James D. Bretz

FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex

To identify CAP3 and CAP4, components of the CD95 (Fas/APO-1) death-inducing signaling complex, we utilized nano-electrospray tandem mass spectrometry, a recently developed technique to sequence femtomole quantities of polyacrylamide gel-separated proteins. Interestingly, CAP4 encodes a novel 55 kDa protein, designated FLICE, which has homology to both FADD and the ICE/CED-3 family of cysteine proteases. FLICE binds to the death effector domain of FADD and upon overexpression induces apoptosis that is blocked by the ICE family inhibitors, CrmA and z-VAD-fmk. CAP3 was identified as the FLICE prodomain which likely remains bound to the receptor after proteolytic activation. Taken together, this is unique biochemical evidence to link a death receptor physically to the proapoptotic proteases of the ICE/CED-3 family.

TRAIL Death Pathway Expression and Induction in Thyroid Follicular Cells

To determine whether programmed cell death in thyroid follicular cells can be related to activation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway, we examined the expression and function of this pathway in primary thyroid follicular cells and a papillary thyroid carcinoma cell line in vitro. Despite the expression of TRAIL receptors death receptor 4 and death receptor 5, purified TRAIL could not induce programmed cell death (PCD) in any of the thyroid follicular cells examined. However, pre-incubation with cycloheximide before TRAIL facilitated the induction of rapid and massive PCD. This suggested that despite the presence of a labile inhibitor of the TRAIL pathway, TRAIL could mediate PCD under appropriate conditions. To determine whether there were sources of TRAIL in the thyroid that could interact with thyroid follicular cell TRAIL receptors, RNase protection assays were used to determine TRAIL mRNA expression. TRAIL message was expressed in intrathyroidal lymphocytes isolated from a patient with thyroiditis, and unexpectedly, thyroid follicular cells themselves could be induced to express abundant TRAIL message in the presence of the inflammatory cytokines interferon γ, tumor necrosis factor α, and interleukin 1β. Furthermore, the papillary thyroid carcinoma cell line could be induced to kill the TRAIL-sensitive lymphoma cell line BJAB through a TRAIL-dependent mechanism.

INFLAMMATORY CYTOKINE REGULATION OF FAS-MEDIATED APOPTOSIS IN THYROID FOLLICULAR CELLS

The occurrence of apoptosis in thyroid follicular cells induced by Fas activation has been a subject of much debate. This is due, in part, to the fact that no physiologically relevant treatment conditions have been reported to cause rapid and extensive Fas-mediated apoptosis in thyroid cells, whereas treatment with the protein synthesis inhibitor cycloheximide prior to Fas activation allows for massive cell death. This indicates that the Fas signaling pathway is present but that its function is blocked in the overwhelming majority of cultured thyroid cells. To reconcile the conflicting reports, we set out to identify physiologically relevant conditions in which rapid, massive thyroid cell apoptosis in response to Fas activation could be demonstrated. We determined that susceptibility to Fas-activated apoptosis could be influenced by certain combinations of inflammatory cytokines. Although no single cytokine was effective, pretreatment of thyroid cells with the combination of γ-interferon and either tumor necrosis factor-α or interleukin 1β allowed for massive Fas-mediated apoptosis. Susceptibility to Fas-induced death correlated with an increase in expression of a tunicamycin-inhibitable high molecular weight form of Fas but not with aggregate expression of Fas.

Fas (APO-1, CD95)-Mediated Apoptosis in Thyroid Cells Is Regulated by a Labile Protein Inhibitor.

To determine whether thyroid cell apoptosis observed in autoimmune thyroid disease could be related to activation of the Fas pathway, we examined the expression and function of Fas on thyroid follicular cells in vitro. Fas messenger RNA was found to be present using two different techniques and was expressed at equal levels in thyrocytes cultured either in the presence or absence of TSH. Fas antigen protein expression was demonstrated by Western blot of thyroid cell lysates and by immunohistochemical staining of thyrocytes, and the amount of Fas protein present did not appear to vary regardless of culture conditions. Despite expressing substantial amounts of Fas protein, thyrocytes treated with anti-Fas monoclonal antibody failed to undergo apoptosis. The addition of either interferon-gamma or interleukin-1beta to the anti-Fas-treated cell cultures also did not promote apoptotic signaling through this pathway. In contrast, the concomitant administration of cycloheximide allowed the induction of apoptosis through the activation of Fas in thyrocytes. These results suggest that Fas is constitutively expressed in thyrocytes, but that the induction of apoptosis through the Fas pathway is blocked by a labile protein inhibitor.

A Unique Combination of Inflammatory Cytokines Enhances Apoptosis of Thyroid Follicular Cells and Transforms Nondestructive to Destructive Thyroiditis in Experimental Autoimmune Thyroiditis

Treatment of cultured primary human thyroid cells with IFN-γ and TNF-α uniquely allows the induction of Fas-mediated apoptosis. To investigate the role of this cytokine combination in vivo, CBA/J mice were immunized with thyroglobulin and then injected with IFN-γ and TNF-α. Compared with control animals, mice treated with IFN-γ and TNF-α showed significantly sustained lymphocytic infiltration in the thyroid, which was associated with the destruction of portions of the follicular architecture at wk 6 after initial immunization. Furthermore, the number of apoptotic thyroid follicular cells was increased only in the thyroids from mice treated with the IFN-γ and TNF-α. We also analyzed the function of the Fas pathway in vivo in cytokine-treated mice by using an agonist anti-Fas Ab injected directly into the thyroid. Minimal apoptosis of thyroid epithelial cells was observed unless the mice were pretreated with IFN-γ and TNF-α. These data demonstrate that this unique combination of inflammatory cytokines facilitates the apoptotic destruction of thyroid follicular cells in experimental autoimmune thyroiditis, in a manner similar to what is observed in Hashimoto’s thyroiditis in humans.

Apoptosis and autoimmune thyroid disease: following a TRAIL to thyroid destruction?

In the past decade, it became apparent that immune mediated cell death in a number of autoimmune endocrine diseases was due to the induction of apoptosis in target organ cells. This was conclusively demonstrated for thyroid follicular cells in Hashimoto’s (destructive autoimmune) thyroiditis, but the mechanisms underlying this cell death were not clear. Several hypotheses were put forth involving the role of deathsignalling molecules expressed on thyroid cells. While many of these hypotheses did not hold up under close scrutiny, this stimulated work on the molecular mechanisms of thyroid destruction. Several apoptosis signalling pathways, initiated by molecules such as Fas ligand (FASL) and tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been shown to be active in thyroid cells and may be involved in destructive thyroiditis. In this review we will attempt to sort out the inconsistencies in published data on the mechanisms of death-receptor mediated thyroid destruction. We will also review recently proposed models of these mechanisms, and outline directions for research that we feel might lead to discoveries of benefit to the clinician in the treatment and prevention of destructive autoimmune thyroiditis.

2-Methoxyestradiol, an endogenous estrogen metabolite, induces thyroid cell apoptosis

The etiology of autoimmune thyroid diseases is unclear; however, the extreme female predominance suggests that sex hormones may have a pathogenic role. 2-Methoxyestradiol (2-ME) is present in the serum of women during the ovulatory and luteal phases of the menstrual cycle, and during pregnancy. We investigated the actions of 2-ME and estrogen on thyroid follicular cells. 2-ME induced dramatic changes in cell morphology and decreased the viability of the cells, as well as disrupted the structural integrity of cultured thyroid follicles. Flow cytometric analysis showed that 2-ME halted cell proliferation by arresting the cells in the G2/M cell-cycle compartment. Prolonged exposure to 2-ME led to apoptosis and to increased release of the autoantigen thyroid peroxidase (TPO). 17beta-estradiol failed to produce a similar effect even in 40-fold molar excess to 2-ME. Co-treatment with estrogen receptor antagonists did not alter the 2-ME effect, indicating that 2-ME was not operating through a classic nuclear estrogen receptor. In conclusion, this study indicates that 2-ME induces G2/M cycle arrest, apoptosis and the disruption of thyroid follicles. This process results in the release of thyroid antigens that may play a role in high incidence of thyroid autoantibodies and autoimmune thyroid disease in women.

The role of Fas-mediated apoptosis in thyroid autoimmune disease.

Apoptosis is a carefully regulated mechanism of cell death that differs from necrosis and plays an important role in normal tissue development and homeostasis, as well as disease processes. Apoptosis also plays an important role in autoimmunity. Defective apoptosis can cause systemic autoimmunity by allowing the survival of autoreactive lymphocytes. It may also be involved in the pathogenesis of organ-specific autoimmune diseases, such as Hashimotos thyroiditis, through altered target organ susceptibility. Apoptosis signaling pathways can be initiated through activation of death receptors. One of these pathways employs the death receptor Fas and its ligand (FasL). Fas expression and death pathway signaling have been demonstrated in the thyroid, but there is controversy surrounding the expression of FasL and its role in thyroid autoimmunity. A number of proteins, including FAP-1, Bcl-2 and I-FLICE may regulate the Fas pathway in the thyroid and provide potential mechanisms for modifying the pathogenesis of autoimmune thyroid disease.

Inflammatory cytokine regulation of TRAIL-mediated apoptosis in thyroid epithelial cells.

Death receptor-mediated apoptosis has been implicated in target organ destruction in chronic autoimmune thyroiditis. Depending on the circumstances, inflammatory cytokines such as IL-1, TNF and IFNγ have been shown to contribute to either the induction, progression or inhibition of this disease. Here we demonstrate that the death ligand TRAIL can induce apoptosis in primary, normal, thyroid epithelial cells under physiologically relevant conditions, specifically, treatment with the combination of inflammatory cytokines IL-1β and TNFα. In contrast, IFNγ is capable of blocking TRAIL-induced apoptosis in these cells. This regulation of TRAIL-mediated apoptosis by inflammatory cytokines appears to be due to alterations of cell surface expression of TRAIL receptor DR5 and not DR4. We also show the in vivo presence of TRAIL and TRAIL receptors DR5 and DcR1 in both normal and inflamed thyroids. Our data suggests TRAIL-mediated apoptosis may contribute to target organ destruction in chronic autoimmune thyroiditis.

Apoptosis and the thyroid: The biology and potential implications for thyroid disease

Apoptosis is a physiologic process of cell death that is central to normal development and occurs in response to a variety of physiologic and pathophysiologic stimuli. In the thyroid, abnormal apoptotic activity may be involved in a variety of diseases such as Hashimoto thyroiditis and Graves disease. Receptor-mediated apoptosis signaling pathways such as those initiated by Fas ligand, tumor necrosis factor, and tumor necrosis factor–related apoptosis-inducing ligand are present and functional in the thyroid. In Hashimoto thyroiditis, which is a typical organ-specific autoimmune disease resulting in thyroid destruction, Fas-mediated apoptosis has been suggested as a mechanism of thyroid follicular cell death. Graves disease is characterized by hyperthyroidism that appears to be caused by autoimmunity. This disorder may be linked to suppression of apoptosis. Apoptosis in thyroid follicular cells may be regulated at multiple levels, including the expression of anti-apoptotic proteins such as Bcl-2. Alterations in the expression of death receptors and ligands by inflammatory cytokines may also play a role in the regulation of apoptosis. These mechanisms may contribute to the pathogenesis of autoimmune thyroid disease.