Marie-Josée Demers

Fak/Src signaling in human intestinal epithelial cell survival and anoikis: differentiation state-specific uncoupling with the PI3-K/Akt-1 and MEK/Erk pathways.

Human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. In the present study, we analyzed the roles of focal adhesion kinase (Fak)/Src signaling to the PI3‐K/Akt‐1 and mitogen‐activated protein kinase (MEK)/extracellular regulated kinases (Erk) pathways, within the context of such differentiation‐state distinctions. Anoikis was induced by inhibition of β1 integrins (antibody blocking), inhibition of Fak (pharmacologic inhibition or overexpression of dominant negative mutants), or by maintaining cells in suspension. Activation parameters of Fak, Src, Akt‐1, and Erk1/2 were analyzed. Activities of Src, Akt‐1, or Erk1/2 were also blocked by pharmacological inhibition or by overexpression of dominant‐negative mutants. We report that: (1) the loss or inhibition of β1 integrin binding activity causes anoikis and results in a down‐activation of Fak, Src, Akt‐1, and Erk1/2 in both undifferentiated, and differentiated cells; (2) the inhibition of Fak likewise causes anoikis and a down‐activation of Src, Akt‐1, and Erk1/2, regardless of the differentiation state; (3) Src, PI3‐K/Akt‐1, and MEK/Erk contribute to the survival of differentiated cells, whereas MEK/Erk does not play a role in the survival of undifferentiated ones; (4) the inhibition/loss of β1 integrin binding and/or Fak activity results in a loss of Src engagement with Fak, regardless of the state of differentiation; and (5) Src contributes to the activation of both the PI3‐K/Akt‐1 and MEK/Erk pathways in undifferentiated cells, but does not influence PI3‐K/Akt‐1 in differentiated ones. Hence, Fak/Src signaling to the PI3‐K/Akt‐1 and MEK/Erk pathways undergoes a differentiation state‐specific uncoupling which ultimately reflects upon the selective engagement of these same pathways in the mediation of intestinal epithelial cell survival. J. Cell. Physiol. 212:717–728, 2007.

Human intestinal epithelial cell survival and anoikis. Differentiation state-distinct regulation and roles of protein kinase B/Akt isoforms.

We have shown previously that human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. Here we analyzed the roles of protein kinase B/Akt isoforms in such differentiation state distinctions. Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of dominant-negative mutants) or β1 integrins (antibody blocking) or by maintaining cells in suspension. Expression/activation parameters of Akt isoforms (Akt-1, Akt-2, and Akt-3) and Fak were analyzed. Activity of Akt isoforms was also blocked by inhibition of phosphatidylinositol 3-kinase or by overexpression of dominant-negative mutants. Here we report the following. 1) The expression/activation levels of Akt-1 increase overall during enterocytic differentiation, and those of Akt-2 decrease, whereas Akt-3 is not expressed. 2) Akt-1 activation is dependent on β1 integrins/Fak signaling, regardless of the differentiation state. 3) Akt-2 activation is dependent on β1 integrins/Fak signaling in undifferentiated cells only. 4) Activation of Akt-1 is phosphatidylinositol 3-kinase-dependent, whereas that of Akt-2 is not. 5) Akt-2 does not promote survival or apoptosis/anoikis. 6) Akt-1 is essential for survival. 7) Akt-2 cannot substitute for Akt-1 in the suppression of anoikis. Hence, the expression and regulation of Akt isoforms show differentiation state-specific distinctions that ultimately reflect upon their selective implication in the mediation of human intestinal epithelial cell survival. These data provide new insights into the synchronized regulation of cell survival/death that is required in the dynamic renewal process of tissues such as the intestinal epithelium.

β1 integrin/Fak/Src signaling in intestinal epithelial crypt cell survival: integration of complex regulatory mechanisms

The molecular determinants which dictate survival and apoptosis/anoikis in human intestinal crypt cells remain to be fully understood. To this effect, the roles of β1 integrin/Fak/Src signaling to the PI3-K/Akt-1, MEK/Erk, and p38 pathways, were investigated. The regulation of six Bcl-2 homologs (Bcl-2, Mcl-1, Bcl-XL, Bax, Bak, Bad) was likewise analyzed. We report that: (1) Anoikis causes a down-activation of Fak, Src, Akt-1 and Erk1/2, a loss of Fak–Src association, and a sustained/enhanced activation of p38β, which is required as apoptosis/anoikis driver; (2) PI3-K/Akt-1 up-regulates the expression of Bcl-XL and Mcl-1, down-regulates Bax and Bak, drives Bad phosphorylation (both serine112/136 residues) and antagonizes p38β activation; (3) MEK/Erk up-regulates Bcl-2, drives Bad phosphorylation (serine112 residue), but does not antagonize p38β activation; (4) PI3-K/Akt-1 is required for survival, whereas MEK/Erk is not; (5) Src acts as a cornerstone in the engagement of both pathways by β1 integrins/Fak, and is crucial for survival; and (6) β1 integrins/Fak and/or Src regulate Bcl-2 homologs as both PI3-K/Atk-1 and MEK/Erk combined. Hence, β1 integrin/Fak/Src signaling translates into integrated mediating functions of p38β activation and regulation of Bcl-2 homologs by PI3-K/Akt-1 and MEK/Erk, consequently determining their requirement (or not) for survival.

Human intestinal epithelial crypt cell survival and death: Complex modulations of Bcl‐2 homologs by Fak, PI3‐K/Akt‐1, MEK/Erk, and p38 signaling pathways

To investigate the mechanisms responsible for survival and apoptosis/anoikis in normal human intestinal epithelial crypt cells, we analyzed the roles of various signaling pathways and cell adhesion on the expression of six Bcl‐2 homologs (Bcl‐2, Bcl‐XL, Mcl‐1, Bax, Bak, Bad) in the well established HIEC‐6 cell model. Pharmacological inhibitors and/or dominant‐negative constructs were used to inhibit focal adhesion kinase (Fak) and p38 isoforms, as well as the phosphatidylinositol 3′‐kinase (PI3‐K)/Akt‐1 and mitogen‐activated protein kinase [MAPK] kinase (MEK)/extracellular regulated kinases (Erk) pathways. Cell adhesion was disrupted by antibody‐inhibition of integrin binding or forced cell suspension. The activation levels of studied kinase pathways were also analyzed. Herein, we report that β1 integrins, Fak, and the PI3‐K/Akt‐1 pathway, but not β4 integrins or the MEK/Erk pathway, are crucial for the survival of HIEC‐6 cells. Conversely, p38β, but not p38α or γ, is required for the induction of apoptosis/anoikis in HIEC‐6 cells. However, each of the signaling molecules/pathways analyzed were found to affect distinctively the individual expression of the Bcl‐2 homologs studied. For example, the inhibition of the PI3‐K/Akt‐1 pathway down‐regulated Bcl‐XL, Mcl‐1, and Bad, while at the same time up‐regulating Bax, whereas the inhibition of Fak up‐regulated both Bax and Bak, down‐regulated Bad, and did not affect the other Bcl‐2 homologs analyzed. These results indicate that integrins, Fak, PI3‐K/Akt‐1, MEK/Erk, and p38 isoforms perform distinct roles in the regulation of HIEC‐6 cell survival and/or death. In addition, our data show that the functions performed by these molecules/pathways in promoting cell survival or apoptosis/anoikis translate into complex, differential modulations of individual Bcl‐2 homologs. J. Cell. Physiol. 198: 209–222, 2004© 2003 Wiley‐Liss, Inc.

Intestinal Epithelial Cancer Cell Anoikis Resistance: EGFR-Mediated Sustained Activation of Src Overrides Fak-Dependent Signaling to MEK/Erk and/or PI3-K/Akt-1

Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3‐K/Akt‐1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down‐activation, cancer cells nevertheless exhibit sustained Fak–Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3‐K/Akt‐1 down‐activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3‐K/Akt‐1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak‐mediated signaling to MEK/Erk and PI3‐K/Akt‐1 in T84 cells, as in undifferentiated IECs, whereas PI3‐K/Akt‐1 is Src‐independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak–Src interactions and down‐activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3‐K/Akt‐1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR‐mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak–Src interactions and MEK/Erk activation, thus not only overriding Fak‐mediated signaling to MEK/Erk and/or PI3‐K/Akt‐1, but also the requirement of Fak and/or PI3‐K/Akt‐1 for survival. J. Cell. Biochem. 107: 639–654, 2009.

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

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.

Merosin (laminin‐2/4)‐driven survival signaling: Complex modulations of Bcl‐2 homologs

We have shown previously that the promotion of myofiber survival by the basement membrane component merosin (laminin‐2 [α2β1γ1]/laminin‐4 [α2β2γ1]) is dependent on the activity of the tyrosine kinase Fyn, whereas myofiber anoikis induced by merosin deficiency is dependent on the stress‐activated protein kinase p38α. To further understand such merosin‐driven survival signaling, we analyzed the expression of five Bcl‐2 homologs (Bcl‐2, Bcl‐XL, Bax, Bak, Bad) and one non‐homologous associated molecule (Bag‐1) in normal and merosin‐deficient myotubes, with or without pharmacological inhibitors for Fyn and p38. Herein, we report that (1) merosin deficiency induces anoikis and causes decreased Bcl‐2, Bcl‐XL, and Bag‐1 levels, increased Bax and Bak levels, and decreased Bad phosphorylation; (2) Bcl‐2, Bcl‐XL, Bag‐1, and Bad phosphorylation are also decreased in anoikis‐dying, Fyn‐inhibited myotubes; (3) the inhibition of p38α in Fyn‐inhibited and/or merosin‐deficient myotubes protects against anoikis and increases Bcl‐2 levels above normal, in addition to restoring Bad phosphorylation and Bag‐1 levels to normal; (4) the overexpression of merosin in deficient myotubes also rescues from anoikis and increases Bcl‐2 levels and Bad phosphorylation above normal, in addition to restoring Bcl‐XL, Bag‐1, Bax, and Bak levels to normal; and (5) Bcl‐2 overexpression is sufficient to rescue merosin‐deficient myotubes from anoikis, even though the expression/phosphorylation levels of the other homologs analyzed are not restored to normal. These results indicate that merosin‐driven myofiber survival signaling affects complex, differential modulations of individual Bcl‐2 homologs. These further suggest that Bcl‐2 can play a major role in suppressing myofiber anoikis.

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

BackgroundRegulation 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.ResultsHuman (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.ConclusionsOur 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.

Characterization of age-associated changes in peripheral organ and brain region weights in C57BL/6 mice.

In order to further understand age-related physiological changes and to have in depth reference values for C57BL/6 mice, we undertook a study to assess the effects of aging on peripheral organ weights, and brain region weights in wild type C57BL/6 male mice. Peripheral organs, body and brain region weights were collected from young (3-4 months), mid (12 months), old (23-28 months) and very old (>30 months) mice. Significant increases are observed with aging in body, liver, heart, kidney and spleen organ weights. A decrease in organ weight is observed with aging in liver and kidney only in the very old mice. In contrast, testes weight decreases with age. Within the brain, hippocampi, striata and olfactory bulbs weight decreases with age. These data further our knowledge of the anatomical and biological changes that occur with aging and provide reference values for physiological-based pharmacokinetic studies in C57BL/6 mice.

Organ-specific alteration in caspase expression and STK3 proteolysis during the aging process.

Caspases and their substrates are key mediators of apoptosis and strongly implicated in various physiological processes. As the serine/threonine kinase family is involved in apoptosis and serine/threonine kinase 3 (STK3) is a recently identified caspase-6 substrate, we assessed the expression and cleavage of STK3 in murine peripheral organs and brain regions during the aging process. We also assessed caspase-3, -6, -7, and -8 expression and activity in order to delineate potential mechanism(s) underlying the generation of the STK3 fragments observed and their relation to the apoptotic pathway. We demonstrate for the first time the cleavage of STK3 by caspase-7 and show that STK3 protein levels globally increase throughout the organism with age. In contrast, caspase-3, -6, -7, and -8 expression and activity vary significantly among the different organs analyzed suggesting differential effects of aging on the apoptotic mechanism and/or nonapoptotic functions of caspases throughout the organism. These results further our understanding of the role of caspases and their substrates in the normal aging process and highlight a potential role for STK3 in neurodegeneration.