear extract) and probe premix were prepared separately. Binding reactions proceeded for 20 min at 4 C. The binding of NF-{kappa}B dimers to the labeled oligo probe was detected on 5% polyacrylamide gel in Tris-glycine electrophoresis buffer (50 mM Tris, pH 8.3; 380 mM glycine; 2.1 mM EDTA). Gels were dried and subjected to autoradiography. The nuclear extract of phorbol, 12-myristate, 13 acetate-treated Jurkat cells (Active Motif Inc.) was used as a positive control. Wild-type cold and mutant oligonucleotides present in excess served as negative controls.

Data analysis

Flow cytometry data were analyzed by WinMDI 2.8 (Joseph Trotter, http://facs.scripps.edu/). Densitometric quantitation of autoradiograms was done using Quantity One (Bio-Rad Laboratories). Statistical analysis was performed using Student’s t test and {chi}2-test. Where significant differences are presented in representative experiments, the results are consistent with data from experiments using other tissues.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased cell surface expression of DR5 in TRAIL-sensitive thyroid cells

We further characterized the sensitization of primary human thyroid epithelial cells to TRAIL by the combination of IL-1ß and TNF{alpha} in this study (Fig. 1AGo). This effect required a long-term pretreatment with IL-1ß/TNF{alpha}, with maximum sensitization observed on the fourth day of cytokine pretreatment, which was followed by a rapid cell death in response to TRAIL (33). The apoptotic signal of TRAIL was transmitted by DR5 and the sensitization of TECs correlated with the increased total and cell surface expression of DR5 (33, 37). This was supported by cell culture experiments, in which IL-1ß/TNF{alpha} treatment resulted in a 3-fold increase in total and 4-fold increase in cell surface expression of DR5 (Fig. 1Go, B and C), both rising from low basal DR5 expression (Fig. 1CGo).



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  		FIG. 1. IL-1ß/TNF{alpha}-induced sensitization of primary thyroid cells to TRAIL correlates with the increased total and cell surface expression of DR5. Primary thyroid cells were treated with 50 ng/ml TNF{alpha} and 50 U/ml IL-1ß for 4 d or left untreated. A, After overnight exposition to TRAIL, cell death was assayed by fluorescein diacetate/propidium iodide staining and 10,000 cells/sample were analyzed by flow cytometry. Data are presented as mean ± SD of triplicate measurements. Asterisk denotes conditions where P < 0.05 ({chi}2 test), compared with the control samples (no cytokines and cytokines without TRAIL). B, DR5 protein was determined by immunoblot analysis, under nonreducing conditions. The blots were subsequently stripped and probed with antiactin antibody. The autoradiograms were quantified by densitometry; the DR5 expression after cytokine treatment was normalized to actin signal and the result from untreated cells. C, Cell surface expression of DR5 was measured by flow cytometry. A representative histogram of anti-DR5 antibody-specific fluorescence for untreated cells (open curve, solid line) and IL-1ß/TNF{alpha}-treated cells (open curve, spotted line) is presented and compared with control goat IgG (filled curve). To quantify the increase in cell surface expression of DR5 after cytokine treatment, the mean fluorescence intensity of the control antibody was subtracted from the anti-DR5 antibody-specific mean fluorescence intensity and was normalized to control cells. *, P < 0.05, compared with the control sample (Student’s t test). These data are representative of results from five independent experiments, each using thyroid cultures from different patient samples.

 
The concentration of procaspase-7 but not other procaspases was regulated by IL-1ß/TNF{alpha}

The signaling pathway of TRAIL-induced apoptosis in normal TECs was investigated with particular attention being paid to changes in proximal and effector caspase protein concentrations in response to IL-1ß/TNF{alpha}. The concentrations of procaspase-10, -8, -9, and -3 were not influenced by the cytokine exposure; however, the level of procaspase-7 was up-regulated by this treatment (Fig. 2Go).



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  		FIG. 2. The effect of IL-1ß/TNF{alpha} treatment on the expression of procaspases. Duplicate samples of normal thyroid cells were treated with IL-1ß/TNF{alpha} as in Fig. 1Go and lysed in Triton X lysis buffer. Immunoblot analysis was performed with the indicated antibodies. The autoradiograms were quantified by densitometry and normalized to results in untreated cells. *, P < 0.05, compared with the control samples (Student’s t test). These are representative results from three independent experiments, each using thyroid cultures from different patient samples.

 
The concentrations of bcl-2 family member mRNA and proteins were altered in response to IL-1ß/TNF{alpha} treatment

Expression of the bcl-2 family mRNA were investigated by RNase protection assay. It appeared that both pro- and antiapoptotic bcl-2 proteins were regulated by IL-1ß/TNF{alpha}. A prominent increase was found in the level of the mRNA for Bid, which demonstrated a 7-fold up-regulation (Fig. 3AGo). A dramatic increase in Bid protein was also identified by immunoblot analysis, which detected a 20-fold increase in the Bid expression (Fig. 3BGo). In contrast, only a moderate increase was observed in the level of message for the antiapoptotic protein bcl-X (Fig. 3BGo), whereas mRNA for Bfl-1, another antiapoptotic bcl-2 family protein, was undetectable in control cells and up-regulated after cytokine treatment. Bfl-1 is induced by the activation of NF-{kappa}B, which occurs through TNF{alpha} and IL-1ß signaling.



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  		FIG. 3. The influence of cytokine treatment on the concentration of bcl-2 family members. A, RNase protection assay using the hAPO-2b template set was performed on 5 µg total RNA isolated from untreated and cytokine-treated thyroid cells. The mRNA expression of the indicated bcl-2 family members was quantified by densitometry and normalized to glyceraldehyde-3 phosphate dehydrogenase band and signal intensity in untreated cells. Bfl-1 mRNA was induced by the cytokine treatment, but because the basal value was zero, it cannot be expressed as a percentage of basal value. B, The protein expression of the bcl-2 family members was assayed by immunoblot analysis from Triton X lysates of thyroid cells. Cells were treated and assayed in duplicates. The autoradiograms were evaluated by densitometry and normalized to actin and protein expression in control cells. This experiment is representative of results from independent experiments using thyroid cells from three normal thyroid tissues. *, P < 0.05, compared with the control samples (Student’s t test).

 
Bid has a central role in apoptosis signaling; after being cleaved by caspase-8, it is transported to the mitochondria and is responsible for mitochondrial activation (38). Given this and the evidence of Bid up-regulation by TNF{alpha} and IL-1ß, we further investigated the regulation of Bid in these cells. We previously reported that IFN{gamma} protects cytokine-sensitized TECs from TRAIL (33). The concentration of Bid was increased by both TNF{alpha} and IL-1ß but decreased by IFN{gamma} (Fig. 4AGo). The role of increased Bid expression in the sensitization of thyroid cells to TRAIL was also supported by the time course of Bid up-regulation (Fig. 4BGo). The sensitization of TECs by IL-1ß/TNF{alpha} treatment started on the second day, and the maximum effect was observed on the fourth day (33), which correlated with the up-regulation of Bid concentration (Fig. 4BGo). TNF{alpha} and IL-1ß are known inducers of NF-{kappa}B; however, the activation of NF-{kappa}B in thyroid cells in response to these cytokines has not been investigated previously. The rapid activation of NF-{kappa}B was documented by EMSA after the exposure of thyroid cells to TNF{alpha} and IL-1ß (Fig. 4CGo).



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  		FIG. 4. Regulation of Bid expression by cytokines and activation of NF-{kappa}B. A, Primary thyroid cells were treated for 4 d with the indicated cytokines, and Bid expression was measured by immunoblot analysis. After densitometry, the signal intensity was normalized to actin and Bid expression in control cells. *, P < 0.05, compared with the control sample (Student’s t test). This experiment is representative of three independent experiments. B, Thyroid cells were treated with IL-1ß/TNF{alpha} for the indicated time, and Bid concentration was assayed as above. This experiment is representative of results from independent experiments using thyroid cells from three normal thyroid tissues. C, After 30 min of incubation with the indicated cytokines, nuclear extract of thyroid cells was prepared, and the activation of NF-{kappa}B was detected by EMSA. Phorbol, 12-myristate, 13 acetate-treated Jurkat cells were used as positive, wild-type cold oligo in excess as negative controls. The retardation of electrophoretic mobility is shown by arrow. This experiment is representative of 10 independent experiments.

 
The TRAIL signaling pathway in cytokine-sensitized TECs

To determine whether the TRAIL signaling pathway induces apoptosis in normal TECs through activation of proximal and effector caspases, we examined the cleavage of these proteins. Caspase-8, caspase-7, and caspase-3 were involved in the induction of apoptosis (Fig. 5AGo), and this activity was observed within 1 h of TRAIL activation. In contrast, no activation of any caspase was detected in TECs not exposed to cytokines,. This supports that TRAIL signal transduction in TEC cells is blocked before caspase-8 activation. A gradual decrease in the concentration of full-length Bid in response to TRAIL is due to the cleavage of Bid by caspase-8 (Fig. 5AGo). As expected, after Bid cleavage mitochondria were activated in the course of TRAIL-induced apoptosis, indicated by the loss of mitochondrial membrane potential (Fig. 5BGo). No mitochondrial activation was seen in control cells after TRAIL treatment (data not shown).



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  		FIG. 5. TRAIL signaling in primary thyroid cells. A, Cells were treated for 4 d with IL-1ß/TNF{alpha} or left untreated and exposed to TRAIL for the indicated time. The activation of caspases was determined by immunoblot analysis using antibodies specific for the active forms of enzymes. Bid was detected with an antibody recognizing the full-length Bid. Actin is shown for comparison of protein loading. B, IL-1ß/TNF{alpha} pretreated cells were exposed to TRAIL for the indicated time, and the mitochondrial activation was detected by ApoAlert mitochondrial membrane sensor kit, using flow cytometric analysis. Results are presented as mean ± SD of triplicate measurements. Asterisk denotes conditions in which P < 0.05 ({chi}2 test), compared with the control sample. These experiments are representative of results from five independent measurements using different patient samples.

 
The involvement of p44/p42 MAPK in TRAIL signaling

It has been reported that p44/p42 MAPK (Erk) has a dominant protective effect over apoptotic signaling from DRs (30). Therefore, the activity of Erk in primary thyroid cells and the effect of cytokines on Erk activity were investigated. Erk activity was detected in untreated TECs, and this was decreased to undetectable levels by IL-1ß/TNF{alpha} treatment (Fig. 6AGo). TRAIL-induced Erk activation was observed in both control and cytokine-pretreated cells (Fig. 6AGo), but the kinetics of Erk activation was delayed in IL-1ß/TNF{alpha}-treated cells (Fig. 6AGo). Cytokine inhibition of the Erk signaling pathway occurred at least at the level of Raf because the activation of Raf, MEK, Erk, and p90RSK (ribosomal S6 kinase) were all significantly decreased in the presence of IL-1ß/TNF{alpha} (Fig. 6BGo). The addition of the MEK (MAPK kinase) inhibitor U0126 did not in and of itself sensitize control thyroid cells to TRAIL-induced apoptosis (Fig. 6CGo) but did increase the sensitivity of cytokine pretreated TECs to TRAIL in a concentration-dependent manner (Fig. 6DGo).



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  		FIG. 6. The role of p44/p42 MAPK activation in TRAIL signaling. A, Cells were treated for 4 d with IL-1ß/TNF{alpha} or left untreated and then subsequently exposed to TRAIL for the indicated time. The activation of p44/p42 MAPK was measured by an antibody specific for the phospho-p44/p42 MAPK. The level of p44/p42 MAPK (inactive form) is shown for comparison. B, The activity of p44/p42 MAPK pathway was determined by antibodies specific for the phosphorylated forms of Raf, MEK, MAPK, and p90RSK. The level of p44/p42 MAPK (inactive form) is shown for comparison and proving the equal protein loading. C, Thyroid cells were exposed to MEK inhibitor at the indicated concentrations 30 min before TRAIL administration. Cell viability was assayed by fluorescein diacetate/propidium iodide staining and 10,000 cells/sample were evaluated by flow cytometry. D, IL-1ß/TNF{alpha} pretreated thyroid cells were exposed to MEK inhibitor at the indicated concentrations 30 min before TRAIL administration. Cell viability was assayed as above. Asterisk denotes conditions in which P < 0.05 ({chi}2 test), compared with the control samples. These are representative results of independent experiments using three normal thyroid tissues.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One of the major goals of apoptosis research is to understand the molecular mechanism of apoptosis signaling. This will identify novel targets for cancer therapy and for the modulation of autoimmune diseases. TRAIL is a promising anticancer agent, and clinical trials with TRAIL are expected in the near future (5). The complexity of TRAIL signaling is apparent from its ubiquitous expression and the facts it has at least five receptors and it can display selective apoptotic effects (39). The signaling events that mediate the tumor selectivity of TRAIL and the functions of various TRAIL receptors are not well characterized. An unexpected sensitization of normal cells to TRAIL-induced apoptosis under physiological conditions could result in serious side effects during cancer therapy involving TRAIL. Therefore, resolving the mechanisms involved in the signaling of apoptosis in normal cells is important.

We determined that a combination of IL-1ß and TNF{alpha} facilitate TRAIL-mediated apoptosis in normal human thyroid cells. IL-1ß/TNF{alpha} treatment results in enhanced expression of TRAIL receptors, increased expression of procaspase-7, considerable up-regulation of the proapoptotic bcl-2 family member Bid, and a significant decrease in the activity of the p44/p42 MAPK pathway. TRAIL was apparently able to induce apoptosis by the activation of caspase-8, caspase-7, and caspase-3. Bid was cleaved in this process and the mitochondrial pathway was subsequently involved in TRAIL-mediated apoptosis of TECs. This is the first description of the regulation of TRAIL signaling in primary, normal thyroid cells, and it demonstrates that Bid expression is extensively regulated by inflammatory cytokines.

In our previous work, we examined the intracellular inhibitors of TRAIL signaling and did not identify a mechanism responsible for the cytokine-induced sensitization of TECs to TRAIL (33, 37). FLIP and X-linked integrin-associated protein were not regulated by cytokines, whereas cIAP-1 and cIAP-2 were up-regulated by IL-1ß/TNF{alpha}, probably as a result of NF-{kappa}B activation (37). The higher concentration of IAPs after exposure to cytokines was paradoxical because IAPs are expected to provide increased protection against the induction of apoptosis. However, the activation of the mitochondrial pathway during TRAIL-mediated apoptosis gives an explanation why the up-regulation of IAPs is insufficient to prevent cell death. The release of Smac/DIABLO from the mitochondria results in the removal of IAPs from caspases by competitive binding and neutralizes the protective effects of increased IAP expression (40, 41). Thus, increased IAP expression would not be effective in blocking TRAIL-mediated apoptosis once cells are exposed to IL-1ß/TNF{alpha} and the mitochondrial pathway is activated.

Proapoptotic proteins also appear to be involved in TRAIL signaling because the increased cell surface expression of DR5 has a role in sensitizing cells to TRAIL-mediated apoptosis. However, increasing evidence supports a central role for Bid in the signaling of DR-mediated apoptosis (38). Bid is activated by caspase-8 and translocates to the mitochondria, inducing cytochrome-c release, which in turn activates downstream caspases (42). Bid-deficient mice are resistant to Fas-mediated apoptosis of hepatocytes (43). Most recently the highly variable expression of Bid in human tissues was published with prominent Bid expression in several types of short-lived cells, and overexpression of this protein made tumors sensitive to apoptosis (44). The low level of Bid expression in normal thyroid cells may be related to the long turnover time of these cells (45) and may contribute to the apoptosis resistance of untreated TECs. The massive increase in Bid expression in TECs in response to TNF{alpha} and IL-1ß is likely due to the activation of NF-{kappa}B because these cytokines are known NF-{kappa}B inducers, and regulation of Bid expression was found to be NF-{kappa}B dependent in rat hepatocytes (46). We documented the activation of NF-{kappa}B by TNF{alpha} and IL-1ß in TECs and NF-{kappa}B was maintained in an active state during the 4 d treatment with cytokines. In contrast, the mechanism of decreased Bid expression by IFN{gamma} is unknown but does correlate with decreased apoptotic potential in TECs. Thus, although it is unclear that Bid is entirely responsible for the cytokine effect, it does appear centrally involved in the regulation of this phenomenon.

We also identify the unique finding that the p44/p42 MAPK pathway is active in cultured normal TECs and its activity is decreased to undetectable level in response to IL-1ß/TNF{alpha}. The reason for this activation is unclear but may result from serum in tissue culture. Despite this, the inactivation of p44/p42 MAPK pathway may have an important role in the process of TRAIL sensitization. The protective effect of Erk activation, if present in vivo, is important for all DR-mediated apoptosis including TRAIL signaling (30). The activation of p44/p42 MAPK inhibits the cleavage of caspase-8, but the exact mechanism of this effect is unknown (30, 47). Similarly, the activation of Erk by DRs is also not well understood. FLIP and 14–3-3 proteins have been suggested as candidates for a role in Erk activation in response to the activation of DRs (48, 49). In the present study, the inhibition of Erk activation alone did not sensitize the untreated TECs to TRAIL, suggesting that multiple protective mechanisms exist in normal cells. However, in IL-1ß/TNF{alpha}-treated cells, the MAPK activity was undetectable; the activation of MAPK in response to TRAIL was delayed but still provided a certain degree of protection because TECs were further sensitized to TRAIL by a MEK inhibitor. In this regard, the nature of the cell death that TRAIL induces, with the activation of effector caspases within 1 h of exposure to the ligand, is remarkable. The speed of this process may help to explain how the insufficient or delayed MAPK activation can facilitate the susceptibility of TECs to TRAIL. Studies examining the role of this pathway and its activation in vivo, potentially in animal models of thyroid disease, will provide information of its biological significance.

In summary, apoptosis signaling in response to TRAIL is blocked before the activation of caspase-8 in normal human thyroid cells. The susceptibility of TECs to TRAIL after IL-1ß/TNF{alpha} treatment correlates with increased expression of DR5, procaspase-7, and Bid and a decrease in the activity of p44/p42 MAPK pathway. The mitochondrial pathway is therefore involved in TRAIL-induced apoptosis in cytokine-exposed cells. These observations may help to understand how the TRAIL-induced apoptosis may be involved in cellular destruction in autoimmune diseases when IL-1ß and TNF{alpha} cytokines are present in the thyroid (50). It also may provide insights into TRAIL regulation in other cell types, especially cancer cells or other endocrine cells involved in organ-specific autoimmunity (51). The sensitization of normal human cells to TRAIL by physiologically relevant factors also implies that a full understanding of TRAIL signaling is required to clarify its therapeutic potential for cancer or other diseases.


   Acknowledgments
 
We gratefully acknowledge Dr. A. Chinnaiyan for the endotoxin-free human recombinant TRAIL.


   Footnotes
 
This work was supported by National Institutes of Health Grants R01 A137141 and P60DK20572.

Abbreviations: cFLIP, Cellular FADD-like IL-1ß converting enzyme inhibitory protein; DcR, decoy receptor; DR, death receptor; IAP, inhibitor of apoptosis protein; IFN, interferon; MEK, MAPK kinase; NF-{kappa}B, nuclear factor {kappa}B; NP-40, Nonidet-P40; p90RSK, ribosomal S6 kinase; TEC, thyroid epithelial cell; TRAIL, TNF-related apoptosis-inducing ligand.

Received April 21, 2003.

Accepted October 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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