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Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation.

Gilbert S, Loranger A, Daigle N, Marceau N - J. Cell Biol. (2001)

Bottom Line: In these cells, the loss of one partner via a targeted mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins.Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8- versus WT hepatocytes.Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche en cancérologie et Département de médecine, Université Laval, Québec, G1K 7P4, Canada.

ABSTRACT
Keratins 8 and 18 belong to the keratin family of intermediate filament (IF) proteins and constitute a hallmark for all simple epithelia, including the liver. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18). In these cells, the loss of one partner via a targeted mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins. Here, we report that K8- mouse hepatocytes in primary culture and in vivo are three- to fourfold more sensitive than wild-type (WT) mouse hepatocytes to Fas-mediated apoptosis after stimulation with Jo2, an agonistic antibody of Fas ligand. This increased sensitivity is associated with a higher and more rapid caspase-3 activation and DNA fragmentation. In contrast, no difference in apoptosis is observed between cultured K8- and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) alpha or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8- and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8- hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8- versus WT hepatocytes. Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.

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A preferential link exists between K8/K18 protection and Fas signaling. (A) The addition of TNF-α (10 ng/ml), TRAIL (1.0 μg/ml), or Jo2 (0.5 μg/ml) alone or in combination with CHX (5 μg/ml) or Act D (0.5 μg/ml) to primary cultured WT or K8- hepatocytes only show differential response to Jo2. (B) Western blot analysis of caspase-3 activation after Jo2 induction shows that procaspase-3 (p32) is cleaved to an active form (p17) more rapidly in K8- than in WT hepatocytes. The tubulin blot used here as control shows no significant variation in the cellular content of this cytoskeletal protein. (C) The DNA ladder appears higher and more rapid in K8- hepatocytes than in WT hepatocytes after Jo2 stimulation.
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fig2: A preferential link exists between K8/K18 protection and Fas signaling. (A) The addition of TNF-α (10 ng/ml), TRAIL (1.0 μg/ml), or Jo2 (0.5 μg/ml) alone or in combination with CHX (5 μg/ml) or Act D (0.5 μg/ml) to primary cultured WT or K8- hepatocytes only show differential response to Jo2. (B) Western blot analysis of caspase-3 activation after Jo2 induction shows that procaspase-3 (p32) is cleaved to an active form (p17) more rapidly in K8- than in WT hepatocytes. The tubulin blot used here as control shows no significant variation in the cellular content of this cytoskeletal protein. (C) The DNA ladder appears higher and more rapid in K8- hepatocytes than in WT hepatocytes after Jo2 stimulation.

Mentions: Apoptosis can be induced by other members of the TNF receptor family, such as those activated by TNF-α and TRAIL, respectively. We thus evaluated whether the protective role played by K8/K18 against Fas-mediated hepatocyte death was also applicable to those death receptors. As shown in Fig. 2 A, the addition of TNF-α to primary cultured hepatocytes induced a low level of apoptosis, but no significant difference was observed between WT and K8- hepatocytes. Similarly, the addition of TRAIL yielded a low apoptotic response of WT hepatocytes on which the loss of K8/K18 had no influence (Fig. 2 A). This lack of response for hepatocytes in primary culture is consistent with previous findings obtained in various established cell lines (Jo et al., 2000).


Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation.

Gilbert S, Loranger A, Daigle N, Marceau N - J. Cell Biol. (2001)

A preferential link exists between K8/K18 protection and Fas signaling. (A) The addition of TNF-α (10 ng/ml), TRAIL (1.0 μg/ml), or Jo2 (0.5 μg/ml) alone or in combination with CHX (5 μg/ml) or Act D (0.5 μg/ml) to primary cultured WT or K8- hepatocytes only show differential response to Jo2. (B) Western blot analysis of caspase-3 activation after Jo2 induction shows that procaspase-3 (p32) is cleaved to an active form (p17) more rapidly in K8- than in WT hepatocytes. The tubulin blot used here as control shows no significant variation in the cellular content of this cytoskeletal protein. (C) The DNA ladder appears higher and more rapid in K8- hepatocytes than in WT hepatocytes after Jo2 stimulation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196458&req=5

fig2: A preferential link exists between K8/K18 protection and Fas signaling. (A) The addition of TNF-α (10 ng/ml), TRAIL (1.0 μg/ml), or Jo2 (0.5 μg/ml) alone or in combination with CHX (5 μg/ml) or Act D (0.5 μg/ml) to primary cultured WT or K8- hepatocytes only show differential response to Jo2. (B) Western blot analysis of caspase-3 activation after Jo2 induction shows that procaspase-3 (p32) is cleaved to an active form (p17) more rapidly in K8- than in WT hepatocytes. The tubulin blot used here as control shows no significant variation in the cellular content of this cytoskeletal protein. (C) The DNA ladder appears higher and more rapid in K8- hepatocytes than in WT hepatocytes after Jo2 stimulation.
Mentions: Apoptosis can be induced by other members of the TNF receptor family, such as those activated by TNF-α and TRAIL, respectively. We thus evaluated whether the protective role played by K8/K18 against Fas-mediated hepatocyte death was also applicable to those death receptors. As shown in Fig. 2 A, the addition of TNF-α to primary cultured hepatocytes induced a low level of apoptosis, but no significant difference was observed between WT and K8- hepatocytes. Similarly, the addition of TRAIL yielded a low apoptotic response of WT hepatocytes on which the loss of K8/K18 had no influence (Fig. 2 A). This lack of response for hepatocytes in primary culture is consistent with previous findings obtained in various established cell lines (Jo et al., 2000).

Bottom Line: In these cells, the loss of one partner via a targeted mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins.Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8- versus WT hepatocytes.Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche en cancérologie et Département de médecine, Université Laval, Québec, G1K 7P4, Canada.

ABSTRACT
Keratins 8 and 18 belong to the keratin family of intermediate filament (IF) proteins and constitute a hallmark for all simple epithelia, including the liver. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18). In these cells, the loss of one partner via a targeted mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins. Here, we report that K8- mouse hepatocytes in primary culture and in vivo are three- to fourfold more sensitive than wild-type (WT) mouse hepatocytes to Fas-mediated apoptosis after stimulation with Jo2, an agonistic antibody of Fas ligand. This increased sensitivity is associated with a higher and more rapid caspase-3 activation and DNA fragmentation. In contrast, no difference in apoptosis is observed between cultured K8- and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) alpha or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8- and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8- hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8- versus WT hepatocytes. Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.

Show MeSH
Related in: MedlinePlus