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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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The protection of hepatocytes by colchicine against Fas-mediated apoptosis is lost when K8/K18 are lacking. (A) Colchicine addition (1 μM; 24 h before Jo2 stimulation) in primary culture protects WT but not K8- hepatocytes against Jo2 (0.5 μg/ml) added in the presence or absence of 0.5 μg/ml Act D for 8 h; the addition of γ-lumicolchicine (1 μM) has no effect. (B) Immunostaining data showing that the colchicine concentration used (1 μM for 24 h) disrupts the microtubule network in both WT and K8- hepatocytes. (C) Immunofluorescence on WT and K8- hepatocytes treated with colchicine (1 μM for 24 h), showing that Fas is maintained in the cytoplasm of WT hepatocytes, whereas it is still mostly localized at surface membrane in K8- hepatocytes. (D) In vivo injection of colchicine (400 μg/kg) 24 h before Jo2 treatment protects WT mice against a lethal dose (200 μg/kg) of Jo2. This protection is reduced in K8- mice. n = 13. Bars, 10 μm.
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fig6: The protection of hepatocytes by colchicine against Fas-mediated apoptosis is lost when K8/K18 are lacking. (A) Colchicine addition (1 μM; 24 h before Jo2 stimulation) in primary culture protects WT but not K8- hepatocytes against Jo2 (0.5 μg/ml) added in the presence or absence of 0.5 μg/ml Act D for 8 h; the addition of γ-lumicolchicine (1 μM) has no effect. (B) Immunostaining data showing that the colchicine concentration used (1 μM for 24 h) disrupts the microtubule network in both WT and K8- hepatocytes. (C) Immunofluorescence on WT and K8- hepatocytes treated with colchicine (1 μM for 24 h), showing that Fas is maintained in the cytoplasm of WT hepatocytes, whereas it is still mostly localized at surface membrane in K8- hepatocytes. (D) In vivo injection of colchicine (400 μg/kg) 24 h before Jo2 treatment protects WT mice against a lethal dose (200 μg/kg) of Jo2. This protection is reduced in K8- mice. n = 13. Bars, 10 μm.

Mentions: The proper trafficking of Fas between the Golgi-sorting compartment and the surface membrane involves microtubules (Feng and Kaplowitz, 2000; Sodeman et al., 2000). Accordingly, treatment of mouse hepatocytes with colchicine in vivo impairs Fas transfer to the surface and results in a protection against Fas-mediated apoptosis (Feng and Kaplowitz, 2000). Here, we observed that the addition of colchicine protects WT but not K8- hepatocytes against Fas-mediated apoptosis in primary cultures, both in the absence or presence of Act D sensitization (Fig. 6 A). The colchicine treatment resulted in a massive microtubule depolymerization (Fig. 6 B). Under these conditions, a reduced Fas density was observed at the surface membrane in association with a major localization in the Golgi area in WT hepatocytes (Fig. 6 C). In contrast, K8- hepatocytes exhibited a large proportion of Fas at the surface membrane, with a low signal in the Golgi area (Fig. 6 C). Finally, since this microtubule-dependent modulation of Fas targeting has been shown recently to reduce the mouse mortality induced by Jo2 (Feng and Kaplowitz, 2000), we assessed K8- versus WT mouse survival as a function of time after an injection of 400 μg/kg colchicine 24 h before an i.p. injection of 200 μg/kg Jo2. As shown in Fig. 6 D, the colchicine pretreatment resulted in a twofold lower survival of K8- mice compared with that of WT mice, confirming the findings observed with hepatocytes in primary culture. On these grounds, both the in vitro and in vivo data suggest an interplay between K8/K18, microtubule integrity, and Fas targeting to 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)

The protection of hepatocytes by colchicine against Fas-mediated apoptosis is lost when K8/K18 are lacking. (A) Colchicine addition (1 μM; 24 h before Jo2 stimulation) in primary culture protects WT but not K8- hepatocytes against Jo2 (0.5 μg/ml) added in the presence or absence of 0.5 μg/ml Act D for 8 h; the addition of γ-lumicolchicine (1 μM) has no effect. (B) Immunostaining data showing that the colchicine concentration used (1 μM for 24 h) disrupts the microtubule network in both WT and K8- hepatocytes. (C) Immunofluorescence on WT and K8- hepatocytes treated with colchicine (1 μM for 24 h), showing that Fas is maintained in the cytoplasm of WT hepatocytes, whereas it is still mostly localized at surface membrane in K8- hepatocytes. (D) In vivo injection of colchicine (400 μg/kg) 24 h before Jo2 treatment protects WT mice against a lethal dose (200 μg/kg) of Jo2. This protection is reduced in K8- mice. n = 13. Bars, 10 μm.
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Related In: Results  -  Collection

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fig6: The protection of hepatocytes by colchicine against Fas-mediated apoptosis is lost when K8/K18 are lacking. (A) Colchicine addition (1 μM; 24 h before Jo2 stimulation) in primary culture protects WT but not K8- hepatocytes against Jo2 (0.5 μg/ml) added in the presence or absence of 0.5 μg/ml Act D for 8 h; the addition of γ-lumicolchicine (1 μM) has no effect. (B) Immunostaining data showing that the colchicine concentration used (1 μM for 24 h) disrupts the microtubule network in both WT and K8- hepatocytes. (C) Immunofluorescence on WT and K8- hepatocytes treated with colchicine (1 μM for 24 h), showing that Fas is maintained in the cytoplasm of WT hepatocytes, whereas it is still mostly localized at surface membrane in K8- hepatocytes. (D) In vivo injection of colchicine (400 μg/kg) 24 h before Jo2 treatment protects WT mice against a lethal dose (200 μg/kg) of Jo2. This protection is reduced in K8- mice. n = 13. Bars, 10 μm.
Mentions: The proper trafficking of Fas between the Golgi-sorting compartment and the surface membrane involves microtubules (Feng and Kaplowitz, 2000; Sodeman et al., 2000). Accordingly, treatment of mouse hepatocytes with colchicine in vivo impairs Fas transfer to the surface and results in a protection against Fas-mediated apoptosis (Feng and Kaplowitz, 2000). Here, we observed that the addition of colchicine protects WT but not K8- hepatocytes against Fas-mediated apoptosis in primary cultures, both in the absence or presence of Act D sensitization (Fig. 6 A). The colchicine treatment resulted in a massive microtubule depolymerization (Fig. 6 B). Under these conditions, a reduced Fas density was observed at the surface membrane in association with a major localization in the Golgi area in WT hepatocytes (Fig. 6 C). In contrast, K8- hepatocytes exhibited a large proportion of Fas at the surface membrane, with a low signal in the Golgi area (Fig. 6 C). Finally, since this microtubule-dependent modulation of Fas targeting has been shown recently to reduce the mouse mortality induced by Jo2 (Feng and Kaplowitz, 2000), we assessed K8- versus WT mouse survival as a function of time after an injection of 400 μg/kg colchicine 24 h before an i.p. injection of 200 μg/kg Jo2. As shown in Fig. 6 D, the colchicine pretreatment resulted in a twofold lower survival of K8- mice compared with that of WT mice, confirming the findings observed with hepatocytes in primary culture. On these grounds, both the in vitro and in vivo data suggest an interplay between K8/K18, microtubule integrity, and Fas targeting to 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