Anne Loranger

Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation

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 null 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-null 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-null and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) α or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8-null and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8-null hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8-null 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.

Keratin-mediated resistance to stress and apoptosis in simple epithelial cells in relation to health and disease

Epithelial cells such as hepatocytes exhibit highly polarized properties as a result of the asymmetric distribution of subsets of receptors at unique portions of the surface membrane. While the proper targeting of these surface receptors and maintenance of the resulting polarity depend on microtubules (MTs), the Golgi sorting compartment, and different actin-filament networks, the contribution of keratin intermediate filaments (IFs) has been unclear. Recent data show that the latter cytoskeletal network plays a predominant role in providing resistance to various forms of stress and to apoptosis targeted to the surface membrane. In this context, we first summarize our knowledge of the domain- or assembly-related features of IF proteins and the dynamic properties of IF networks that may explain how the same keratin pair K8/K18 can exert multiple resistance-related functions in simple epithelial cells. We then examine the contribution of linker protein(s) that integrate interactions of keratin IFs with MTs and the actin-cytoskeleton network, polarity-dependent surface receptors and cytoplasmic organelles. We next address likely molecular mechanisms by which K8/K18 can selectively provide resistance to a mechanical or toxic stress, or to Fas-mediated apoptosis. Finally, these issues on keratin structure-function are examined within a context of pathological anomalies emerging in tissue architecture as a result of natural or targeted mutations, or posttranslational modifications at specific amino acid residues. Clearly. the data accumulated in recent years provide new and significant insights on the role of K8/K18, particularly under conditions where polarized cells resist to stressful or apoptotic insults.

Keratins Modulate c-Flip/Extracellular Signal-Regulated Kinase 1 and 2 Antiapoptotic Signaling in Simple Epithelial Cells

ABSTRACT Among the large family of intermediate filament proteins, the keratin 8 and 18 (K8/K18) pair constitutes a hallmark for all simple epithelial cells, such as hepatocytes and mammary cells. Functional studies with different cell models have suggested that K8/K18 are involved in simple epithelial cell resistance to several forms of stress that may lead to cell death. We have reported recently that K8/K18-deprived hepatocytes from K8-null mice are more sensitive to Fas-mediated apoptosis. Here we show that upon Fas, tumor necrosis factor alpha receptor, or tumor necrosis factor alpha-related apoptosis-inducing ligand receptor stimulation, an inhibition of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation sensitizes wild-type but not K8-null mouse hepatocytes to apoptosis and that a much weaker ERK1/2 activation occurs in K8-null hepatocytes. In turn, this impaired ERK1/2 activation in K8-null hepatocytes is associated with a drastic reduction in c-Flip protein, an event that also holds in a K8-null mouse mammary cell line. c-Flip, along with Raf-1, is part of a K8/K18-immunoisolated complex from wild-type hepatocytes, and Fas stimulation leads to further c-Flip and Raf-1 recruitment in the complex. This points to a new regulatory role of simple epithelium keratins in the c-Flip/ERK1/2 antiapoptotic signaling pathway.

Keratin 8/18 Modulation of Protein Kinase C-mediated Integrin-dependent Adhesion and Migration of Liver Epithelial Cells

Hepatocyte and hepatoma cell IFs are made solely of keratins 8/18 (K8/K18). Cell adhesion and migration involve integrin interactions with focal adhesion kinase (FAK) and protein kinase C (PKC). Here we report a new regulatory function for K8/K18 IFs in the PKC-mediated integrin/FAK-dependent adhesion and migration of simple epithelial cells.

Switch in Fas-activated death signaling pathway as result of keratin 8/18-intermediate filament loss

Fas-induced apoptosis is initiated through the recruitment of FADD and procaspase 8 to form the death-inducing signaling complex (DISC). In some cells (type I cells) the initiator caspase 8 directly activates effector caspases such as procaspase 3, whereas in others (type II cells) the death signal is amplified through mitochondria. In epithelial cells, Fas-induced hierarchic caspase activation is also linked with DEDD, a member of the DED family that binds to keratin (K) intermediate filaments (IFs). Hepatocytes are type II cells and their IFs are made exclusively of K8/K18. We have shown previously that K8-null mouse hepatocytes, lacking K8/K18 IFs, are more sensitive than their wild-type counterparts to Fas-induced apoptosis. Here, by examining the cell-death kinetics and death-signaling ordering, we found that K8-null hepatocytes exhibited prominent DISC formation, higher procaspase 8 activation and direct procaspase 3 activation as reported for type I cells; however they experienced a reduced Bid cleavage and a stronger procaspase 9 activation. In addition, the K8/K18 loss altered the DEDD ubiquitination status and nuclear/cytoplasmic distribution. Together, the results suggest that the K8/K18 loss induces a switch in Fas-induced death signaling, likely through a DEDD involvement.

Cytoskeleton keratin regulation of FasR signaling through modulation of actin/ezrin interplay at lipid rafts in hepatocytes

FasR stimulation by Fas ligand leads to rapid formation of FasR microaggregates, which become signaling protein oligomerization transduction structures (SPOTS), through interactions with actin and ezrin, a structural step that triggers death-inducing signaling complex formation, in association with procaspase-8 activation. In some cells, designated as type I, caspase 8 directly activates effector caspases, whereas in others, known as type II, the caspase-mediated death signaling is amplified through mitochondria. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatocyte IFs are made solely of keratins 8/18 (K8/K18), the hallmark of all simple epithelia. We have shown recently that in comparison to type II wild-type (WT) mouse hepatocytes, the absence of K8/K18 IFs in K8-null hepatocytes leads to more efficient FasR-mediated apoptosis, in link with a type II/type I-like switch in FasR-death signaling. Here, we demonstrate that the apoptotic process occurring in type I-like K8-null hepatocytes is associated with accelerated SPOTS elaboration at surface membrane, along with manifestation of FasR cap formation and internalization. In addition, the lipid raft organization is altered in K8-null hepatocytes. While lipid raft inhibition impairs SPOTS formation in both WT and K8-null hepatocytes, the absence of K8/K18 IFs in the latter sensitizes SPOTS to actin de-polymerization, and perturbs ezrin compartmentalization. Overall, the results indicate that the K8/K18 IF loss in hepatocytes alters the initial FasR activation steps through perturbation of ezrin/actin interplay and lipid raft organization, which leads to a type II/type I switch in FasR-death signaling.

Keratin–protein kinase C interaction in reactive oxygen species-induced hepatic cell death through mitochondrial signaling

Keratins (Ks), the intermediate filament (IF) proteins of epithelia, constitute at least 20 cytoskeletal proteins subdivided into type I (K9-20) and type II (K1-K8) and expressed as type I/type II pairs in a cell differentiation manner. Hepatocyte IFs are made only of K8/K18, the hallmark of simple epithelial cells. We have shown previously that a K8/K18 loss leads to a modulation of apoptosis in Fas-stimulated mouse hepatocytes. Here we report that K8-knockout mouse hepatocytes and K8-knockdown H4-II-E-C3 (shK8b1) rat hepatoma cells were much more resistant than their K8/K18-containing counterparts, wild-type hepatocytes, and H4ev hepatoma cells, in response to excess H2O2 or tert-butyl hydroperoxide, a ROS generator. While excess H2O2 altered glutathione (GSH) and ROS levels in H4ev versus shK8b1 cells, the differential death response was largely GSH level independent. Assessment of key cell death features revealed that hepatic cells exposed to H2O2 die through a mitochondrial involvement. Similarly, administration of the GSH depletor L-buthionine-sulfoximine to generate mitochondrial ROS-sensitized H4-II-E-C3 cells but not shK8b1 cells to death. Treatment with protein kinase C (PKC) inhibitors yielded a resistance of H2O2-treated H4-II-E-C3 cells comparable to that of nontreated shK8b1 cells, which in turn were not affected by the treatment. In addition, this differential death response was associated with altered PKCdelta activation and surface-membrane/mitochondria distribution in H2O2-treated shK8b1 cells. Together, these results point to a key regulatory function for K8/K18 in ROS-induced mitochondria-mediated death through PKCdelta involvement in hepatic cells.

Keratin 8/18 regulation of glucose metabolism in normal versus cancerous hepatic cells through differential modulation of hexokinase status and insulin signaling

As differentiated cells, hepatocytes primarily metabolize glucose for ATP production through oxidative phosphorylation of glycolytic pyruvate, whereas proliferative hepatocellular carcinoma (HCC) cells undergo a metabolic shift to aerobic glycolysis despite oxygen availability. Keratins, the intermediate filament (IF) proteins of epithelial cells, are expressed as pairs in a lineage/differentiation manner. Hepatocyte and HCC (hepatoma) cell IFs are made solely of keratins 8/18 (K8/K18), thus providing models of choice to address K8/K18 IF functions in normal and cancerous epithelial cells. Here, we demonstrate distinctive increases in glucose uptake, glucose-6-phosphate formation, lactate release, and glycogen formation in K8/K18 IF-lacking hepatocytes and/or hepatoma cells versus their respective IF-containing counterparts. We also show that the K8/K18-dependent glucose uptake/G6P formation is linked to alterations in hexokinase I/II/IV content and localization at mitochondria, with little effect on GLUT1 status. In addition, we find that the insulin-stimulated glycogen formation in normal hepatocytes involves the main PI-3 kinase-dependent signaling pathway and that the K8/K18 IF loss makes them more efficient glycogen producers. In comparison, the higher insulin-dependent glycogen formation in K8/K18 IF-lacking hepatoma cells is associated with a signaling occurring through a mTOR-dependent pathway, along with an augmentation in cell proliferative activity. Together, the results uncover a key K8/K18 regulation of glucose metabolism in normal and cancerous hepatic cells through differential modulations of mitochondrial HK status and insulin-mediated signaling.

Uncovering the roles of intermediate filaments in apoptosis.

Publisher Summary Intermediate filament (IF) genes are differentially expressed according to cell lineage establishment and terminal cell differentiation, and in this context IF proteins constitute reliable sets of cell markers in normal tissues, except K8/K18 pair—the first cytoplasmic IF proteins to be expressed in the embryo. This chapter focuses on the analysis of the roles fulfilled by the events that provide cell resistance to apoptosis stimulation before commitment or those that are part of the execution phase in the committed cells. Although a prominent role for cytoskeleton is to maintain cellular integrity, it seems that IFs constitute a protein scaffold that is largely responsible for the capacity of cells to sustain mechanical and nonmechanical stresses including those that can lead to apoptosis. Because of their accessibility and their specialization—the availability of both tissue and primary culture based approaches and the severe IF-dependent diseases associated with them—keratinocytes and hepatocytes are the two cell models that are most frequently used, particularly to investigate IF involvement in cell stress and apoptosis under culture conditions. The chapter presents four main points on the role of IF: (1) IF proteins can readily be used as the markers of cell lineage establishment and cell differentiation, (2) apoptosis can occur by the stimulation of death receptors (DR) at the surface membrane or perturbation of mitochondria by toxic agents or oxidative stress, (3) the strongest experimental evidence for IF involvement in apoptosis has come from the work performed on simple epithelial cells, particularly for the K8/K18 pair, and (4) K8/K18 have been shown to be the modulators of apoptosis by acting both upstream and downstream of the caspase machinery activation.

The mechanism whereby heat shock induces apoptosis depends on the innate sensitivity of cells to stress

The cellular response to heat shock (HS) is a paradigm for many human diseases collectively known as “protein conformation diseases” in which the accumulation of misfolded proteins induces cell death. Here, we analyzed how cells having a different apoptotic threshold die subsequent to a treatment with HS. Cells with a low apoptotic threshold mainly induced apoptosis through activation of conventional stress kinase signaling pathways. By contrast, cells with a high apoptotic threshold also died by apoptosis but likely after the accumulation of heat-aggregated proteins as revealed by the formation of aggresomes in these cells, which were associated with the generation of atypical nuclear deformations. Inhibition of the proteasome or expression of an aggregation prone protein produced similar nuclear alterations. Furthermore, elevated levels of chaperones markedly suppressed both HS-induced nuclear deformations and apoptosis induced upon protein aggregation whereas they had little effect on stress kinase-mediated apoptosis. We conclude that the relative contribution of stress signaling pathways and the accumulation of protein aggregates to cell death by apoptosis is related to the innate sensitivity of cells to deadly insults.