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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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Schematic representation of the relevant components of the Fas-signaling pathways in relation to the cytoskeletal networks, the Golgi-sorting compartment, and the surface membrane. In simple epithelial cells like hepatocytes K8/K18, IFs and fibrillar actin (FA) are largely localized underneath the surface membrane, whereas microtubules (MT) extend throughout the cytoplasm. The Golgi compartment (GC), next to the nucleus (N) and the endoplasmic reticulum (not shown), is involved in the sorting out of the newly synthesized receptors, such as Fas and EGF Receptor (EGFR), before their targeting to appropriate membrane portions, via a microtubule-dependent process. A look at the steps of Fas-mediated apoptosis indicates that, once the receptor has properly reached the surface membrane, the intensity of the Fas activation becomes dependent on the density of the receptor and on its degree of clustering, an actin-dependent process. Stimulation by FasL leads to Fas trimerization and death-inducing signaling complex formation, which in turn can activate two distinct caspase-signaling pathways classified as type I and type II, respectively. Hepatocytes correspond to type II cells, and accordingly, a large part of the death signaling occurs via the release of cytochrome C (Cyto C) and subsequently the activation of procaspase-9, and so on. EGF provides protection against Fas-mediated apoptosis via an activation of the Akt pathway that leads to the inhibition of caspase-9. c-FLIP, a labile protective protein homologous to caspase but exhibiting an inactive catalytic site, regulates death receptor–mediated apoptosis. Significantly, the results reported here suggest that K8/K18 largely modulate the Fas density at the hepatocyte surface, in a manner that depends on the participation of microtubules. This dynamic interplay may involve a contribution of plectin, a known integrator of the cytoskeletal networks in many cell types.
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fig7: Schematic representation of the relevant components of the Fas-signaling pathways in relation to the cytoskeletal networks, the Golgi-sorting compartment, and the surface membrane. In simple epithelial cells like hepatocytes K8/K18, IFs and fibrillar actin (FA) are largely localized underneath the surface membrane, whereas microtubules (MT) extend throughout the cytoplasm. The Golgi compartment (GC), next to the nucleus (N) and the endoplasmic reticulum (not shown), is involved in the sorting out of the newly synthesized receptors, such as Fas and EGF Receptor (EGFR), before their targeting to appropriate membrane portions, via a microtubule-dependent process. A look at the steps of Fas-mediated apoptosis indicates that, once the receptor has properly reached the surface membrane, the intensity of the Fas activation becomes dependent on the density of the receptor and on its degree of clustering, an actin-dependent process. Stimulation by FasL leads to Fas trimerization and death-inducing signaling complex formation, which in turn can activate two distinct caspase-signaling pathways classified as type I and type II, respectively. Hepatocytes correspond to type II cells, and accordingly, a large part of the death signaling occurs via the release of cytochrome C (Cyto C) and subsequently the activation of procaspase-9, and so on. EGF provides protection against Fas-mediated apoptosis via an activation of the Akt pathway that leads to the inhibition of caspase-9. c-FLIP, a labile protective protein homologous to caspase but exhibiting an inactive catalytic site, regulates death receptor–mediated apoptosis. Significantly, the results reported here suggest that K8/K18 largely modulate the Fas density at the hepatocyte surface, in a manner that depends on the participation of microtubules. This dynamic interplay may involve a contribution of plectin, a known integrator of the cytoskeletal networks in many cell types.

Mentions: We have reported previously that simple epithelium K8/K18 IFs are required for the maintenance of the mechanical integrity of the hepatocyte surface membrane (Loranger et al., 1997). Much of the knowledge accumulated so far on the nonmechanical role of K8/K18 has come from biochemical observations, e.g., keratin phosphorylation status, made in nonhepatic simple epithelium-derived cell lines in culture and also in hepatocytes in vivo in response to various insults, including chemical stresses (Ku et al., 1996). Here, we provide the first direct evidence for a functional link between Fas and K8/K18 in hepatocytes, in a manner that excludes the participation of the TNF-α and TRAIL receptors. This protective resistance provided by K8/K18 is mechanistically linked to a microtubule-dependent modulation of Fas targeting to the surface membrane. The relevant components of the Fas-signaling pathways in relation to the relevant cytoskeletal networks, the Golgi compartment, and the surface membrane are depicted in Fig. 7 .


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)

Schematic representation of the relevant components of the Fas-signaling pathways in relation to the cytoskeletal networks, the Golgi-sorting compartment, and the surface membrane. In simple epithelial cells like hepatocytes K8/K18, IFs and fibrillar actin (FA) are largely localized underneath the surface membrane, whereas microtubules (MT) extend throughout the cytoplasm. The Golgi compartment (GC), next to the nucleus (N) and the endoplasmic reticulum (not shown), is involved in the sorting out of the newly synthesized receptors, such as Fas and EGF Receptor (EGFR), before their targeting to appropriate membrane portions, via a microtubule-dependent process. A look at the steps of Fas-mediated apoptosis indicates that, once the receptor has properly reached the surface membrane, the intensity of the Fas activation becomes dependent on the density of the receptor and on its degree of clustering, an actin-dependent process. Stimulation by FasL leads to Fas trimerization and death-inducing signaling complex formation, which in turn can activate two distinct caspase-signaling pathways classified as type I and type II, respectively. Hepatocytes correspond to type II cells, and accordingly, a large part of the death signaling occurs via the release of cytochrome C (Cyto C) and subsequently the activation of procaspase-9, and so on. EGF provides protection against Fas-mediated apoptosis via an activation of the Akt pathway that leads to the inhibition of caspase-9. c-FLIP, a labile protective protein homologous to caspase but exhibiting an inactive catalytic site, regulates death receptor–mediated apoptosis. Significantly, the results reported here suggest that K8/K18 largely modulate the Fas density at the hepatocyte surface, in a manner that depends on the participation of microtubules. This dynamic interplay may involve a contribution of plectin, a known integrator of the cytoskeletal networks in many cell types.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Schematic representation of the relevant components of the Fas-signaling pathways in relation to the cytoskeletal networks, the Golgi-sorting compartment, and the surface membrane. In simple epithelial cells like hepatocytes K8/K18, IFs and fibrillar actin (FA) are largely localized underneath the surface membrane, whereas microtubules (MT) extend throughout the cytoplasm. The Golgi compartment (GC), next to the nucleus (N) and the endoplasmic reticulum (not shown), is involved in the sorting out of the newly synthesized receptors, such as Fas and EGF Receptor (EGFR), before their targeting to appropriate membrane portions, via a microtubule-dependent process. A look at the steps of Fas-mediated apoptosis indicates that, once the receptor has properly reached the surface membrane, the intensity of the Fas activation becomes dependent on the density of the receptor and on its degree of clustering, an actin-dependent process. Stimulation by FasL leads to Fas trimerization and death-inducing signaling complex formation, which in turn can activate two distinct caspase-signaling pathways classified as type I and type II, respectively. Hepatocytes correspond to type II cells, and accordingly, a large part of the death signaling occurs via the release of cytochrome C (Cyto C) and subsequently the activation of procaspase-9, and so on. EGF provides protection against Fas-mediated apoptosis via an activation of the Akt pathway that leads to the inhibition of caspase-9. c-FLIP, a labile protective protein homologous to caspase but exhibiting an inactive catalytic site, regulates death receptor–mediated apoptosis. Significantly, the results reported here suggest that K8/K18 largely modulate the Fas density at the hepatocyte surface, in a manner that depends on the participation of microtubules. This dynamic interplay may involve a contribution of plectin, a known integrator of the cytoskeletal networks in many cell types.
Mentions: We have reported previously that simple epithelium K8/K18 IFs are required for the maintenance of the mechanical integrity of the hepatocyte surface membrane (Loranger et al., 1997). Much of the knowledge accumulated so far on the nonmechanical role of K8/K18 has come from biochemical observations, e.g., keratin phosphorylation status, made in nonhepatic simple epithelium-derived cell lines in culture and also in hepatocytes in vivo in response to various insults, including chemical stresses (Ku et al., 1996). Here, we provide the first direct evidence for a functional link between Fas and K8/K18 in hepatocytes, in a manner that excludes the participation of the TNF-α and TRAIL receptors. This protective resistance provided by K8/K18 is mechanistically linked to a microtubule-dependent modulation of Fas targeting to the surface membrane. The relevant components of the Fas-signaling pathways in relation to the relevant cytoskeletal networks, the Golgi compartment, and the surface membrane are depicted in Fig. 7 .

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