Limits...
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.

Show MeSH

Related in: MedlinePlus

Differentiation state-selective contributions of α2β1, α3β1, α5β1, and α6β4 integrins in the suppression of anoikis in human IECs. Schematic drawing of an undifferentiated IEC (A) and its differentiated counterpart (B), illustrating how α2β1, α3β1, α5β1, and α6β4 integrins contribute in the engagement of Fak/Src signaling for the suppression of anoikis. A. In undifferentiated IECs, α2β1 and α5β1 (but not α3β1) contribute in the engagement and activation of Fak (assessed by Y397 residue phosphorylation) via the classical requirement of both α and β subunits for integrin functionality and signalling. Fak then engages and activates Src (assessed by Y418 residue phosphorylation) which, in turn, enacts functional Fak-Src interactions (assessed by phosphorylation of Y576/577 residues of Fak). In parallel, α6β4ctd- engages and activates a distinct pool of Src that does not interact functionally with Fak. Furthermore, such Src engagement by α6β4ctd- is enacted in a primarily α6 subunit-dependent manner. B. In differentiated IECs, α3β1 (but not α2β1 or α5β1) engages and activates Fak, again via the classical requirement of both α and β subunits. Fak then engages and activates Src which, in turn, enacts functional Fak-Src interactions. However, in this context, Src contributes reciprocally to Fak activation. In parallel, α6β4 engages and activates a distinct pool of Src that does not interact functionally with Fak. Additionally, the engagement of Src by α6β4 is now enacted via the classical requirement of both α and β subunits. A-B. Such integrin subunit- and differentiation state-selective roles of α2β1, α3β1, α5β1, and α6β4 integrins, in the engagement of Fak and/or Src, are likely to contribute to the outcome of differentiated IECs being more sensitive to anoikis than their undifferentiated counterparts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4219346&req=5

Figure 11: Differentiation state-selective contributions of α2β1, α3β1, α5β1, and α6β4 integrins in the suppression of anoikis in human IECs. Schematic drawing of an undifferentiated IEC (A) and its differentiated counterpart (B), illustrating how α2β1, α3β1, α5β1, and α6β4 integrins contribute in the engagement of Fak/Src signaling for the suppression of anoikis. A. In undifferentiated IECs, α2β1 and α5β1 (but not α3β1) contribute in the engagement and activation of Fak (assessed by Y397 residue phosphorylation) via the classical requirement of both α and β subunits for integrin functionality and signalling. Fak then engages and activates Src (assessed by Y418 residue phosphorylation) which, in turn, enacts functional Fak-Src interactions (assessed by phosphorylation of Y576/577 residues of Fak). In parallel, α6β4ctd- engages and activates a distinct pool of Src that does not interact functionally with Fak. Furthermore, such Src engagement by α6β4ctd- is enacted in a primarily α6 subunit-dependent manner. B. In differentiated IECs, α3β1 (but not α2β1 or α5β1) engages and activates Fak, again via the classical requirement of both α and β subunits. Fak then engages and activates Src which, in turn, enacts functional Fak-Src interactions. However, in this context, Src contributes reciprocally to Fak activation. In parallel, α6β4 engages and activates a distinct pool of Src that does not interact functionally with Fak. Additionally, the engagement of Src by α6β4 is now enacted via the classical requirement of both α and β subunits. A-B. Such integrin subunit- and differentiation state-selective roles of α2β1, α3β1, α5β1, and α6β4 integrins, in the engagement of Fak and/or Src, are likely to contribute to the outcome of differentiated IECs being more sensitive to anoikis than their undifferentiated counterparts.

Mentions: In the present study, we investigated the differentiation state-specific roles of the α2, α3, α5, α6, β1, and β4 integrin subunits in the suppression of IEC anoikis, including with regards to their contributions in the activation of Fak and/or Src. Human undifferentiated/crypt and differentiated/villus IECs express distinct repertoires of integrins (and variants) [4,22-24,26,38-41]. Particularly, undifferentiated IECs predominantly express α2β1, α5β1 and α6β4ctd-, whereas differentiated ones predominantly express α3β1 and α6β4 [4,22-24,26,38-41]. Herein, we report that differentiated IECs exhibit a greater sensitivity to anoikis than undifferentiated ones. This implicates 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. Accordingly, Fak performs significantly greater contributions in the suppression of anoikis in differentiated cells. We also show that α2β1 and α5β1 suppress anoikis in undifferentiated cells, whereas α3β1 does so in differentiated ones (Figure 11). Furthermore, we provide evidence that α6β4ctd-, which is expressed in undifferentiated IECs and is non-functional for anchorage [26,38-41], contributes nevertheless to the suppression of anoikis in a primarily α6 subunit-dependent manner. Additionally, we show that α6β4, which is expressed in differentiated cells and is anchorage-functional [26,38-41], not only performs significantly greater contributions than its anchorage non-functional counterpart in the suppression of anoikis, but does so through a dependence on both of its subunits (Figure 11). Hence, 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.


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)

Differentiation state-selective contributions of α2β1, α3β1, α5β1, and α6β4 integrins in the suppression of anoikis in human IECs. Schematic drawing of an undifferentiated IEC (A) and its differentiated counterpart (B), illustrating how α2β1, α3β1, α5β1, and α6β4 integrins contribute in the engagement of Fak/Src signaling for the suppression of anoikis. A. In undifferentiated IECs, α2β1 and α5β1 (but not α3β1) contribute in the engagement and activation of Fak (assessed by Y397 residue phosphorylation) via the classical requirement of both α and β subunits for integrin functionality and signalling. Fak then engages and activates Src (assessed by Y418 residue phosphorylation) which, in turn, enacts functional Fak-Src interactions (assessed by phosphorylation of Y576/577 residues of Fak). In parallel, α6β4ctd- engages and activates a distinct pool of Src that does not interact functionally with Fak. Furthermore, such Src engagement by α6β4ctd- is enacted in a primarily α6 subunit-dependent manner. B. In differentiated IECs, α3β1 (but not α2β1 or α5β1) engages and activates Fak, again via the classical requirement of both α and β subunits. Fak then engages and activates Src which, in turn, enacts functional Fak-Src interactions. However, in this context, Src contributes reciprocally to Fak activation. In parallel, α6β4 engages and activates a distinct pool of Src that does not interact functionally with Fak. Additionally, the engagement of Src by α6β4 is now enacted via the classical requirement of both α and β subunits. A-B. Such integrin subunit- and differentiation state-selective roles of α2β1, α3β1, α5β1, and α6β4 integrins, in the engagement of Fak and/or Src, are likely to contribute to the outcome of differentiated IECs being more sensitive to anoikis than their undifferentiated counterparts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219346&req=5

Figure 11: Differentiation state-selective contributions of α2β1, α3β1, α5β1, and α6β4 integrins in the suppression of anoikis in human IECs. Schematic drawing of an undifferentiated IEC (A) and its differentiated counterpart (B), illustrating how α2β1, α3β1, α5β1, and α6β4 integrins contribute in the engagement of Fak/Src signaling for the suppression of anoikis. A. In undifferentiated IECs, α2β1 and α5β1 (but not α3β1) contribute in the engagement and activation of Fak (assessed by Y397 residue phosphorylation) via the classical requirement of both α and β subunits for integrin functionality and signalling. Fak then engages and activates Src (assessed by Y418 residue phosphorylation) which, in turn, enacts functional Fak-Src interactions (assessed by phosphorylation of Y576/577 residues of Fak). In parallel, α6β4ctd- engages and activates a distinct pool of Src that does not interact functionally with Fak. Furthermore, such Src engagement by α6β4ctd- is enacted in a primarily α6 subunit-dependent manner. B. In differentiated IECs, α3β1 (but not α2β1 or α5β1) engages and activates Fak, again via the classical requirement of both α and β subunits. Fak then engages and activates Src which, in turn, enacts functional Fak-Src interactions. However, in this context, Src contributes reciprocally to Fak activation. In parallel, α6β4 engages and activates a distinct pool of Src that does not interact functionally with Fak. Additionally, the engagement of Src by α6β4 is now enacted via the classical requirement of both α and β subunits. A-B. Such integrin subunit- and differentiation state-selective roles of α2β1, α3β1, α5β1, and α6β4 integrins, in the engagement of Fak and/or Src, are likely to contribute to the outcome of differentiated IECs being more sensitive to anoikis than their undifferentiated counterparts.
Mentions: In the present study, we investigated the differentiation state-specific roles of the α2, α3, α5, α6, β1, and β4 integrin subunits in the suppression of IEC anoikis, including with regards to their contributions in the activation of Fak and/or Src. Human undifferentiated/crypt and differentiated/villus IECs express distinct repertoires of integrins (and variants) [4,22-24,26,38-41]. Particularly, undifferentiated IECs predominantly express α2β1, α5β1 and α6β4ctd-, whereas differentiated ones predominantly express α3β1 and α6β4 [4,22-24,26,38-41]. Herein, we report that differentiated IECs exhibit a greater sensitivity to anoikis than undifferentiated ones. This implicates 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. Accordingly, Fak performs significantly greater contributions in the suppression of anoikis in differentiated cells. We also show that α2β1 and α5β1 suppress anoikis in undifferentiated cells, whereas α3β1 does so in differentiated ones (Figure 11). Furthermore, we provide evidence that α6β4ctd-, which is expressed in undifferentiated IECs and is non-functional for anchorage [26,38-41], contributes nevertheless to the suppression of anoikis in a primarily α6 subunit-dependent manner. Additionally, we show that α6β4, which is expressed in differentiated cells and is anchorage-functional [26,38-41], not only performs significantly greater contributions than its anchorage non-functional counterpart in the suppression of anoikis, but does so through a dependence on both of its subunits (Figure 11). Hence, 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.

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