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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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Fas targeting is affected by the loss of K8/K18 in cultured hepatocytes. (A) Immunostaining of WT hepatocytes showing that K8 (green in the merged picture) is localized at the surface membrane, whereas Fas (red in the merge) is mostly localized in the Golgi area (blue in the merge). The identity of the Golgi compartment was confirmed by detecting a standard marker, mannosidase II (blue in the merge). In contrast, Fas is largely present at the surface membrane in K8- hepatocytes, with little in the Golgi area. (B) Western blot showing that the Fas content does not vary between WT and K8- hepatocytes. Bars, 10 μm.
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fig4: Fas targeting is affected by the loss of K8/K18 in cultured hepatocytes. (A) Immunostaining of WT hepatocytes showing that K8 (green in the merged picture) is localized at the surface membrane, whereas Fas (red in the merge) is mostly localized in the Golgi area (blue in the merge). The identity of the Golgi compartment was confirmed by detecting a standard marker, mannosidase II (blue in the merge). In contrast, Fas is largely present at the surface membrane in K8- hepatocytes, with little in the Golgi area. (B) Western blot showing that the Fas content does not vary between WT and K8- hepatocytes. Bars, 10 μm.

Mentions: Previous work performed on mouse hepatocytes in situ and in monolayer culture have shown that cell sensitivity to Fas-mediated apoptosis is linked to the receptor density at the surface (Sodeman et al., 2000). Here, our immunofluorescence staining analyses in culture indicated that in contrast to the regional localization found at the surface membrane of WT hepatocytes, Fas was largely distributed along large portion of the membrane in K8- hepatocytes. Notably, in WT hepatocytes, where K8/K18 were shown to be mainly localized at the surface membrane (Fig. 4 A), Fas was largely localized in the Golgi area (Fig. 4 A, merged images), whereas in K8- hepatocytes Fas was preferentially detected at the surface. In light of this shift in Fas targeting to the surface membrane, we next analyzed the Fas content. As demonstrated in Fig. 4 B, the total content of the death receptor was not affected by the K8- mutation, thus suggesting that K8/K18 modulate Fas targeting to the surface membrane.


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)

Fas targeting is affected by the loss of K8/K18 in cultured hepatocytes. (A) Immunostaining of WT hepatocytes showing that K8 (green in the merged picture) is localized at the surface membrane, whereas Fas (red in the merge) is mostly localized in the Golgi area (blue in the merge). The identity of the Golgi compartment was confirmed by detecting a standard marker, mannosidase II (blue in the merge). In contrast, Fas is largely present at the surface membrane in K8- hepatocytes, with little in the Golgi area. (B) Western blot showing that the Fas content does not vary between WT and K8- hepatocytes. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196458&req=5

fig4: Fas targeting is affected by the loss of K8/K18 in cultured hepatocytes. (A) Immunostaining of WT hepatocytes showing that K8 (green in the merged picture) is localized at the surface membrane, whereas Fas (red in the merge) is mostly localized in the Golgi area (blue in the merge). The identity of the Golgi compartment was confirmed by detecting a standard marker, mannosidase II (blue in the merge). In contrast, Fas is largely present at the surface membrane in K8- hepatocytes, with little in the Golgi area. (B) Western blot showing that the Fas content does not vary between WT and K8- hepatocytes. Bars, 10 μm.
Mentions: Previous work performed on mouse hepatocytes in situ and in monolayer culture have shown that cell sensitivity to Fas-mediated apoptosis is linked to the receptor density at the surface (Sodeman et al., 2000). Here, our immunofluorescence staining analyses in culture indicated that in contrast to the regional localization found at the surface membrane of WT hepatocytes, Fas was largely distributed along large portion of the membrane in K8- hepatocytes. Notably, in WT hepatocytes, where K8/K18 were shown to be mainly localized at the surface membrane (Fig. 4 A), Fas was largely localized in the Golgi area (Fig. 4 A, merged images), whereas in K8- hepatocytes Fas was preferentially detected at the surface. In light of this shift in Fas targeting to the surface membrane, we next analyzed the Fas content. As demonstrated in Fig. 4 B, the total content of the death receptor was not affected by the K8- mutation, thus suggesting that K8/K18 modulate Fas targeting to the surface membrane.

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