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. 2008 Jul;149(7):3321-9.
doi: 10.1210/en.2008-0080. Epub 2008 Mar 20.

Cultured murine thyroid epithelial cells expressing transgenic Fas-associated death domain-like interleukin-1beta converting enzyme inhibitory protein are protected from fas-mediated apoptosis

Yujiang Fang  1 , Helen Braley-Mullen
Affiliations

Cultured murine thyroid epithelial cells expressing transgenic Fas-associated death domain-like interleukin-1beta converting enzyme inhibitory protein are protected from fas-mediated apoptosis

Yujiang Fang et al. Endocrinology. 2008 Jul.

Abstract

The antiapoptotic molecule Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein (FLIP) inhibits Fas-mediated apoptosis by blocking activation of caspase-8. We previously showed that expression of transgenic FLIP on thyroid epithelial cells (TECs) of DBA/1 and CBA/J mice promoted earlier resolution of granulomatous experimental autoimmune thyroiditis in vivo. This study was undertaken to directly determine whether transgenic FLIP expressed on cultured TECs can protect TECs from Fas-mediated apoptosis in vitro. The results indicate that cultured TECs from DBA/1 and CBA/J mice can be sensitized in vitro by interferon-gamma and TNF-alpha to undergo Fas-mediated apoptosis. Transgenic overexpression of FLIP protected cultured TECs of FLIP transgene (Tg)+ DBA/1 and CBA/J mice from Fas-mediated apoptosis, and FLIP small interfering RNA transfection of cultured TECs of FLIP Tg+ DBA/1 and CBA/J mice abolished the protective effect. These in vitro results are consistent with our previous in vivo studies using DBA/1 and CBA/J FLIP Tg+ mice and provide direct support for the hypothesis that transgenic expression of FLIP promotes resolution of granulomatous experimental autoimmune thyroiditis by protecting TECs from apoptosis.

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Figures

Figure 1
Figure 1
Generation of PCK+TG+ murine TEC cultures Most cultured cells detected by confocal laser-scanning immunofluorescence microscopy were PCK+ and TG+ in both FLIP Tg− and Tg+ groups (A–D). FLIP and FLAG were not detected by IHC in cultured TECs of FLIP Tg− mice (E and G) but were highly expressed on cultured TECs of FLIP Tg+ DBA/1 mice (F and H). Original magnification, A–D, ×600; E and F, ×400.
Figure 2
Figure 2
Sensitization of primary TECs to Fas-mediated apoptosis is blocked by transgenic expression of FLIP. Cell treatments are shown on the left. Sixty percent to 70% confluent cultured TECs from Tg− and Tg+ mice were treated with or without agonist anti-Fas (1 μg/ml) in the presence or absence of IFN-γ (100 IU/ml) and TNF-α (50 IU/ml). Apoptosis was detected by TUNEL staining (A–H). TUNEL+ cells (red) in five to six randomly selected high-power fields of three wells/group were manually counted (magnification, ×400), and the results are summarized (I). Bars (a–h) correspond to A–H. Shown are representative areas on slides. A significant difference in the percentage of TUNEL+ cells between Tg− and Tg+ TEC is indicated by the asterisk (P < 0.05). Original magnification, A–H, ×400.
Figure 3
Figure 3
Expression of active caspase-8 and active capase-3 on cultured TECs of FLIP Tg+ and Tg− mice treated with IFN-γ and TNF-α and anti-Fas. Cell treatments are shown on the left. The staining pattern of active caspsase-8 (A–H) was identical with that of active caspase-3 (I–P). Active caspase-8+ or caspase-3+ cells (red) in five to six randomly selected high-power fields of three wells/group (magnification, ×400) were manually counted, and the results are summarized (Q and R; bars a–p correspond to A–P). Shown are representative areas on slides. A significant difference in the percentage of caspase-8+ or caspase-3+ cells between Tg− and Tg+ TEC is indicated by the asterisk (P < 0.05). Original magnification, A–H, ×400.
Figure 4
Figure 4
Transfection of cultured TECs of FLIP Tg+ mice with FLIP siRNA abolishes the protective effect of FLIP on Fas-mediated apoptosis 60–70% confluent cultured TECs of FLIP Tg+ mice were transfected with 10 pmol murine FLIP siRNA (right columns) or control siRNA (left columns). Three days after transfection, TECs were cultured for 4 d with 100 IU/ml IFN-γ and 50 IU/ml TNF-α and then cultured overnight with 1 μg/ml anti-Fas (the time corresponds to 8 d after transfection). Shown are IHC and TUNEL staining 8 d after transfection with control siRNA and FLIP siRNA. FLIP and FLAG staining is shown (A–D), and TUNEL staining is shown (E and F). IHC of activecaspase-8 and active caspase-3 is shown (G–J). FLIP+, FLAG+, TUNEL+, active caspsase-8+, or active caspsase-3+ cells (red) in five to six randomly selected high-power fields of three wells/group were manually counted, and the results are summarized (K, bars a–j correspond to A–J). A significant difference in the percentage of positive cells between Tg− and Tg+ TEC is indicated by the asterisk (P < 0.05). Shown are representative areas on slides. Original magnification, A–H, ×400.
Figure 5
Figure 5
mRNA expression of pro- and antiapoptotic molecules on cultured TECs of FLIP Tg+ and Tg− mice treated with IFN-γ and TNF-α and anti-Fas. mRNA was extracted from cultured TEC of FLIP Tg+ or Tg− mice as described in Materials and Methods. FLIP, FasL, Fas, FADD, Bcl-xL, and Bcl-2 mRNAs are shown (D–F). Results are expressed as the mean ratio of molecule densitometric U to HPRT ± sem (×100) of four to five plates/group and are representative of three independent experiments. A significant difference between cultured TECs of Tg+ and Tg− mice is indicated by the diamond (P < 0.05). A significant difference between cultured TECs of Tg− mice pretreated with and without cytokines is indicated by the club (P < 0.05). A significant difference between cultured TECs of Tg+ mice pretreated with and without cytokines is indicated by the asterisk (P < 0.05).
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