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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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Expression of PI3-K isoforms and isoform complexes in HIEC cells. a Representative (n ≥ 3) RT-PCR (left side of panel) and WB (right side of panel) analyses of the expression of the known class I PI3-K C (p110α, p110β, p110γ, p110δ) and R (p85α, p85β, p55γ) isoforms, using isoform-specific primers (for RT-PCR) or antibodies (for WB). Actin expression was used as a reference. b Same as in (a), except that amplified bands (for mRNAs; open columns) and immunoreactive bands (for proteins; filled columns) were semi-quantified and compared to those of actin, in order to establish the relative expression levels for each isoform analyzed (n ≥ 3). c Representative (n ≥ 5) WB analyses of the IP of the PI3-K R isoforms p85α, p85β, and p55γ, for the verification of association by co-IP of the PI3-K C isoforms p110α and p110β, and consequent determination of the predominant PI3-K R/C isoform complexes expressed in HIEC cells. d Representative (n ≥ 3) WB analyses of the reciprocal validation of the IP/co-IP analyses in (c), this time via the IP of p110α and p110β, and verification of association of p85α, p85β, and/or p55γ
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Fig1: Expression of PI3-K isoforms and isoform complexes in HIEC cells. a Representative (n ≥ 3) RT-PCR (left side of panel) and WB (right side of panel) analyses of the expression of the known class I PI3-K C (p110α, p110β, p110γ, p110δ) and R (p85α, p85β, p55γ) isoforms, using isoform-specific primers (for RT-PCR) or antibodies (for WB). Actin expression was used as a reference. b Same as in (a), except that amplified bands (for mRNAs; open columns) and immunoreactive bands (for proteins; filled columns) were semi-quantified and compared to those of actin, in order to establish the relative expression levels for each isoform analyzed (n ≥ 3). c Representative (n ≥ 5) WB analyses of the IP of the PI3-K R isoforms p85α, p85β, and p55γ, for the verification of association by co-IP of the PI3-K C isoforms p110α and p110β, and consequent determination of the predominant PI3-K R/C isoform complexes expressed in HIEC cells. d Representative (n ≥ 3) WB analyses of the reciprocal validation of the IP/co-IP analyses in (c), this time via the IP of p110α and p110β, and verification of association of p85α, p85β, and/or p55γ

Mentions: We first established the expression profile of class I PI3-K R and C isoforms in HIEC-6 cells. Semi-quantitative RT-PCR analyses indicate that these cells predominantly express the mRNAs for p85β, p55γ, p110α and p110δ, whereas those for p85α, p110β and p110γ are weakly expressed (Fig. 1a, left side of panel; 1b, open columns). Verification of protein expression by WB confirmed that p85β, p55γ and p110α are indeed predominantly expressed, and that p85α and p110β are effectively weakly expressed (Fig. 1a, right side of panel; 1b, filled columns). However, no protein products for either p110γ or p110δ were detected in HIEC-6 cells (Fig. 1a, right side of panel; 1b, filled columns), as confirmed by the strong detection of these two isoforms in GMCSF-stimulated human neutrophils using the same specific antibodies (not shown).Fig. 1


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)

Expression of PI3-K isoforms and isoform complexes in HIEC cells. a Representative (n ≥ 3) RT-PCR (left side of panel) and WB (right side of panel) analyses of the expression of the known class I PI3-K C (p110α, p110β, p110γ, p110δ) and R (p85α, p85β, p55γ) isoforms, using isoform-specific primers (for RT-PCR) or antibodies (for WB). Actin expression was used as a reference. b Same as in (a), except that amplified bands (for mRNAs; open columns) and immunoreactive bands (for proteins; filled columns) were semi-quantified and compared to those of actin, in order to establish the relative expression levels for each isoform analyzed (n ≥ 3). c Representative (n ≥ 5) WB analyses of the IP of the PI3-K R isoforms p85α, p85β, and p55γ, for the verification of association by co-IP of the PI3-K C isoforms p110α and p110β, and consequent determination of the predominant PI3-K R/C isoform complexes expressed in HIEC cells. d Representative (n ≥ 3) WB analyses of the reciprocal validation of the IP/co-IP analyses in (c), this time via the IP of p110α and p110β, and verification of association of p85α, p85β, and/or p55γ
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Fig1: Expression of PI3-K isoforms and isoform complexes in HIEC cells. a Representative (n ≥ 3) RT-PCR (left side of panel) and WB (right side of panel) analyses of the expression of the known class I PI3-K C (p110α, p110β, p110γ, p110δ) and R (p85α, p85β, p55γ) isoforms, using isoform-specific primers (for RT-PCR) or antibodies (for WB). Actin expression was used as a reference. b Same as in (a), except that amplified bands (for mRNAs; open columns) and immunoreactive bands (for proteins; filled columns) were semi-quantified and compared to those of actin, in order to establish the relative expression levels for each isoform analyzed (n ≥ 3). c Representative (n ≥ 5) WB analyses of the IP of the PI3-K R isoforms p85α, p85β, and p55γ, for the verification of association by co-IP of the PI3-K C isoforms p110α and p110β, and consequent determination of the predominant PI3-K R/C isoform complexes expressed in HIEC cells. d Representative (n ≥ 3) WB analyses of the reciprocal validation of the IP/co-IP analyses in (c), this time via the IP of p110α and p110β, and verification of association of p85α, p85β, and/or p55γ
Mentions: We first established the expression profile of class I PI3-K R and C isoforms in HIEC-6 cells. Semi-quantitative RT-PCR analyses indicate that these cells predominantly express the mRNAs for p85β, p55γ, p110α and p110δ, whereas those for p85α, p110β and p110γ are weakly expressed (Fig. 1a, left side of panel; 1b, open columns). Verification of protein expression by WB confirmed that p85β, p55γ and p110α are indeed predominantly expressed, and that p85α and p110β are effectively weakly expressed (Fig. 1a, right side of panel; 1b, filled columns). However, no protein products for either p110γ or p110δ were detected in HIEC-6 cells (Fig. 1a, right side of panel; 1b, filled columns), as confirmed by the strong detection of these two isoforms in GMCSF-stimulated human neutrophils using the same specific antibodies (not shown).Fig. 1

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