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

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 alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 4 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 (alpha 2, alpha 3, alpha 5, alpha 6, beta 1 or beta 4) to verify whether or not their inhibition induced anoikis. The knockdown of alpha 6 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 beta 1 and beta 4 integrins in the suppression of anoikis in differentiated cells, and functional distinctions between beta 1 and beta 4 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 alpha 2 beta 1 and alpha 5 beta 1 suppress anoikis in undifferentiated cells, whereas alpha 3 beta 1 does so in differentiated ones. Furthermore, we provide evidence that alpha 6 beta 4 contributes to the suppression of anoikis in a primarily alpha 6 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.