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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 9 4264-4272
Copyright © 2002 by The Endocrine Society

Other Original Article

Aberrant Apoptosis in Thyroid Epithelial Cells from Goiter Nodules

Emese Mezosi, Hiroko Yamazaki, James D. Bretz, Su He Wang, Patricia L. Arscott, Saho Utsugi, 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., H.Y., J.D.B., S.H.W., P.L.A., S.U., J.R.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 MSRB III, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0648. E-mail: . jbakerjr{at}umich.edu

Abstract

The specific pathogenesis of nodular goiter and the role of apoptosis in goitrogenesis are not known. We sought to examine the regulation of the TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL)-induced apoptosis pathways in primary thyroid cells from 17 patients with nodular goiter, using 10 normal thyroids as controls. Both goitrous and normal thyroid cells were resistant to recombinant human TRAIL and an agonist anti-Fas antibody under basal conditions. However, all normal thyrocytes could be sensitized by TNF{alpha}/IL-1ß or interferon {gamma}/IL-1ß to undergo apoptosis in response to TRAIL or FasL, respectively. In contrast, the majority of goiter-derived cells remained resistant to TRAIL (12 of 17 samples) or FasL (9 of 17 samples) after cytokine pretreatment; 14 of 17 goiter nodules were resistant to at least one death ligand. Goiter size was inversely correlated with the sensitivity to TRAIL-mediated apoptosis. The resistance of goiter cells to TRAIL did not appear to be due to transcriptional regulation or cell surface expression of death and decoy receptors. However, increased proteasome activity was found in a subset of goiter cells resistant to both death ligands, and proteasome inhibitors could sensitize these goiter cells to TRAIL-mediated apoptosis. In conclusion, goiter-derived thyroid cells are resistant to TRAIL and/or Fas-induced apoptosis in vitro, and this may represent a new aspect of aberrant growth regulation in goiter nodules. The increased proteasome activity associated with this resistance suggests that the proteasome may be an important regulator of apoptosis in nodular goiter.

NODULAR GOITER IS one of the most common endocrine diseases. The pathogenesis of nodule formation has been intensively studied and recently activating mutations in the TSH receptor and Gs{alpha} genes were identified in the development of toxic adenomas (1). However, the majority of nonhyperthyroid nodules do not demonstrate protooncogene mutations, and the primary events in the pathogenesis of these nodular goiters are still unknown (2). The most widely accepted hypothesis of nodule formation argues that there is heterogeneity in the growth potential and function of individual thyrocytes (3). Increasing evidence suggest a role for growth factor production in the thyroid, leading to TSH-independent growth of thyroid nodules (4, 5, 6). A lack of growth inhibition may also participate in the tissue imbalance that results in nodular goiter (7).

Tissue homeostasis requires a proper balance between cell proliferation and cell death. Apoptosis occurs through an evolutionarily conserved cellular program that eliminates infected or unnecessary cells and cells produced in excess or having genetic damage (8). Aberrant apoptosis is involved in the pathogenesis of many human diseases: Abnormal cell death results in excessive parenchymal cell loss, but decreased cell death contributes to the development of hyperplasias and neoplasias (9).

The two most frequently investigated apoptosis signaling pathways with relevance to thyroid homeostasis are the Fas Ligand (FasL) and the TNF-related apoptosis-inducing ligand (TRAIL) pathways (10, 11, 12). FasL and TRAIL are members of the TNF family and act through type I membrane proteins called death receptors (13, 14). FasL is expressed in activated T lymphocytes and in cells of immune-privileged organs (15). It participates in cell-mediated cytotoxicity and maintenance of immune homeostasis by eliminating activated immune cells at the end of inflammatory reactions (16). A role for FasL-mediated apoptosis has been proposed for autoimmune thyroiditis (17, 18, 19) and goiter involution (20). In contrast to FasL, TRAIL is expressed in a wide variety of normal tissues, suggesting that this pathway is highly regulated and protective mechanisms exist in normal cells (21). Although normal cells are resistant to TRAIL, it has been reported to selectively kill the majority of cancer cells (22). Recent studies suggest that the physiologic role of TRAIL is to remove virus-infected and cancer cells (23). TRAIL induces apoptosis by interacting with either of two death receptors, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) (24, 25). Three additional decoy receptors for TRAIL, which cannot transduce an apoptotic signal but can competitively block signal transduction, have also been identified: DcR1 (TRID, TRAIL-R3), DcR2 (TRUNDD, TRAIL-R4), and osteoprotegerin (21). During death receptor signaling, the intracellular death domains of the receptors bind to an adapter protein that activates an apical caspase (25). This protein complex is known as death-inducing signal complex. The initiator caspase cleaves and activates effector caspases that cleave other proteins, death substrates. This proteolytic cascade leads to apoptosis.

The death receptor pathways are regulated at several levels including the expression of death and decoy receptors, inhibitors of death-inducing signal complex formation, and apical caspase activation such as cFLIP, caspase inhibitors like the inhibitor of apoptosis (IAP) protein family and bcl-2 family members (11). Most recently, the important role of the proteasome was established in the regulation of programed cell death (26, 27). The proteasome is an adenosine triphosphate-dependent multisubunit proteolytic complex, responsible for the degradation of most intracellular proteins. Recent findings indicate that the proteasome also plays a key role in cell cycle progression and in the activation of nuclear factor {kappa}B, which is an important modulator of cell survival during stress and immune responses (28). Proteasome inhibitors have been used to induce apoptosis in various cell types (30, 31), whereas in others, these compounds were able to prevent apoptosis induced by different stimuli (32).

In the normal thyroid gland, the rate of apoptosis is very low (33), and in multinodular goiters and thyroid adenomas, similarly low apoptosis rates have been detected by immunohistochemistry (34, 35, 36). However, these studies do not examine the function of apoptosis pathways. Normal primary thyrocytes are resistant to both TRAIL and FasL- induced apoptosis, despite the constitutive expression of their respective death receptors (37, 38). Thyroid cells can be sensitized by TNF{alpha}/IL-1ß or interferon (IFN){gamma}/IL-1ß to undergo apoptosis in response to TRAIL or FasL, respectively (39, 40). In the present study, we examined the regulation of FasL- and TRAIL-mediated apoptosis in primary thyroid cells from goiter nodules.

Materials and Methods

Cell culture

These studies and tissue procurement were 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 [n = 10, age 42.3 ± 14.3 yr, female/male (F/M) 9/1]. Goiter cells were derived from well-defined nodules of multinodular goiters (n = 17, age 51.5 ± 13.4 yr, F/M 17/0). The histological diagnosis was nodular hyperplasia in every case. The nonnodular part of multinodular goiters was also obtained and the primary cultures from these tissues were usable in 10 cases. Three papillary cancers were used for screening of proteasome activity in tumor tissue (age 45.1 ± 9.7 y, F/M 3/0). All excised tissues were prepared for cell culture as previously described (37). 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% FCS, so the final concentration of FCS 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, agonist anti-DR5, anti-Fas antibody and proteasome inhibitor treatment

Primary thyroid cells were treated for 4 d with cytokines at the following concentrations: 100U/ml IFN{gamma} (Roche Molecular Biochemicals), 50 ng/ml TNF{alpha} (Collaborative Biomedical Products), 50 U/ml IL-1ß (Sigma). Cells were then treated overnight with 800 ng/ml TRAIL, 0.1 µg/ml agonist goat polyclonal anti-DR5 antibody (R&D Systems, Minneapolis, MN) or 1 µg/ml agonist mouse monoclonal IgM anti-Fas antibody (clone CH11, Upstate Biotechnology, Inc., Waltham, MA). The concentration dependence and time course of the effect of TRAIL and anti-Fas antibody were published in our previous papers (37, 40). TRAIL (a kind gift from A. Chinnaiyan, University of Michigan) was affinity purified as described (41) from bacterial lysates of cells 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 (41). Cell death was measured 20 h after TRAIL, anti-DR5, or anti-Fas antibody administration. To detect the effect of proteasome inhibition on TRAIL susceptibility, two different proteasome inhibitors, lactacystin and MG132 (Calbiochem, La Jolla, CA), were used at 1 µM and 10 µM concentrations, respectively, at the same time of TRAIL administration. Cell death was measured 5 h later to avoid the toxicity of proteasome inhibition.

Determination of cell viability and apoptosis

Cell viability was determined by staining with fluorescein diacetate (FDA) and propidium iodide (PI), and quantitated by flow cytometry as described by Killinger et al. (42). Living cells are FDA positive and PI negative. During the apoptosis process, cells lose the capability of FDA uptake and maintain membrane integrity and become FDA negative and PI negative. In the late phase of apoptosis, parallel with the increase of membrane permeability, cells will be FDA negative and PI positive.

RNase protection assay

RNA was isolated from cells using Trizol reagent according to the manufacturer’s protocol (Life Technologies, Inc., Grand Island, NY). RiboQuant MultiProbe RNase protection assay system (PharMingen, San Diego, CA) was used for the detection and quantitation of TRAIL receptors (hAPO-3d template set) and IAPs [human apoptosis (hAPO)-5 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, Inc., 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.

Immunoblot analysis

Radioimmuoprecipitation assay lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) was used for the detection of DR5 and cFLIP, Triton-X lysis buffer [150 mM NaCl, 10 mM Tris (pH 7.4), 5 mM EDTA, 1% Triton-X-100] to determine bcl-2 protein level, with protease inhibitors (Complete, Roche Molecular Biochemicals). Insoluble material was removed by centrifugation and supernatants were stored frozen at -20 C until used for Western analysis. Total protein concentration was quantitated 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 nitrocellulose membrane. Goat polyclonal anti-DR5 (R&D Systems), rabbit polyclonal anti-cFLIP (Alexis), and hamster monoclonal anti-bcl-2 antibodies (BD, PharMingen) were used according to the manufacturer’s protocol. The results were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL) followed by auto-radiography.

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, washed in PBS, and incubated for 15 min in blocking buffer (2% FBS, 0.1% Tween 20 in PBS). Blocking buffer was replaced with anti-cytokeratin 18 antibody diluted to 2.0 µg/ml in blocking buffer and incubated for 1 h. Cells were then washed in blocking buffer and resuspended in antimouse fluorescein isothiocyanate conjugate (Jackson ImmunoResearch Laboratories, Inc., 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, DR4, DcR2, and DcR1 surface expression

Cytokine-treated thyroid cells were made nonadherent by incubation with 0.265 mM EDTA in PBS, washed in PBS, and incubated for 15 min in blocking solution (2% normal horse serum, 1% BSA in PBS) at 4 C. Goat polyclonal antihuman DR5, DR4, DcR2, or DcR1 antibodies (R&D Systems) were diluted in blocking solution to 5 µg/ml, and cells were incubated with the antibodies for 20 min at 4 C. After washing in the blocking solution, cells were incubated with fluorescein isothiocyanate-conjugated antigoat F(ab')2 fragment (Jackson ImmunoResearch Laboratories, Inc.) at 1:100 dilution for 20 min at 4 C. Purified goat IgG (R&D Systems) served as control. The 2 x 104 cells were acquired for each sample and quantitated on a FACScalibur flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ).

Fluorogenic peptide substrate assay for proteasome activity

Thyroid cells were lysed and homogenized in ice-cold buffer A [50 mM Tris-Hcl (pH 7.5), 25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA]. Protein concentration was quantitated by BCA protein assay kit (Pierce Chemical Co.). Proteasome substrate Suc-LLVY-MCA (Calbiochem) was incubated at a final concentration of 0.1 mM with thyroid cell lysates containing 5 µg protein at 37 C for 30 min. The reaction was stopped by buffer F [30 mM NaAc (pH 4.3), 100 mM CH2ClCOOH, 70 mM acetic acid]. Hydrolysis of peptides was determined with a RF-5001 PC spectrofluorometer (Shimadzu, Tokyo, Japan) at 380 nm excitation/460 nm emission for MCA. The measurements were made in triplicate. Results were standardized to hydrolysis of Suc-LLVY-MCA by 5 µg recombinant proteasome (Calbiochem), which was used as a positive control (100%).

Computer software

Flow cytometry data were analyzed by WinMDI 2.8 (Joseph Trotter, http://facs.scripps.edu/). Densitometric quantitation of autoradiograms was performed using Quantity One (Bio-Rad Laboratories, Inc.). Statistical analysis was performed using t test and {chi}2 test.

Results

Inflammatory cytokines induce susceptibility to TRAIL- and FasL-mediated cell death in normal thyrocytes but not in the majority of goiter-derived cells

In agreement with our previous observations, untreated normal primary thyroid cells were resistant to TRAIL and agonist anti-Fas antibody (Fig. 1Go, A and B) (38, 39). Thyrocytes from 17 goiter nodules were also resistant to both death ligands under basal conditions (Fig. 1Go, C and D). Normal thyroid cells could be sensitized to TRAIL and FasL by unique combinations of inflammatory cytokines (39, 40). In Fig. 2Go a typical response of one normal (A and B) and one goiter-derived sample (C and D) to cytokine pretreatment and death induction is demonstrated. The sensitivity of goiter cells to TRAIL- and Fas-mediated apoptosis after cytokine pretreatment was significantly decreased, compared with normal cells (Fig. 2Go, A and B). The normal range was defined as mean ± 2 SD of apoptosis rate in normal cells. According to their response to death ligands after cytokine pretreatment, the goiter cell populations were divided into sensitive (cell death within the normal range) and resistant (cell death below the normal range) groups. The susceptibility of cytokine pretreated goiter cells to TRAIL and anti-Fas antibody was diverse in several cases: Seven goiters were resistant to death signaling through both pathways, five were resistant to TRAIL but sensitive to CH11, two were sensitive to TRAIL but resistant to CH11, and only three goiters were sensitive to both death ligands. In summary, the majority of goiter cells (14 of 17) remained resistant to at least one death ligand after pretreatment with inflammatory cytokines.



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  		Figure 1. Flow cytometric analysis of TRAIL- and Fas-mediated apoptosis after cytokine pretreatment in a normal (A and B) and goiter sample (C and D). A, Normal thyroid cells were exposed to 800 ng/ml TRAIL after 4 d of pretreatment with the indicated cytokines. I/T/I: IFN{gamma}/TNF{alpha}/IL-1ß treatment. Cell death was determined by FDA and PI staining and 10,000 cells/sample were acquired by flow cytometry. Asterisk denotes conditions in which P is less than 0.0001 ({chi}2 test), compared with the control samples (no cytokines and cytokines without TRAIL). B, Normal thyroid cells were treated with 1 µg/ml agonist anti-Fas antibody (CH11) after 4 d of pretreatment with the indicated cytokines. Cell death was assayed as above. *, P < 0.0001 ({chi}2 test), compared with the control samples (no cytokines and cytokines without CH11). C, Primary thyroid cells from a goiter nodule were treated and assayed as in A. D, Primary thyroid cells from a goiter nodule were treated and assayed as in B. This experiment is representative of results from independent experiments using thyroid cells from 10 normal thyroid tissues and seven resistant goiters.

 


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  		Figure 2. Distribution of death receptor-mediated apoptosis after cytokine pretreatment in normal and goiter-derived cells. A, Primary thyroid cells from 10 normal thyroid glands and 17 nodular goiters were treated with 800 ng/ml TRAIL after 4 d of pretreatment with TNF{alpha}/IL-1ß. Cell death was measured by FDA and PI staining and quantitated by flow cytometry. The 10,000 cells/sample were assayed; individual data points represent TRAIL-mediated percent cell death. Mean ± SD is shown for each group. B, Normal and goiter-derived primary thyroid cells from the same patients as above were treated with 1 µg/ml anti-Fas antibody (clone CH11) after 4 d of pretreatment with IFN{gamma}/IL-1ß. Cell death was assayed as above; individual data points represent Fas-mediated percent cell death. Mean ± SD is shown for both groups.

 
The comparison of TRAIL sensitivity in nonnodular and nodular thyroid cells from the same patients with multinodular goiter confirmed that the decreased death ligand sensitivity is characteristic for nodule-forming goiter cells (Fig. 3Go).



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  		Figure 3. TRAIL-mediated apoptosis in nonnodular and nodular thyroid cells from patients with multinodular goiter. Primary thyroid cells were derived from relatively normal, nonnodular parts of multinodular goiters and from well-defined nodules of the same patients (n = 10). Data from the same patient are connected by dotted line. Cells were treated with 800 ng/ml TRAIL after 4 d of pretreatment with TNF{alpha}/IL-1ß. Cell death was measured by FDA and PI staining and quantitated by flow cytometry. The 10,000 cells/sample were assayed; individual data points represent TRAIL-mediated percent cell death. Mean ± SD is shown for each group.

 
Sensitivity to TRAIL-mediated apoptosis inversely correlates with goiter size

Goiter size was approximated by the weight of surgically removed thyroid as reported by pathological records. The mean weight of TRAIL-sensitive goiters was significantly lower than the mean weight of the TRAIL-resistant group (P < 0.05). The TRAIL-sensitive goiters were small (<25 g) in every case, but the TRAIL-resistant goiters formed a heterogeneous group, containing small and large goiters (reflected by the high SD value) (Fig. 4AGo). The susceptibility to anti-Fas antibody did not show a relationship with goiter size (Fig. 4BGo).



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  		Figure 4. Comparison of goiter size between TRAIL and FasL sensitive and resistant goiters. A, Goiter-derived thyroid cell cultures were divided into two groups according to their sensitivity to TRAIL after cytokine pretreatment. The normal range was defined as mean ± 2 SD of apoptosis rate in normal cells. Cell populations responding to a lesser extent than this normal range, were categorized as TRAIL-resistant goiters. Cultures responding similar to normal cells were categorized as TRAIL-sensitive goiters. Results are presented for each group as mean ± SD weight of thyroid glands removed during thyroidectomy. B, Primary thyroid cell cultures from goiter nodules were divided into sensitive and resistant groups as above, according to their response to CH11. Data are presented as mean ± SD weight of thyroid glands removed during thyroidectomy.

 
TRAIL receptor expression and regulation in goiter cells

To define the mechanism of resistance to TRAIL-mediated cell death, the mRNA expression of TRAIL receptors was compared between five normal and three resistant goiter cell cultures after cytokine pretreatment, using RNase protection assays (Fig. 5Go). The expression of TRAIL receptors was moderately regulated by cytokines. TNF{alpha} treatment resulted in an approximate 2-fold increase in DR5 and DcR1 (Fig. 6Go). A similar up-regulation of DcR2 was found after TNF{alpha}/IL-1ß treatment. Nevertheless, this up-regulation did not correlate with enhanced TRAIL-mediated apoptosis because TNF{alpha} alone did not sensitize the cells to TRAIL, and the expected result of DcR2 up-regulation would be the protection of cells from TRAIL. Also of note, these changes did not differ between the normal and goiter groups. The mRNA level of DcR1 was significantly decreased by TNF{alpha}/IL-1ß treatment in normal cells, compared with controls and goiters (P < 0.05); however, further experiments excluded the involvement of DcR1 in goiter resistance (Figs. 7Go and 8Go).



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  		Figure 5. Cytokine regulation of TRAIL receptor mRNA expression. A, RNase protection assay using the hAPO-3d template set was performed on 5 µg total RNA isolated from normal and goiter-derived thyroid cells after 4 d of pretreatment with the indicated cytokines. This experiment is representative of results from independent experiments using thyroid cells from five normal thyroid tissues and three resistant goiters. B, The mRNA expression of TRAIL receptors was quantitated by densitometry and normalized to GAPDH signal and TRAIL receptor expression in untreated cells. Data are presented as mean ± SD of mRNA expression from five normal thyroid cell cultures. Asterisk denotes significant alteration of mRNA expression between the normal and the goiter group. C, The mRNA expression of TRAIL receptors was quantitated as above and normalized to GAPDH signal and results in untreated cells. Data are present as the mean ± SD of mRNA expression from three resistant goiters.

 


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  		Figure 6. Cytokine regulation of DR5 protein expression. Western analysis of DR5 was performed from cell lysates of five normal, two TRAIL-sensitive, and six TRAIL-resistant goiter cell cultures after 4 d of pretreatment with the indicated cytokines. The autoradiograms were quantitated by densitometry and the DR5 expression after cytokine treatment was normalized to the result from untreated cells. Representative results are shown for each category.

 


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  		Figure 7. Flow cytometric determination of DR5 and DcR1 cell surface expression. A, The analysis of DR5 and DcR1 cell surface expression was performed on eight normal thyroid cell populations with and without 4 d of TNF{alpha}/IL-1ß treatment. Representative histograms using DR5 and DcR1 antibodies are demonstrated (open curve), compared with control antibody (filled curve). B, DR5 and DcR1 surface expression is presented as above in a TRAIL-resistant goiter cell population, which is representative of six resistant goiters.

 


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  		Figure 8. The effect of an agonist anti-DR5 antibody in normal and TRAIL-resistant goiter cells. Normal and goiter-derived cells were treated with 100 ng/ml goat control IgG, 100 ng/ml goat agonist anti-DR5 antibody, or 800 ng/ml TRAIL after 4 d of TNF{alpha}/IL-1ß pretreatment. Cell death was measured by FDA and PI staining and 10,000 cells/sample quantitated by flow cytometry. Data are presented as the mean ± SD percent cell death of triplicate measurements.

 
The death signal from TRAIL is transmitted by DR5 in normal thyroid cells (40). Despite the lack of DR5 mRNA regulation in the TRAIL-sensitive conditions, the total DR5 protein expression was up-regulated by IL-1ß and TNF{alpha}/IL-1ß treatment and positively correlated with the sensitization of cell to TRAIL from five investigated normal thyroid tissues (Fig. 6Go). In sensitive goiter cells the up-regulation of total DR5 expression was similar to that in normal cells; however, in six resistant goiters, these changes were diminished or absent (Fig. 6Go).

The surface expression of TRAIL receptors was investigated in eight normal, four sensitive, and six resistant goiter-derived cell populations. IL-1ß and TNF{alpha}/IL-1ß treatment uniformly resulted in a significant increase in the cell surface expression of DR5, both in normal and goiter cells, suggesting that the resistance of goiter cells to TRAIL was not caused by a lack of DR5 cell surface expression (Fig. 7Go). DR4 and DcR2 were not detected on either normal or resistant cells (data not shown). DcR1 surface expression was minimal in control cells and remained unchanged after TNF{alpha}/IL-1ß treatment (Fig. 7Go).

TRAIL-resistant goiter cells are resistant to an agonist anti-DR5 antibody

To exclude a role for decoy receptors in mediating resistance to TRAIL, an agonist anti-DR5 antibody without cross-reactivity to DcR1 and DcR2 was used. The TRAIL-resistant goiter cells were also resistant to the agonist anti-DR5 antibody (Fig. 8Go). This confirms that DR5 activation does not induce apoptosis in these cells, despite the presence of DR5 on the cell surface. The loss of DR5 signal transduction makes the participation of decoy receptors unnecessary for the expression of goiter cell resistance.

Intracellular inhibitors of apoptosis in normal and goiter-derived cells

Five normal and five TRAIL-resistant goiter cells were compared for the expression of cFLIP and bcl-2 by Western blot analysis. No difference in baseline and cytokine-induced expression was observed for these proteins (data not shown). Members of IAP family were also unchanged (XIAP) or up-regulated (cIAP-1, cIAP-2, NAIP) by pretreatment with TNF{alpha} and IL-1ß (Fig. 9Go). These changes would be expected to increase protection instead of sensitization to death ligands.



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  		Figure 9. Cytokine regulation of IAPs mRNA expression. RNase protection assay using hAPO-5 template set was performed on 5 µg total RNA isolated from normal and goiter-derived thyroid cells after 4 d of pretreatment with the indicated cytokines. The results are representative of three normal and three resistant goiter cells.

 
Proteasome activity positively correlates with the resistance to death ligands

Recent findings indicated that the proteasome system plays an important role in the regulation of apoptosis (26, 27, 28, 29, 30, 31). Normal thyroid cells from 10 individuals were characterized by low proteasome activity (mean ± SD 12.9% ± 5.2%) (Fig. 10AGo). Lysates from six goiter cell cultures resistant to both death ligands demonstrated proteasome activity that was significantly increased over normal thyroid cells (87.2% ± 37.9%) and was similar to the proteasome activity observed in papillary cancers (Fig. 10AGo). In contrast, death ligand-sensitive goiters and goiters resistant to only one of the two death ligands showed low proteasome activity, similar to normal cells (Fig. 10AGo). The proteasome activity was also low in thyroid cells derived from the nonnodular part of multinodular goiters (data not shown). The treatment with proteasome inhibitor lactacystin restored sensitivity of goiter cells to TRAIL-mediated cell death (Fig. 10BGo). Restoration of TRAIL susceptibility in these cells by proteasome inhibition was confirmed by another inhibitor, MG132 (data not shown).



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  		Figure 10. Proteasome activity in thyroid cells and the effect of proteasome inhibition on TRAIL susceptibility. A, The proteasome activity of thyroid cells was measured by the hydrolysis of a fluorogenic substrate Suc-LLVY-MCA. The individual values were measured in triplicates with SD of less than 5% for all data points and standardized to the hydrolysis of substrate by 5 µg recombinant proteasome as a positive control (100%). Results are presented as mean ± SD of proteasome activity in the indicated number of patients, grouped according to histological diagnosis and susceptibility to death ligands. The proteasome activity in papillary cancers is shown for comparison. *, P < 0.05. B, Normal and goiter-derived thyroid cells were pretreated for 4 d by TNF{alpha}/IL-1ß and incubated with 800 ng/ml TRAIL with or without 1 µM lactacystin for 5 h. Cell death was measured by FDA and PI staining and 10,000 cells/sample were assayed by flow cytometry. Data are presented as percent cell death with or without lactacystin. *, P < 0.05 (t test).

 
Discussion

A proper balance between cell proliferation and cell death is required to maintain tissue homeostasis. Goitrogenesis is a very slow process, unlike cancer, which is caused by increased cell proliferation. It is possible that a decrease in cell death can contribute to the accumulation of cells in goiter nodules. Normal thyroid cells are resistant to all the known death ligands, despite the constitutive expression of their respective death receptors (37, 38, 39, 40) but can be sensitized to death induction by these ligands using proinflammatory cytokines (39, 40). In contrast to normal cells, the majority of goiter cell populations are not sensitized to TRAIL- or Fas-mediated apoptosis by cytokine pretreatment. This suggests a functional decrease in death receptor-mediated apoptotic activity in goiter-derived primary thyroid cells and indicates that there is an altered regulation of these death pathways in goiter cells. Moreover, the sensitivity to TRAIL-induced cell death inversely correlated with the goiter size. This raises the possibility that TRAIL is important in maintaining normal thyroid cell populations and TRAIL resistance contributes to the development of nodular goiter.

The death receptor pathways are controlled by a variety of mechanisms, from the expression of death and decoy receptors to the control of intracellular signaling. In various cell types, the resistance to TRAIL has been reported to be mediated by distinct mechanisms (43). The lack of surface death receptor expression was decisive in the resistance of melanocytes to TRAIL, and expression of a decoy receptor (DcR1) was reported to be important in the protection of endothelial cells (43). Fibroblasts were protected from TRAIL-induced apoptosis by intracellular inhibitors (43). In our studies, TRAIL resistance in untreated normal thyroid cells likely is due to the low level of death receptor surface expression (40). However, the presence of intracellular inhibitors to mediate TRAIL resistance cannot be ruled out because the inhibition of protein synthesis makes normal thyroid cells susceptible to TRAIL, possibly through selective loss of labile inhibitors (37).

In TNF{alpha}/IL-1ß pretreated normal thyrocytes, TRAIL signal is transmitted by DR5 (40). In these cells, the treatment with TNF{alpha}/IL-1ß up-regulated overall levels of DR5 protein and cell surface expression, providing a possible mechanism for TRAIL sensitization. In TRAIL-resistant goiter cells, there was no change in total DR5 protein expression after TNF{alpha}/IL-1ß treatment, but DR5 surface protein expression was increased similar to normal cells. Despite the presence of cell surface receptor, DR5 signal transmission was not accomplished in these cells as confirmed in studies using an agonist anti-DR5 antibody. Thus, in goiter cells that did not respond to TNF{alpha}/IL-1ß, intracellular mechanisms appeared to play a role in TRAIL resistance. A screening of well-known intracellular inhibitors (cFLIP, bcl-2, IAPs) did not identify the source for goiter cell resistance, raising the possibility that other inhibitors or a down-regulation of proapoptotic proteins may be responsible.

One potential mechanism affecting the down-regulation of proapoptotic proteins in resistant goiters could be an increase in protein degradation. Most intracellular proteins are degraded by the proteasome complex. In a subset of goiters increased proteasome activity was found, which was correlated with the resistance of goiter cells to both death ligands. Low doses of proteasome inhibitors sensitized the resistant goiters to TRAIL, supporting a causal relationship between elevated proteasome activity and apoptosis resistance. The essential role of the proteasome in cell survival is well known, and many studies showed that proteasome inhibitors can induce apoptosis or sensitize cells to TRAIL and FasL (26, 27, 28, 29, 30, 31). However, this is the first study of proteasome activity in normal and goiter-derived thyroid cells and the first demonstration of a positive correlation between the proteasome activity and resistance to death receptor-mediated apoptosis.

Primary cell cultures provide a unique opportunity to investigate the normal regulation of death receptor-mediated apoptosis and altered regulation in specific disease conditions. Concerns might be raised that the in vitro culture of these cells might alter their resistance to apoptosis. This is controlled because normal and goiter cells were cultured simultaneously for similar time periods and under identical conditions. However, there might be differences in serum-dependent signaling between normal and nodular tissue. The relationship of growth-promoting signaling and resistance to apoptosis should be addressed in future examinations. These in vitro results need to be confirmed in forthcoming in vivo studies.

In summary, although normal thyroid cells in vitro can be sensitized to TRAIL and FasL by inflammatory cytokines, the majority of goiter samples show resistance to death ligands after cytokine pretreatment. The resistance of goiter cells to TRAIL is not caused by the altered surface expression of death receptors or the overexpression of cFLIP, bcl-2, or IAPs. The increased proteasome activity in a subset of resistant goiters and the ability of proteasome inhibitors to sensitize resistant cells to TRAIL suggests that the proteasome is an important regulator of apoptosis resistance. Moreover, the inverse correlation of goiter size with the sensitivity to TRAIL-mediated apoptosis suggests a relationship to goiter pathogenesis.

Acknowledgments

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

Footnotes

This work was supported by the NIH Grants R01 A137141, P60DK20572, and DK58771.

Abbreviations: FasL, Fas ligand; FDA, fluorescein diacetate; F/M, female/male; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hAPO, human apoptosis; IAP, inhibitor of apoptosis; IFN, interferon; PI, propidium iodide; TRAIL, TNF-related apoptosis-inducing ligand.

Received January 28, 2002.

Accepted May 28, 2002.

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