Role of endothelin-1 and its receptors, ETA and ETB, in the survival of human vascular endothelial cells.

Our previous work showed the presence of endothelin-1 (ET-1) receptors, ETA and ETB, in human vascular endothelial cells (hVECs). In this study, we wanted to verify whether ET-1 plays a role in the survival of hVECs via the activation of its receptors ETA and (or) ETB (ETAR and ETBR, respectively). Our results showed that treatment of hVECs with ET-1 prevented apoptosis induced by genistein, an effect that was mimicked by treatment with ETBR-specific agonist IRL1620. Furthermore, blockade of ETBR with the selective ETBR antagonist A-192621 prevented the anti-apoptotic effect of ET-1 in hVECs. However, activation of ETA receptor alone did not seem to contribute to the anti-apoptotic effect of ET-1. In addition, the anti-apoptotic effect of ETBR was found to be associated with caspase 3 inhibition and does not depend on the density of this type of receptor. In conclusion, our results showed that ET-1 possesses an anti-apoptotic effect in hVECs and that this effect is mediated, to a great extent, via the activation of ETBR. This study revealed a new role for ETBR in the survival of hVECs.

Histone deacetylase inhibition impairs normal intestinal cell proliferation and promotes specific gene expression.

Mechanisms that maintain proliferation and delay cell differentiation in the intestinal crypt are not yet fully understood. We have previously shown the implication of histone methylation in the regulation of enterocytic differentiation. In this study, we investigated the role of histone deacetylation as an important epigenetic mechanism that controls proliferation and differentiation of intestinal cells using the histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) on the proliferation and differentiation of human and mouse intestinal cells. Treatment of newly confluent Caco-2/15 cells with SAHA resulted in growth arrest, increased histone acetylation and up-regulation of the expression of intestine-specific genes such as those encoding sucrase-isomaltase, villin and the ion exchanger SLC26A3. Although SAHA has been recently used in clinical trials for cancer treatment, its effect on normal intestinal cells has not been documented. Analyses of small and large intestines of mice treated with SAHA revealed a repression of crypt cell proliferation and a higher expression of sucrase-isomaltase in both segments compared to control mice. Expression of SLC26A3 was also significantly up-regulated in the colons of mice after SAHA administration. Finally, SAHA was also found to strongly inhibit normal human intestinal crypt cell proliferation in vitro. These results demonstrate the important implication of epigenetic mechanisms such as histone acetylation/deacetylation in the regulation of normal intestinal cell fate and proliferation.

Met receptor-induced Grb2 or Shc signals both promote transformation of intestinal epithelial cells, albeit they are required for distinct oncogenic functions.

BACKGROUND: Deregulation of receptor tyrosine kinases (RTK) contributes to the initiation and progression of intestinal-derived epithelial cancers, including colorectal cancer (CRC). However, the roles of the proximal signaling molecules engaged by RTKs in different oncogenic functions of CRC remain unclear. METHODS: Herein, the functional impact of expressing variant forms of the oncogenic Met receptor (Tpr-Met) that selectively recruit the adaptor proteins Grb2 or Shc was investigated in a model derived from normal intestinal epithelial cells (IEC-6). An RNA interference (RNAi) approach was used to define the requirement of Grb2 or Shc in Tpr-Met-transformed IEC-6 cells. Since Grb2 and Shc couple RTKs to the activation of the Ras/MEK/Erk and PI3K/Akt pathways, Erk and Akt phosphorylation/activation states were monitored in transformed IEC-6 cells, and a pharmacological approach was employed to provide insights into the roles of these pathways in oncogenic processes evoked by activated Met, and downstream of Grb2 and Shc. RESULTS: We show, for the first time, that constitutive activation of either Grb2 or Shc signals in IEC-6 cells, promotes morphological transformation associated with down-regulation of E-cadherin, as well as increased cell growth, loss of growth contact inhibition, anchorage-independent growth, and resistance to serum deprivation and anoikis. Oncogenic activation of Met was revealed to induce morphological transformation, E-cadherin down-regulation, and protection against anoikis by mechanisms dependent on Grb2, while Shc was shown to be partly required for enhanced cell growth. The coupling of activated Met to the Ras/MEK/Erk and PI3K/Akt pathways, and the sustained engagement of Grb2 or Shc in IECs, was shown to trigger negative feedback, limiting the extent of activation of these pathways. Nonetheless, morphological alterations and E-cadherin down-regulation induced by the oncogenic Tpr-Met, and by Grb2 or Shc signals, were blocked by MEK, but not PI3K, inhibitors while the enhanced growth and resistance to anoikis induced by Tpr-Met were nearly abolished by co-treatment with both inhibitors. CONCLUSION: Overall, these results identify Grb2 and Shc as central signaling effectors of Met-driven progression of intestinal epithelial-derived cancers. Notably, they suggest that Grb2 may represent a promising target for the design of novel CRC therapies.

Suppression of anoikis in human intestinal epithelial cells: differentiation state-selective roles of α2ß1, α3ß1, α5ß1, and α6ß4 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 α2ß1, α3ß1, α5ß1, and α6ß4 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, ß1 or ß4) 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 ß1 and ß4 integrins in the suppression of anoikis in differentiated cells, and functional distinctions between ß1 and ß4 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 α2ß1 and α5ß1 suppress anoikis in undifferentiated cells, whereas α3ß1 does so in differentiated ones. Furthermore, we provide evidence that α6ß4 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.

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, ß, and p55γ) and four C (p110α, ß, 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 ß1 integrin/Fak/Src-mediated suppression of anoikis. We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85ß and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110ß, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85ß 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 p85ß; and (4) both the p110α/p85ß and p110α/p55γ complexes are engaged by ß1 integrin/Fak/Src signaling; however, the engagement of p110α/p85ß 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 ß1 integrin/Fak/Src-mediated suppression of anoikis.

Integrin-linked kinase regulates migration and proliferation of human intestinal cells under a fibronectin-dependent mechanism.

Integrin-linked kinase (ILK) plays a role in integrin signaling-mediated extracellular matrix (ECM)-cell interactions and also acts as a scaffold protein in functional focal adhesion points. In the present study, we investigated the expression and roles of ILK in human intestinal epithelial cells (IECs) in vivo and in vitro. Herein, we report that ILK and its scaffold-function interacting partners, PINCH-1, alpha-parvin, and beta-parvin, are expressed according to a decreasing gradient from the bottom of the crypt (proliferative/undifferentiated) compartment to the tip of the villus (non-proliferative/differentiated) compartment, closely following the expression pattern of the ECM/basement membrane component fibronectin. The siRNA knockdown of ILK in human IECs caused a loss of PINCH-1, alpha-parvin, and beta-parvin expression, along with a significant decrease in cell proliferation via a loss of cyclin D1 and an increase in p27 and hypophosphorylated pRb expression levels. ILK knockdown severely affected cell spreading, migration, and restitution abilities, which were shown to be directly related to a decrease in fibronectin deposition. All ILK knockdown-induced defects were rescued with exogenously deposited fibronectin. Altogether, our results indicate that ILK performs crucial roles in the control of human intestinal cell and crypt-villus axis homeostasis-especially with regard to basement membrane fibronectin deposition-as well as cell proliferation, spreading, and migration.

Degeneration of the pericryptal myofibroblast sheath by proinflammatory cytokines in inflammatory bowel diseases.

BACKGROUND & AIMS: Inflammatory bowel diseases (IBDs) are characterized by remodeling of the intestinal mucosa, which is associated with excessive cytokine release. Previous studies have shown that the epithelium in the crypt region of the mucosa in patients with Crohn's disease is susceptible to proinflammatory cytokines. We investigated whether the subepithelial myofibroblasts in this region were affected by these inflammatory conditions. METHODS: Immunofluorescence and immunohistochemistry were performed on inflamed and uninflamed specimens from patients with IBD to detect alpha-smooth muscle actin (alphaSMA), desmin, and tenascin-C. The effects of the proinflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were analyzed in human intestinal myofibroblast cultures by immunoblotting and apoptosis assays. RESULTS: Immunofluorescence analysis revealed decreased levels of the extracellular matrix molecule tenascin-C in pericryptal sheaths and alphaSMA in the immediate vicinity of the crypts in the inflamed specimens, indicating that the myofibroblast pericryptal sheath is affected by proinflammatory cytokines. Although individual cytokines did not affect myofibroblast proliferation or survival, cytokine combinations triggered caspase-dependent apoptosis. alphaSMA levels were reduced significantly in cells exposed to cytokines, either alone or in combination, suggesting dedifferentiation of myofibroblasts. Proinflammatory cytokines did not affect tenascin-C expression, suggesting that the decrease observed in the inflamed mucosa resulted from myofibroblast apoptosis. CONCLUSIONS: The subepithelial myofibroblasts of the epithelial sheath are disrupted in the intestinal mucosa of patients with IBD. A loss of myofibroblasts appears to result from the susceptibility of these cells to proinflammatory cytokines.

Integrin alpha8beta1 confers anoikis susceptibility to human intestinal epithelial crypt cells.

We previously reported that integrin alpha8beta1 is expressed in human intestinal epithelial crypt cells (HIECs) and represents one of the major RGD-binding integrins expressed by these cells. Moreover, the depletion of alpha8beta1 affects vinculin, but not paxillin, localization at focal adhesion points. In the present study, we show that the integrin alpha8 shRNA-mediated knockdown in HIECs leads to a decrease in anoikis susceptibility under cell suspension culture conditions, marked by a reduction in PARP cleavage and propidium iodide incorporation. Moreover, alpha8beta1-depleted HIECs exhibited an illicitly sustained activation of Fak and PI3-K/Akt-1 under anoikis conditions, rendering them refractory to anoikis. To this effect, colon cancer cells exhibiting resistance to anoikis not only displayed a loss of alpha8beta1 expression, but forced expression of alpha8beta1 in these cells decreased their resistance to anoikis. Consequently, alpha8beta1 is a prerequisite for the proper conduct of anoikis in normal HIECs, whereas its loss contributes to the illicit acquisition of anoikis resistance.

ILK supports RhoA/ROCK-mediated contractility of human intestinal epithelial crypt cells by inducing the fibrillogenesis of endogenous soluble fibronectin during the spreading process.

BACKGROUND: Fibronectin (FN) assembly into an insoluble fibrillar matrix is a crucial step in many cell responses to extracellular matrix (ECM) properties, especially with regards to the integrin-related mechanosensitive signaling pathway. We have previously reported that the silencing of expression of integrin-linked kinase (ILK) in human intestinal epithelial crypt (HIEC) cells causes significant reductions in proliferation and spreading through concomitantly acquired impairment of soluble FN deposition. These defects in ILK-depleted cells are rescued by growth on exogenous FN. In the present study we investigated the contribution of ILK in the fibrillogenesis of FN and its relation to integrin-actin axis signaling and organization. RESULTS: We show that de novo fibrillogenesis of endogenous soluble FN is ILK-dependent. This function seemingly induces the assembly of an ECM that supports increased cytoskeletal tension and the development of a fully spread contractile cell phenotype. We observed that HIEC cell adhesion to exogenous FN or collagen-I (Col-I) is sufficient to restore fibrillogenesis of endogenous FN in ILK-depleted cells. We also found that optimal engagement of the Ras homolog gene family member A (RhoA) GTPase/Rho-associated kinase (ROCK-1, ROCK-2)/myosin light chain (MLC) pathway, actin ventral stress fiber formation, and integrin adhesion complex (IAC) maturation rely primarily upon the cell's capacity to execute FN fibrillogenesis, independent of any significant ILK input. Lastly, we confirm the integrin α5ß1 as the main integrin responsible for FN assembly, although in ILK-depleted cells αV-class integrins expression is needed to allow the rescue of FN fibrillogenesis on exogenous substrate. CONCLUSION: Our study demonstrates that ILK specifically induces the initiation of FN fibrillogenesis during cell spreading, which promotes RhoA/ROCK-dependent cell contractility and maturation of the integrin-actin axis structures. However, the fibrillogenesis process and its downstream effect on RhoA signaling, cell contractility and spreading are ILK-independent in human intestinal epithelial crypt cells.

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.

B1 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 beta1 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-X(L), 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 p38beta, which is required as apoptosis/anoikis driver; (2) PI3-K/Akt-1 up-regulates the expression of Bcl-X(L) and Mcl-1, down-regulates Bax and Bak, drives Bad phosphorylation (both serine112/136 residues) and antagonizes p38beta activation; (3) MEK/Erk up-regulates Bcl-2, drives Bad phosphorylation (serine112 residue), but does not antagonize p38bactivation; (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 beta1 integrins/Fak, and is crucial for survival; and (6) beta1 integrins/Fak and/or Src regulate Bcl-2 homologs as both PI3-K/Atk-1 and MEK/Erk combined. Hence, beta1 integrin/Fak/Src signaling translates into integrated mediating functions of p38beta activation and regulation of Bcl-2 homologs by PI3-K/Akt-1 and MEK/Erk, consequently determining their requirement (or not) for survival.

Methods for Assessing Apoptosis and Anoikis in Normal Intestine/Colon and Colorectal Cancer.

Caspase-dependent apoptosis, including its distinct cell death subroutine known as anoikis, perform essential roles during organogenesis, as well as in the maintenance and repair of tissues. To this effect, the continuous renewal of the human intestinal/colon epithelium is characterized by the exfoliation by anoikis of differentiated cells, whereas immature/undifferentiated cells may occasionally undergo apoptosis in order to evacuate daughter cells that are damaged or defective. Dysregulated epithelial apoptosis is a significant component of inflammatory bowel diseases. Conversely, the acquisition of a resistance to apoptosis represents one of the hallmarks of cancer initiation and progression, including for colorectal cancer (CRC). Furthermore, the emergence of anoikis resistance constitutes a critical step in cancer progression (including CRC), as well as a limiting one that enables invasion and metastasis.Considering the implications of apoptosis/anoikis dysregulation in gut physiopathology, it therefore becomes incumbent to understand the functional determinants that underlie such dysregulation-all the while having to monitor, assess, or evidence apoptosis and/or anoikis. In this chapter, methodologies that are typically used to assess caspase-dependent apoptosis and anoikis in intestinal/colonic normal and CRC cells, whether in vivo, ex vivo, or in cellulo, are provided.

Involvement of the Integrin α1ß1 in the Progression of Colorectal Cancer.

Integrins are a family of heterodimeric glycoproteins involved in bidirectional cell signaling that participate in the regulation of cell shape, adhesion, migration, survival and proliferation. The integrin α1ß1 is known to be involved in RAS/ERK proliferative pathway activation and plays an important role in fibroblast proliferation. In the small intestine, the integrin α1 subunit is present in the crypt proliferative compartment and absent in the villus. We have recently shown that the integrin α1 protein and transcript (ITGA1) are present in a large proportion of colorectal cancers (CRC) and that their expression is controlled by the MYC oncogenic factor. Considering that α1 subunit/ITGA1 expression is correlated with MYC in more than 70% of colon adenocarcinomas, we postulated that the integrin α1ß1 has a pro-tumoral contribution to CRC. In HT29, T84 and SW480 CRC cells, α1 subunit/ITGA1 knockdown resulted in a reduction of cell proliferation associated with an impaired resistance to anoikis and an altered cell migration in HT29 and T84 cells. Moreover, tumor development in xenografts was reduced in HT29 and T84 sh-ITGA1 cells, associated with extensive necrosis, a low mitotic index and a reduced number of blood vessels. Our results show that α1ß1 is involved in tumor cell proliferation, survival and migration. This finding suggests that α1ß1 contributes to CRC progression.

Upregulation of a functional form of the beta4 integrin subunit in colorectal cancers correlates with c-Myc expression.

The integrin beta4 subunit has been shown to be involved in various aspects of cancer progression. The aim of the present work was to evaluate the expression of beta4 in primary colon cancers and to investigate the occurrence of a previously identified intestinal nonfunctional variant of beta4 (beta4ctd-) for adhesion to laminin. Immunodetection of beta4 using a panel of antibodies and RT-PCR analyses were performed on series of paired primary colon tumors and corresponding resection margins. The beta4 subunit was found to be significantly overexpressed in cancer specimens at both the protein and transcript levels. Surprisingly, beta4 levels of expression were closely correlated with those of the oncogene c-Myc in individual specimens. In vitro studies of c-Myc overexpression showed an upregulation of beta4 promoter activity. Finally, the beta4ctd- form was identified in the normal proliferative colonic cells but was found to be predominantly absent in colon cancer cells, both in situ and in vitro. We concluded that the beta4ctd- form is lost from colon cancer cells, while the level of the wild-type form of beta4, which is functional for adhesion to laminin, is increased in primary tumors in relation with the expression of c-Myc.

[Cell survival: differences and differentiation]. / Survie cellulaire: différences et différenciation.

The regulatory mechanisms of cell survival and apoptosis are very complex in nature, implicating numerous players and signaling pathways not only in the decision-making process of surviving (or dying), but as well as in the execution of apoptosis itself. The same complex nature applies with regards to anoikis, a form of apoptosis that is largely regulated by integrin-mediated, cell-extracellular matrix interactions. However, cell survival, apoptosis and anoikis also happen to implicate further mechanistic distinctions according to the specific tissue and/or cell type concerned. Incidentally, recent studies in a particular tissue, the human intestinal epithelium, have unearthed yet another layer of complexity in the regulation of these three cellular processes, namely the implication of differentiation state-specific mechanisms. Although our understanding of the molecular underpinnings of this new concept of differentiation state-distinct regulation of cell survival, apoptosis and/or anoikis is in its infancy, there is already evidence that such principle applies as well to cell types other than intestinal epithelial cells. Further studies on the differentiation state-specific regulation of these three cellular processes, either under normal or physiopathological situations, should prove crucial in increasing our understanding of pathologies which implicate a dysregulation of apoptosis and/or anoikis - such as cancer.

Integrin signaling, cell survival, and anoikis: distinctions, differences, and differentiation.

Cell survival and apoptosis implicate an increasing complexity of players and signaling pathways which regulate not only the decision-making process of surviving (or dying), but as well the execution of cell death proper. The same complex nature applies to anoikis, a form of caspase-dependent apoptosis that is largely regulated by integrin-mediated, cell-extracellular matrix interactions. Not surprisingly, the regulation of cell survival, apoptosis, and anoikis furthermore implicates additional mechanistic distinctions according to the specific tissue, cell type, and species. Incidentally, studies in recent years have unearthed yet another layer of complexity in the regulation of these cell processes, namely, the implication of cell differentiation state-specific mechanisms. Further analyses of such differentiation state-distinct mechanisms, either under normal or physiopathological contexts, should increase our understanding of diseases which implicate a deregulation of integrin function, cell survival, and anoikis.

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 beta1 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 beta1 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 beta1 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.

Gene expression profiles of normal proliferating and differentiating human intestinal epithelial cells: a comparison with the Caco-2 cell model.

cDNA microarray technology enables detailed analysis of gene expression throughout complex processes such as differentiation. The aim of this study was to analyze the gene expression profile of normal human intestinal epithelial cells using cell models that recapitulate the crypt-villus axis of intestinal differentiation in comparison with the widely used Caco-2 cell model. cDNA microarrays (19,200 human genes) and a clustering algorithm were used to identify patterns of gene expression in the crypt-like proliferative HIEC and tsFHI cells, and villus epithelial cells as well as Caco-2/15 cells at two distinct stages of differentiation. Unsupervised hierarchical clustering analysis of global gene expression among the cell lines identified two branches: one for the HIEC cells versus a second comprised of two sub-groups: (a) the proliferative Caco-2 cells and (b) the differentiated Caco-2 cells and closely related villus epithelial cells. At the gene level, supervised hierarchical clustering with 272 differentially expressed genes revealed distinct expression patterns specific to each cell phenotype. We identified several upregulated genes that could lead to the identification of new regulatory pathways involved in cell differentiation and carcinogenesis. The combined use of microarray analysis and human intestinal cell models thus provides a powerful tool for establishing detailed gene expression profiles of proliferative to terminally differentiated intestinal cells. Furthermore, the molecular differences between the normal human intestinal cell models and Caco-2 cells clearly point out the strengths and limitations of this widely used experimental model for studying intestinal cell proliferation and differentiation.

Repressed E-cadherin expression in the lower crypt of human small intestine: a cell marker of functional relevance.

In epithelia, abnormal expression of E-cadherin is related to pathologies involving a loss of cell polarization and/or differentiation. However, recent observations suggest that E-cadherin could also be repressed under physiological conditions, such as in some epithelial stem cell lineages. In the present work, we have analyzed E-cadherin expression in human intestinal epithelial cell progenitors and investigated its potential role. E-cadherin expression was analyzed along the crypt-villus axis by immunofluorescence on cryosections of small intestine. E-cadherin was found to be differentially expressed, being significantly weaker in the cells located at the bottom of the crypts. Surprisingly, neither the E-cadherin protein nor transcript were detected in a normal human intestinal epithelial (HIEC) crypt cell model isolated in our laboratory, whereas other E-cadherin-related components such as catenins and APC were present. Forced expression of E-cadherin in HIEC cells increased membrane-associated beta-catenin and was accompanied by the appearance of junction-like structures at the cell-cell interface. Functionally, cell kinetics and p21Cip levels were found to be altered in the E-cadherin expressing HIEC cells as compared to controls. Furthermore, a significant reduction of the migration abilities and an increase in sensitivity to anoikis were also observed. These results suggest that down-regulated expression of E-cadherin is a human intestinal crypt base cell-related feature that appears to be of functional relevance for the maintenance of the progenitor cell population.

Merosin-integrin promotion of skeletal myofiber cell survival: Differentiation state-distinct involvement of p60Fyn tyrosine kinase and p38alpha stress-activated MAP kinase.

Myofiber survival and suppression of anoikis depend in large part on the merosin (laminin-2/-4)-integrin alpha7beta1D cell adhesion system; however, the question remains as to the nature of the signaling molecules/pathways involved. In the present study, we investigated this question using the C2C12 cell model of myogenic differentiation and its merosin- and laminin-deficient derivatives. Herein, we report that: 1) of four members of the Src family of tyrosine kinases studied (p60Src, p53/56Lyn, p59Yes, or p60Fyn), the expression and activity of p60Fyn are found in myotubes exclusively; 2) a severe decrease of p60Fyn activity correlates with myotube apoptosis/anoikis induced by pharmocological compounds (herbimycin A or PP2) which inhibit tyrosine kinases of the Src family, by merosin deficiency and by beta1 integrin inhibition; 3) myoblast survival depends on Fak and the MEK/Erk pathway, in contrast to myotubes; 4) the PI3-K pathway is not involved in either myoblast or myotube survival; and 5) p38alpha SAPK stimulation and activity (but not that of p38beta) are required in the progression of myotube apoptosis/anoikis induced by p60Fyn inhibition, merosin deficiency or beta1 integrin-inhibition; however, p38 is not involved in myoblast apoptosis. Taken together, these results suggest that the promotion of myotube survival by the merosin-alpha7beta1D adhesion system involves p60Fyn, and that disruptions in this cell adhesion system induce myotube apoptosis/anoikis through a p38alpha SAPK-dependent pathway.