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

Abstract

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.

Involvement of Protein Kinase Cε (PKCε) in Thyroid Cell Death

“…Growth factor or TSH deprivation and protein synthesis inhibitors can trigger apoptosis in thyrocytes (31). The role of interleukin-1␤ and Fas is more controversial (56,57). Downstream of these initiating events are a variety of intermediates that either positively or negatively regulate apoptosis.…”

Specificity questions concerning the clone 33 anti-fas ligand antibody

“…We were confused by these findings because others and we had observed constitutive expression of Fas antigen on normal thyroid cells. 13 The simultaneous, constitutive expression of FasL therefore should have led to thyroid destruction even in normal tissue. We decided to assay for FasL mRNA expression in normal thyroid cells and did not observe it either by nuclease protection assay or RT ± PCR.…”

Enhanced sensitivity to apoptosis in Ras-transformed thyroid cells

“…Although primary (Di Matola et al, 2000) and established (Vitale et al, 1999) human thyroid cells reportedly undergo more extensive anoikis than established rat thyroid cell lines, the e ects of Ras on anoikis have not been reported. However, primary human thyrocytes, like WRT cells, are resistant to a variety of apoptotic insults including activation of death receptors (Arscott et al, 1997;Bretz et al, 1999). Intriguingly, Ras transformation renders human thyroid cells more sensitive to apoptosis induced by treatment with PI3K inhibitors , suggesting that the potentiating e ects of Ras on apoptosis in thyroid cells are physiologically signi®cant.…”

“…Fas ligation or addition of TRAIL, robust stimulators of apoptosis in many cells, required the concerted action of cycloheximide to stimulate apoptosis in human thyroid cells (Arscott et al, 1997;Bretz et al, 1999). Even following 12 days in the absence of TSH, insulin and serum, WRT cells retain a normal morphology and reenter the cell cycle upon growth factor addition (Meinkoth, unpublished).…”