1 Canadian Institutes of Health Research Group on the Functional Development and Physiopathology of the Digestive Tract, Département d'Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4; 2 The Burnham Institute, La Jolla Cancer Research Center, La Jolla, California 92037; and 3 Thématique de Recherche en Physiopathololgie Digestive du Centre de Recherches Cliniques du CHUS, Fleurimont, Quebec, Canada J1H 5N4
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ABSTRACT |
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To investigate whether human
intestinal epithelial cell survival involves distinct control
mechanisms depending on the state of differentiation, we analyzed the
in vitro effects of insulin, pharmacological inhibitors of Fak,
MEK/Erk, and PI3-K/Akt, and integrin (1,
4)-blocking antibodies
on the survival of the well-established human Caco-2 enterocyte-like
and HIEC-6 cryptlike cell models. In addition, relative expression
levels of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax,
Bak, and Bad) and activation levels of Fak, Erk-2, and Akt were
analyzed. Herein, we report that 1) the enterocytic
differentiation process results in the establishment of distinct
profiles of Bcl-2 homolog expression levels, as well as
p125Fak, p42Erk-2, and p57Akt
activated levels; 2) the inhibition of Fak, of the MEK/Erk
pathway, or of PI3-K, have distinct impacts on enterocytic cell
survival in undifferentiated (subconfluent Caco-2, confluent HIEC-6)
and differentiated (30 days postconfluent Caco-2) cells; 3)
exposure to insulin and the inhibition of Fak, MEK, and PI3-K resulted in differentiation state-distinct modulations in the expression of each
Bcl-2 homolog analyzed; and 4) Fak,
1 and
4 integrins, as well as the MEK/Erk and PI3-K/Akt pathways, are distinctively involved in cell survival depending on the state of cell
differentiation. Taken together, these data indicate that human
intestinal epithelial cell survival is regulated according to
differentiation state-specific control mechanisms.
anoikis; apoptosis; gut; intestine; signal transduction
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INTRODUCTION |
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PROGRAMMED CELL DEATH, or apoptosis, is a complex and tightly regulated process that performs crucial functions in tissue homeostasis (1, 25). The Bcl-2 family of proteins constitutes a critical decisional checkpoint in cell survival, regulating the common downstream effector pathway of apoptosis (1, 47). At least 15 family members have been identified so far, functioning either as anti-apoptotic (e.g., Bcl-2, Bcl-XL, Mcl-1) or pro-apoptotic (e.g., Bax, Bad, Bak) regulators (1, 20, 48). It is now well acknowledged that regulation of cell survival in different cell types does not depend on the activity of single Bcl-2 homologs but, rather, on a balance of anti- and pro-apoptotic activities from multiple homologs (1, 47). To this effect, extracellular signals determine in large part if a cell lives or dies, involving signaling events that ultimately affect the expression and/or functions of multiple anti- and pro-apoptotic Bcl-2 homologs (1, 4, 13, 15, 19-21). Such survival signals originate from growth factors (e.g., insulin) and cell adhesion, often implicating similar signaling pathways (4, 13, 15, 19-21, 25). In this respect, the roles of focal adhesion kinase (p125Fak), the phosphatidylinositol 3'-kinase (PI3-K)/Akt (PKB; p57Akt) pathway, and the MEK [mitogen-activated protein kinase (MAPK) kinase]/Erk (extracellular regulated kinases 1 and 2; p42Erk-2/p44Erk-1 MAPK) pathway have received much attention in recent years (4, 11, 13, 15, 21). However, it is becoming increasingly evident that the involvement of these signaling molecules/pathways in cell survival varies depending on the cell type that is receiving such signals, thus underlying the complexity in the regulation of apoptosis among various tissues (1, 4, 11, 13, 15, 19-21, 25).
The intestinal epithelium is a useful model for the study of the working mechanics of tissue renewal processes, including apoptosis. Its rapid, continuous cell renewal consists of spatially separated stem cells, proliferative and differentiated compartments located, respectively, in the lower regions of the crypts and on the villi (26, 45). Such a "gradient" of cell differentiation is further defined by functional properties of the fully differentiated villus enterocytes, which distinguish them from crypt cells (26, 37, 41, 45). Hormonal responses as well as cell adhesion components, such as integrins, underlie additional distinctions between crypt and villus enterocytes (7, 16, 37, 41, 43-45). Although the predominant means to remove obsolete enterocytes is through apoptosis and shedding at the villus apex, "spontaneous" crypt cell apoptosis is a less frequent process that serves to remove defective/injured progeny cells (22, 24, 26, 40, 44, 45, 53). In addition to this apparent "duality of fate" between undifferentiated and differentiated intestinal cells, some Bcl-2 homologs have been shown individually to exhibit gradients of expression along the crypt-villus axis (29-32, 38-40, 44, 45, 59, 60). By analyzing six homologs at the same time in the proximal-distal axis of the developing human gut (jejunum, ileum, and colon), we have previously shown that villus cells gradually come to exhibit a Bcl-2 homolog expression profile that clearly differs from the one observed in crypt cells by midgestation, once the crypt-villus axis has matured (52, 53). These observations altogether suggest that intestinal epithelial cell survival may be regulated distinctively according to the state of cell differentiation.
In the present study, we investigated this hypothesis by using the human enterocyte-like cell line Caco-2, a widely used model of intestinal epithelial cell maturation and functions (8, 27, 37, 41, 42, 50). Although cancerous in origin, these cells are unique in their property to undergo a gradual differentiation process that takes place spontaneously once confluence has been reached and that is completed after 25-30 days of postconfluent culture (8, 27, 37, 41, 42, 50). Consequently, Caco-2 cells acquire a morphological polarity and show enzymatic activities as well as protein and mRNA levels of brush-border membrane enzymes that are highly comparable to those of mature enterocytes (8, 27, 37, 41, 42, 50). In this respect, Caco-2 cells as a single cell culture system have provided an important tool to study and further understand the regulation of various human intestinal epithelial cellular processes, such as enterocytic differentiation (2, 12, 37, 41, 42, 50, 51), proliferation (2, 17, 41-43), digestive functions (8, 12, 27, 37, 41), and cell adhesion (5-7, 35, 41, 51, 61). We also used the normal human fetal cryptlike HIEC-6 cells (41), which do not differentiate even when in postconfluent culture, as an additional model of undifferentiated intestinal epithelial cells to complement the one represented by undifferentiated Caco-2 cells.
Herein, Caco-2 and HIEC-6 cells were exposed to growth factors (namely,
insulin) or pharmacological compounds that inhibit signal transduction
molecules (namely, tyrosine kinases, Fak, PI3-K/Akt, and/or MEK/Erk) to
evaluate their impact on cell survival and Bcl-2 homolog expression. We
find that undifferentiated and differentiated cells display strikingly
distinct Bcl-2 homolog expression profiles, such profiles being
established gradually during the enterocytic differentiation process.
In addition, we find that exposure to insulin, or the inhibition of
signaling molecules/pathways, modulates the expression of Bcl-2
homologs regardless of the state of differentiation but that the
modulatory effects and homologs affected vary between undifferentiated
and differentiated cells. Furthermore, we find that 1 and
4
integrins, as well as the PI3-K/Akt and MEK/Erk pathways, are
distinctively involved in the survival of undifferentiated
(subconfluent Caco-2, confluent HIEC-6) and differentiated (30 days
postconfluent Caco-2) enterocytes. Altogether, these data indicate that
human intestinal cell survival is subject to distinct regulatory
mechanisms according to the state of differentiation.
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MATERIALS AND METHODS |
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Cell culture.
The Caco-2/15 cell line, a stable clone of the parent Caco-2 cells
(42), has been characterized elsewhere (8,
50). Caco-2/15 cells are known to be poor secretors of growth
factors (2, 8, 12, 17). Cells between passages 53 and 70 were cultured in plastic dishes (60 or 100 mm; Falcon Plastics, Los Angeles, CA), or on 13-mm coverslips, at 37°C in an atmosphere of
95% air-5% CO2. The medium used was Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS;
GIBCO BRL, Burlington, ON, Canada), 4 mM glutamine, 20 mM HEPES (pH 7.5), 50 U/ml penicillin, and 50 µg/ml streptomycin. Cultures were
refed every 48 h and subcultured serially as described previously (8, 50). Studies were performed on cultures at 2 days
(subconfluence; 50-70% confluence), 0 days (confluence), and/or
5-30 days postconfluence. The expression of sucrase-isomaltase, a
major enterocytic differentiation marker, was monitored by Western blot
to ensure proper differentiation of cultures (not shown) as previously
described (8, 50, 51). In some experiments, the intestinal
cryptlike human fetal HIEC-6 cells (41) were used in
parallel with undifferentiated Caco-2 cell cultures; HIEC-6 cells do
not differentiate on reaching confluence, and their undifferentiated
"cryptlike" status does not change with postconfluence
(41).
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Antibodies. Primary rabbit polyclonal antibodies used in the present study were Ab1682, directed against human Mcl-1 (29); Ab1695, directed to human/mouse Bcl-XL (28, 32); Ab1701 (29, 31) and AbPC68 (Calbiochem), both directed against human Bcl-2; Ab1712 (31) and AbPC66 (Calbiochem), both directed to human Bax; Ab1764, directed against human Bak (28, 30); AbI-19 (Santa Cruz Biotechnology, Santa Cruz, CA), directed to human/mouse Bak; AbPC67 (Calbiochem), directed to human Bcl-XL; AbK-20 (Santa Cruz Biotechnology), directed against human/mouse Mcl-1; and Ab9292 (New England Biolabs, Beverly, MA) and AbR-20 (Santa Cruz Biotechnology), both directed to human Bad. Primary mouse monoclonal antibodies used were mAbCY-90 (Sigma-Aldrich Canada, Oakville, ON), directed against human cytokeratin 18 (K18).
Note that antibodies Ab1682, Ab1695, Ab1701, Ab1712, and Ab1764 were developed in the laboratory of one of the authors (J. C. Reed) of the present study; these, as well as the commercial antibodies used herein, have been characterized in previous studies (10, 23, 28-32, 39, 52, 53).In situ detection of apoptosis-associated DNA strand
breaks.
For some experiments, in situ terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling (ISEL) was carried out as
already described (52, 54, 55) on coverslip-grown cultures after treatments, using the ApopTag apoptosis detection kit
(Oncor, Gaithersburg, MD). Preparations where then counterstained with Evans blue and mounted and viewed with a Reichart Polyvar 2 microscope (Leica, St. Laurent, QC, Canada) equipped for epifluorescence. Evaluation of ISEL-positive cells was performed as previously described
(54, 55). A minimum of 300 cells was counted in at least
three (n 3) separate experiments and/or cultures.
The apoptotic indexes were expressed as the percentage of
apoptotic (ISEL-positive) cells over the total number of cells
counted (±SE); statistically significant (0.001
P
0.01) differences were determined with
Student's t-test.
DNA laddering assays. After treatments (see above), DNA was isolated according to the modified method of Frisch (18) as previously described (54). DNA contents of all samples were estimated by optical density at 260 nm. For the visualization of apoptosis-associated DNA internucleosomal fragmentation (DNA laddering), each sample was then resolved by electrophoresis (20 µg DNA/lane) on 2% agarose gels stained with ethidium bromide, using a 100-bp ladder (GIBCO BRL) as standard. Note that the method used for DNA extraction uses Triton rather than SDS, thus leaving behind intact genomic DNA (18); consequently, nonapoptotic cell cultures produce near-empty lanes on the gel as a result (for example, see Fig. 2, A and B, lanes 1 and 2).
Protein expression levels.
For analyses of protein expression levels of Bcl-2 homologs, total
protein from cell lysates was obtained after medium removal, PBS
washes, and scrapping of cells in 1× solubilization buffer [2.3%
SDS, 10% glycerol, and 0.001% bromphenol blue in 62.5 mM Tris · HCl (pH 6.8) containing 5% -mercaptoethanol].
Samples were then boiled (105°C, 5 min), cleared by centrifugation
(15,000 g, 5 min, room temperature), and processed for
storage as described previously (51-55). SDS-PAGE on
15% acrylamide Tris · HCl gels (Bio-Rad, Hercules, CA) was
performed as described previously (51-55).
Broad-range molecular mass markers (6.8-209 kDa range; Bio-Rad)
were used as standards. Total proteins (50 µg/well) were separated by
electrophoresis and then electrotransferred to nitrocellulose membranes
(Supported NitroCellulose-1; GIBCO BRL) for subsequent immunoblotting
(51-55). Rabbit antisera were used at
1:200-1:2,000 dilutions, and mouse monoclonal antibodies were used
at 1:5,000 dilutions. Immunoreactive bands were visualized by the
enhanced chemiluminescence method (ECL system; Amersham/Pharmacia
Biotechnology, Baie D'Urfé, QC, Canada) according to the
manufacturer's instructions. Band intensities were quantified by laser
densitometry using an Alpha Imager 1200 documentation and analysis
system (Alpha Innotech, San Leondro, CA).
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Immunoprecipitation.
For some experiments, cell cultures were harvested in cold RIPA buffer
[50 mM Tris · HCl (pH 7.2), 150 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µM Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 0.5 µg/ml leupeptin, 0.5 µg/ml aprotinin, 0.7 µg/ml
pestadin, 40 mM -glycerophosphate, and 10 mM
Na2P4O7] and lysed in the buffer
(30 min on ice). Lysates were cleared by centrifugation (15,000 rpm, 15 min, 4°C) and aliquoted for storage as previously described (6,
54). Immunoprecipitation of Fak was carried out according to the
protocol already described (54), using 1 µg of the
rabbit polyclonal AbC-903 (Santa Cruz Biotechnology) directed against
human/mouse/rat p125Fak.
Assay of Fak, Erk-1/Erk-2, and Akt relative activation levels by immunoblotting. Total proteins were resolved by SDS-PAGE and electrotransferred as described above. Assays of p125Fak, p42Erk-2/p44Erk-1, or p57Akt relative activation levels were performed as described elsewhere (2, 10, 33, 34, 61). For Fak assays, membranes were first probed with the rabbit polyclonal Ab07-012 (Upstate Biotechnology, Lake Placid, NY) directed to the activated phospho-Tyr-397 form of Fak (11, 21, 61) and then reprobed with the AbC-903 for normalization purposes. For some experiments, Fak immunoprecipitates were used instead (see above). For Erk-1/Erk-2 assays, membranes were probed with the rabbit polyclonal Ab9101s (New England Biolabs) directed to the doubly phosphorylated (activated) forms of Erk-1/Erk-2 (2, 4, 10, 34) and then reprobed with the rabbit polyclonal Ab9102 (New England Biolabs) directed to total Erk-1/Erk-2. Finally, assays of p57Akt relative activation levels were performed by first probing membranes with the rabbit polyclonal Ab9271s (New England Biolabs) directed to the activated phospho-Ser-473-Akt form (4, 15, 33, 34, 48, 56) and then reprobed with the rabbit polyclonal Ab9272 (New England Biolabs) directed to total Akt.
The relative activation levels of p125Fak, p42Erk-2/p44Erk-1, or p57Akt were evaluated by determining the total peak areas (AU × mm) for the phosphorylated forms and for the corresponding total protein, to establish the ratios pp125Fak/p125Fak, pp42Erk-2/p42Erk-2, and pp57Akt/p57Akt. In the case of Erk-1/Erk-2, analyses focused on p42Erk-2 (2, 10). Ratios were in turn compared with those of control cultures, ×100 (expressed as % of control). Values shown represent means ± SE for at least three (n ![]() |
RESULTS |
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Establishment of distinct Bcl-2 expression profiles during enterocytic differentiation. To determine whether human intestinal epithelial cell survival is subject to differentiation state-specific regulatory mechanisms, we first investigated the steady-state expression levels of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) during the enterocytic differentiation process of Caco-2/15 cells. Immunoblot analyses of cell lysates demonstrated the protein expression of all molecules analyzed herein (Fig. 1A). Thus Bcl-2 (~26 kDa), Bcl-XL (~28-30 kDa), Mcl-1 (~39-42 kDa), Bax (~21 kDa), Bak (~ 25-28 kDa), and Bad (~28-32 kDa) were detected at all differentiation stages studied as protein bands migrating at their previously reported relative molecular weights (1, 10, 28-32, 39, 47-49, 50, 52, 53).
To examine the modulations of Bcl-2 homolog expression in relation to Caco-2/15 cell enterocytic differentiation, the relative expression levels of Bcl-2 homologs were determined by comparison with a reference protein, K18. The densitometric data presented in Fig. 1, B and C, show that the relative expression levels of all Bcl-2 homologs analyzed were distinctively modulated in parallel to the differentiation process of Caco-2/15 cells. In the case of anti-apoptotic homologs (Fig. 1B), the steady-state levels of Bcl-2 gradually increased between 0 and 15 days postconfluence, to stabilize more or less thereafter as the cells completed their differentiation (Fig. 1A, Bcl-2; Fig. 1B, open squares). Similarly, Mcl-1 levels gradually increased throughout the differentiation process of Caco-2/15 cells (Fig. 1A, Mcl-1; Fig. 1B, filled triangles). On the other hand, Bcl-XL levels decreased during enterocytic differentiation, particularly between the 10- and 30-day postconfluent stages (Fig. 1A, Bcl-XL; Fig. 1B, filled circles). In the case of proapoptotic homologs (Fig. 1C), the steady-state levels of Bax were found to increase gradually during Caco-2/15 cell differentiation (Fig. 1A, Bax; Fig. 1C, filled squares). On the other hand, Bak levels decreased sharply between theEnterocytes display distinct susceptibilites to apoptosis according to their differentiation state. Because undifferentiated and differentiated Caco-2/15 cells exhibit distinct Bcl-2 homolog expression profiles, we then sought to ascertain whether this translated into differential susceptibilities to apoptosis as well. Using DNA laddering assays to visualize the internucleosomal DNA fragmentation associated with apoptosis, we evaluated the presence of apoptosis in undifferentiated and differentiated cultures maintained for 48 h with or without FBS, or without serum but with the addition of insulin or pharmacological inhibitors of signal transduction molecules/pathways. Apoptotic indexes in treated cultures were also determined using the ISEL method.
We first found that maintenance of Caco-2/15 cells in the absence of serum for 48 h did not impact significantly on their survival compared with their maintenance in the presence of FBS. Indeed, no DNA laddering was evidenced in either culture condition for undifferentiated (Fig. 2A, lane 1 vs. 2) and differentiated cells (Fig. 2B, lane 1 vs. 2), whereas the "basic" apoptotic indexes found in the presence of FBS (Table 1, +FBS) did not differ significantly from those obtained in absence of serum (Table 1, control) for either differentiation states. Incidentally, we (51) and others (27, 41) have previously reported that absence of serum for 24-48 h was not detrimental to enterocytic functions of Caco-2 cells.
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Differentiation state distinct involvement of Fak and integrins in
enterocytic cell survival.
To verify whether the observed differentiation state distinctions in
apoptosis susceptibility were linked to a differential involvement of signaling pathways, we first investigated specifically the role of cell adhesion signaling in Caco-2/15 cell survival, focusing on p125Fak activation (Fig.
3). As previously reported in the
parental Caco-2 cell line (35), we found that levels of
activated Fak (pp125Fak) were downregulated while those of
the protein (p125Fak) increased during the differentiation
process (Fig. 3A). This resulted in distinct enterocytic
p125Fak activation profiles, whereby Fak activation was at
minimal levels in differentiated cells as opposed to undifferentiated
ones (Fig. 3, A and B). Nonetheless, relative
p125Fak activated levels were equally inhibited in the two
differentiated states when either genistein (Fig. 3C, +G) or
cytochalasin D (Fig. 3C, +CD) was used as a treatment. The
nonspecific inhibition of p125Fak activation by genistein,
a wide-spectrum inhibitor of tyrosine kinases, was expected (35,
36, 58, 61), whereas cytochalasin D can act as a more specific
inhibitor of p125Fak at the concentration range used
(36, 58). We also observed that the inhibition of the
downstream MEK/Erk pathway (Fig. 3C, +PD) or PI3-K (Fig.
3C, +Ly), as well as exposure to insulin (Fig. 3C, +I), did not impact on the relative activation levels of
p125Fak itself in either undifferentiated or differentiated
Caco-2/15 cells, as expected from previous studies in other cell types
and tissues (4, 11, 33, 34, 36, 58).
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Differentiation state-specific involvement of MEK/Erk signaling in
enterocytic cell survival.
To further dissect the observed distinctions in apoptosis
susceptibility between undifferentiated and differentiated Caco-2/15 cells, we then examined more closely the involvement of the MEK/Erk pathway by focusing on p42Erk-2 activation (Fig.
5). As in the case of p125Fak
(see above and Ref. 35) and as previously reported
(2, 35), we found that levels of activated Erk-2
(pp42Erk-2) levels were downregulated while those of the
Erk-2 protein (p42Erk-2) increased during the
differentiation process (Fig. 5A). This resulted in distinct
enterocytic p42Erk-2 activated profiles, whereby Erk-2
activation was at minimal levels in differentiated Caco-2/15 cells as
opposed to undifferentiated ones (Fig. 5, A and
B). Nonetheless, the inhibition of upstream MEK with
PD-98059 significantly lowered the activated levels of p42Erk-2 in the two differentiated states (Fig.
5C, +PD) as expected (2, 3, 10, 34, 61), while
the inhibition of PI3-K had no effect (Fig. 5C, +Ly). Also
as expected (4, 11, 13, 21, 34), the inhibition of
p125Fak (Fig. 5C, +CD) and of tyrosine kinase
activities (Fig. 5C, +G) significantly lowered the relative
activation levels of p42Erk-2 in both undifferentiated and
differentiated Caco-2/15 cells. Conversely, exposure to insulin
produced similar results (Fig. 5C, +I).
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Differentiation state-distinct regulation of the PI3-K/Akt pathway
and involvement in enterocytic cell survival.
Because the MEK/Erk pathway displayed differentiation state
distinctions in its involvement in enterocytic cell survival but not in
its regulation of activation, we then verified whether this was also
the case for the PI3-K/Akt pathway by focusing on p57Akt
activation (Fig. 6). As in the case of
p125Fak (see above and Ref. 35) and
p42Erk-2 (see above and Refs. 2 and 35), we
found that levels of activated Akt (pp57Akt) were
downregulated while those of the Akt protein (p57Akt)
increased during the differentiation process of Caco-2/15 cells (Fig.
6A). This resulted in distinct p57Akt activated
profiles, whereby Akt activation was at minimal levels in
differentiated Caco-2/15 cells as opposed to undifferentiated ones
(Fig. 6, A and B). Conversely, we found that the
inhibition of p125Fak (Fig. 6C, +CD) and
tyrosine kinase activities (Fig. 6C, +G) lowered p57Akt relative activation levels in both undifferentiated
and differentiated Caco-2/15 cells, also as observed for
p42Erk-2 (see above) and as expected from previous studies
in other cell types and tissues (11, 15, 21, 56).
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Differentiation state-specific regulation of Bcl-2 homolog
steady-state levels in enterocytes.
To ascertain whether the distinctions in susceptibility to
apoptosis and involvement of signaling pathways between
undifferentiated and differentiated Caco-2/15 cells are linked with
differentiation state-specific regulatory mechanisms of Bcl-2 homolog
expression, we then investigated the steady-state expression levels of
six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad)
following the various treatments. The densitometric analyses presented
in Figs. 7 and
8 show that the relative expression
levels of each Bcl-2 homolog analyzed were distinctively modulated in
both undifferentiated and differentiated Caco-2/15 cells.
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DISCUSSION |
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In this study, we investigated the question of whether the
regulation of human intestinal epithelial cell survival involves distinct control mechanisms depending on the state of differentiation, using the well-established human Caco-2/15 enterocyte-like and HIEC-6
cryptlike in vitro models. We found that the enterocytic differentiation process of Caco-2/15 cells results in the gradual establishment of differentiation state-distinct profiles of
1) Bcl-2 homolog steady-state levels and 2)
p125Fak, p42Erk-2, and p57Akt
activated levels. Accordingly, we found that the inhibition of tyrosine
kinase activities, Fak, the MEK/Erk pathway, or PI3-K have distinct
impacts on enterocytic cell survival in undifferentiated (subconfluent
Caco-2/15, confluent HIEC-6) and differentiated (30 days postconfluent
Caco-2/15) cells. We also observed that exposure to insulin and the
inhibition of these various signaling molecules/pathways modulated
distinctively the expression of each Bcl-2 homolog analyzed in both
undifferentiated and differentiated Caco-2/15 cells; however, sharp
distinctions were noted between the two states of differentiation in
the resulting effects of the same treatments on the expression of Bcl-2
homologs. Furthermore, we found that the PI3-K/Akt pathway is
distinctively regulated in undifferentiated and differentiated
enterocytes. Finally, we have shown that Fak and 1 and
4
integrins, as well as the MEK/Erk and PI3-K/Akt pathways, are
distinctively involved in enterocytic cell survival, depending on the
state of differentiation. Therefore, these data altogether indicate
that human intestinal epithelial cell survival is characterized by
differentiation state-specific susceptibilities to apoptosis,
which in turn are linked with distinctions in both the regulation of
Bcl-2 homologs and the involvement of signaling molecules/pathways.
The intestinal crypt-villus axis is defined by proliferative and
functional properties of the crypt cells that distinguish them from the
fully differentiated villus cells (26, 37, 41, 44, 45).
Previous in vivo studies in the adult rodent (26, 29-32,
38-40, 44, 45, 60) and human (26, 28-32, 39, 44, 45, 52, 53, 59) intestine have reported that Bcl-2 homologs exhibit gradients of expression along this crypt-villus axis of enterocytic differentiation. To this effect, the data reported herein
show for the first time that such distinct Bcl-2 homolog expression
profiles are gradually established during the enterocytic differentiation process. Because Bcl-2 homologs constitute a critical checkpoint in the regulation of apoptosis, it has been
suggested that intestinal epithelial cell survival may be regulated
distinctively according to the state of cell differentiation (22,
24, 26, 40, 44-46, 53). In support of this, it is now well
established that the predominant means to remove obsolete
differentiated enterocytes is through apoptosis and shedding at
the villus apex, whereas spontaneous crypt cell apoptosis, a
rarer (less frequent) process, serves to remove defective/injured
progeny cells (22, 26, 40, 44, 45, 53). Hence, in vivo
studies have consistently reported a dramatic increase of
apoptosis in crypt cells, but little or no increase in villus
cells, after irradiation or chemotherapeutic drug exposure (24,
38, 44-46). Likewise, analyses of enterocytic apoptosis in bcl-2/
and
bax
/
knockout mice have reported
differential consequences for crypt and villus cells, with regard to
resistance and/or susceptibility to apoptosis after irradiation
(44-46, 60). Incidentally, the present study provides
further documentation on the distinct susceptibilities to
apoptosis between undifferentiated and differentiated
enterocytes in vitro. In addition, our data demonstrate for the first
time that such distinctions are linked with a differentiation
state-specific regulation of Bcl-2 homolog expression and involvement
of integrins and signal transduction pathways. Finally, it has been
recently reported (22) that villus cells exhibit distinct
expression and activation profiles of caspases, the major components of
the apoptotic effector machinery (1, 25, 47).
Consequently, and in light of these considerations, our data altogether
clearly establish that human intestinal epithelial cell survival is
subject to differentiation state-specific control mechanisms in vitro.
External stimuli responsible for the promotion of cell proliferation,
differentiation, and/or survival largely come from hormones/growth factors and cell adhesion (1, 4, 11, 13, 15, 19-21, 56). As with other cell types (4, 10, 13, 19, 21, 25, 54,
55), the differentiation process of enterocytes is accompanied
by the establishment of differentiation state distinctions in hormonal
responses (12, 16, 17, 27, 37, 43-45) as well as in
the expression of cell cycle regulators (2, 17) and cell
adhesion components, such as integrins (7, 35, 37, 41,
43-45), in addition to morphological and functional
differentiated characteristics. For example, while both crypt and
villus cells express 1 integrins, differentiation state-distinct
profiles of specific
1 integrins are nonetheless expressed
(6-7, 33, 35, 51); also, the
4 integrin subunit is
functional in differentiated enterocytes but not in crypt cells
(5, 7). In addition, signaling molecules/pathways have
been shown to play dual roles in enterocytes: for instance, the MEK/Erk
pathway is required for proliferation of undifferentiated cells and the
onset of enterocytic differentiation (2). Similarly, PI3-K
performs a major role in enterocytic de novo DNA synthesis and
differentiation (9, 35). Taking these observations into
consideration, it therefore follows that 1) undifferentiated
and differentiated intestinal cells exhibit distinct profiles of
activated levels of Fak (this study and Ref. 35),
Erk-1/Erk-2 (this study and Refs. 2 and 35) and Akt (this
study) which, in the case at least of the PI3-K/Akt pathway (this study
and Refs. 9 and 35), underlie as well distinct modes of
regulation depending on the differentiation state; and 2)
Fak and integrins
1 and
4, as well as the MEK/Erk and PI3-K/Akt
pathways, exert distinct influences on intestinal epithelial cell
survival depending on the state of cell differentiation (this study).
In this respect, the establishment of differentiation state-specific
profiles of MEK/Erk and PI3-K/Akt activation has been observed in other
cell types (10, 13, 15, 56). Furthermore, a
differentiation state-distinct involvement of integrins and/or signaling pathways in cell survival has been demonstrated in other cell
types as well (4, 11, 13, 15, 19, 21), such as neural
(4, 13) and skeletal muscle (54, 55) cells.
In the past few years, it has become increasingly evident that the regulation of individual Bcl-2 homologs can involve numerous pathways acting in synergy or independently and that the specific pathways involved in the regulation of a single homolog can differ depending on the cell type and the differentiation state studied (1, 4, 10, 11, 13-15, 19-21, 47, 49, 56). It now appears that intestinal epithelial cells are no exception to this (this study and Refs. 23, 33, 39, 48). Indeed, exposure to butyrate induces apoptosis in undifferentiated intestinal cells by decreasing Bcl-2 levels without affecting those of Bax or Bak (48), whereas the same treatment induces apoptosis in differentiated cells by increasing Bak levels (39, 48). Similarly, ras transformation of rat crypt cells stimulates the PI3-K/Akt pathway but not the MEK/Erk pathway, resulting in decreased Bak levels (33). In addition to these, our findings that Fak, MEK/Erk, and PI3-K/Akt play distinct roles in the modulation of individual Bcl-2 homolog expression either within undifferentiated and differentiated intestinal cells or between the two differentiation states illustrate well the complexity that is required in the regulation of the expression and functions of Bcl-2 homologs, depending on the cell type and state of cell differentiation. This in turn further stresses the concept that cell survival is not regulated by the activity of single Bcl-2 homologs but, rather, by a balance of activities from numerous homologs that arises from the input of multiple stimuli and pathways (1, 4, 13, 14, 21, 25, 34, 47, 49).
It is now well acknowledged that signaling pathways can cross talk with
each other to act in tight coordination, although the exact mechanisms
involved remain unclear (4, 13, 21, 56). For example, a
close cooperation has been reported between the MEK/Erk and PI3-K/Akt
pathways in suppressing apoptosis in some cell types, even to a
point where both pathways can be required to effectively sustain cell
survival (4, 13, 14, 21, 49, 56). On the other hand, it is
becoming increasingly evident that cell survival is furthermore
regulated through cell type-specific machineries of integration and
fine-tuning of signaling pathways, which remain to be fully understood
(1, 4, 13, 15, 20, 21, 56). For instance, recent
observations indicate that the MEK/Erk and PI3-K/Akt pathways can also
act coindependently in maintaining cell survival, with one being able
to compensate for the inhibition of the other (4, 13-15, 34,
49, 56). To this effect, several lines of evidence now indicate
that the relationship between the MEK/Erk and PI3-K/Akt pathways, as
well as their integration and fine-tuning, are distinct depending on
the state of enterocytic differentiation: 1) the inhibition
of Fak impacts equally on the MEK/Erk and PI3-K/Akt pathways in
undifferentiated and differentiated enterocytes, and yet Fak and these
two pathways nonetheless display striking differentiation
state-specific distinctions in their involvement in enterocytic cell
survival (this study); 2) the inhibition of the MEK/Erk
pathway does not impact on the survival of undifferentiated cells (this
study and Ref. 33) but does so for differentiated cells
(this study); 3) the inhibition of PI3-K causes
apoptosis in both undifferentiated (this study and Refs.
9 and 33) and differentiated (this study) cells, albeit to
a greater extent in the former differentiation state (this study);
4) the inhibition of 1 integrins causes a downactivation of Fak in both undifferentiated and differentiated enterocytes but, as
for the inhibition of Fak (this study), causes apoptosis to a
greater extent in the latter cell state (this study); 5) the
1 integrin-mediated survival signaling in undifferentiated enterocytes appears to be PI3-K/Akt dependent but not MEK/Erk dependent
(33); 6) overexpression of ras protects crypt
cells from anoikis by stimulating the PI3-K/Akt pathway and not the Raf/MEK/Erk pathway (33), even though ras is
well known for its role in usually stimulating the latter pathway
(2, 4, 11, 13, 21, 34); 7)
4 integrins,
which are known for their role in maintaining the activation of the
MEK/Erk pathway through the recruitment of Shc and Grb2-SOS (7,
11, 21), are involved in enterocytic cell survival only in the
differentiated state (this study); and 8) p57Akt
activation appears to be PI3-K independent in undifferentiated enterocytes only (this study), an uncoupling situation already shown to
exist in other cell systems (13, 15, 56). Consequently, these considerations altogether suggest that both the MEK/Erk and
PI3-K/Akt pathways are required for the survival of differentiated enterocytes, whereas PI3-K (with or without p57Akt) is
sufficient and able to compensate for the inhibition of the MEK/Erk
pathway in the survival of undifferentiated enterocytes. Alternately,
the MEK/Erk pathway may not play any role in the survival of
undifferentiated intestinal cells.
In conclusion, the present findings provide new insights into the
complex regulatory mechanisms that are responsible for the survival of
human intestinal epithelial cells. Such mechanisms are likely to vary
somewhat in their specifics along the proximal-distal axis of the gut,
considering the differences in Bcl-2 homolog expression profiles
(28-32, 38, 40, 52, 53, 60), susceptibilities to
apoptosis (24, 26, 38, 40, 44-46), and
regulation of cellular functions (7, 16, 37, 41, 43)
between jejunum, ileum, and colon enterocytes in vivo and in vitro.
Nonetheless, it is now clear that intestinal epithelial cells exhibit a
differentiation state-specific susceptibility to apoptosis
through distinctions in the involvement of signaling
molecules/pathways, such as Fak, MEK/Erk, and PI3-K/Akt, which in turn
impact distinctively on the expression of Bcl-2 homologs. Accordingly,
cell adhesion components such as 1 and
4 integrins also
participate in the regulation of intestinal cell survival in a
differentiation state-specific manner. However, the exact molecular
processes responsible for such distinct control mechanisms of survival
between undifferentiated and differentiated cells remain to be fully
understood. For example, the question is open as to why Akt is
seemingly independent of PI3-K for its activation in undifferentiated
intestinal cells. Conversely, further analyses are required to dissect
the exact molecular relationships and functions of the MEK/Erk and
PI3-K/Akt pathways in the regulation of enterocytic cell survival and
Bcl-2 homolog expression. Increasing our knowledge on the specific
roles of these pathways in intestinal epithelial cell survival should provide a better understanding of the role of apoptosis/anoikis in the maintenance and repair of the intestinal epithelium, as well as
in the pathogenesis of intestinal disorders with dysregulation of
apoptosis, such as cancer.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. E. Ruoslahti and E. Engvall (The Burnham Institute, LaJolla, CA) for the kind gifts of the P4C10 and 3E1 antibodies, Dr. J.-F. Beaulieu for the gift of Caco-2/15 and HIEC-6 cells and useful discussions, and Dr. N. Rivard also for useful discussions.
![]() |
FOOTNOTES |
---|
This work was supported by Canadian Institutes of Health Research Grant MGC-15186.
P. H. Vachon is a Chercheur-Boursier du Fonds de la Recherche en Santé du Québec and a Chercheur de la Fondation Canadienne Pour l'Innovation.
Address for reprint requests and other correspondence: P. H. Vachon, Département d'Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4 (E-mail: phvachon{at}courrier.usherb.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 December 2000; accepted in final form 17 January 2001.
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