Human Intestinal Epithelial Cell Survival and Anoikis

We have shown previously that human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. Here we analyzed the roles of protein kinase B/Akt isoforms in such differentiation state distinctions. Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of dominant-negative mutants) or β1 integrins (antibody blocking) or by maintaining cells in suspension. Expression/activation parameters of Akt isoforms (Akt-1, Akt-2, and Akt-3) and Fak were analyzed. Activity of Akt isoforms was also blocked by inhibition of phosphatidylinositol 3-kinase or by overexpression of dominant-negative mutants. Here we report the following. 1) The expression/activation levels of Akt-1 increase overall during enterocytic differentiation, and those of Akt-2 decrease, whereas Akt-3 is not expressed. 2) Akt-1 activation is dependent on β1 integrins/Fak signaling, regardless of the differentiation state. 3) Akt-2 activation is dependent on β1 integrins/Fak signaling in undifferentiated cells only. 4) Activation of Akt-1 is phosphatidylinositol 3-kinase-dependent, whereas that of Akt-2 is not. 5) Akt-2 does not promote survival or apoptosis/anoikis. 6) Akt-1 is essential for survival. 7) Akt-2 cannot substitute for Akt-1 in the suppression of anoikis. Hence, the expression and regulation of Akt isoforms show differentiation state-specific distinctions that ultimately reflect upon their selective implication in the mediation of human intestinal epithelial cell survival. These data provide new insights into the synchronized regulation of cell survival/death that is required in the dynamic renewal process of tissues such as the intestinal epithelium.


We have shown previously that human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. Here we analyzed the roles of protein kinase B/Akt isoforms in such differentiation state distinctions. Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of dominantnegative mutants) or ␤ 1 integrins (antibody blocking) or by maintaining cells in suspension. Expression/activation parameters of Akt isoforms (Akt-1, Akt-2, and Akt-3) and Fak were analyzed. Activity of Akt isoforms was also blocked by inhibition of phosphatidylinositol 3-kinase or by overexpression of dominant-negative mutants.
Here we report the following. 1) The expression/activation levels of Akt-1 increase overall during enterocytic differentiation, and those of Akt-2 decrease, whereas Akt-3 is not expressed. 2) Akt-1 activation is dependent on ␤ 1 integrins/Fak signaling, regardless of the differentiation state. 3) Akt-2 activation is dependent on ␤ 1 integrins/Fak signaling in undifferentiated cells only. 4) Activation of Akt-1 is phosphatidylinositol 3-kinase-dependent, whereas that of Akt-2 is not. 5) Akt-2 does not promote survival or apoptosis/anoikis. 6) Akt-1 is essential for survival. 7) Akt-2 cannot substitute for Akt-1 in the suppression of anoikis. Hence, the expression and regulation of Akt isoforms show differentiation statespecific distinctions that ultimately reflect upon their selective implication in the mediation of human intestinal epithelial cell survival. These data provide new insights into the synchronized regulation of cell survival/ death that is required in the dynamic renewal process of tissues such as the intestinal epithelium.
The human intestinal epithelium is a useful biological model for the study of the cell dynamics involved in tissue homeostasis. Its continuous renewal essentially consists of the production of enterocytes in the crypts, which differentiate and then migrate toward the apex of the villi in order to enter anoikis (24 -28). In addition, proliferative crypt cells may enter "spontaneous" apoptosis, a less frequent process that serves to remove defective or injured undifferentiated progeny cells (24 -26). Although both crypt and villus cells are susceptible to anoikis, it is now becoming well established that human intestinal epithelial cell survival and death are subjected to differentiation state-specific control mechanisms (12, 24 -27, 29 -33). For instance, undifferentiated/crypt cells exhibit a susceptibility to apoptosis and/or anoikis that is distinct from the differentiated/villus cells as follows: 1) their expression and regulation of cell death regulators/effectors (e.g. Bcl-2 homologs and caspases) (27, 29 -34); 2) a differential involvement of the PI3K/ Akt and MEK/Erk pathways in their survival (30,31,34); 3) distinct roles enacted by integrins and Fak in the suppression of anoikis (12,30,31,34); and 4) a differentiation state-selective involvement of p38 stress-activated MAPK isoforms in the induction of apoptosis/anoikis (12,34). This in turn fits well with the fact that intestinal epithelial cells express differential integrin profiles and interact with specific basement membrane components along the crypt-villus axis, depending on their state of differentiation (35).
Considering that the regulation of human enterocytic cell survival involves distinct mechanisms according to the state of differentiation, the question as to the specific implication of individual Akt isoforms in this process therefore remains open. In the present study, we investigated the expression, regulation, and roles of Akt-1, Akt-2, and Akt-3 in the suppression of human intestinal epithelial cell anoikis within the context of undifferentiated versus differentiated cells. By using several approaches, we provide evidence that the expression and regulation of Akt isoforms by cell adhesion and cell adhesion signaling show differentiation state-specific distinctions in human intestinal epithelial cells. Furthermore, we identify the ␤ 1 integrin/Fak/PI3K/Akt-1 signaling pathway as a crucial determinant in the suppression of enterocytic anoikis, whereas Akt-2 is independent of PI3K and does not play a role in enterocytic cell survival or death.
It is of note that the strict specificity of the antibodies used herein for immunoprecipitation and/or Western blot for p57 Akt-1 and p57 Akt-2 , as well as for pser473 p57 Akt-1 , was verified. As shown in Fig. 2A, immunoprecipitated p57 Akt-1 was not recognized by the anti-Akt-2 antibody, whereas immunoprecipitated p57 Akt-2 was not recognized by the anti-Akt-1 antibody. Furthermore, the anti-pser473 p57 Akt-1 antibody reacted with immunoprecipitated Akt-1 but not with Akt-2 ( Fig. 2A). Conversely, the anti-phosphoserine antibody reacted with both p57 Akt-1 and p57 Akt-2 ( Fig. 2A), as expected. Antibodies strictly specific for the phosphorylated serine 474 residue of Akt-2 were not available at the time of the present study.
Cell Culture-The human intestinal epithelial cell line Caco-2/15, a clone of the parental Caco-2 cell line, has been fully characterized in previous studies (36,37) and used extensively to study the regulation of various enterocytic cellular processes (for example, see Refs. 36 -42), including cell survival/death (12,30). Caco-2/15 cells undergo a full enterocytic differentiation process that takes place spontaneously once confluence (0 PC) has been reached and is completed after 25-30 PC. Cells were routinely cultured and monitored for enterocytic differentiation as described previously (12,30,37). Cells between passages 53 and 70 were cultured in plastic dishes (100 mm; Falcon Plastics, Los Angeles) or on 13-mm coverslips. Studies were performed on cultures at 50% subconfluence (Ϫ2 PC; proliferating, undifferentiated enterocytes), between 0 and 20 PC and/or at 30 PC (nonproliferating, differentiated enterocytes).
For treatments, cells were maintained 24 -48 h in either of the following conditions: 1) with 1 M cytochalasin (CD), which has been shown to inhibit Fak at this concentration range without affecting actin filament organization (12,30,31,34,43,44); 2) with 50 g/ml of the monoclonal antibody P4C10 (Invitrogen and Chemicon), which inhibits the binding activities of the ␤ 1 integrin subunit (10,12,30,45,46); 3) with 100 g/ml nonimmune mouse IgGs, as control for experiments using the P4C10 antibody; or 4) with 30 M Ly294002 (Calbiochem), a specific inhibitor of PI3K (13)(14)(15). The working concentrations of the reagents used were established previously (10 -12, 30, 31, 34, 46, 47). Cells were also kept in suspension by either seeding freshly trypsinized undifferentiated cells onto poly-2-hydroxyethyl methacrylate-coated dishes or by detaching intact monolayers of differentiated cells by gentle flushing below the monolayer with serum-free medium as described previously (12,30,34). In all cases, experiments were performed in medium without serum. It is noteworthy that control cultures included exposure to the same solvent as that used for pharmacological inhibitors and showed no significant differences with cultures maintained in serum-free medium only (not shown; see Refs. 10 -12, 30, 31, 34, 47).
DNA Laddering Assays and in Situ Terminal Deoxynucleotidyltransferase-dUTP-mediated Nick-end Labeling (ISEL)-DNA was isolated according to the method described previously (12,30,34,48). DNA contents of all samples were estimated by A 260 . The visualization of apoptosis/anoikis-associated internucleosomal DNA fragmentation (DNA laddering) was performed on 2% agarose gels (20 g of DNA/lane) as described (10 -12, 30, 34, 47, 48). Note that the method used for DNA extraction uses Triton rather than SDS, thus leaving behind most intact genomic DNA (49). Consequently, nonapoptotic cell cultures often produce near-empty lanes on the gel as a result. For the in situ detection of DNA laddering, treated (see above) or transfected (see below) coverslip-grown cells were washed twice in cold phosphatebuffered saline (PBS) and fixed in 2% formaldehyde/PBS for 30 min at 4°C. Following permeabilization with 0.1% Triton X-100 in PBS for 5 min, free aldehyde groups were quenched with 100 mM glycine/PBS (pH 7.4). ISEL was then carried out as described previously (10,12,27,30,31,34,46,48), using the fluorescein isothiocyanate (FITC)-ApopTag apoptosis detection kit (Intergen, Purchase, NY). Negative staining controls included omission of the terminal deoxyribonucleotidyltransferase enzyme in all experiments performed. Preparations were viewed with a Leica DM-RxA microscope (Leica), and evaluation of ISELpositive cells was performed as described previously (12,27,30,31,34,46,48). Apoptotic indices were expressed as the percentage (%) of apoptotic (ISEL-positive) cells over the total number of cells counted (n Ն 300 cells). Alternatively, counts were compared with those of control cultures, ϫ100 (expressed as % of control).
Indirect Immunofluorescence Double Labeling-ISEL performed on transfected cells (see above) was immediately followed by incubation (45 min, room temperature) with an anti-FLAG (for Akt-1 constructs) or anti-HA (for Akt-2 constructs) primary antibody diluted in 10% powdered skim milk/PBS (pH 7.4). After washing with PBS, a secondary antibody coupled to lissamine-rhodamine (RHD) was likewise incubated. Preparations were thereafter mounted and viewed with a Leica DM-RxA microscope (Leica). Negative staining controls included incubation without primary antibody in all experiments performed. Transfected (RHD-stained/FLAG-or HA-positive) cells that were induced to enter apoptosis (FITC-stained/ISEL-positive) as a consequence show up in shades of yellow when the RHD and FITC stainings are overlaid.
Data Processing-Results and values shown represent the mean Ϯ S.E. for at least three (n Ն 3) separate experiments and/or cultures. Statistically significant differences were determined with the Student's t test. Data were compiled, analyzed, and processed with Excel (Microsoft, Redmond, WA) and Cricket Graph (Computer Associates, Islandia, NY). Unless specified otherwise, images from blots, gels, and scans were processed with Vistascan (Umax), Photoshop (Adobe, San Jose, CA), and PowerPoint (Microsoft).

Establishment of Distinct Expression/Activation Profiles of Akt Isoforms during Human Enterocytic Differentiation-We
first analyzed the mRNA and protein expression levels of Akt-1, -2, and -3 by RT-PCR and Western blot, in relation to the human enterocytic differentiation process. Undifferentiated Caco-2/15 cells undergo a gradual differentiation process that takes place spontaneously once confluence (0 PC) has been reached and that is completed after 25-30 PC (36,37,41). This is well evidenced by a sharp drop of proliferation at confluence (not shown; see Refs. 36 -38, 40 -42, and 73) coincident with the post-confluent appearance and/or gradual increase in the expression of brush border membrane hydrolases such as SI (Fig. 1B, filled columns; Fig. 1D, dark-gray columns), lactasephlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV (not shown; see Refs. 36 -38, 40 -42, and 73).
The relative levels of Akt-1 mRNA were found to decrease overall throughout the enterocytic differentiation process (Fig.  1, A, Akt-1, and B, gray columns). However, such patterns of Akt-1 mRNA expression did not reflect the relative expression levels of the protein; to this effect, Akt-1 steady-state protein levels remained more or less stable throughout the enterocytic differentiation process (Fig. 1, C, p57 Akt-1 , and D, gray columns). Akt-2 mRNA levels were also found to decrease overall as a function of the state of differentiation (Fig. 1, A, Akt-2, and B, open columns). Furthermore, and in contrast to Akt-1, such patterns of Akt-2 mRNA expression reflected the relative expression levels of the protein (Fig. 1, C, p57 Akt-2 , and D, open columns). Finally, Akt-3 mRNA was not detected by RT-PCR in Caco-2/15 cells, regardless of their state of differentiation (data not shown).
It is well acknowledged that the expression of a kinase is not necessarily a reflection of its activation/activity levels (3,8,9,14,17). Fak is a good example of this in human intestinal epithelial cells, and although its protein expression levels increase according to the state of differentiation ( Fig. 1, C, p125 Fak , and D, filled columns; see also Refs. 12, 28, and 51), its relative activation levels (as assessed by phosphorylation on its tyrosine 397 residue) are nonetheless significantly lower in differentiated cells than in undifferentiated ones (Fig. 2, B, IP: Fak, and C, gray columns). Therefore, we investigated whether human intestinal epithelial cells also display variations of activation levels of Akt-1 and Akt-2 in relation to their state of differentiation. In contrast to Fak, the relative activation levels of Akt-1 (as assessed by phosphorylation on its serine 473 residue) were found significantly higher in differentiated cells than in undifferentiated ones (Fig. 2, B, IP: Akt-1, and C, open columns). On the other hand, and similarly to Fak, the activation levels of Akt-2 (as assessed by phosphorylation on its serine residues) were significantly higher in undifferentiated cells than in their differentiated counterparts (Fig. 2, B, IP: Akt-2, and C, filled columns). Altogether, these results indicate that human intestinal epithelial cells display differentiation state-distinct profiles of expression and activation of Fak, Akt-1, and Akt-2.
Distinct Regulation of Akt-1 and Akt-2 Activation within the Context of Human Intestinal Epithelial Apoptosis/ Anoikis-We then analyzed the regulation of activation of Akt-1 and Akt-2 in intestinal epithelial cells, specifically within the context of loss of cell adhesion signaling. In contrast to adherent/control cultures (Fig. 3, A, lanes 1 and 5, and B, lanes  1, 2, 4, and 5), apoptosis/anoikis-associated internucleosomal DNA laddering was observed in both undifferentiated (Ϫ2 PC) and differentiated (30 PC) cells when Fak was inhibited with CD ( Fig. 3A, lanes 2 and 6), when PI3K activity was inhibited with Ly294002 (Fig. 3A, lanes 3 and 7), when ␤ 1 integrin binding activity was blocked with the P4C10 antibody (Fig. 3B,  lanes 3 and 6), or when cells were kept in suspension (Fig. 3A,  lanes 4 and 8). Likewise, elevated apoptotic indices (Fig. 3C), as well as high cleavage/activation of CASP-3 (see Fig. 3D as example) and CASP-7 (not shown), were noted when undifferentiated and differentiated cultures were exposed to the same treatments, as opposed to controls.
In contrast to Akt-1, the regulation of Akt-2 activation was found to be more complex (Fig. 6). The inhibition of PI3K failed to affect significantly the relative activated levels of Akt-2 in both undifferentiated and differentiated cells (Fig. 6A,  Total RNA was extracted and reversed-transcribed, and then 20 cycles of amplification were performed using primers specific for human Akt-1/PKB␣ and Akt-2/PKB␤. S14 mRNA expression was likewise analyzed for normalization purposes. L, 100-bp DNA size markers. B, same as in A except that the mRNA expression of SI, an enterocytic differentiation marker, was also analyzed. Furthermore, amplified bands were semi-quantified, and the relative levels of Akt-1 (gray columns), Akt-2 (open columns), and SI (filled columns) were determined as ratios relative to S14. C, representative WB analyses of the expression of p57 Akt-1 , p57 Akt-2 , and p125 Fak in Caco-2/15 cells at the same time points as in A. Total proteins (50 g/well) were separated by SDS-PAGE under reducing conditions, electrotransferred onto nitrocellulose membranes, and then probed with specific antibodies for the detection of Akt-1, Akt-2, and Fak. Detection of K18 was likewise analyzed for normalization purposes. D, same as in C, except that the expression of p220 SI was also analyzed. Furthermore, amplified bands were semi-quantified, and the relative levels of Akt-1 (gray columns), Akt-2 (open columns), Fak (filled columns), and SI (dark-gray columns) were determined as ratios relative to K18. B and D, statistically significant (0.001 Յ p Յ 0.01) differences between Ϫ2 (Undifferentiated) and 30 (Differentiated) PC Caco-2/15 cells are indicated by *. versus 1 and 6 versus 2; Fig. 6B, Ly294002). On the other hand, Akt-2 activation dropped significantly following Fak inhibition and cell suspension in undifferentiated cells (Fig. 6A, lanes 3   versus 1 and 7 versus 1; Fig. 6B, Ϫ2 PC: Cytochalasin D and Suspension) but was not affected significantly in differentiated cells following these same treatments (Fig. 6A, lanes 4 versus 2  and 8 versus 2; Fig. 6B, 30 PC: Cytochalasin D and Suspension). It is also noteworthy that the amounts of Akt-2 immunoprecipitated from cells undergoing true anoikis were barely affected as compared with those from control cultures (Fig. 6C,  lanes 7 versus 1 and 8 versus 2), in sharp contrast to Fak and Akt-1 (see above). In any event, these results show that Akt-2 activation is dependent on cell adhesion/␤ 1 integrins and Fak in undifferentiated human intestinal epithelial cells only. Furthermore, these indicate that Akt-2 activation is not dependent on PI3K in human intestinal epithelial cells, regardless of their state of differentiation. Consequently, these data altogether show that the activated levels of Akt-1 and Akt-2 are distinctively regulated by cell adhesion/␤ 1 integrins, Fak, and PI3K in human enterocytes.
Distinct Roles for Akt-1 and Akt-2 in the Survival of Human Intestinal Epithelial Cells-To verify whether Akt-1 and/or Akt-2 contribute in the promotion of human intestinal epithelial cell survival, Caco-2/15 cells were transiently transfected with wild type or mutant cDNA constructs of these kinases and assayed for apoptosis in comparison to control cells (i.e. shamtransfected or transfected with an empty vector). Fak cDNA constructs were also tested. The forced expression of the nonkinase, dominant-negative Fak isoform p45 FRNK significantly induced anoikis, as opposed to cells transfected with wild type Fak and/or control cells (Fig. 7, pCMV-myc-p45 FRNK versus pCMV-myc-FakWT). Likewise, the forced expression of the nonactivable, dominant-negative mutant FakY397F also induced anoikis in a significant manner (Fig. 7, pCMV-myc-FakY397F versus pCMV-myc-FakWT). Hence, these data confirm that Fak is crucial for the survival of human intestinal epithelial cells (12,30,31,34).
As in the case of Fak, Akt-1 was identified as a major promoter of the suppression of apoptosis/anoikis in human intestinal epithelial cells. Indeed, the forced expression of a kinasedead, dominant-negative mutant of Akt-1 significantly induced apoptosis, as opposed to cells transfected with wild type Akt-1 and/or control cells (Fig. 7, pCMV-2-Flag-Akt-1DN versus pCMV-2-Flag-Akt-1WT). Double IF studies were then performed on cultures transfected with either the Flag-Akt-1WT or Flag-Akt-1DN constructs, in order to verify any correlation between apoptotic (i.e. ISEL-positive, in green) and constructexpressing (i.e. FLAG-positive, in red) cells (Fig. 8). For cells transfected with the Flag-Akt-1WT construct (Fig. 8, A-C), we found that the ISEL-positive (Fig. 8A) and FLAG-positive (Fig.  8B) stainings correlated poorly when overlaid (Fig. 8C). On the other hand, for cells transfected with the Flag-Akt-1DN construct (Fig. 8, D--F), the ISEL-positive (Fig. 8D) and FLAGpositive (Fig. 8E) stainings exhibited a near-perfect correlation when overlaid (Fig. 8F). Therefore, these results demonstrate a role for Akt-1 in the survival of human enterocytes.
In sharp contrast to Akt-1, Akt-2 was not found to play a role in human enterocytic cell survival. Indeed, the forced expression of a kinase-dead, dominant-negative mutant of Akt-2 did not induce apoptosis, as compared with cells transfected with wild type Akt-2 and/or control cells (Fig. 7,  pCDNA3-HA-Akt-2DN versus pCDNA3-HA-Akt-2WT). Likewise, the forced expression of a constitutive-active mutant of Akt-2 had no significant effect on cell survival/death (Fig. 7,  pCDNA3-HA-myrAkt-2 versus pCDNA3-HA-Akt-2WT). Double IF studies were also performed on cultures transfected with either the HA-Akt-2WT, HA-Akt-2DN, or HA-myrAkt-2 constructs, in order to verify any lack of correlation between apoptotic (i.e. ISEL-positive) and construct-expressing (i.e. HA-

Restrictions and Distinctions in the Expression/Activation of Akt Isoforms in Human Intestinal Epithelial Cells-Although
Akt isoforms are widely expressed, and often overexpressed in cancers (13-17, 19, 23, 50, 51), Akt-3 is recognized as being more restricted in its expression patterns than Akt-1 and Akt-2 (13-17, 19, 56). This is also the case for human intestinal epithelial cells, because Akt-3 is not detected in such cells regardless of their state of differentiation. However, previous studies (19,56) have shown the presence of Akt-3 mRNA in the human small intestinal and colonic mucosae, as well as in colon cancers. This apparent discrepancy is explained by the fact that whole-tissue extracts were used in these studies and therefore included not only intestinal epithelial cells as a source of biological material but also cells from the underlying stromal tissue. To this effect, Akt-3 mRNA is indeed detected in pure cultures of human intestinal mesenchymal cells and yet absent in pure cultures of human crypt or villus cells, 2 thus confirming a lack of Akt-3 expression in intestinal epithelial cells.
Aside from such restriction in the expression of Akt isoforms in human enterocytes, our study also showed that the specific expression and/or activation profiles of Akt-1 and Akt-2 are distinct according to the state of enterocytic differentiation. Indeed, the activation of Akt-1 increases with differentiation whereas that of Akt-2 decreases sharply. It is noteworthy that these expression/activation profiles of Akt-1 and Akt-2 are somewhat complementary in relation to the state of differentiation and corroborate our previous findings concerning the expression/activation of Akt in human enterocytes, using nonisoform-specific antibodies (30). It is germane that differentiation state-distinct profiles of expression and activation of Akt isoforms have been observed as well in skeletal muscle cells, although not in similar patterns as we noted here for intestinal epithelial cells. Specifically, Akt-1 expression/activation remains more or less stable during myogenic differentiation, whereas that of Akt-2 gradually increases to even supercede that of Akt-1 (57,58). Consequently, it is now becoming  lanes 7 and 8). Cells were lysed, and Fak (IP: Fak) was immunoprecipitated with specific antibodies. Total proteins from immunoprecipitates (50 g/well) were separated by SDS-PAGE under reducing conditions, electrotransferred onto nitrocellulose membranes, and probed with specific antibodies for the detection of the activated phosphorylated form of Fak (WB: ptyr397 p125 Fak ). Membranes were thereafter reprobed with the antibodies used for IP in order to detect the Fak (WB: p125 Fak ) protein. B, same as in A except that cells were also exposed to 100 g/ml nonimmune mouse IgGs or 50 g/ml of the ␤ 1 integrin-blocking mAb P4C10 (P4C10). Immunoreactive bands were then semi-quantified, and the relative activated levels of Fak were established, which in turn were compared with those of control cultures, ϫ100 (expressed as % of control). Statistically significant (0.001 Յ p Յ 0.01) differences between apoptosis/ anoikis-inducing treatments and control cultures are indicated by *.  7 and 8). Cells were lysed, and Akt-1 (IP: Akt-1) was immunoprecipitated with specific antibodies. Total proteins from immunoprecipitates (50 g/well) were separated by SDS-PAGE under reducing conditions, electrotransferred onto nitrocellulose membranes, and probed with specific antibodies for the detection of the activated phosphorylated form of Akt-1 (WB: pser473 p57 Akt-1 ). Membranes were thereafter reprobed with the antibodies used for IP in order to detect the Akt-1 (WB: p57 Akt-1 ) protein. B, same as in A except that cells were also exposed to 100 g/ml nonimmune mouse IgGs or 50 g/ml of the ␤ 1 integrin-blocking mAb P4C10 (P4C10). Immunoreactive bands were then semi-quantified, and the relative activated levels of Akt-1 were established, which in turn were compared with those of control cultures, ϫ100 (expressed as % of Control). Statistically significant (0.001 Յ p Յ 0.01) differences between apoptosis/anoikis-inducing treatments and control cultures are indicated by *.
increasingly evident that the expression/activation of Akt isoforms may not only vary from one tissue type to the next but may also show distinct profiles according to the state of differentiation within the same given cell type.

Distinctions in the Regulation of Akt-1 and Akt-2 Activation by PI3K and ␤ 1 Integrins/Fak in Human Intestinal Epithelial
Cells-In addition to differences in the expression/activation profiles between Akt isoforms, there is now mounting evidence that the regulation proper of their activation may also involve isoform-specific mechanisms (13)(14)(15)(16)(17). As an example, one Akt isoform and two or all three known isoforms may be stimulated alone or simultaneously by epidermal growth factor or insulin, depending on the cell type being analyzed (13-17, 19, 59 -61). Furthermore, several studies (14,17,62,63,74) have reported that Akt isoforms may be independent of PI3K for their activation, again depending on the tissue or cell type context being studied. To this effect, our observation that the activation of Akt-1, but not that of Akt-2, is dependent on PI3K in human intestinal epithelial cells not only provides another example of PI3K independence for an Akt isoform ( Fig. 9) but also corroborates our previous findings (30,31) concerning the apparent PI3K independence of Akt in enterocytes by using nonisoformspecific antibodies. More surprising was our finding that ␤ 1 integrins/Fak regulate Akt-1 activation regardless of the state of enterocytic differentiation, whereas such ␤ 1 integrins/Fak dependence for Akt-2 activation occurred in undifferentiated enterocytes only (Fig. 9). Much remains to be understood of the mechanisms implicated in the regulation of the activation of Akt isoforms. In addition to other PI3K effectors such as 3-phosphoinositide-dependent kinase 1 or integrin-linked kinase, several signaling molecules have been identified as being susceptible to also influence Akt activation (13-17, 42, 64, 65). To this effect, such additional players are thought to be potentially responsible for the isoform-selective and/or cell typespecific differences in the regulation of Akt activation (13)(14)(15)(16)(17). In addition, several cell types such as intestinal epithelial cells are known to express differentiation state-distinct profiles of integrins, which in turn are thought to provide yet another level of complexity and specificity in the regulation of the activation of signaling pathways (4 -6, 30, 34 -35, 38, 40 -41, 46, 66, 67). Consequently, further studies will be required to identify which integrin receptors and upstream kinases selectively contribute to the differentiation state-distinct stimulation of Akt isoforms in human enterocytes. Nonetheless, our data herein provide a striking example of isoform-selective, as well as differentiation state-specific, distinctions in the regulation of the expression and activation of Akt-1 and Akt-2.
Selective Roles of Akt-1 and Akt-2 in Human Enterocytic Cell Survival and Anoikis-Akt isoforms have been found to perform diverse functions in the control of various cellular processes such as cell proliferation, differentiation, and survival (13)(14)(15)(16)(17)68). There is now mounting evidence that this diversity of function may be defined by the cell type and differentiation state contexts in which stimuli influence Akt activation. For example, Akt-1 drives the proliferation of myoblasts but hinders myogenic differentiation and does not play a role in the  lanes 7 and 8). Cells were lysed, and Akt-2 (IP: Akt-2) was immunoprecipitated with specific antibodies. Total proteins from immunoprecipitates (50 g/well) were separated by SDS-PAGE under reducing conditions, electrotransferred onto nitrocellulose membranes, and probed with specific antibodies for the detection of the activated phosphorylated form of Akt-2 (WB: pser p57 Akt-2 ). Membranes were thereafter reprobed with the antibodies used for IP in order to detect the Akt-2 (WB: p57 Akt-2 ) protein. B, same as in A except that cells were also exposed to 100 g/ml nonimmune mouse IgGs or 50 g/ml of the ␤ 1 integrin-blocking mAb P4C10 (P4C10). Immunoreactive bands were then semi-quantified, and the relative activated levels of Akt-1 were established, which in turn were compared with those of control cultures, ϫ100 (expressed as % of control). Statistically significant (0.001 Յ p Յ 0.01) differences between apoptosis/anoikis-inducing treatments and control cultures are indicated by *. FIG. 7. Akt isoforms and Fak in the survival and apoptosis/ anoikis of human enterocytes. Caco-2/15 cells were transfected with either one of the following cDNA constructs: wild type Fak (pCMV-myc-FakWT); FRNK, a dominant-negative, kinase domain-lacking isoform of Fak (pCMV-myc-p45 FRNK ); a nonactivable, dominant-negative mutant of Fak (pCMV-myc-FakY397F); wild type Akt-1 (pCMV-2-Flag-Akt-1WT); a dominant-negative, kinase-dead mutant of Akt-1 (pCMV-2-Flag-Akt-1DN); wild type Akt-2 (pCDNA3-HA-Akt-2WT); a constitutiveactive mutant of Akt-2 (pCDNA3-HA-myrAkt-2); and/or a dominantnegative, kinase-dead mutant of Akt-2 (pCDNA3-HA-Akt-2DN). Shamtransfected (empty expression vectors) cells were used as controls. Cells were thereafter kept in serum-free medium for 24 h. ISEL was then performed to establish the apoptotic indices, which in turn were compared with those of control cultures, ϫ100 (expressed as % of control). Statistically significant (0.001 Յ p Յ 0.01) differences between treated and control (sham-transfected) cultures are indicated by *.
survival/death of either myoblasts or myotubes (22,58). Conversely, Akt-2 plays a major role in driving myogenic differentiation and promoting survival during myoblast fusion but does not play a role in myoblast proliferation (21,22,58). In addition, recent studies using single and double gene knock-out mice have revealed that Akt isoforms can at best compensate partially for each other and therefore perform specific, crucial roles depending on the tissue type (68 -72). To this effect, our demonstration that Akt-1 is crucial for the survival of human intestinal epithelial cells, whereas Akt-2 does not play a role in either cell survival or induction of apoptosis/anoikis (Fig. 9), therefore provides a novel instance whereby the functions of Akt isoforms within a given cell type are not redundant and cannot be compensated/substituted by one or another. Given this, our study nonetheless raises the question as to what roles Akt-2 may perform in human intestinal epithelial cellular processes, other than promoting survival or apoptosis/anoikis.
In conclusion, this study has provided evidence that the expression and regulation of Akt isoforms by cell adhesion and cell adhesion signaling show differentiation state-specific distinctions in human intestinal epithelial cells. Furthermore, we identify the ␤ 1 integrin/Fak/PI3K/Akt-1 signaling pathway as a crucial determinant in the suppression of enterocytic anoikis, whereas Akt-2 is independent of PI3K and does not play a role in enterocytic cell survival or death. However, the exact molecular processes responsible for such differentiation state-distinct controls on the roles and regulation of Akt-1 and Akt-2 remain to be understood. For example, the question is open as to why Akt-2 does not participate in the regulation of enterocytic cell survival, although Akt-1 does. Likewise, and considering that human intestinal epithelial cell survival and death are subjected to differentiation state-distinct control mechanisms (this study and see Refs. 12,[24][25][26][27][29][30][31][32][33][34], the survival-promoting functions specifically enacted by Akt-1 remain to be fully elucidated in both undifferentiated and differentiated cells. Such further studies, in addition to the present findings, will provide a greater insight into the complex, synchronized governance of cell survival and death that is required for the maintenance and renewal of tissues such as the intestinal epithelium.  9. Distinct roles and regulation of Akt isoforms in human enterocytic cell survival and apoptosis/anoikis. Schematic drawing of an undifferentiated, proliferating enterocyte (left) and its nonproliferating, differentiated counterpart (right), illustrating how integrinmediated cell adhesion can stimulate the Fak/PI3K/Akt-1 pathway to promote cell survival and suppression of enterocytic apoptosis/anoikis, whereas Akt-2 does not play a role in either the survival or death of enterocytes. The drawing also summarizes the main results of the present study concerning the differentiation state distinctions in the regulation of the activation of Fak, Akt-1, and/or Akt-2 by cell adhesion, as well as the independence of Akt-2 activation with regard to PI3K. The low levels of expression and activation of Akt-2 in differentiated enterocytes is shown by parentheses. Finally, note that Akt-3 is not expressed by enterocytes, regardless of their state of differentiation.