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Molecular Endocrinology 19 (3): 804-811
Copyright © 2005 by The Endocrine Society

Induction and Regulation of Fas-Mediated Apoptosis in Human Thyroid Epithelial Cells

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 48109-0648

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fas-mediated apoptosis has been proposed to play an important role in the pathogenesis of Hashimoto’s thyroiditis. Normal thyroid cells are resistant to Fas-mediated apoptosis in vitro but can be sensitized by the unique combination of interferon-{gamma} and IL-1ß cytokines. We sought to examine the mechanism of this sensitization and apoptosis signaling in primary human thyroid cells. Without the addition of cytokines, agonist anti-Fas antibody treatment of the thyroid cells resulted in the cleavage of proximal caspases, but this did not lead to the activation of caspase 7 and caspase 3. Apoptosis associated with the cleavage of caspases 7, 3, and Bid, and the activation of mitochondria in response to anti-Fas antibody occurred only after cytokine pretreatment. Cell surface expression of Fas, the cytoplasmic concentrations of procaspases 7, 8, and 10, and the proapoptotic molecule Bid were markedly enhanced by the presence of the cytokines. In contrast, P44/p42 MAPK (Erk) appeared to provide protection from Fas-mediated apoptosis because an MAPK kinase inhibitor (U0126) sensitized thyroid cells to anti-Fas antibody. In conclusion, Fas signaling is blocked in normal thyroid cells at a point after the activation of proximal caspases. Interferon-{gamma}/IL-1ß pretreatment sensitizes human thyroid cells to Fas-mediated apoptosis in a complex manner that overcomes this blockade through increased expression of cell surface Fas receptor, increases in proapoptotic molecules that result in mitochondrial activation, and late caspase cleavage. This process involves Bcl-2 family proteins and appears to be compatible with type II apoptosis regulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
APOPTOSIS, OR PROGRAMMED cell death, is a physiological mechanism of cell elimination based on a highly regulated intrinsic cell program (1). Apoptosis plays an important role in the pathogenesis of autoimmune diseases including the central pathogenic events in the destruction of thyroid follicular cells in Hashimoto’s thyroiditis (2, 3), and increasing evidence supports the role for FasL-mediated apoptosis in Hashimoto’s thyroiditis (4, 5, 6, 7, 8, 9). FasL is a member of the tumor necrosis factor family that is expressed on activated T lymphocytes and acts through death receptor Fas (10, 11). Fas (CD95) is a widely expressed type I transmembrane protein, a member of the TNF receptor family (12) and is expressed on the thyroid. However, normal thyroid cells are resistant to Fas-mediated apoptosis. This has led to a conundrum as to how T cells might activate this pathway to kill thyroid cells in thyroiditis.

Recent work has shown that inflammatory cytokines are involved in the regulation of apoptosis (13). Cytokines can influence the expression of death receptors, apoptotic signaling components, and inhibitors in target cells, as well as control the expression of death ligands in many immune effector cells (14, 15). Our work has documented that thyrocytes can be sensitized in vitro by the combination of interferon (IFN) {gamma} and IL-1ß or TNF{alpha} (14, 16). In addition, injecting these cytokines into the thyroids of mice with experimental autoimmune thyroiditis induces destructive thyroiditis (17). Inflammatory cytokines are known to be present in the thyroid gland in Hashimoto’s thyroiditis (18), and T cell clones isolated from intrathyroidal lymphocytic infiltrates of Hashimoto’s thyroiditis produce high levels of Th1 cytokines (19). Therefore, it would appear that inflammatory cytokine facilitation of the Fas pathway is crucial to the pathogenesis of destructive thyroiditis.

How these cytokines regulate the Fas pathway is important because it can provide clues to the origins of thyroiditis and potentially identify therapeutic targets to block destruction of target cells in autoimmunity. However, the regulation of Fas-mediated apoptosis can occur at multiple levels including the expression of the Fas receptor, the inhibition of intracellular signaling, and the modulation of bcl-2 family members (20). During Fas signaling, the intracellular death domains of Fas bind to an adapter protein that activates proximal caspases (caspase 8 and 10) (21). In type I cells, the activated proximal caspases can directly cleave apical caspases (caspase 7 and 3) that cleave other proteins: death substrates leading to apoptosis (22). In type II cells, other signals causing mitochondrial activation are required to initiate the programmed cell death (22). Bid, a proapoptotic bcl-2 family member, is one of these signals and is normally cleaved by proximal caspases, translocates to the mitochondria that results in the loss of mitochondrial membrane potential, release of mitochondrial proapoptotic factors (23, 24). Some of these factors, including Smac/DIABLO, are known to be important in the activation of caspase 3 and other effector caspases (25). The activation of caspase-3 is inhibited by XIAP and Smac/DIABLO released from the mitochondria removes XIAP from caspase-3 and overcomes this inhibition (26). In addition, apoptosis in type II cells is also blocked by antiapoptotic bcl-2 family members (22) and the activation of p44/p42 MAPK (also called Erk1/2) in response to death ligands (27). Thus, the aim of the this study was to investigate the complex regulation of the Fas signaling pathway in thyroid cells and clarify the mechanism of cytokine-induced sensitization to FasL in primary human thyroid cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IFN{gamma}/IL-1ß-Induced Sensitization of Primary Thyroid Cells to Fas-Mediated Apoptosis Correlates with the Increased Expression of Fas
Normal thyroid epithelial cells are resistant to agonist anti-Fas antibody but can be sensitized by the combination of IFN{gamma} and TNF{alpha} or IL-1ß (15). The cell death in response to anti-Fas antibody in cytokine pretreated thyroid cells correlated with the increased mRNA expression of Fas (Fig. 1Go, A and B). Only a mild increase in Fas protein expression was detected and appeared to be due to a higher molecular weight form of Fas (Fig. 1BGo), in agreement with our previous results (15). In contrast, the surface expression of Fas was markedly enhanced by the cytokine pretreatment (Fig. 1CGo), suggesting either enhanced membrane transport or decreased degradation.



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  		Fig. 1. IFN{gamma}/IL-1ß-Induced Sensitization of Primary Thyroid Cells to Anti-Fas Antibody Correlates with the Increased Expression of Fas

Primary thyroid cells were treated with 100 U/ml IFN{gamma} and 50 U/ml IL-1ß for 4 d or left untreated. A, After overnight exposure to anti-Fas antibody, cell death was assayed by FDA/PI staining and 10,000 cells per sample were analyzed by flow cytometry. Data are presented as mean ± SD of triplicate measurements. B, Fas mRNA expression was measured by an RNase protection assay using the hAPO-2b template set on 5 µg total RNA isolated from untreated and cytokine-treated thyroid cells. The Fas mRNA expression was quantitated by densitometry and normalized to the GAPDH band and to the signal intensity in untreated cells. Fas protein was determined by immunoblot analysis. The blots were reprobed with antiactin antibody. The autoradiograms were quantitated by densitometry. The Fas expression after cytokine treatment was normalized to the actin signal and to the result from untreated cells. C, Cell surface expression of Fas was measured by flow cytometry. A representative histogram of anti-Fas antibody-specific fluorescence for untreated cells (open curve, solid line) and IFN{gamma}/IL-1ß-treated cells (open curve, spotted line) is presented and compared with control IgG (filled curve). To quantitate the increase in cell surface expression of Fas after cytokine treatment, the mean fluorescence intensity of the control antibody was subtracted from the anti-Fas antibody-specific mean fluorescence intensity and was normalized to the control cells. Asterisk denotes conditions where P < 0.05 compared with the control samples. These data are representative of results from five independent experiments each using thyroid cultures from different patient samples.

 
The Effect of IFN{gamma}/IL-1ß Treatment on the Expression of Procaspases
Fas-mediated apoptosis is controlled at several levels from the death receptor to the activation of caspases. The effect of cytokines on the level of procaspases was examined (Fig. 2Go.) The protein concentrations of procaspase 8, 10, and 7 were significantly increased by the IFN{gamma}/IL-1ß pretreatment (Fig. 2CGo). The expression of caspase 8 and 10 mRNA was also markedly increased detected (Fig. 2Go, A and B). The level of caspase 9 and caspase 3 was unchanged after cytokine treatment (Fig. 2CGo).



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  		Fig. 2. The Effect of IFN{gamma}/IL-1ß Treatment on the Expression of Procaspases

Normal thyroid cells were treated in duplicate with IFN{gamma}/IL-1ß as above. A, Five micrograms of total RNA from untreated and cytokine-treated cells were used for the quantitation of caspase-8 mRNA expression (RNase protection assay, hAPO-3d template set). The signal intensity was normalized to the GAPDH bands and to the control cells. B, Caspase-10 mRNA expression was measured by RT-PCR. Amplified messages were specific for caspase 10/a (230 bp), caspase 10/c (260 bp), caspase 10/d (350 bp), and ß-actin. The expression of caspase-10 isoforms in cytokine-treated cells was normalized to actin and the control cells. C, Thyroid cells were lysed in Triton X-100 lysis buffer and an immunoblot analysis was performed with the indicated antibodies. The autoradiograms were quantitated by densitometry and normalized to the 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 Influence of Cytokine Treatment on the Concentration of bcl-2 Family Members
Bcl-2 family members are known regulators of Fas-mediated apoptosis. Ribonuclease (RNase) protection assay revealed the complex transcriptional regulation of these genes by IFN{gamma}/IL-1ß (Fig. 3Go). The pro- and antiapoptotic members of the bcl-2 family can form heterodimers and the final decision about apoptosis depends on the ratio of these proteins. The mRNA levels of the proapoptotic Bid, bak, and bax, and of the antiapoptotic bcl-X, bfl-1, and mcl-1 were increased by various amounts (Fig. 3AGo). The mRNA and protein level of Bid increased to the largest extent, with the protein concentration enhanced about 15-fold after cytokine treatment (Fig. 3Go). The concentrations of bcl-X and Bak protein was also significantly increased (Fig. 3BGo). The bcl-2 protein concentration was slightly decreased; however, the difference was not significant (Fig. 3BGo).



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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 quantitated by densitometry and normalized to the GAPDH band and to the signal intensity in untreated cells. B, The protein expression of the bcl-2 family members was assayed by immunoblot analysis from Triton X-100 lysates of thyroid cells. Cells were treated and assayed in duplicate. The autoradiograms were quantitated by densitometry and normalized to actin and the controls. 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).

 
Fas Signaling in Primary Thyroid Cells
To further analyze the factors involved in the cytokine-induced sensitization of thyroid cells to Fas-mediated apoptosis, Fas signaling was investigated. Surprisingly, caspase 8 and caspase 10 were cleaved and activated in response to anti-Fas antibody even in the control cells that are resistant to Fas-mediated apoptosis. An early cleavage product (22 kDa) of caspase 3 was also detected in the control cells (Fig. 4AGo). The activation of proximal caspases means that the death signal in the control cells passed the DISC and was inhibited after the proximal caspases. XIAP can bind to this cleavage product and effectively inhibits its activation by further cleavage to 17- and 19-kDa products (26). XIAP is removed from caspase 3 by the mitochondrial release of Smac/DIABLO as a result of mitochondrial activation (26). In cytokine-pretreated thyroid cells, these latter cleavage products can be seen in response to anti-Fas antibody (Fig. 4AGo), and this is associated with mitochondrial activation in cytokine pretreated cells but not in the control cells (Fig. 4BGo). The mediator of mitochondrial activation is the Bid protein (28). Bid is cleaved by caspase-8, transported to the mitochondria, and results in the loss of mitochondrial membrane potential (29). The disappearance of full-length Bid can be seen in cytokine-pretreated cells in response to anti-Fas antibody (Fig. 4AGo).



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  		Fig. 4. Fas Signaling in Primary Thyroid Cells

Cells were treated for 4 d with IFN{gamma}/IL-1ß or left untreated. All cells were exposed to anti-Fas antibody for the indicated time. A, The activation of caspases was determined by immunoblot analysis using antibodies detecting the active forms of the enzymes (caspase-8: 54/55 kDa procaspase, 41/43 kDa intermediate cleavage product, 26 kDa cleavage product (inactive), 18 kDa active enzyme; caspase-10: 57/58 kDa procaspase, 25 kDa active enzyme; caspase-7: 35 kDa procaspase, 20 kDa active enzyme; caspase-3: 22 kDa cleavage product (inactive), 19/17 kDa active enzyme). Bid was detected with an antibody recognizing the full-length Bid (the cleavage of Bid is shown by the disappearance of signal). B, The mitochondrial activation was detected by ApoAlert mitochondrial membrane sensor kit, using flow cytometric analysis. Results are presented as mean ± SD of triplicate measurements. *, P < 0.05 compared with the control samples. These experiments are representative of result from three independent measurements using different patient samples.

 
The Role of p44/p42 MAPK Activation in Fas Signaling
The activation of p44/p42 MAPK is another cellular defense against apoptosis (27, 30). P44/p42 MAPK is constitutively active in cultured thyroid cells, and it is further activated in control cells after anti-Fas antibody administration (Fig. 5Go.) MAPK kinase (MEK) is responsible for the activation of p44/p42 MAPK. The MEK inhibitor UO126 sensitized the control cells to the anti-Fas antibody, indicating that p44/p42 MAPK activity is an important factor in the resistance of thyroid cells to Fas-mediated apoptosis. After cytokine pretreatment, the Fas-mediated apoptosis was further enhanced by the MEK inhibitor UO126 (Fig. 5CGo).



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  		Fig. 5. The Role of p44/p42 MAPK Activation in Fas Signaling

A, Cells were treated for 4 d with IL-1ß/TNF{alpha} or left untreated and exposed to anti-Fas antibody 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, Thyroid cells were exposed to MEK inhibitor at the indicated concentrations 30 min before anti-Fas antibody administration. Cell viability was assayed by FDA/PI staining and 10,000 cells per sample were evaluated by flow cytometry. C, IFN{gamma}/IL-1ß pretreated thyroid cells were exposed to MEK inhibitor at the indicated concentrations 30 min before anti-Fas antibody administration. Cell viability was assayed as above. *, P < 0.05 compared with the control samples. These are representative results of independent experiments using three normal thyroid tissues.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
There is increasing evidence for Fas-mediated thyroid cell apoptosis in the pathogenesis of Hashimoto’s thyroiditis. However, normal thyroid follicular cells are resistant to Fas-mediated apoptosis (15, 17, 31), suggesting that these cells are altered during thyroiditis. Th1 cytokines, which are present in the thyroid during thyroiditis (32), have been shown to sensitize thyroid cells to Fas-mediated apoptosis, but the molecular mechanism of this action is largely unknown (15, 17, 31). The current studies examined the complex regulation of the Fas signaling pathway by IFN{gamma}/IL-1ß. The results suggest that in normal thyroid follicular cells some Fas is expressed on the cell surface and agonist anti-Fas antibody can activate caspases 8 and 10. Caspase 3 is also cleaved, but the complete activation of caspase 3 does not occur, possibly due to XIAP inhibition. Mitochondria are not activated, presumably due to the lack of sufficient Bid cleavage and the dominance of antiapoptotic bcl-2 proteins. The p44/p42 MAPK is also activated in normal thyroid cells and can inhibit the cleavage of caspase 8 further preventing the induction of apoptosis. The inhibition of p44/p42 MAPK by MEK inhibitor can sensitize the thyroid cells to the anti-Fas antibody, suggesting that p44/p42 MAPK activity is involved in the protection of thyroid cells to Fas-mediated apoptosis. However, the precise mechanism of MEK inhibitor is not clear and need to be further clarified.

Exposing thyroid follicular cells to the combination of IFN{gamma}/IL-1ß cytokines results in dramatic changes in the signaling pathway, which involves everything from the receptor to the caspase enzymes that induce apoptosis. Death receptors are stored in intracellular pools, but the surface expression determines the response to death ligands. A greater than 4-fold increase in the surface expression of Fas was observed after cytokine pretreatment, and this can significantly contribute to the sensitization of thyroid cells to Fas-mediated apoptosis. The concentrations of the proximal caspase 8 and caspase 10 are elevated, and this may also be important in inducing apoptosis. However, the absolute change that occurs after cytokine activation is the induction of mitochondrial activation. The mechanism for this appears to involve massive increases in the concentration of Bid protein, without similar changes in antiapoptotic Bcl-2 family members, along with much more effective cleavage of Bid. The release of cytochrome c and Smac/DIABLO from the mitochondria then results in the activation of caspases 7 and 3 and the final induction of apoptosis. In general, this work demonstrates that IFN{gamma}/IL-1ß treatment sensitizes thyroid cells to Fas-mediated apoptosis by up-regulating the proapoptotic proteins. Also, mitochondrial activation appears essential in this process, so thyroid cells demonstrate type II regulation regarding Fas-mediated apoptosis facilitated by cytokines.

The requirement for the combination of cytokines is unique, but it becomes understandable when evaluating the changes that facilitate apoptosis. A significant increase in the cell surface expression of Fas was detected after single cytokine pretreatment, in accordance with other reports (16, 31, 33, 34). However, although this has been reported to in and of itself facilitate Fas-mediated apoptosis, it cannot be responsible for sensitization of thyroid cells because mitochondrial activation remains inhibited. In addition, whereas IFN{gamma} increases the expression of the Fas receptor and the procaspases, it decreases the concentration of Bid, which works against mitochondrial activation. Bid appears to be essential for apoptosis induction in many cells (35, 36), and therefore this may explain why IFN{gamma} alone is not sensitizing. The combination of cytokines increases in the concentrations of Fas and proximal caspases and massive increases in Bid expression achieved with the combination IFN{gamma}/IL-1ß must be crucial for the sensitizing effect of cytokines.

TNF-related apoptosis-inducing ligand (TRAIL), another death ligand, can kill thyroid cells after pretreatment with IL-1ß and TNF{alpha}, a combination that does not sensitize to Fas, whereas TRAIL cannot be activated by IFN{gamma}/IL-1ß. Whereas both of these combinations cause massive increases in the level of Bid due to the activation of nuclear factor-{kappa}B, the block in each pathway appeared different because TRAIL induction did not activate caspase 8 or 10 without cytokines (37). The regulation of the Fas signaling pathway also reveals that the sensitizing effect of the cytokines on thyroid cells, based primarily on the up-regulation of proapoptotic molecules, is in direct contrast to the effect of cycloheximide, which sensitizes the thyroid cells to Fas-mediated apoptosis by removing a labile protein inhibitor (16). However, the participation of other, unknown inhibitors of the Fas signaling pathway cannot be ruled out.

In summary, normal thyroid cells are resistant to Fas-mediated apoptosis, but the combination of IFN{gamma} and IL-1ß sensitizes them to FasL. The sensitizing effect of IFN{gamma}/IL-1ß involves the increases in the concentrations of Fas receptor and caspases 8, 10, and 7 and Bid and mitochondrial activation. Th1 cytokines, therefore, have a crucial role in determining target cell sensitivity in autoimmune thyroiditis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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. All excised tissues were prepared for cell culture as previously described (19). The primary cultures were passaged 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 that contains 25% fetal calf serum, so the final concentration of fetal calf serum in the culture medium was 5%. The purity of the thyroid cell population was verified by staining with anticytokeratin 18 antibody (a marker for epithelial cells), and quantitated by flow cytometry. Only cultures that were more than 90% cytokeratin 18 positive were used for experiments.

Cytokines, Anti-Fas Antibody, and MEK Inhibitor Treatment
Primary thyroid cells were treated for 4 d with 100 IU/ml IFN{gamma} (Roche Molecular Biochemicals, Indianapolis, IN) and 50 IU/ml IL-1ß (Sigma). Cells were then treated overnight with 1 µg/ml agonist anti-Fas antibody (clone CH11, Upstate Biotechnology, Lake Placid, NY). The activation of 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 the anti-Fas antibody administration.

Determination of Cell Viability
Cell viability was measured 20 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. (38).

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 bovine 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 resuspended in antimouse fluorescein isothiocyanate conjugate (Jackson ImmunoResearch) 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 Fas Surface Expression
Thyroid cells were made nonadherent by incubation with 0.265 mM EDTA in PBS, incubated for 15 min in blocking solution (2% normal horse serum, 1% BSA in PBS) at 4 C and exposed to 5 µg/ml mouse monoclonal anti-Fas antibody (clone UB2, MBL, Watertown, MA) for 30 min at 4 C. Then the cells were incubated with fluorescein isothiocyanate-conjugated antimouse F(ab')2 fragment (Jackson ImmunoResearch) at 1:100 dilution for 30 min at 4 C. Isotype-matched mouse IgG served as control. Cells (2 x 104) 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 (Invitrogen Life Technologies, Grand Island, NY). The RiboQuant MultiProbe RNase Protection Assay System (Pharmingen, San Diego, CA) was used for the detection and quantitation of Fas (hAPO-3d template set) and bcl-2 family members (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. We quantified transcripts 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 (GAPDH) band intensity for each sample.

RT-PCR Analysis
The expression of caspase-10 mRNA was measured by RT-PCR. RNA isolated from the control and cytokine-treated cells was converted to cDNA by reverse transcription using Superscript II reverse transcriptase (Invitrogen Life Technologies) and the oligo-deoxythymidine primer. The cDNA was then amplified by PCR. The forward and backward primers for caspase-10 were previously described (39). Actin was also measured by RT-PCR from the same RNA samples and used as an internal control.

Immunoblot 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], RIPA lysis buffer (1% Nonidet P-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% CHAPS, 5 mM dithiothreitol] with protease inhibitors (Complete, Roche Molecular Biochemicals), depending on the primary antibodies. The protein fraction of the Trizol lysates was used for the determination of the Fas concentration. Insoluble material was removed by centrifugation and supernatants were stored frozen at –20 C until used for Western analysis. The total protein concentration was quantitated with a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). Equivalent amounts of each sample were electrophoretically separated on a 12.5% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Rabbit polyclonal anti-Fas (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit polyclonal anti-bcl-X, rabbit polyclonal anti-bax, mouse monoclonal anti-Bid, mouse monoclonal anti-bak, and hamster monoclonal anti-bcl-2 (BD Pharmingen), rabbit polyclonal anticaspase 9, rabbit polyclonal anticaspase 7, rabbit polyclonal anticaspase 3 (cleaved), mouse monoclonal anticaspase 8 (clone 1C12) (Cell Signaling Technology, Beverly, MA) and anticaspase 10 (MBL, Naka-ku, Nagoya, Japan) antibodies were used according to the manufacturer’s protocol. The vendor of the rabbit polyclonal anti-p44/p42 MAPK and phospho-p44/p42 was Cell Signaling Technology. Mouse monoclonal antiactin (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 anti-Fas antibody was detected with an ApoAlert Mitochondrial Membrane Sensor Kit (CLONTECH Laboratories, Palo Alto, CA), according to the manufacturer’s protocol and was analyzed by flow cytometry.

Data Analysis
Flow cytometry data were analyzed by WinMDI 2.8 (Joseph Trotter URL http://facs.scripps.edu/). Densitometric quantitation of autoradiograms was performed using Quantity One (Bio-Rad). Statistical analysis was performed using Student’s t test and {chi}2 test.


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

First Published Online November 24, 2004

Abbreviations: FDA, Fluorescein diacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN, interferon; MEK, MAPK kinase; PI, propidium iodide; RNase, ribonuclease; TRAIL, TNF-related apoptosis-inducing ligand.

Received for publication July 15, 2004. Accepted for publication November 15, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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