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Suppression of anoikis in human intestinal epithelial cells: differentiation state-selective roles of α2β1, α3β1, α5β1, and α6β4 integrins.

Beauséjour M, Thibodeau S, Demers MJ, Bouchard V, Gauthier R, Beaulieu JF, Vachon PH - BMC Cell Biol. (2013)

Bottom Line: Activation levels of Fak and Src, as well as functional Fak-Src interactions, were also assessed.We report herein that differentiated IECs exhibit a greater sensitivity to anoikis than undifferentiated ones.Additionally, we show that α2β1 and α5β1 suppress anoikis in undifferentiated cells, whereas α3β1 does so in differentiated ones.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département d'anatomie et de biologie cellulaire, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, J1H5N4 Sherbrooke, Québec, Canada. Pierre.H.Vachon@USherbrooke.ca.

ABSTRACT

Background: Regulation of anoikis in human intestinal epithelial cells (IECs) implicates differentiation state-specific mechanisms. Human IECs express distinct repertoires of integrins according to their state of differentiation. Therefore, we investigated whether α2β1, α3β1, α5β1, and α6β4 integrins perform differentiation state-specific roles in the suppression of IEC anoikis.

Results: Human (HIEC, Caco-2/15) IECs were exposed to specific antibodies that block the binding activity of integrin subunits (α2, α3, α5, α6, β1 or β4) to verify whether or not their inhibition induced anoikis. The knockdown of α6 was also performed by shRNA. Additionally, apoptosis/anoikis was induced by pharmacological inhibition of Fak (PF573228) or Src (PP2). Anoikis/apoptosis was assayed by DNA laddering, ISEL, and/or caspase activity (CASP-8, -9, or -3). Activation levels of Fak and Src, as well as functional Fak-Src interactions, were also assessed. We report herein that differentiated IECs exhibit a greater sensitivity to anoikis than undifferentiated ones. This involves an earlier onset of anoikis when kept in suspension, as well as significantly greater contributions from β1 and β4 integrins in the suppression of anoikis in differentiated cells, and functional distinctions between β1 and β4 integrins in engaging both Fak and Src, or Src only, respectively. Likewise, Fak performs significantly greater contributions in the suppression of anoikis in differentiated cells. Additionally, we show that α2β1 and α5β1 suppress anoikis in undifferentiated cells, whereas α3β1 does so in differentiated ones. Furthermore, we provide evidence that α6β4 contributes to the suppression of anoikis in a primarily α6 subunit-dependent manner in undifferentiated cells, whereas this same integrin in differentiated cells performs significantly greater contributions in anoikis suppression than its undifferentiated state-counterpart, in addition to doing so through a dependence on both of its subunits.

Conclusions: Our findings indicate that the suppression of human IEC anoikis implicates differentiation state-selective repertoires of integrins, which in turn results into distinctions in anoikis regulation, and sensitivity, between undifferentiated and differentiated IECs. These data further the functional understanding of the concept that the suppression of anoikis is subjected to cell differentiation state-selective mechanisms.

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Engagement of Fak and Src by β1 and β4 integrins in the suppression of human IEC anoikis. A. Representative (n ≥ 4) WB analyses of the activation of Src and Fak, and verifications of Fak-Src interactions, from -2PC (Undifferentiated) and 30PC (Differentiated) Caco-2/15 adhering cell cultures maintained 24 h serum-free (control) with mouse IgG’s, P4C10 (β1 binding activity-blocking mAb), or 3E1 (β4 binding activity-blocking mAb). Specific antibodies for pY576/577p125Fak, pY418p60Src and pY397p125Fak, as well as for respective total protein forms, were used. B-D. -2PC (Undifferentiated; filled columns) and 30PC (Differentiated; open columns) Caco-2/15 cell cultures were maintained and processed as in (A), except that the relative pY576/577 levels of Fak (B), as well as the relative activation levels of Src (C) and Fak (D), were established in comparison to controls. A-D. Results obtained with HIEC cells were highly similar to those shown here for -2PC Caco-2/15 cells. B-D. Statistically significant (0.0001 ≤ P ≤ 0.001; n ≥ 4) differences between treated and control cultures are indicated by (*). Statistically significant (0.0005 ≤ P ≤ 0.005; n ≥ 4) differences between differentiated and undifferentiated IECs are indicated by (#).
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Figure 4: Engagement of Fak and Src by β1 and β4 integrins in the suppression of human IEC anoikis. A. Representative (n ≥ 4) WB analyses of the activation of Src and Fak, and verifications of Fak-Src interactions, from -2PC (Undifferentiated) and 30PC (Differentiated) Caco-2/15 adhering cell cultures maintained 24 h serum-free (control) with mouse IgG’s, P4C10 (β1 binding activity-blocking mAb), or 3E1 (β4 binding activity-blocking mAb). Specific antibodies for pY576/577p125Fak, pY418p60Src and pY397p125Fak, as well as for respective total protein forms, were used. B-D. -2PC (Undifferentiated; filled columns) and 30PC (Differentiated; open columns) Caco-2/15 cell cultures were maintained and processed as in (A), except that the relative pY576/577 levels of Fak (B), as well as the relative activation levels of Src (C) and Fak (D), were established in comparison to controls. A-D. Results obtained with HIEC cells were highly similar to those shown here for -2PC Caco-2/15 cells. B-D. Statistically significant (0.0001 ≤ P ≤ 0.001; n ≥ 4) differences between treated and control cultures are indicated by (*). Statistically significant (0.0005 ≤ P ≤ 0.005; n ≥ 4) differences between differentiated and undifferentiated IECs are indicated by (#).

Mentions: We then analyzed the relative activation levels of Fak and Src, as well as functional Fak-Src interactions, in the same treated and untreated cell cultures. As expected [29,32], the exposure to IgG’s did not affect the activation of either Fak or Src, and did not influence Fak-Src interactions (Figure 4A-D). However, the inhibition of β1 resulted in a significant down-activation of both Fak and Src, as well as in a significant decrease in Fak-Src interactions, regardless of the state of differentiation (Figure 4A-D). These effects were similar as those observed when cells were maintained in suspension (see previous section). By contrast, the inhibition of β4 did not affect the activation of Fak in undifferentiated or differentiated cells (Figure 4A, D), as expected [4,6,12,13,29]. Interestingly, the inhibition of this subunit did result in a significant down-activation of Src in differentiated IECs only (Figure 4A, C), while at the same time failing to affect functional Fak-Src interactions regardless of the state of differentiation (Figure 4A-B).


Suppression of anoikis in human intestinal epithelial cells: differentiation state-selective roles of α2β1, α3β1, α5β1, and α6β4 integrins.

Beauséjour M, Thibodeau S, Demers MJ, Bouchard V, Gauthier R, Beaulieu JF, Vachon PH - BMC Cell Biol. (2013)

Engagement of Fak and Src by β1 and β4 integrins in the suppression of human IEC anoikis. A. Representative (n ≥ 4) WB analyses of the activation of Src and Fak, and verifications of Fak-Src interactions, from -2PC (Undifferentiated) and 30PC (Differentiated) Caco-2/15 adhering cell cultures maintained 24 h serum-free (control) with mouse IgG’s, P4C10 (β1 binding activity-blocking mAb), or 3E1 (β4 binding activity-blocking mAb). Specific antibodies for pY576/577p125Fak, pY418p60Src and pY397p125Fak, as well as for respective total protein forms, were used. B-D. -2PC (Undifferentiated; filled columns) and 30PC (Differentiated; open columns) Caco-2/15 cell cultures were maintained and processed as in (A), except that the relative pY576/577 levels of Fak (B), as well as the relative activation levels of Src (C) and Fak (D), were established in comparison to controls. A-D. Results obtained with HIEC cells were highly similar to those shown here for -2PC Caco-2/15 cells. B-D. Statistically significant (0.0001 ≤ P ≤ 0.001; n ≥ 4) differences between treated and control cultures are indicated by (*). Statistically significant (0.0005 ≤ P ≤ 0.005; n ≥ 4) differences between differentiated and undifferentiated IECs are indicated by (#).
© Copyright Policy - open-access
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Figure 4: Engagement of Fak and Src by β1 and β4 integrins in the suppression of human IEC anoikis. A. Representative (n ≥ 4) WB analyses of the activation of Src and Fak, and verifications of Fak-Src interactions, from -2PC (Undifferentiated) and 30PC (Differentiated) Caco-2/15 adhering cell cultures maintained 24 h serum-free (control) with mouse IgG’s, P4C10 (β1 binding activity-blocking mAb), or 3E1 (β4 binding activity-blocking mAb). Specific antibodies for pY576/577p125Fak, pY418p60Src and pY397p125Fak, as well as for respective total protein forms, were used. B-D. -2PC (Undifferentiated; filled columns) and 30PC (Differentiated; open columns) Caco-2/15 cell cultures were maintained and processed as in (A), except that the relative pY576/577 levels of Fak (B), as well as the relative activation levels of Src (C) and Fak (D), were established in comparison to controls. A-D. Results obtained with HIEC cells were highly similar to those shown here for -2PC Caco-2/15 cells. B-D. Statistically significant (0.0001 ≤ P ≤ 0.001; n ≥ 4) differences between treated and control cultures are indicated by (*). Statistically significant (0.0005 ≤ P ≤ 0.005; n ≥ 4) differences between differentiated and undifferentiated IECs are indicated by (#).
Mentions: We then analyzed the relative activation levels of Fak and Src, as well as functional Fak-Src interactions, in the same treated and untreated cell cultures. As expected [29,32], the exposure to IgG’s did not affect the activation of either Fak or Src, and did not influence Fak-Src interactions (Figure 4A-D). However, the inhibition of β1 resulted in a significant down-activation of both Fak and Src, as well as in a significant decrease in Fak-Src interactions, regardless of the state of differentiation (Figure 4A-D). These effects were similar as those observed when cells were maintained in suspension (see previous section). By contrast, the inhibition of β4 did not affect the activation of Fak in undifferentiated or differentiated cells (Figure 4A, D), as expected [4,6,12,13,29]. Interestingly, the inhibition of this subunit did result in a significant down-activation of Src in differentiated IECs only (Figure 4A, C), while at the same time failing to affect functional Fak-Src interactions regardless of the state of differentiation (Figure 4A-B).

Bottom Line: Activation levels of Fak and Src, as well as functional Fak-Src interactions, were also assessed.We report herein that differentiated IECs exhibit a greater sensitivity to anoikis than undifferentiated ones.Additionally, we show that α2β1 and α5β1 suppress anoikis in undifferentiated cells, whereas α3β1 does so in differentiated ones.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département d'anatomie et de biologie cellulaire, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, J1H5N4 Sherbrooke, Québec, Canada. Pierre.H.Vachon@USherbrooke.ca.

ABSTRACT

Background: Regulation of anoikis in human intestinal epithelial cells (IECs) implicates differentiation state-specific mechanisms. Human IECs express distinct repertoires of integrins according to their state of differentiation. Therefore, we investigated whether α2β1, α3β1, α5β1, and α6β4 integrins perform differentiation state-specific roles in the suppression of IEC anoikis.

Results: Human (HIEC, Caco-2/15) IECs were exposed to specific antibodies that block the binding activity of integrin subunits (α2, α3, α5, α6, β1 or β4) to verify whether or not their inhibition induced anoikis. The knockdown of α6 was also performed by shRNA. Additionally, apoptosis/anoikis was induced by pharmacological inhibition of Fak (PF573228) or Src (PP2). Anoikis/apoptosis was assayed by DNA laddering, ISEL, and/or caspase activity (CASP-8, -9, or -3). Activation levels of Fak and Src, as well as functional Fak-Src interactions, were also assessed. We report herein that differentiated IECs exhibit a greater sensitivity to anoikis than undifferentiated ones. This involves an earlier onset of anoikis when kept in suspension, as well as significantly greater contributions from β1 and β4 integrins in the suppression of anoikis in differentiated cells, and functional distinctions between β1 and β4 integrins in engaging both Fak and Src, or Src only, respectively. Likewise, Fak performs significantly greater contributions in the suppression of anoikis in differentiated cells. Additionally, we show that α2β1 and α5β1 suppress anoikis in undifferentiated cells, whereas α3β1 does so in differentiated ones. Furthermore, we provide evidence that α6β4 contributes to the suppression of anoikis in a primarily α6 subunit-dependent manner in undifferentiated cells, whereas this same integrin in differentiated cells performs significantly greater contributions in anoikis suppression than its undifferentiated state-counterpart, in addition to doing so through a dependence on both of its subunits.

Conclusions: Our findings indicate that the suppression of human IEC anoikis implicates differentiation state-selective repertoires of integrins, which in turn results into distinctions in anoikis regulation, and sensitivity, between undifferentiated and differentiated IECs. These data further the functional understanding of the concept that the suppression of anoikis is subjected to cell differentiation state-selective mechanisms.

Show MeSH
Related in: MedlinePlus