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Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes.

Beauséjour M, Noël D, Thibodeau S, Bouchard V, Harnois C, Beaulieu JF, Demers MJ, Vachon PH - Apoptosis (2012)

Bottom Line: Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit.Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known.We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent).

View Article: PubMed Central - PubMed

Affiliation: Département d'anatomie et de Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.

ABSTRACT
In human intestinal epithelial crypt (HIEC) cells, the PI3-K/Akt-1 pathway is crucial for the promotion of cell survival and suppression of anoikis. Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit. Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known. Herein, we analyzed the expression of PI3-K isoforms in HIEC cells and determined their roles in cell survival, as well as in the β1 integrin/Fak/Src-mediated suppression of anoikis. We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent). Hence, HIEC cells selectively express PI3-K isoform complexes, translating into distinct roles in Akt-1 activation and cell survival, as well as in a selective engagement by Fak and/or Src within the context of β1 integrin/Fak/Src-mediated suppression of anoikis.

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Selective roles of PI3-K C isoforms in the maintenance of HIEC cell survival. a Representative (n ≥ 4) double labeling-merged immunofluorescence micrographs of untreated HIEC cell cultures (control) and of cultures treated with Ly292002, PIK-75 or TGX221. ISEL (green) was thereafter performed, with DAPI (blue) counterstaining of nuclei. Original magnification: 20×. b HIEC cell cultures were maintained as in (a), in addition to being also treated with AS605240. ISEL was then performed. Statistically significant (0.0001 ≤ P ≤ 0.001) differences between treated and control cultures are indicated by (*). c HIEC cell cultures were maintained as in (a), in addition to being also treated with IC87114. CASP-3 relative activity was then established, using the substrate Ac-DEVD-AMC, by comparison to controls. Statistically significant (0.0005 ≤ P ≤ 0.005) differences between treated and control cultures are indicated by (*) (Color figure online)
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Fig2: Selective roles of PI3-K C isoforms in the maintenance of HIEC cell survival. a Representative (n ≥ 4) double labeling-merged immunofluorescence micrographs of untreated HIEC cell cultures (control) and of cultures treated with Ly292002, PIK-75 or TGX221. ISEL (green) was thereafter performed, with DAPI (blue) counterstaining of nuclei. Original magnification: 20×. b HIEC cell cultures were maintained as in (a), in addition to being also treated with AS605240. ISEL was then performed. Statistically significant (0.0001 ≤ P ≤ 0.001) differences between treated and control cultures are indicated by (*). c HIEC cell cultures were maintained as in (a), in addition to being also treated with IC87114. CASP-3 relative activity was then established, using the substrate Ac-DEVD-AMC, by comparison to controls. Statistically significant (0.0005 ≤ P ≤ 0.005) differences between treated and control cultures are indicated by (*) (Color figure online)

Mentions: We functionally analyzed the roles of PI3-K isoforms in the maintenance of survival in HIEC-6 cells, considering that PI3-K and its main effector Akt-1 are critical for their survival [28–32]. Indeed, the “pan” inhibition of PI3-K activity results in extensive caspase-dependent apoptosis as assessed by ISEL (Fig. 2a, control vs. Ly294002; Fig. 2b, Ly294002) and CASP-3 activity (Fig. 2c, Ly294002), in addition to causing a sharp drop of Akt-1 activation (Fig. 3a, b, Ly294002). As expected from our PI3-K isoform expression profiling (see previous section), the specific inhibition of p110α similarly impacted upon HIEC cell survival (Fig. 2a–c, PIK-75) and Akt-1 activation (Fig. 3a, b, PIK-75), whereas the specific inhibition of p110β did not affect either (Figs. 2a–c, 3a–b, TGX221). Likewise, the specific inhibition of p110γ or p110δ failed to impact upon the survival of HIEC-6 cells (respectively Fig. 2b, AS60540; 2c, IC87114).Fig. 2


Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes.

Beauséjour M, Noël D, Thibodeau S, Bouchard V, Harnois C, Beaulieu JF, Demers MJ, Vachon PH - Apoptosis (2012)

Selective roles of PI3-K C isoforms in the maintenance of HIEC cell survival. a Representative (n ≥ 4) double labeling-merged immunofluorescence micrographs of untreated HIEC cell cultures (control) and of cultures treated with Ly292002, PIK-75 or TGX221. ISEL (green) was thereafter performed, with DAPI (blue) counterstaining of nuclei. Original magnification: 20×. b HIEC cell cultures were maintained as in (a), in addition to being also treated with AS605240. ISEL was then performed. Statistically significant (0.0001 ≤ P ≤ 0.001) differences between treated and control cultures are indicated by (*). c HIEC cell cultures were maintained as in (a), in addition to being also treated with IC87114. CASP-3 relative activity was then established, using the substrate Ac-DEVD-AMC, by comparison to controls. Statistically significant (0.0005 ≤ P ≤ 0.005) differences between treated and control cultures are indicated by (*) (Color figure online)
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Related In: Results  -  Collection

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Fig2: Selective roles of PI3-K C isoforms in the maintenance of HIEC cell survival. a Representative (n ≥ 4) double labeling-merged immunofluorescence micrographs of untreated HIEC cell cultures (control) and of cultures treated with Ly292002, PIK-75 or TGX221. ISEL (green) was thereafter performed, with DAPI (blue) counterstaining of nuclei. Original magnification: 20×. b HIEC cell cultures were maintained as in (a), in addition to being also treated with AS605240. ISEL was then performed. Statistically significant (0.0001 ≤ P ≤ 0.001) differences between treated and control cultures are indicated by (*). c HIEC cell cultures were maintained as in (a), in addition to being also treated with IC87114. CASP-3 relative activity was then established, using the substrate Ac-DEVD-AMC, by comparison to controls. Statistically significant (0.0005 ≤ P ≤ 0.005) differences between treated and control cultures are indicated by (*) (Color figure online)
Mentions: We functionally analyzed the roles of PI3-K isoforms in the maintenance of survival in HIEC-6 cells, considering that PI3-K and its main effector Akt-1 are critical for their survival [28–32]. Indeed, the “pan” inhibition of PI3-K activity results in extensive caspase-dependent apoptosis as assessed by ISEL (Fig. 2a, control vs. Ly294002; Fig. 2b, Ly294002) and CASP-3 activity (Fig. 2c, Ly294002), in addition to causing a sharp drop of Akt-1 activation (Fig. 3a, b, Ly294002). As expected from our PI3-K isoform expression profiling (see previous section), the specific inhibition of p110α similarly impacted upon HIEC cell survival (Fig. 2a–c, PIK-75) and Akt-1 activation (Fig. 3a, b, PIK-75), whereas the specific inhibition of p110β did not affect either (Figs. 2a–c, 3a–b, TGX221). Likewise, the specific inhibition of p110γ or p110δ failed to impact upon the survival of HIEC-6 cells (respectively Fig. 2b, AS60540; 2c, IC87114).Fig. 2

Bottom Line: Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit.Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known.We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent).

View Article: PubMed Central - PubMed

Affiliation: Département d'anatomie et de Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.

ABSTRACT
In human intestinal epithelial crypt (HIEC) cells, the PI3-K/Akt-1 pathway is crucial for the promotion of cell survival and suppression of anoikis. Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit. Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known. Herein, we analyzed the expression of PI3-K isoforms in HIEC cells and determined their roles in cell survival, as well as in the β1 integrin/Fak/Src-mediated suppression of anoikis. We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent). Hence, HIEC cells selectively express PI3-K isoform complexes, translating into distinct roles in Akt-1 activation and cell survival, as well as in a selective engagement by Fak and/or Src within the context of β1 integrin/Fak/Src-mediated suppression of anoikis.

Show MeSH
Related in: MedlinePlus