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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 loss of K8/K18 leads to a prominent increase in hepatocyte sensitivity to Fas-mediated apoptosis both in culture and in vivo. (A) After 24 h in the presence of 0.5 μg/ml Jo2, K8- hepatocytes in primary culture are three to four times more responsive to Jo2- induced death than WT, as measured by Acridine orange staining of DNA fragmentation (inset). The differential response reaches saturation at a Jo2 dose >2.5 μg/ml. C, control. (B) Time– response curves obtained at a dose of 0.5 μg/ml demonstrate that the differential response between K8- and WT hepatocytes reaches a maximum at 8 h of induction. (C) The transfer of a complete K8 cDNA into 48-h K8- hepatocyte cultures using a retroviral vector yields a resistance to apoptosis in response to 0.5 μg/ml Jo2 equivalent to that observed with WT hepatocytes. The expression of a TK cDNA does not change the K8- hepatocyte response to Jo2, confirming that the rescue observed above is due to K8 reexpression. Note that the apoptotic response was assessed on GFP-expressing hepatocytes only. (D) The assessment of the Fas dose–response curve in vivo yields a LD50 (dashed line) of ∼150 μg/kg. At a dose of 75 μg/kg, most of the WT mice remain alive at 24 h after injection. (E) Measurements of ALT activity levels at 8 h after Jo2 injection (75 μg/kg) showing that K8- hepatocytes are three times more affected than WT hepatocytes. n = 11 mice; *p-value < 0.02.
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fig1: The loss of K8/K18 leads to a prominent increase in hepatocyte sensitivity to Fas-mediated apoptosis both in culture and in vivo. (A) After 24 h in the presence of 0.5 μg/ml Jo2, K8- hepatocytes in primary culture are three to four times more responsive to Jo2- induced death than WT, as measured by Acridine orange staining of DNA fragmentation (inset). The differential response reaches saturation at a Jo2 dose >2.5 μg/ml. C, control. (B) Time– response curves obtained at a dose of 0.5 μg/ml demonstrate that the differential response between K8- and WT hepatocytes reaches a maximum at 8 h of induction. (C) The transfer of a complete K8 cDNA into 48-h K8- hepatocyte cultures using a retroviral vector yields a resistance to apoptosis in response to 0.5 μg/ml Jo2 equivalent to that observed with WT hepatocytes. The expression of a TK cDNA does not change the K8- hepatocyte response to Jo2, confirming that the rescue observed above is due to K8 reexpression. Note that the apoptotic response was assessed on GFP-expressing hepatocytes only. (D) The assessment of the Fas dose–response curve in vivo yields a LD50 (dashed line) of ∼150 μg/kg. At a dose of 75 μg/kg, most of the WT mice remain alive at 24 h after injection. (E) Measurements of ALT activity levels at 8 h after Jo2 injection (75 μg/kg) showing that K8- hepatocytes are three times more affected than WT hepatocytes. n = 11 mice; *p-value < 0.02.

Mentions: We determined the sensitivity of K8- and WT mouse hepatocytes to Jo2, using the two complementary experimental approaches provided by primary culture and whole liver in vivo. After 18 h in culture, hepatocytes formed a dense cell monolayer exhibiting canaliculi equivalent to those formed by hepatocytes in vivo (not shown). The hepatocyte's sensitivity to Jo2 was assessed quantitatively by counting apoptotic nuclei. At day 2 after seeding, K8- hepatocytes were three- to fourfold more sensitive than WT hepatocytes to the addition of Jo2 in the range of 0.05–0.5 μg/ml (Fig. 1 A), and the response reached a maximum at 8 h after treatment (Fig. 1 B). At that time, essentially all the apoptotic hepatocytes remained attached to the culture substratum. Beyond that timepoint, a low percentage of the apoptotic cells gradually detached, but even at 24 h after treatment, the difference in cell death between K8- and WT hepatocytes remained comparable whether or not the detached apoptotic cells were included in the counting (not shown). At a high Jo2 concentration, the loss of K8/K18 had a reduced effect on Fas-mediated apoptosis, suggesting that the protective resistance provided by K8/K18 is less efficient upon a massive Fas stimulation (Fig. 1 A). The same three- to fourfold differential response of K8- versus WT hepatocytes to Fas-mediated apoptosis was found at 3 h after seeding when most of the cells were still dispersed on the culture substratum, indicating that the increased sensitivity of K8- hepatocytes was not dependent on cell–cell adhesion (not shown). A relevant control was to determine whether the observed phenotype in these K8- hepatocytes was rescued after the reinsertion of the WT K8 protein. As shown in Fig. 1 C, the transfer of a complete K8 cDNA in 48-h K8- hepatocyte cultures with a retroviral vector yielded a resistance to Fas-mediated apoptosis at 5 d after seeding equivalent to that observed with WT hepatocytes (Fig. 1 A). Assessment of apoptosis of nontransduced WT hepatocytes at 5 d after seeding (not shown) yielded a response essentially identical to that obtained at 3 d (Fig. 1 A). The transfer of a nonrelated cDNA (thymidine kinase [TK]) confirmed that the rescue obtained above was due solely to K8 reexpression (Fig. 1 C).


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 loss of K8/K18 leads to a prominent increase in hepatocyte sensitivity to Fas-mediated apoptosis both in culture and in vivo. (A) After 24 h in the presence of 0.5 μg/ml Jo2, K8- hepatocytes in primary culture are three to four times more responsive to Jo2- induced death than WT, as measured by Acridine orange staining of DNA fragmentation (inset). The differential response reaches saturation at a Jo2 dose >2.5 μg/ml. C, control. (B) Time– response curves obtained at a dose of 0.5 μg/ml demonstrate that the differential response between K8- and WT hepatocytes reaches a maximum at 8 h of induction. (C) The transfer of a complete K8 cDNA into 48-h K8- hepatocyte cultures using a retroviral vector yields a resistance to apoptosis in response to 0.5 μg/ml Jo2 equivalent to that observed with WT hepatocytes. The expression of a TK cDNA does not change the K8- hepatocyte response to Jo2, confirming that the rescue observed above is due to K8 reexpression. Note that the apoptotic response was assessed on GFP-expressing hepatocytes only. (D) The assessment of the Fas dose–response curve in vivo yields a LD50 (dashed line) of ∼150 μg/kg. At a dose of 75 μg/kg, most of the WT mice remain alive at 24 h after injection. (E) Measurements of ALT activity levels at 8 h after Jo2 injection (75 μg/kg) showing that K8- hepatocytes are three times more affected than WT hepatocytes. n = 11 mice; *p-value < 0.02.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: The loss of K8/K18 leads to a prominent increase in hepatocyte sensitivity to Fas-mediated apoptosis both in culture and in vivo. (A) After 24 h in the presence of 0.5 μg/ml Jo2, K8- hepatocytes in primary culture are three to four times more responsive to Jo2- induced death than WT, as measured by Acridine orange staining of DNA fragmentation (inset). The differential response reaches saturation at a Jo2 dose >2.5 μg/ml. C, control. (B) Time– response curves obtained at a dose of 0.5 μg/ml demonstrate that the differential response between K8- and WT hepatocytes reaches a maximum at 8 h of induction. (C) The transfer of a complete K8 cDNA into 48-h K8- hepatocyte cultures using a retroviral vector yields a resistance to apoptosis in response to 0.5 μg/ml Jo2 equivalent to that observed with WT hepatocytes. The expression of a TK cDNA does not change the K8- hepatocyte response to Jo2, confirming that the rescue observed above is due to K8 reexpression. Note that the apoptotic response was assessed on GFP-expressing hepatocytes only. (D) The assessment of the Fas dose–response curve in vivo yields a LD50 (dashed line) of ∼150 μg/kg. At a dose of 75 μg/kg, most of the WT mice remain alive at 24 h after injection. (E) Measurements of ALT activity levels at 8 h after Jo2 injection (75 μg/kg) showing that K8- hepatocytes are three times more affected than WT hepatocytes. n = 11 mice; *p-value < 0.02.
Mentions: We determined the sensitivity of K8- and WT mouse hepatocytes to Jo2, using the two complementary experimental approaches provided by primary culture and whole liver in vivo. After 18 h in culture, hepatocytes formed a dense cell monolayer exhibiting canaliculi equivalent to those formed by hepatocytes in vivo (not shown). The hepatocyte's sensitivity to Jo2 was assessed quantitatively by counting apoptotic nuclei. At day 2 after seeding, K8- hepatocytes were three- to fourfold more sensitive than WT hepatocytes to the addition of Jo2 in the range of 0.05–0.5 μg/ml (Fig. 1 A), and the response reached a maximum at 8 h after treatment (Fig. 1 B). At that time, essentially all the apoptotic hepatocytes remained attached to the culture substratum. Beyond that timepoint, a low percentage of the apoptotic cells gradually detached, but even at 24 h after treatment, the difference in cell death between K8- and WT hepatocytes remained comparable whether or not the detached apoptotic cells were included in the counting (not shown). At a high Jo2 concentration, the loss of K8/K18 had a reduced effect on Fas-mediated apoptosis, suggesting that the protective resistance provided by K8/K18 is less efficient upon a massive Fas stimulation (Fig. 1 A). The same three- to fourfold differential response of K8- versus WT hepatocytes to Fas-mediated apoptosis was found at 3 h after seeding when most of the cells were still dispersed on the culture substratum, indicating that the increased sensitivity of K8- hepatocytes was not dependent on cell–cell adhesion (not shown). A relevant control was to determine whether the observed phenotype in these K8- hepatocytes was rescued after the reinsertion of the WT K8 protein. As shown in Fig. 1 C, the transfer of a complete K8 cDNA in 48-h K8- hepatocyte cultures with a retroviral vector yielded a resistance to Fas-mediated apoptosis at 5 d after seeding equivalent to that observed with WT hepatocytes (Fig. 1 A). Assessment of apoptosis of nontransduced WT hepatocytes at 5 d after seeding (not shown) yielded a response essentially identical to that obtained at 3 d (Fig. 1 A). The transfer of a nonrelated cDNA (thymidine kinase [TK]) confirmed that the rescue obtained above was due solely to K8 reexpression (Fig. 1 C).

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