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Endocrinology, doi:10.1210/en.2007-0126
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Endocrinology Vol. 148, No. 10 4844-4852
Copyright © 2007 by The Endocrine Society

Microarray Analysis of Cytokine Activation of Apoptosis Pathways in the Thyroid

Su He Wang, Mary Van Antwerp, Rork Kuick, Paul G. Gauger, Gerard M. Doherty, Yang Yi Fan and James R. Baker, Jr.

Departments of Internal Medicine (S.H.W., M.V.A., Y.Y.F., J.R.B.) and Surgery (P.G.G., G.M.D.) and Comprehensive Cancer Center (R.K.), Medical School, University of Michigan Medical Center, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: James R. Baker, Jr., M.D., Department of Medicine, University of Michigan Medical School, 9240 MSRB III, Ann Arbor, Michigan 48109-0648. E-mail: jbakerjr{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been suggested that Fas-mediated apoptosis plays an important role in the pathogenesis of autoimmune thyroid diseases. Our previous studies have demonstrated that normal primary thyroid epithelial cells are resistant to Fas-mediated apoptosis, but the resistance can be overcome by pretreatment with a combination of interferon-{gamma} (IFN-{gamma}) and IL-1ß. To understand the molecular mechanism responsible for the IFN-{gamma}/IL-1ß effects, we profiled changes in the transcription induced by these two cytokines in normal human thyroid cells, using cDNA microarrays. We found that IFN-{gamma}/IL-1ß showed a significant increase in apoptosis-related genes such as inducible nitric oxide synthase (iNOS), receptor-interacting protein 2 (RIP2), and caspases 10. These increases were confirmed by other methods, including real-time PCR and Western blot. Furthermore, the sensitization of primary thyroid epithelial cells to Fas-mediated apoptosis by IFN-{gamma}/IL-1ß was significantly blocked by a general caspase inhibitor, z-VAD, or by the combination of two specific individual caspase inhibitors. In addition, our results showed that IFN-{gamma}/IL-1ß enhance p38 MAPK phosphorylation and that SB 203580, a p38 MAPK inhibitor, can inhibit IFN-{gamma}/IL-1ß-induced p38 MAPK phosphorylation. SB 203580 also significantly prevented cytokine-induced iNOS expression and caspase activation and thus blocked Fas-mediated apoptosis of thyroid cells sensitized by IFN-{gamma}/IL-1ß. In conclusion, our data suggest that both p38 MAPK and iNOS are involved in IFN-{gamma}/IL-1ß-induced sensitization of the thyroid cells to Fas-mediated apoptosis via the activation of caspases 3, 7, and 10 and that this pathway may be further activated by BID. This hints that inflammatory cytokines regulate death-receptor-mediated apoptosis at multiple points in the process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOPTOSIS IS A PROCESS of cellular self-destruction observed in all eukaryotes. This process has also been called programmed cell death because it requires controlled gene expression, which is activated in response to a variety of external or internal stimuli. Receptor-mediated apoptosis involves cell activation through the engagement of cell surface death receptors with subsequent intracellular signaling initiated by the interaction. It has been proposed that receptor-mediated apoptosis plays an important role in a number of autoimmune disorders including the pathogenesis of Hashimoto’s thyroiditis (1). Fas is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on the thyroid cells. Normal thyroid cells are resistant to Fas-mediated apoptosis but have been shown to be sensitized to Fas-induced cell death by treatment with a combination of interferon-{gamma} (IFN-{gamma}) and IL-1ß or TNF-{alpha} (2, 3, 4). Although these cytokines are known to be present in the thyroid gland during thyroiditis, the mechanism of this sensitization is still unclear.

The signal pathway of Fas-mediated apoptosis in cells is believed to transduce through one of two general cell types, type I or type II. In general, mesenchymal cells belong to type I cells, whereas epithelial cells are type II cells (5). A recent study has revealed that internalization of the Fas receptor is required to initiate Fas-mediated apoptosis in type I cells (6). Internalized Fas recruits an adapter molecule, Fas-associated death domain (FADD), which further recruits the inactive procaspase-8 and/or -10 and assembles the death-induced signaling complex (DISC). After engagement of Fas, type I cells exhibit rapid receptor internalization and form bulk quantities of DISC, whereas type II cells are more reliant on the expansion of the mitochondrial pathway and display slower and lower amounts of DISC assembly. Therefore, mitochondrial activation is essential in type II cells, and not surprisingly, apoptosis in type II cells can be blocked by antiapoptotic bcl-2 family members (7, 8). Also, the activation of the MAPK and nuclear factor (NF)-{kappa}B signaling pathway responds to death ligands (9, 10). MAPKs, a family of protein kinases, play a crucial role in relaying signals from the plasma membrane to the nucleus. They are activated by a wide range of proliferation- or differentiation-inducing signals, being strongly activated by agonists such as polypeptide growth factors and tumor-promoting phorbol esters but more weakly by stress stimuli. Thus, the induction and regulation of apoptosis in type II cells is very complex.

Our studies have demonstrated that thyroid epithelial cells can be sensitized to Fas-mediated apoptosis by the combination of IFN-{gamma} and IL-1ß or TNF-{alpha} in vitro (2, 3) and also in vivo (4). The effect of the cytokine combination on the sensitization of thyroid cell to Fas-mediated apoptosis may be through the facilitation of receptor internalization, alternation of bcl-2 family members, and inactivation of the NF-{kappa}B signal pathway. Although these effects are likely involved in the regulation of apoptosis, the effects of cytokines on thyroid cells have not been studied in detail. This is likely because there are too many molecules affected by cytokines in target cells to be simultaneously analyzed by conventional methods. The cDNA microarray technique, which is a powerful tool for investigating the differential expression of thousands of genes simultaneously, meets this challenge. Thus, we used the cDNA microarrays to screen for molecules induced by the cytokines and explored the possibility that these molecules are relevant in the regulation of Fas-mediated apoptosis in thyroid cells. Therefore, the purpose of the current study was to define the complex regulation of the Fas signaling pathway and elucidate the mechanism of cytokine-induced sensitization to Fas-mediated apoptosis in thyroid cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Normal thyroid tissue was obtained during thyroidectomy from patients from the uninvolved, contralateral lobes of thyroids resected for tumors. All excised tissues were prepared for cell culture as previously described (2, 3). The purity of the thyroid cell population was verified by staining with anti-cytokeratin 18 antibody (an epithelial cell marker) and analyzed using a Coulter EPICS-XL MCL Beckman-Coulter (Fullerton, CA) flow cytometer as previously described (2). Only cultures that were more than 95% cytokeratin 18 positive were used in experiments.

Human genome U133A arrays
Normal primary thyroid cells were incubated with the vehicle, 100 IU/ml IFN-{gamma} (Roche Molecular Biochemicals, Indianapolis, IN), 50 IU/ml IL-1ß (Sigma Chemical Co., St. Louis, MO), or IFN-{gamma}/ IL-1ß for 24 or 72 h. Total RNA was isolated using Trizol reagent (GIBCO-BRL, Carlsbad, CA), followed by clean-up on an RNeasy spin column (QIAGEN, Valencia, CA). It was then used to generate cRNA probes. Preparation of cRNA, hybridization, and scanning of the Human Genome U133A Arrays were performed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA). Probe-set intensities were obtained and normalized as previously described using publicly available software (11, 12). Data were log-transformed using Y = log[max(x + 50,0) + 50]. Fold changes between groups were computed based on the averages of the log-transformed data. The array data are available from NCBI’s Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) using series accession number GSE5054.

RT-PCR analysis
The expression of BID mRNA was measured by real-time RT-PCR. RNA isolated from the control and cytokine-treated cells was converted to cDNA by RT using M-MLV reverse transcriptase. The cDNA was then amplified by PCR using the Cepheid Smart Cycler System (Cepheid, Sunnyvale, CA). The forward and backward primers for BID were 5'-aagaaggtggccagtcacac-3' and 5'-gtccatcccatttctggcta-3', respectively. ß-Actin was also measured by RT-PCR from the same RNA samples and used as an internal control. PCR was performed in a total volume of 25 µl, containing 2.5 µl cDNA, 5 mM MgCl2. 0.2 mM dNTPs, 0.25 µM each primer, 1.25 U AmpliTaq polymerase, and 1 µl 800x diluted SYBR Green I stock. The PCR program was hold at 95 C for 2 min and a three-temperature cycle repeating for a total of 45 times, 94 C for 15 sec, 66 C for 20 sec, and 72 C for 25 sec. Melting curve analysis was conducted from 60–95 C at 0.2 C/sec with Optics Ch1 On. PCR products were visualized by 1% agarose gel electrophoresis. The PCR bands were isolated from the gel and then sequenced to confirm authentic sequences of BID. The mRNA expression was quantified using the comparative cross threshold (CT, the PCR cycle number that crosses the signal threshold) method. The CT of the housekeeping gene ß-actin was subtracted from the CT of the target gene (BID) to obtain {Delta}CT. The normalized fold changes of the BID mRNA expression were expressed as 2–{Delta}{Delta}CT, where {Delta}{Delta}CT is equal to {Delta}CT sample – {Delta}CT control.

Treatment of cells with caspase and p38 MAPK inhibitors
Cultured thyroid cells were treated for 3 d with IFN-{gamma} and IL-1ß. Cells were then treated overnight with 1 µg/ml agonist anti-Fas antibody (clone CH11; Upstate Biotechnology, Lake Placid, NY). To block the cytokine-induced sensitization of thyroid cells to Fas-mediated apoptosis, a series of caspase inhibitors purchased from BioVision (Fremont, CA) were used. In some experiments, we added the following inhibitors singly or in pairs 60 min before IFN-{gamma} and IL-1ß administration: general caspase inhibitor (z-VAD), caspase 1 inhibitor (IC1), caspase 3/7 inhibitor (IC3), caspase 4 inhibitor (IC4), caspase 8 inhibitor (IC8), caspase 10 inhibitor (IC10), a p38 MAPK inhibitor (SB 203580), or a protesome inhibitor (MG132).

Determination of cell viability
Cell viability was measured 16 h after the anti-Fas antibody administration by staining with fluorescein diacetate (FDA) and propidium iodide (PI) and then quantitating by flow cytometry as described by Killinger et al. (13).

Western blot analysis
Cytokine-pretreated and untreated primary thyroid cells were lysed in Triton X-100 lysis buffer [150 mM NaCl, 10 mM Tris (pH 7.4), 5 mM EDTA, 1% Triton X-100], with protease inhibitors. Equivalent amounts of each sample were electrophoretically separated on a polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Rabbit polyclonal anti-RIP2 (also called RIPK2; AnaSpec, San Jose, CA), goat polyclonal anti-CARD15 (Novus Biologicals, Littleton, CO), rabbit polyclonal anti-iNOS (Cayman Chemical), rabbit anti-cIAP2 (also called BIRC3; R&D Systems, Minneapolis, MN), mouse monoclonal anti-caspase 4 (BD PharMingen, San Diego, CA), and anti-caspase 10 (Naka-Ku, Yokohama, Japan) antibodies were used according to the manufacturers’ protocols.

Detection of caspase activation
The caspase activation in response to anti-Fas antibody was detected with a CaspaTag Caspase-3/7 in situ assay kit (Chemicon, Temecula, CA). The assay was conducted by flow cytometry according to the manufacturer’s protocol. With or without z-VAD pretreatment, primary thyroid cells treated by IFN-{gamma}/IL-1ß, anti-Fas antibody, or both were lysed in CHAPS buffer with protease inhibitors (Complete; Roche). Rabbit polyclonal anti-caspase 7, rabbit polyclonal anti-caspase 3, mouse monoclonal anti-caspase 8 (Cell Signaling Technology, Beverly, MA), and anti-caspase 10 (Naka-Ku) antibodies were used to detect cleavage of caspase by Western blot analysis according to the manufacturers’ protocol.

Determination of p38 MAPK and NF-{kappa}B activities
For detecting the phosphorylation of p38 MAPK, normal thyroid cells with or without SB203580 (6 µM) pretreatment were incubated with IFN-{gamma} and IL-1ß for 30 min. The treated cells were fixed with 4% formaldehyde. Then, phosphorylated and total p38 MAPK were measured by FACE p38 ELISA Kits (Active Motif, Rockford, IL). The activation of NF-{kappa}B in response to IFN-{gamma} and IL-1ß in thyroid cells was detected by LightShift chemiluminescent EMSA kit according to the manufacturers’ protocol.

Data analysis
Flow cytometry data were analyzed by Expo32 software (Beckman-Coulter, Miami, FL). Densitometric quantitation of autoradiograms was calculated using Quantity One (Bio-Rad, Richmond, CA). Statistical analysis was performed using Student’s t test analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Genes regulated in response to IFN-{gamma} and IL-1ß
To delineate specific alterations in gene expression induced by IFN-{gamma} and IL-1ß, normal cells isolated from five separate thyroids were treated with media, IFN-{gamma}, IL-1ß, or IFN-{gamma} and IL-1ß in combination. RNA was extracted and subjected to oligonucleotide microarray profiling using Affymetrix HG-U133A arrays having 22,283 probe sets representing transcripts. Significant differences were determined by fitting two-way ANOVA (on log-transformed data) modeling experiment (five individual thyroids) and treatment effects. Using the criteria of P < 0.01 and 2-fold change in expression, we found 1774, 696, and 568 qualifying probe sets when comparing combined cytokines vs. media control, IFN-{gamma}, or IL-1ß, respectively. To estimate the fraction of these selected probe sets expected due to chance (the false discovery rate), we performed permutation testing by analyzing 1000 data sets in which the treatment labels within each of the five experiments were randomly permuted. On average, we obtained fewer than 25 qualifying probe sets in the permuted data sets for each of these three selections, obtaining estimated false discovery rates of 1.0, 3.6, and 4.2%, respectively. The intersection of these three lists contained 151 probe sets, whereas we obtained fewer than one such qualifying probe set on average in the permuted data sets. Table 1Go shows a smaller subset of 43 distinct genes that met the more stringent criteria of P < 0.01 and 3-fold change in expression for all three comparisons. The entire data set, including the selections above, are given in supplemental Table S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Our results demonstrate that treatment of primary normal thyroid cells with IFN-{gamma} and IL-1ß alters expression of genes encoding apoptotic signaling and antiapoptotic signal proteins, cell surface proteins, and also proteins involved in intracellular transport and signaling via both endoplasmic reticulum and mitochondria.


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  		TABLE 1. Gene expression profiles in normal primary thyroid cells after IFN-{gamma}/IL-1ß treatment

 
Effects of IFN-{gamma} and IL-1ß on the apoptotic pathway
Among the differentially expressed genes, we focused on the molecules associated with apoptosis. IFN-{gamma} and IL-1ß showed a synergistic regulation of apoptosis-related genes, including receptor and adaptor molecules, caspase family members, Bcl-2-related genes, and BIRC family members (Fig. 1Go). Expression of caspase family members was particular affected. In the caspase family, caspases 4 and 10 were identified as transcripts increased in the combined treatment (P < 0.01, 2-fold changes), compared with each of the other three conditions. Caspases 1, 7, and 8 were increased in the combined treatment compared with the control condition but did not meet our selection criteria for being increased above the levels of both individual treatments. In the case of caspase 3, although both IL-1ß alone and the combined treatment gave significant increases compared with the controls (P values of 1.5 x 10–6 and 6.9 x 10–8, respectively), the estimated fold changes were less than 2 (1.74 and 1.75, respectively). We further confirmed the microarray data by real-time RT-PCR or Western blot analysis. Real-time RT-PCR showed that the mRNA level of BID in four samples tested was significantly increased by an average of 4.32 ± 2.97-fold when treatment with cytokines in combination was compared with the controls (Table 2Go). The expression of receptor-interacting protein 2 (RIP2), cellular inhibitor of apoptosis protein 2 (cIAP2), caspase recruitment domain 15 (CARD15), and caspase 10 proteins was also higher in the samples with IFN-{gamma}/IL-1ß treatment than in those without or with only one cytokine treatment (Fig. 2Go); however, the expression of caspase 4 protein was similar in the samples with IFN-{gamma}/IL-1ß and only one cytokine treatment (data not shown).


Figure 1
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  		FIG. 1. Apoptosis-related gene expression profiles in normal primary thyroid cells after IFN-{gamma}/IL-1ß treatment. The expression level of some apoptosis-related genes, such as death receptor and adaptor molecules, caspase family members, Bcl2, and BIRC family members regulated by IFN-{gamma}/IL-1ß, are shown with a colorimetric intensity. An X is placed to the right of the single IFN-{gamma} and IL-1ß treatments for genes that gave P < 0.01 and a fold-change of at least 2 compared with the controls, and is placed to the right of the combined treatment only for probe sets for which the combined treatment gave P < 0.01 and at least a 2-fold change compared with all of the other three conditions. An O is placed to the right of the combined treatment for probe sets where the combined treatment gave P < 0.01 and at least a 2-fold change compared with the controls.

 

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  		TABLE 2. BID mRNA expression

 

Figure 2
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  		FIG. 2. Primary thyroid cells were treated with media, IFN-{gamma}, IL-1ß, or IFN-{gamma}/IL-1ß for 3 d. The expression of RIP2, cIAP2, CARD15, and caspase 10 proteins was determined by immunoblot analysis. The blots were reprobed with anti-actin antibody.

 
Blocking of Fas-mediated apoptosis in primary thyroid cells
To further determine the molecules involved in the cytokine-induced sensitization of thyroid cells to Fas-mediated apoptosis, we tested the ability of various inhibitors to block the sensitization effect of the combination of cytokines. First, the individual caspase inhibitors were chosen based on the fold change of individual caspase gene vs. control (Fig. 3AGo). We found that the sensitization of primary thyroid epithelial cells to Fas-mediated apoptosis by IFN-{gamma} and IL-1ß could be significantly blocked by a general caspase inhibitor, z-VAD, in a dose-dependent manner (Fig. 3Go, B and C). Furthermore, the combination of specific inhibitors of caspase 3/7 (IC3) and caspase 10 (IC10) also prevented Fas-mediated apoptosis in the cells treated with anti-Fas antibody plus IFN-{gamma}/IL-1ß (Fig. 4AGo). The combination of the other pairs of specific inhibitors (IC3 and IC4, IC3 and IC8) also increased living cell percentage, but the effect was not statistically significant (data not shown). In addition to caspase inhibitors, a p38 MAPK inhibitor and a proteasome inhibitor were also applied to block the thyrocyte apoptosis induced by IFN-{gamma}/IL-1ß and anti-Fas antibody. SB 203580, a p38 MAPK inhibitor, did have a blocking effect (Fig. 4AGo). However, MG132, a proteasome inhibitor, did not show the same effect (data not shown). The specific caspase inhibitors or p38 MAPK inhibitor with IFN-{gamma}/IL-1ß did not affect cell viability (Fig. 4BGo). SB 203580 also inhibited that IFN-{gamma}/IL-1ß-induced p38 MAPK phosphorylation (Fig. 4CGo). These results suggest that both caspase and p38 MAPK are involved in the apoptosis of normal thyrocytes treated with IFN-{gamma}/IL-1ß and anti-Fas antibody.


Figure 3
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  		FIG. 3. Effect of caspase inhibition. A, Summary of the effect of cytokines on caspase family members measured by microarrays. *, P < 0.01 vs. control; **, P < 0.001 for combined cytokine treatment compared with controls and both single cytokine treatments. B, Primary thyroid cells were treated with media, IFN-{gamma}/IL-1ß, or IFN-{gamma}/IL-1ß plus CH11, with or without 10 µM z-VAD for 3 d. After overnight exposure to anti-Fas antibody, cell viability was assayed by FDA/PI staining, and 10,000 cells per sample were analyzed by flow cytometry. C, Thyroid cells were treated by IFN-{gamma}/IL-1ß plus CH11 with different concentrations of z-VAD. Data are presented as mean ± SD of triplicate measurements. *, P < 0.05 compared with IFN-{gamma}/IL-1ß plus CH11.

 

Figure 4
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  		FIG. 4. Effects of individual caspase inhibitors and p38 MAPK inhibitor. A, Primary thyroid cells were treated by media, IFN-{gamma}/IL-1ß with or without inhibitors for caspase 3/7 and 10, or SB203580 for 3 d. After overnight exposure to anti-Fas antibody, FDA/PI-stained cells were analyzed by flow cytometry. B, Some primary thyroid cells were treated with IC3, IC10, or SB 203580 and IFN-{gamma}/IL-1ß for 3 d, and FDA/PI-stained cells were measured by flow cytometry. *, P < 0.05 compared with the IFN-{gamma}/IL-1ß plus CH11. C, A total of 9000 thyroid cells per well in a 96-well plate were treated with or without SB 203580 for 30 min and incubated with or without IFN-{gamma} and IL-1ß for an additional 30 min. After fixation of cells, phospho-p38 antibody or total p38 antibody was added to each well. The results were expressed as OD450 divided by OD595. Data are presented as mean ± SD of triplicate measurements. *, P < 0.05 compared with IFN-{gamma}/IL-1ß.

 
Blocking of Fas signaling in primary thyroid cells
The appearance of cleaved forms of caspase 3, 7, 8, and 10 under apoptotic conditions in thyroid cells have been reported in our previous study. However, under apoptosis-blocking conditions, the cleaved pattern of the caspases is unknown. As shown in Fig. 3BGo, the caspase general inhibitor z-VAD was able to markedly block Fas-mediated apoptosis of the thyroid cells. Here, we demonstrate that the occurrence of cleaved caspase 7 and 10 was completely blocked by z-VAD, whereas that of cleaved caspase 3 was only partially blocked by z-VAD, but the amount of cleaved caspase 8 was not reduced by z-VAD (Fig. 5AGo). Such a pattern of caspase blockage indicates for the first time that the activation of caspase 8 is not required for thyroid cell response to cytokine treatment. Moreover, the activities of caspase 3/7 induced by IFN-{gamma}/IL-1ß plus anti-Fas antibody were also significantly inhibited by z-VAD (Fig. 5BGo). In addition to z-VAD, the inhibitors of caspase 3/7, caspase 10, and p38 MAPK also suppressed the activity of caspase 3/7 induced by IFN-{gamma}/ IL-1ß and anti-Fas antibody (Fig. 5BGo). Therefore, this result further supports that the activation of caspase 3/7 and caspase 10 is crucial for thyrocyte apoptosis induced by IFN-{gamma}/IL-1ß and anti-Fas antibody.


Figure 5
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  		FIG. 5. Determination of caspase activity. A, Primary thyroid cells were treated with media and IFN-{gamma}/IL-1ß with or without z-VAD for 3 d. Some of cells were exposed to anti-Fas antibody for 6 h, and then cell lysates were prepared. The activation of caspases was determined by immunoblot analysis, using antibodies detecting the active forms of the enzymes. The bands for the active enzymes are marked with arrows. B, Thyroid cells were treated with media, IFN-{gamma}/IL-1ß with or without z-VAD, inhibitors for caspase 3 and 10, or SB 203580 for 3 d. After overnight exposure to anti-Fas antibody, the caspase 3/7 activation was detected by CaspaTag caspase 3/7 in situ assay kit, using flow cytometric analysis. Results are presented as mean ± SD of triplicate measurements. *, P < 0.05 compared with the IFN-{gamma}/IL-1ß plus CH11 condition.

 
The microarray data for inducible nitric oxide synthase (iNOS) regulatory genes are summarized in Fig. 6AGo, which illustrates that iNOS is the only gene significantly up-regulated by the combination of the two cytokines. In Fig. 6BGo, we show that both the RIP2 and iNOS proteins are markedly up-regulated by IFN-{gamma}/ IL-1ß in thyrocytes. However, such an up-regulation was significantly suppressed by the p38 MAPK inhibitor SB 203580 (Fig. 6BGo). In addition, NF-{kappa}B, which is directly downstream of p38 MAPK, was also activated by IFN{gamma}/IL-1ß as shown in Fig. 6CGo. These results suggest that the activation of NF-{kappa}B may be responsible for the increase in iNOS expression.


Figure 6
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  		FIG. 6. iNOS regulatory molecules. A, Summary of microarray data for iNOS regulatory genes. *, P < 0.01 vs. control; **, P < 0.001 for combined cytokine treatment compared with controls and both single cytokine treatments. B, Primary thyroid cells were treated with media, SB203580, IFN-{gamma}/IL-1ß, or SB 203580 plus IFN-{gamma}/IL-1ß for 3 d. Cell lysates were prepared for immunoblot analysis of iNOS and RIP2 protein expression as well as actin as a loading control. C, After 30 min of incubation of the indicated cytokines, nuclear extracts of thyroid cells were prepared, and the activation of NF-{kappa}B was detected by EMSA. The band containing active NF-{kappa}B is marked by an arrow.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Immune-mediated damage to target cells is an important process in autoimmunity, tumor immunity, and immunity against intracellular pathogens and viruses. The regulation of programmed cell death in these situations is complex and not fully understood. We have previously demonstrated the biological significance of inflammatory cytokines in the regulation of apoptosis in autoimmunity. The development of human Hashimoto’s thyroiditis is associated with a change of the cytokine microenvironment in the thyroid that initiates the apoptosis resulting in damage to the thyroid (1). It is interesting that the combination of IFN-{gamma} and IL-1ß is uniquely necessary for the induction of apoptosis (2, 3, 4). Several groups have verified that the mechanism of apoptosis facilitation by the combination of IFN-{gamma} and IL-1ß or TNF-{alpha} is via a Fas-mediated pathway (2, 4, 14, 15); however, the precise details of the molecules involved are still not completely known. The current studies examine the complex regulation of the Fas signaling pathway in thyrocytes by the combination of IFN-{gamma} and IL-1ß. To the best of our knowledge, this was the first attempt to thoroughly survey the molecules involved in apoptosis of human thyroid cells treated by IFN-{gamma}/IL-1ß using microarrays. The present studies showed that treatment of primary normal thyroid cells with IFN-{gamma} and IL-1ß altered the expression of genes that encode apoptotic signaling proteins, antiapoptotic signal proteins, cell surface proteins, intracellular transport proteins, and signaling proteins related to both endoplasmic reticulum and mitochondria. Thus, the effect of these cytokines on thyroid cells is extensive and affects multiple pathways and molecules.

Fas is expressed on normal thyroid follicular cells (1, 2). Fas-mediated apoptosis is related to the activation of a variety of caspases including caspases 1, 2, 3, 7, 8, 9, and 10 (16, 17). In this study, we showed that IFN-{gamma} and IL-1ß treatment enhanced the levels of caspases 1, 3, 4, 7, 8, and 10 in thyrocytes. The caspases exist within the cell as inactive zymogens and are activated via proteolytic cascades. The first phase of activation targets the initiator caspases, such as caspase 8, 9, and 10, which can transduce apoptotic signals by directly activating the downstream executioner or effector caspases, such as caspase 3, 6, and 7. The inflammatory caspases include caspase 1, 4, 11, and 12. Therefore, it appears that the combination of IFN-{gamma} and IL-1ß treatment first induces initiator caspases, followed by effector caspases. The central role of caspases in cell death induced by IFN-{gamma} and IL-1ß treatment in thyrocytes was further demonstrated by employing caspase inhibitors. The cell death of thyrocytes induced by cytokines was partially prevented by specific inhibitors of caspases 3, 7, and 10, applied individually, but was completely blocked by a combination of caspase 3/7 and caspase 10 inhibitors. This inhibition was also achieved with a general caspase inhibitor, z-VAD. Although the cleavage of caspase 3, 7, 8, and 10 under these apoptotic conditions in the thyroid cell has been studied, the cleavage pattern of the caspases under blocking conditions is unclear. We found that the general caspase inhibitor (z-VAD) inhibited cleavage of caspases 3, 7, and 10 induced by IFN-{gamma}/IL-1ß and the anti-Fas antibody but not caspase 8. Although the reason that z-VAD failed to inhibit caspase 8 is unknown at present, these findings suggest that the cytokine activation of caspases 3, 7, and 10 are adequate to facilitate the Fas-mediated cell death of thyrocytes.

It is well known that apoptosis is induced by two different pathways. One is the death receptor-mediated pathway, and the other is the mitochondrial-mediated pathway. The two pathways intersect each other via a proapoptotic Bcl-2 family member, BID (18, 19). The outcome of these two intersected pathways should efficiently amplify the apoptotic signals. Our studies show that the level of BID was significantly increased by IL-1ß treatment as well as by the combined IFN-{gamma}/IL-1ß treatment. The activated BID performs its function in mitochondria. However, as shown in our previous work, mitochondria are not significantly activated in thyroid cells treated with the agonist anti-Fas antibody. Unlike type I cells, thyroid cells are type II cells and are dependent on the activation of the mitochondrial pathway and display slower and lower amounts of DISC assembly (5, 6). Our earlier work has revealed that mitochondria are not activated in thyroid cells treated with the agonist anti-Fas antibody, likely due to the small amount of activated BID and incomplete caspase 3’s being cleaved (2). However, after IFN-{gamma}/IL-1ß treatment, the agonist anti-Fas antibody has been shown to markedly enhance the cleaved BID, which is probably due to increased DISC assembly and the availability of a higher level of BID. Thus, the cross-talk mechanism via BID can contribute to cytokine-mediated sensitizing of thyroid cells to apoptosis.

Another important molecule identified by the present study is iNOS (NOS2A), an enzyme responsible for the production of nitric oxide. NO is an important bioregulatory molecule that mediates a variety of actions such as vasodilatation, neurotransmission, host defense, and apoptosis. It is now widely accepted that iNOS is expressed in response to several stresses, including inflammatory cytokines and bacterial endotoxin. Our microarray result identified that iNOS was one of the genes that was significantly elevated in thyrocytes by IFN-{gamma}/IL-1ß treatment, and the result was further confirmed at the protein level by Western blot. NO induces apoptosis in a variety of cell types, including macrophages, neurons, pancreatic islet cells, thymocytes, chondrocytes, hepatocytes, and dendritic cells (20, 21, 22, 23). However, there is no report on the expression of iNOS or the production of NO in normal thyrocytes. Our study not only provides evidence that normal thyrocytes can express a high level of iNOS upon cytokine treatment but also suggests that an increase in iNOS may play a central role in the apoptosis of thyrocytes. First, NO is able to activate BID (24, 25), the level of which is increased in cytokine-treated thyrocytes. Second, NO-induced apoptosis is mediated by downstream caspases including caspases 3 and 10 (26, 27) and is blocked by caspase inhibitors (28). Interestingly, the levels of both caspases 3 and 10 were increased in thyrocytes treated by cytokines, and the up-regulation of both could be blocked by the inhibitors of caspase. Finally, the activation of p38 MAPK appears to be crucial for stimulation of the NO-mediated apoptosis because such apoptosis can be significantly abrogated by the p38 MAPK inhibitor SB 203580 (29, 30). Our current study supports the proapoptotic role of p38 MAPK in thyrocytes treated by cytokines because IFN-{gamma}/IL-1ß-induced phosphorylated p38 MAPK could be blocked by SB 203580 and the elevated level of iNOS could also be significantly blocked by the p38 MAPK inhibitor SB 203580. These results suggest that the activation of p38 MAPK is upstream of iNOS expression. It has been reported that the activation of NF-{kappa}B increases iNOS expression (31). We also demonstrated that the combination of IFN-{gamma}/IL-1ß can activate NF-{kappa}B in normal thyroid cells. Collectively, these findings indicate that the induction of iNOS by IFN-{gamma}/IL-1ß treatment may occur through activation of NF-{kappa}B. However, the precise molecular mechanism by which iNOS expression is altered by p38 MAPK requires further study.

In addition to the regulation of iNOS expression, p38 MAPK also plays a role in the activation of caspases 3 and 7 induced by IFN{gamma}/IL-1ß treatment because the cotreatment with the p38 MAPK inhibitor SB203580 blocks the activity of caspases 3 and 7 in thyrocytes. This result is in line with findings in other cell types such as lymphocytes, endothelial cells, and neurons (32, 33, 34), in which the activation of p38 MAPK is associated with the activities of caspases 3, 8, and 9. Our studies also revealed two additional molecules that are significantly induced by cytokine treatment in thyrocytes. The first one is RIP2, which can facilitate apoptosis by interacting with members of TNF receptor-1 signaling complex (35). RIP2 is a potent activator of NF-{kappa}B and inducer of apoptosis in response to various stimuli. The activity of RIP2 may also be related to p38 MAPK because the inhibitory mechanism of RIP2 is similar to that of p38 MAPK (36). In current study, the elevated level of RIP2 protein by cytokines can be suppressed to a certain degree by the p38 MAPK inhibitor SB203580. Fas sensitization induced by p38 MAPK is possibly controlled by a RIP2-related mechanism because RIP2 facilitates apoptosis by interacting with members of the TNF receptor-1 signaling complex. Nevertheless, the exact role of RIP2 in the sensitization of thyrocytes by cytokines needs further investigation. The other molecule identified is CARD15, which could induce NF-{kappa}B via RIP2 (37). The expression of CARD15 was markedly up-regulated by the combined IFN-{gamma}/IL-1ß treatment. Both RIP2 and CARD15 may influence the iNOS expression via NF-{kappa}B.

Among members of the IAP family, IFN-{gamma}/IL-1ß treatment increased the protein level only of cIAP2. The function of cIAP2 is still not clear. The most well understood member of IAP family is XIAP, a tight binding inhibitor of both executioner caspase 3/7 and the initiator caspase 9. Unlike XIAP, cIAP1 and cIAP2 are very weak inhibitors of the caspase 3, 7, and 9 (38). This previous study also demonstrated that cIAP1 and cIAP2 retained the binding exosites, but they lost the key interacting surface for caspase inhibition. We predict that the increase of cIAP2 may influence apoptosis through an interaction with other members of the IAP family.

In summary, the combination of IFN-{gamma} and IL-1ß treatment of normal thyroid cells can alter a range of molecules related to apoptotic signaling, cell surface proteins, intracellular transport proteins, and signaling proteins of both the endoplasmic reticulum and the mitochondria. Together, these alterations may reverse the resistance of normal thyrocytes to apoptotic stimulation. IFN-{gamma}/IL-1ß pretreatment sensitizes human thyroid cells to Fas-mediated apoptosis and is associated with the activation of p38 MAPK and increased iNOS expression. Both the activation of p38 MAPK and increased iNOS expression are thought to activate their downstream molecules such as BID and caspases. Among the caspases, both initiator caspase (caspase 10) and effector caspase (caspases 3 and 7) are involved in the apoptosis induced. These data provide insight into the mechanism of the cytokine-sensitive effect on thyroid cells of the Fas-mediated apoptotic pathway and may help clarify apoptosis regulation in other type II cells.


   Acknowledgments
 
We thank David Misek and Barbara Lamb for technical assistance.


   Footnotes
 
This work was supported by the National Institutes of Health Grant 2 R01 AI 37141 and the MDRTC Cellular and Molecular Biology Core at the University of Michigan.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 19, 2007

Abbreviations: CARD15, Caspase recruitment domain 15; cIAP2, cellular inhibitor of apoptosis protein 2; CT, cross threshold; DISC, death-induced signaling complex; FDA, fluorescein diacetate; IFN, interferon; iNOS, inducible nitric oxide synthase; NF, nuclear factor; PI, propidium iodide; RIP2, receptor-interacting protein 2.

Received January 29, 2007.

Accepted for publication July 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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