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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 the liver in vivo. (A) Immunostaining showing that K8 is localized at the surface membrane in WT hepatocytes, whereas Fas is mostly detected in the cytoplasm. In contrast, Fas is largely localized at the surface membrane in K8- hepatocytes. (B) Western blot demonstrating that no difference in Fas content exists between livers from three WT mice and three K8- mice, before or even after Jo2 injection (75 μg/kg). (C) FACS® analysis showing a shift in Fas density on the surface of freshly isolated living K8- hepatocytes over that of WT hepatocytes. (D) Fluorescence means derived from data in C, showing a significant (30%) increase in Fas density at the surface of K8- hepatocytes; error bars represent the standard deviation of two trials with three different mice. *p-value < 0.01. Bar, 10 μm.
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fig5: Fas targeting is affected by the loss of K8/K18 in the liver in vivo. (A) Immunostaining showing that K8 is localized at the surface membrane in WT hepatocytes, whereas Fas is mostly detected in the cytoplasm. In contrast, Fas is largely localized at the surface membrane in K8- hepatocytes. (B) Western blot demonstrating that no difference in Fas content exists between livers from three WT mice and three K8- mice, before or even after Jo2 injection (75 μg/kg). (C) FACS® analysis showing a shift in Fas density on the surface of freshly isolated living K8- hepatocytes over that of WT hepatocytes. (D) Fluorescence means derived from data in C, showing a significant (30%) increase in Fas density at the surface of K8- hepatocytes; error bars represent the standard deviation of two trials with three different mice. *p-value < 0.01. Bar, 10 μm.

Mentions: In light of these new findings obtained with hepatocytes in monolayer culture, we next used immunofluorescence microscopy to verify the Fas distribution at the surface of hepatocytes in situ. As in culture, a higher Fas density was observed in K8- hepatocytes (Fig. 5 A); this change was not associated with a variation in Fas content (Fig. 5 B). Moreover, our FACS® analysis of freshly isolated hepatocytes (Fig. 5 C) allowed a quantification of the fluorescence mean on live cells, and overall, the data demonstrated a significant (30%) increase in Fas density at the surface of K8- hepatocytes (Fig. 5 D).


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 the liver in vivo. (A) Immunostaining showing that K8 is localized at the surface membrane in WT hepatocytes, whereas Fas is mostly detected in the cytoplasm. In contrast, Fas is largely localized at the surface membrane in K8- hepatocytes. (B) Western blot demonstrating that no difference in Fas content exists between livers from three WT mice and three K8- mice, before or even after Jo2 injection (75 μg/kg). (C) FACS® analysis showing a shift in Fas density on the surface of freshly isolated living K8- hepatocytes over that of WT hepatocytes. (D) Fluorescence means derived from data in C, showing a significant (30%) increase in Fas density at the surface of K8- hepatocytes; error bars represent the standard deviation of two trials with three different mice. *p-value < 0.01. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Fas targeting is affected by the loss of K8/K18 in the liver in vivo. (A) Immunostaining showing that K8 is localized at the surface membrane in WT hepatocytes, whereas Fas is mostly detected in the cytoplasm. In contrast, Fas is largely localized at the surface membrane in K8- hepatocytes. (B) Western blot demonstrating that no difference in Fas content exists between livers from three WT mice and three K8- mice, before or even after Jo2 injection (75 μg/kg). (C) FACS® analysis showing a shift in Fas density on the surface of freshly isolated living K8- hepatocytes over that of WT hepatocytes. (D) Fluorescence means derived from data in C, showing a significant (30%) increase in Fas density at the surface of K8- hepatocytes; error bars represent the standard deviation of two trials with three different mice. *p-value < 0.01. Bar, 10 μm.
Mentions: In light of these new findings obtained with hepatocytes in monolayer culture, we next used immunofluorescence microscopy to verify the Fas distribution at the surface of hepatocytes in situ. As in culture, a higher Fas density was observed in K8- hepatocytes (Fig. 5 A); this change was not associated with a variation in Fas content (Fig. 5 B). Moreover, our FACS® analysis of freshly isolated hepatocytes (Fig. 5 C) allowed a quantification of the fluorescence mean on live cells, and overall, the data demonstrated a significant (30%) increase in Fas density at the surface of K8- hepatocytes (Fig. 5 D).

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