1 Institut de Génétique Moléculaire, CNRS UMR5535, IFR122, 1919 route de Mende, 34293 Montpellier Cedex 5, France
2 UMR 5539 Centre National de la Recherche Scientifique, Dynamique Moléculaire des Interactions Membranaires, Université Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France
3 Service d'Anatomie Pathologique, Groupe Hospitalo-Universitaire Caremeau, Place du Pr Robert Debré, 30029 Nîmes Cedex 9, France
4 CRBM, CNRS FRE2593, IFR122, 1919 Route de Mende, 34293 Montpellier Cedex 5, France
5 UMR 5539 CNRS/UM2, IFR122, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France
* Author for correspondence (e-mail: ula.hibner{at}igmm.cnrs.fr)
Accepted 23 March 2005
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Summary |
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Key words: Apoptosis, Cell polarity, FAS/CD95, Hepatocyte, NF-B
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Introduction |
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One important aspect of an epithelial cell's physiology is its apico-basolateral polarization. Even though many epithelial cells adopt a partially polarized morphology in a monolayer culture, the establishment and maintenance of a strongly polarized state requires appropriate adhesive substrata and cell-cell contacts, and the correct geometrical orientation (Chen et al., 1997; Knust and Bossinger, 2002
; O'Brien et al., 2002
). Epithelial cells cultured in a physiologically relevant three-dimensional (3D) matrix form polarized organoids, with the basal cell surfaces in contact with the matrix and the apical membrane domains, delimited by tight junctions between adjacent cells, pointing into a lumen. For example, thyroid cells embedded in collagen organize into follicles (Chambard et al., 1981
), Madin-Darby canine kidney (MDCK) cells form cysts and tubules in both collagen and Matrigel [a 3D matrix derived from the Enggelbreth-Holm-Swarm mouse sarcoma (Zegers et al., 2003
)], and mammary epithelium grown in Matrigel forms hollow acini composed of polarized cells that retain many characteristics of mammary tissue in vivo (Weaver et al., 1997
), including the resistance to a range of apoptotic stimuli (Weaver et al., 2002
).
Hepatocytes are epithelial cells of complex polarity, characterized by the presence of several distinct apical membrane domains. In vivo, the apical surfaces of adjacent cells form microvillus-lined lumens called bile canaliculi. Primary hepatocytes can be cultured in vitro over extensive periods of time. Although they rapidly dedifferentiate in standard monolayer cultures (for a review, see LeCluyse et al., 1996), several culture conditions have been described that allow long-term maintenance of the differentiated phenotype (Dunn et al., 1989
; Lazaro et al., 2003
; Michalopoulos et al., 2001
; Moghe et al., 1996
; Rojkind et al., 1980
). For example, cells in collagen gels remain cuboidal and polarized, and maintain the production of hepatocyte-specific markers (Dunn et al., 1989
; LeCluyse et al., 1996
; Moghe et al., 1996
; Richert et al., 2002
). Matrigel, a tumour-derived laminin-rich basal-membrane extracellular matrix, provides an especially appropriate environment for hepatocyte culture, as judged by the cells' morphology and metabolism (LeCluyse et al., 1996
; Semler and Moghe, 2001
; Semler et al., 2000
). Interestingly, in addition to the chemical composition of the matrix, the physical properties of the gels (especially the pore size and the pliability) profoundly influence both the spatial organization and the differentiation of the cells (Moghe et al., 1996
; Ranucci et al., 2000
; Semler and Moghe, 2001
).
It is widely accepted that the physiological context of a cell is a strong determinant of the outcome of an apoptotic stimulation (Danial and Korsmeyer, 2004). Although the influence of culture conditions on hepatocyte viability and function has been extensively studied (Allen et al., 2001
; Block et al., 1996
; Dunn et al., 1989
; Khalil et al., 2001
; LeCluyse et al., 1996
; Michalopoulos and Pitot, 1975
; Richert et al., 2002
; Rojkind et al., 1980
; Semler et al., 2000
), little is known about the effect of hepatocyte morphology and physiology on the modulation of apoptosis. One well-described pathway of apoptosis induction originates from the engagement of death receptors, such as tumour necrosis factor receptor (TNFR) or Fas (CD95), expressed on the surface of many cell types (Debatin and Krammer, 2004
). Upon binding of their respective ligands, the receptors recruit multiprotein complexes, in which the initiator procaspase 8 undergoes activation mediated by proteolytic cleavages. Recruitment of other proteins and activation of additional signalling pathways control this first step of apoptotic signalling. In particular, NF-
B activation, frequently associated with death receptor stimulation, constitutes a potent restraint of the cell's apoptotic response (Baud and Karin, 2001
). The importance of NF-
B signalling in the liver is exemplified by the phenotype of mice invalidated for the p65/RelA subunit of NF-
B: the animals die at embryonic day 15-16 of liver destruction (Beg et al., 1995
), owing to TNFR1-dependent apoptosis (Rosenfeld et al., 2000
).
Liver cells are exquisitely sensitive to Fas stimulation in vivo: fulminant hepatitis is a frequently lethal consequence of massive hepatocyte apoptosis following Fas activation (Ryo et al., 2000; Song et al., 2003
). In mice, intravenous injection of an agonistic anti-Fas antibody kills the animals through liver destruction and haemorrhage (Ogasawara et al., 1993
). However, in monolayer cultures of both immortalized and primary hepatocytes, strong pro-survival signalling accompanies death-receptor signal transduction, rendering the cells resistant to Fas- or TNFR-mediated apoptosis in the absence of NF-
B inhibition.
In the present study, we have used mhAT3F, a differentiated murine hepatocyte cell line (Antoine et al., 1992; Levrat et al., 1993
), to assess the possible links between the cells' polarity and function with their sensitivity to apoptosis induced by stimulation of death receptors. Contrary to the reported resistance to apoptosis of polarized mammary epithelium (Weaver et al., 2002
), strong polarization of hepatocytes in a three-dimensional culture diminished NF-
B activation and restored the cells' in vivo sensitivity to the stimulation of the Fas pathway.
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Materials and Methods |
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Cell culture
MhAT3F cells were cultured in DMEM supplemented with 5% foetal calf serum, 5 µg ml1 insulin, 1 µM dexamethasone, 1 µM triiodothyronine, 250 ng ml1 fungizone, 20 µg ml1 streptomycin and 25 U ml1 penicillin.
For the 3D Matrigel culture, monolayer grown cells were harvested by trypsinization, taken up in the complete medium and separated into single-cell suspension by several passages through a Pasteur pipette. 50 µl complete medium containing 100,000 cells was added to 250 µl Matrigel, gently mixed, deposited into a Falcon cell culture insert and incubated at 37°C until Matrigel solidified. The cells were then covered with complete medium and grown at 37°C under 5% CO2 for up to 8 days with the change of medium every 2 days. For statistical analysis of organoid formation, 40-70 structures in randomly chosen fields were counted in two independent experiments.
For the collagen sandwich cultures, 100 µl collagen (2.5 mg ml1) in complete medium was left to solidify in a culture insert at 37°C for 30 minutes. The culture was seeded with 8000 cells in 500 µl complete medium for 2 hours and covered with a second layer of 100 µl collagen and the complete medium. The cells were cultured for up to 8 days with medium change every 2 days.
Sample preparation and immunofluorescence
Floating and attached cells from monolayer cultures were harvested, pooled and deposited on slides by cytospin centrifugation. Matrigel or collagen cultures were included in Cryomatrix (Shandon), snap frozen in liquid N2 and kept at 80°C. For analysis, 20 µm sections were deposited on slides, fixed in 4% paraformaldehyde for 10 minutes, permeabilized for 2 minutes in 0.1% Triton X-100, rinsed in PBS and blocked with 10% foetal calf serum. Alternatively, caspase-3 staining was performed on whole collagen inserts, in which case the fixation period was 15 minutes. Samples were incubated with primary antibodies followed by the appropriate fluorochrome-labelled secondary antibodies (both for 1 hour at room temperature). Control sections were stained with secondary antibodies only. Actin was visualized by TRITC-phalloidin labelling. Nuclei were counterstained with Hoechst 33258 (2 µM). The mounting media were Mowiol (Calbiochem) for the two-dimensional (2D) samples or glycerol/PBS (9:1) containing p-phenylenediamine for the Matrigel and collagen sections.
Confocal microscopy
Immunofluorescence confocal images were acquired using a Zeiss LSM 510 inverted laser-scanning confocal microscope equipped with an external argon laser. Images were captured at 0.5 µm intervals using a Zeiss fluor 63x objective, deconvoluted with Huygens Pro and reconstructed in 3D with Imaris. Individual confocal stacks are shown, unless otherwise indicated.
Transmission electron microscopy and histology
Matrigel cultures were fixed in situ with 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.3 for 1 hour, washed for 15 minutes in the same buffer with 6.84% sucrose, post-fixed for 1 hour in 2% osmium tetroxide, dehydrated in a graded ethanol series followed by propylene oxide treatment for 3D culture and another ethanol-dehydration step for 2D samples, and embedded in Epon 812. Semi-thin sections were stained with toluidine blue and ultra-thin sections were contrasted with uranyl acetate and lead citrate, and observed with a Jeol 1200X transmission electron microscope.
Histochemistry
Hall's bilirubin staining was performed according to a standard protocol (Sheehan and Hrapchak, 1980).
Apoptosis assay
Cells were incubated with Jo2 antibody (1 µg ml1) or TNF (100 ng ml1) for 22 hours, cycloheximide (1 µg ml1) or actinomycin D (2 µg ml1) were added, as indicated. Apoptotic cells were identified by immunofluorescence staining of activated caspase 3, the total number of cells was assessed by nuclear staining with the Hoechst 33258 dye. For quantification, at least 400 cells were counted in randomly chosen fields. The exact number of apoptotic cells in 3D organoids being more difficult to estimate, apoptosis in 3D cultures was quantified by counting caspase-3-positive organoids rather than cells. At least 80 organoids were analysed for each Matrigel section. Typically, only a few cells were caspase-3 positive in untreated organoids in the course of their maturation, whereas, in the course of the response to the stimulation of the death receptors, a caspase-3-positive organoid was composed mostly of cells with activated caspase 3.
NF-B reporter assay
Subconfluent monolayer cells were co-transfected with the NF-B-dependent luciferase reporter (Ig
3-conaluc) (Munoz et al., 1994
) or a control plasmid lacking the NF-
B binding sites, and a constitutive Renilla luciferase construct. 18 hours later, cells were collected, split in two and either plated as a monolayer culture or embedded in Matrigel. After 24 hours or 8 days, respectively, for the 2D and 3D cultures, the cells were treated with Jo2 antibody (1 µg ml1) or TNF
(100 ng ml1) for 7 hours and harvested by scraping and by MatriSperse treatment, respectively, for 2D and 3D cultures. The cells were lysed and luciferase activities were assayed using the Promega dual luciferase kit according to the manufacturer's instructions. In all cases, and specifically in the samples derived from the 3D culture, unstimulated activities of both Renilla and firefly luciferases (measured in lysates of transfected cells without any death-receptor stimulation) were at least 30 times higher than the low non-specific background values of untransfected samples. The unstimulated firefly luciferase activity, normalized to Renilla luciferase, was arbitrarily set as 1 and the activation of NF-
B signalling was presented as a fold increase over this value.
Western-blot analysis
Matrigel or collagen inserts were recovered, rinsed in PBS and digested at 37°C in the presence of protease inhibitors, with Matrisperse for 1 hour or collagenase H for 30 minutes. The cells were centrifuged, lysed in 1x SDS sample buffer, analysed by PAGE and blotted onto nitrocellulose membranes. Secretion of albumin was allowed to proceed for 24 hours in serum-free medium and an aliquot of medium was analysed. Cell-free matrices or cultures of murine fibroblasts were used as a control for detection of bovine albumin; both gave rise to a negligible signal.
Statistical analysis
Results are expressed as means ± s.d. Each assay, performed in triplicate, was repeated at least twice. Statistical significance was analysed by Fisher's test: ****, P0.0001; ***, P
0.001; *, P
0.05.
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Results |
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Transmission-electron-microscopy analysis confirmed that both monolayer cells and organoids contained polarized cells with visible tight junctions (Fig. 2Ca,b, monolayer, Fig. 2Cc,d, 3D). However, we estimate that only about 50% of monolayer-grown hepatocytes contained tight junctions and distinct apical, microvillus-lined, membrane domains. These extended apical domains facing the medium contained short, sparse microvilli, whereas abundant, well-formed microvilli lined the intercellular lumens of 3D organoids (Fig. 2C). Furthermore, the cells in 3D structures and in monolayers had drastically different shapes: a cuboid geometry was observed in the 3D culture, whereas the monolayer cells were flat (estimated height/width ratio=0.90±0.16 and 0.32±0.06, respectively). Thus, mhAT3F cells were flattened and weakly polarized in monolayer cultures and acquired a cuboid shape and strong polarization when arranged into 3D organoids in the Matrigel.
Collagen-sandwich culture is an alternative support compatible with hepatocyte polarization (Dunn et al., 1989; LeCluyse et al., 1996
; Moghe et al., 1996
). Under our experimental conditions of low-density seeding, mhAT3F cells adopted two distinct morphological organizations in collagen: spheroid organoids similar to those formed in the Matrigel and extended sheets of cells (Fig. 2D). Whereas the distribution of radixin was very similar in organoids formed in either 3D matrix (Fig. 2A,E), the cells in the extended configuration showed more uniform labelling.
In order to ascertain whether the cells grown under different conditions retained functional characteristics of differentiated hepatocytes, we first measured their capacity to secrete albumin (Fig. 3A). Monolayer-grown cells synthesized and secreted albumin that was detectable in the conditioned medium. The secretion was slightly increased in immature (day 4) organoids and strongly increased both in the mature Matrigel-grown organoids and in collagen-grown cells. Bilirubin accumulation is another stringent test for hepatocyte function. In contrast to the cells grown as a monolayer (Fig. 3Ba), the organoids produced histologically detectable bilirubin (Fig. 3Bb), as judged by a green colour after Hall's histological staining (Sheehan and Hrapchak, 1980). Cholestatic liver section, characterized by excess bile accumulation, served as a positive control in this experiment (Fig. 3Bc).
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Hepatocyte organoids are sensitive to Fas stimulation
Intravenous injection of an agonistic anti-Fas antibody (Jo2) is strongly hepatotoxic, leading to massive apoptotic death of hepatocytes and liver haemorrhage (Ogasawara et al., 1993). By contrast, both primary and immortalized murine hepatocytes in monolayer culture survived treatment with even very high doses of the same antibody (Fig. 4A and data not shown), even though their apoptotic signal transduction pathway was fully functional, as revealed by the efficient induction of apoptosis following Fas stimulation in the presence of an inhibitor of protein synthesis (Fig. 4A-C). In order to account for the different sensitivities of hepatocytes to Fas stimulation in vivo and in vitro, we assessed the effects of polarization of mhAT3F on their response to death-receptor stimulation. Treatment of 3D organoids by Jo2 resulted in massive apoptosis, as shown both by morphological changes (Fig. 4A) and by activation of caspase 3, detected in over 80% of organoids (Fig. 4B,D). The same treatment performed in parallel on cells in a monolayer culture gave rise to no apoptotic response above the low background (Fig. 4B,C).
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These data suggested that the maintenance of cell shape and polarization might lead to an increased sensitivity to apoptosis. To settle this point unambiguously, we took advantage of the fact that the mhAT3F cells adopted two distinct configurations in a collagen sandwich spherical organoids and extended sheets of cells (Fig. 2D). Upon Fas stimulation of the collagen-grown cells, only the spherical organoids underwent apoptosis, whereas the same treatment performed in the presence of cycloheximide killed all the cells, irrespective of their 3D-organisation (Fig. 4G). To gain a more quantitative insight into this effect, we counted all structures present in a culture insert. Before treatment, intact spherical organoids represented 39% of all structures (n=117) but, after incubation with Jo2, they dropped to 7% (n=128).
One major difference between subconfluent monolayers versus cells organized into mature 3D organoids is the cells' proliferative status. However, quiescence was not responsible for the increased sensitivity to Fas stimulation, because the fully confluent mhAT3F monolayer cultures, characterized by a drastically reduced BrdU labelling index (not shown), did not die upon treatment with Jo2 (see Fig. S2 in supplementary material). Thus, cell density and proliferative status did not account for the apoptotic response of the 3D organoids.
Interestingly, organoids in the Matrigel were not significantly more sensitive to the stimulation of a related death-receptor pathway. As shown in Fig. 4F, treatment of 3D cultures with TNF did not give rise to significant caspase-3 activation. However, the full apoptotic response to TNF
was obtained by simultaneously blocking macromolecular synthesis in both 2D and 3D cultures (Fig. 4E,F).
NF-B regulates the apoptotic response to death-receptor stimulation
Activation of NF-B by the ligand-bound death receptor is a widely accepted paradigm of survival signalling (for a review, see Baud and Karin, 2001
). Accordingly, we asked whether interfering with NF-
B activation was sufficient to promote Fas-mediated apoptosis in mhAT3F cells. Monolayer-grown cells were co-transfected with vectors encoding a truncated rat CD2 antigen and a mutant `super-repressor' I
B protein, whose production efficiently inhibits p65/p50 NF-
B activation (Munoz et al., 1994
). After treatment with the Jo2 anti-Fas antibody, transfected cells were labelled with the anti-CD2 antibody and apoptotic cells were detected by staining of activated caspase 3. No caspase activation was seen in untransfected (CD2-negative) cells, whereas 25% of transfected cells responded to Fas stimulation by activating caspase 3 (Fig. 5A,B). Thus, NF-
B activation contributed to survival signalling in this model of Fas-induced apoptosis.
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Discussion |
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We have shown that the Matrigel embedded mhAT3F cells organized into compact spheroids that matured, through apoptotic elimination of internally localized cells, into structures containing one, or several, intercellular lumens (Figs 1, 2). Large lumens are usually absent from 3D structures formed by primary hepatocytes in 3D Matrigel cultures (Moghe et al., 1996). A possible explanation for this discrepancy might be the difference in the mechanism of organoid formation: whereas the primary cells aggregate into organoids, the mhAT3F structures are formed by several cell divisions in the first days of culture.
Relatively large lumens and a series of adjacent small lumens could both be detected in mhAT3F organoids (Fig. 2C). The latter suggest a possible mechanism of lumen formation by the fusion of small apical domains, reminiscent of tubulogenesis in tracheal morphogenesis in Drosophila (Lubarsky and Krasnow, 2003). In addition, apoptosis clearly participated in the organoid lumen formation, as witnessed by the transient caspase-3 activation during the process of maturation and the presence of apoptotic bodies in some of the lumens (Fig. 1).
Lumen formation by apoptosis that occurs in 3D cultures of kidney (O'Brien et al., 2002) and mammary (Debnath et al., 2002
; Muthuswamy et al., 2001
) epithelium is believed to reflect the physiological death occurring during development and homeostasis of these tissues. Apoptosis as a mechanism for lumen formation is more surprising in hepatocyte organoids. Even though an acinar arrangement of partially depolarized hepatocytes has been described in vivo during organogenesis and regeneration, and in some cases of well-differentiated hepatocellular adenomas (Stamatoglou and Hughes, 1994
), hepatocytes do not usually form large tubular structures but rather small canaliculi. However, in contrast to the 3D culture conditions, the extracellular matrix in a healthy liver is not deposited uniformly but lines the tracts of hepatocytes. The geometrical constraints imposed on cells striving to maintain contacts with both the matrix and the neighbouring cells, and to preserve a free apical surface are thus different in vivo and in 3D cell culture. This could be sufficient to account for an altered arrangement of cells in the organoids. In any case, the highly polarized localization of radixin, a marker of hepatocytic bile canaliculi (Kikuchi et al., 2002
), argues in favour of strong cellular polarization of cells in the organoids.
It is noteworthy that, in contrast to the reported structures of mature MDCK or mammary epithelium organoids (O'Brien et al., 2002; Weaver et al., 1997
), the hepatocyte organoids often contained several intercellular lumens, apparently bordered by as few as two or three cells (Fig. 2; see Fig. S3 in supplementary material). Such organization is reminiscent of a morphology of a genuine bile canaliculus in the liver.
Organoids undergo apoptosis upon Fas stimulation
Hepatocytes are a major physiological target of Fas/CD95 signalling in vivo in both mice and humans. Intravenous injection of an agonistic anti-Fas antibody kills the mouse through liver destruction owing to hepatocyte apoptosis and haemorrhage (Ogasawara et al., 1993), and animals with a homozygous invalidation of the Fas gene, in addition to immunological disorders, suffer from hepatomegaly, presumably caused by inefficient hepatocyte apoptosis (Adachi et al., 1995
). Similarly, both acute hepatitis and fulminant hepatic failure in human patients appear to be related to strong pro-apoptotic Fas signalling (Ryo et al., 2000
). By contrast, neither primary nor immortalized hepatocytes grown in traditional monolayer cultures are killed by Fas-receptor engagement unless survival signalling, which follows the death-receptor stimulation (Baud and Karin, 2001
), is simultaneously inhibited.
This difference of behaviour could be due partly to the in vivo effect of Fas stimulation on the liver endothelial cells (Jodo et al., 2003). Our data show that, alternatively but not exclusively, the altered geometry of the cells in a monolayer culture can affect survival signalling and lead to resistance to Fas-induced apoptosis. Assembly into 3D organoids preserved the hepatocyte-specific morphology and function, and strongly influenced the cells' apoptotic responses. The observed sensitivity to Fas stimulation was due neither to the proliferation arrest (see Fig. S2 in supplementary material) nor to the simple contact with Matrigel, because the sensitization to Fas stimulation only occurred after 3 days of culture (i.e. was coincident with the organoid formation) (data not shown). Thus, sensitivity to Fas-induced apoptosis correlated with the establishment of strong cellular polarization and correct geometry. This conclusion is further strengthened by the results obtained in the collagen culture, where two types of cell organization were present simultaneously. Cells that formed compact organoids, similar to those observed in the Matrigel culture, underwent massive apoptosis, whereas those arranged into extended sheets survived Fas stimulation. Because these strikingly different behaviours co-existed in the same culture, they provide a strong argument for a causal relationship between cellular geometry and the apoptotic response.
Interestingly, in contrast to the mammary epithelium, in which 3D polar acini were resistant to a range of apoptotic stimuli, including death-receptor engagement (Weaver et al., 2002), maintenance of polarity in hepatocytes correlated with an increased sensitivity to Fas stimulation. This apparent discrepancy could be a reflection of differences in the physiology of the two cell types because, in contrast to many cell types, healthy hepatocytes are a major target of Fas-induced apoptosis in vivo (Ogasawara et al., 1993
; Ryo et al., 2000
; Song et al., 2003
).
NF-B regulates the apoptotic response to death-receptor stimulation
The apoptotic signal-transduction pathway leading from the engagement of death receptors to caspase-3 activation has been described in considerable detail (for a review, see Wallach et al., 1999). It involves the recruitment of DISC (death-inducing signalling complex) to the intracytoplasmic domain of the trimeric receptor, resulting in proteolytic activation of the initiator caspase 8. In hepatocytes, which are type II cells in respect to Fas signalling (Scaffidi et al., 1998
), the resulting apoptotic signalling is strictly dependent on caspase-8-mediated cleavage of Bid, a pro-apoptotic member of the Bcl2 family (Disson et al., 2004
; Yin et al., 1999
).
Large numbers of cells are required for a purification of DISC. This is incompatible with 3D culture: as a result we could not analyse it biochemically in our experimental system. As a consequence, we cannot formally exclude the possibility that cellular polarization altered the production and/or the subcellular localization of the receptors and their associated proteins. However, the inhibition of macromolecular synthesis gave rise to an efficient activation of the apoptotic cascade that was indistinguishable in monolayer and 3D cultures. This finding argues against major differences in the composition or arrangement of the apoptotic machinery in cells cultured under the two experimental conditions.
Activation of NF-B by the ligand-bound death receptor constitutes a potent survival signal (for a review, see Baud and Karin, 2001
). Although this has been well described for TNFR engagement in several cell types, its involvement in Fas signalling is less well documented. Our data confirm a previous report of the inhibition of NF-
B signalling in hepatocytes that conferred an increased sensitivity to Fas-induced apoptosis (Hatano et al., 2000
). Moreover, we have shown that the NF-
B activation by death-receptor engagement in polarized hepatocytes was much reduced in comparison to unpolarized cells. The molecular mechanisms responsible for this difference are under investigation. It is neither due to altered production of NF-
B itself (Fig. 5C) nor its regulators I
B
and IKKß (not shown). Furthermore, the decrease, or the lack of NF-
B activation following TNFR or Fas stimulation, respectively, was unlikely to be due to a lower accessibility of the receptors to the ligands in the 3D matrix, because the apoptotic response to the same treatments was actually increased under these conditions.
Taken together, our data support the idea that the maintenance of polarized cell morphology has a strong impact on the cellular interpretation of an apoptotic signal. Interestingly, our results argue against the notion that cell polarity is an universal anti-apoptotic feature of an epithelial cell (Weaver et al., 2002) but, rather, suggest that it allows to recapitulate important aspects of a cell physiology.
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Acknowledgments |
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Footnotes |
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