Dual role of MEK/ERK signaling in senescence and transformation of intestinal epithelial cells

The mitogen-activated protein kinase cascade operates downstream of Ras to convey cell-surface signals to the nucleus via nuclear translocation of ERK1 and ERK2. We and others have recently demonstrated that activation of ERK1/2 by growth factors is required for proliferation of intestinal epithelial crypt cells. However, it remained to be established whether ERK1/2 activation alone was sufficient to trigger intestinal epithelial cell (IEC) proliferation. To this aim, retrovirus encoding the hemagglutinin-tagged MAPK/ERK kinase (MEK)1 wild type (wtMEK), the upstream activator of ERK1/2, or a constitutively active mutant of MEK1 (MEK1-S218D/S222D; caMEK) were used to infect nonimmortalized human normal intestinal epithelial crypt cell cultures [human intestinal epithelial cells (HIEC)] and rodent immortalized intestinal crypt cells (IEC-6). Stable expression of caMEK but not wtMEK in HIEC led to the irreversible arrest of cellular proliferation (premature senescence). Concomitant with the onset of cell-cycle arrest was the induction of the cyclin-dependent kinase inhibitors p21(Cip), p53, and p16(INK4A). By contrast, overexpression of caMEK in IEC-6 cells induced growth factor relaxation for DNA synthesis, promoted morphological transformation and growth in soft agar, and did not affect expression of p21(Cip), p53, and p16(INK4A). We provided evidences that ERK1b, an alternatively spliced isoform of ERK1, is activated and may contribute to the deregulation of contact inhibition cell growth and transformation of these cells. Constitutive activation of MEK in IECs can produce either premature senescence or forced mitogenesis depending on the integrity of a senescence program controlled by the cell cycle inhibitors p53, p16(INK4A), and p21(CIP).     

The critical role of protein kinase C-theta in Fas/Fas ligand-mediated apoptosis

A functional immune system not only requires rapid expansion of antigenic specific T cells, but also requires efficient deletion of clonally expanded T cells to avoid accumulation of T cells. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen-induced expansion and subsequent deletion of T cells. In this study, we show that in the absence of protein kinase C-theta (PKC-theta), superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) CD4(+) T cells was defective in PKC-theta(-/-) mice. In response to staphylococcal enterotoxin B challenge, up-regulation of FasL, but not Fas, was significantly reduced in PKC-theta(-/-) mice. PKC-theta is thus required for maximum up-regulation of FasL in vivo. We further show that stimulation of FasL expression depends on PKC-theta-mediated activation of NF-AT pathway. In addition, PKC-theta(-/-) T cells displayed resistance to Fas-mediated apoptosis as well as activation-induced cell death (AICD). In the absence of PKC-theta, Fas-induced activation of apoptotic molecules such as caspase-8, caspase-3, and Bid was not efficient. However, AICD as well as Fas-mediated apoptosis of PKC-theta(-/-) T cells were restored in the presence of high concentration of IL-2, a critical factor required for potentiating T cells for AICD. PKC-theta is thus required for promoting FasL expression and for potentiating Fas-mediated apoptosis.     

Oxidative stress induces the expression of Fas and Fas ligand and apoptosis in murine intestinal epithelial cells

Intestinal epithelial cell function is compromised by local immune and inflammatory responses. In this study, we examined the possibility that intestinal epithelial cell injury occurs in the presence of activated inflammatory cells, such as neutrophils and macrophages, via production of reactive oxygen species (ROS). Following exposure to 50–150 μM H₂O₂, levels of mRNA and protein for Fas and, to a lesser degree, Fas-L were increased and intestinal epithelial cells underwent apoptosis. Treatment of H₂O₂-exposed cells with agonistic anti-Fas antibody, but not isotype control antibody, significantly enhanced apoptosis. Apoptosis was associated with the activation of caspase 8, while Z-IETD, an inhibitor of caspase 8, blocked apoptosis of H₂O₂-exposed intestinal epithelial cells. Thus, ROS induced Fas and Fas-L expression in association with intestinal epithelial cell apoptosis. These data support the hypothesis that, following exposure to oxidative stress, enterocytes are primed for cell death via Fas-mediated pathways.     

Normal breast epithelial cells induce apoptosis of breast cancer cells via Fas signaling

Fas/Fas ligand (Fas L) death pathway is an important mediator of apoptosis. Deregulation of Fas pathway is reported to be involved in the immune escape of breast cancer and the resistance to anti-cancer drugs. In this study, we demonstrated that conditioned medium by normal breast epithelial cells (NBEC-CM) induced apoptosis of MCF-7 and T-47D Fas-sensitive cells but had no effect on MDA-MB-231 Fas-resistant cells. Inhibition of PI3 kinase or NF-kappaB by specific inhibitors or transient transfections restored the sensitivity of MDA-MB-231 cells to NBEC-induced apoptosis. Moreover, the constitutive activation of NF-kappaB was controlled by PI3 kinase because inhibition of PI3 kinase reduced NF-kappaB activity. Inducible activation of NF-kappaB rendered MCF-7 cells resistant to NBEC-CM- and Fas agonist antibody-triggered apoptosis. Therefore, constitutive or inducible activation of PI3 kinase and/or NF-kappaB in breast cancer cells rendered them resistant to NBEC-triggered apoptosis. In addition, Fas neutralizing antibody and dominant negative Fas abolished NBEC-triggered apoptosis. Western blot and confocal microscopy analysis showed an increase of membrane Fas/Fas L when cells were induced into apoptotis by NBEC-CM. Taken together, these data show that NBEC induced apoptosis in breast cancer cells via Fas signaling.     

Protein kinase D1 mediates stimulation of DNA synthesis and proliferation in intestinal epithelial IEC-18 cells and in mouse intestinal crypts

We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p < 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p < 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.     

Normal rat intestinal cells IEC-18: characterization and transfection with immortalizing oncogenes

IEC-18 cells, a cell line derived from the ileum of rat intestine, have the characteristics of normal cells since they have a contact inhibited cell growth, do not form colonies in soft agar and are not tumorigenic when injected in nude mice. IEC-18 cells were transfected with nuclear oncogenes, c-myc, v-myc and SV40 T antigen in order to obtain immortal cell lines. Independent clones were isolated and characterized for the growth properties. Expression of v-myc altered the morphology of the cells and shortened the doubling time. A slow growth together with a low cloning efficiency was associated with the expression of SV40 T antigen. No changes either in growth or in morphology were observed in c-myc-expressing IEC-18 cells. Expression of these nuclear oncogenes did not result in the neoplastic transformation of the IEC-18 cells, since none of the clones lost the anchorage dependence or were able to form tumors in vivo. The c-myc-containing IEC-18 cells were unable to secrete in the growth medium TGF and exposure to TGF inhibited the growth rate by 30%. All these observations are consistent with the conclusion that the expression of nuclear oncogenes does not lead to the neoplastic transformation of these cells.     

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

Background and aims

The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour.

Methods

Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence.

Results

We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids.

Conclusions

The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.     

Hydrogen peroxide induces oxidative stress and the mitochondrial pathway of apoptosis in RAT intestinal epithelial cells (IEC-6)

In order to investigate the mechanism of apoptosis in rat intestinal epithelial cells (IEC-6) induced by hydrogen peroxide (H₂O₂), IEC-6 cells were subjected to 20 μmol/L H₂O₂ and cell proliferation activity was determined using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide. Cell morphology was observed by microscopy and cell apoptosis was detected by acridine orange and ethidium bromide staining and the portion of apoptotic cells was measured by flow cytometry. Genes and proteins related to cell apoptosis were detected by RT-PCR and Western blotting, and the mitochondrial membrane potential was evaluated by fluorescence probes. Results: Significant morphology damage was caused by exposure to H₂O₂, and results showed that ROS generation significantly increased (P < 0.01). The activity of superoxide dismutase decreased significantly (P < 0.05), malondialdehyde content increased (P < 0.05), and expression of both catalase and glutathione peroxidase decreased significantly (P < 0.05) in the H₂O₂ treatment group. Mitochondrion membrane potential was reduced, cytochrome released into the cytoplasm and caspase-9 and caspase-3 were significantly increased (P < 0.01) after treatment with H₂O₂. Moreover, the ratio of Bax/Bcl-2 and apoptosis were significantly increased (P < 0.01) in the H₂O₂ group. In conclusion, the present study indicated that the mitochondrial pathway plays a vital role in H₂O₂ induced IEC-6 cell apoptosis.     

Bacterial lipopolysaccharide reduced intestinal barrier function and altered localization of 7H6 antigen in IEC-6 rat intestinal crypt cells

The intestinal epithelial barrier restricts the passage of potentially toxic substances into the systemic circulation and is considered to be mostly mediated by tight junctions, though the mechanisms involved in the regulation of intestinal tight junctions are not yet fully understood. In the present study, we examined whether bacterial lipopolysaccharide (LPS) altered the barrier function of tight junction and localization of tight junctional proteins, ZO-1 and 7H6 antigen, in IEC-6 intestinal cells. Administration of LPS to the basolateral surface of IEC-6 cells disrupted the barrier function and caused the disappearance of 7H6 antigen from the cell border, whereas LPS administered to the apical surface altered neither the barrier function nor the localization of 7H6 antigen in IEC-6 cells. On the other hand, the localization of ZO-1 was not influenced by these treatments of LPS. These results suggest that the interaction of LPS with the basolateral surface of intestinal epithelial cells disrupts the barrier function and 7H6 antigen take part in the maintenance of the barrier function in IEC-6 cells. J. Cell. Physiol. 171:284–290, 1997. © 1997 Wiley-Liss, Inc.     

p66SHC promotes apoptosis and antagonizes mitogenic signaling in T cells

Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc(-/-) T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.     

Proliferation and mRNA expression of absorptive villous cell markers and mineral transporters in prolactin-exposed IEC-6 intestinal crypt cells

During pregnancy and lactation, prolactin (PRL) enhances intestinal absorption of calcium and other minerals for fetal development and milk production. Although an enhanced absorptive efficiency is believed to mainly result from the upregulation of mineral transporters in the absorptive villous cells, some other possibilities, such as PRL-enhanced crypt cell proliferation and differentiation to increase the absorptive area, have never been ruled out. Here, we investigated cell proliferation and mRNA expression of mineral absorption-related genes in the PRL-exposed IEC-6 crypt cells. As expected, the cell proliferation was not altered by PRL. Inasmuch as the mRNA expressions of villous cell markers, including dipeptidylpeptidase-4, lactase and glucose transporter-5, were not increased, PRL was not likely to enhance crypt cell differentiation into the absorptive villous cells. In contrast to the previous findings in villous cells, PRL was found to downregulate the expression of calbindin-D(9k), claudin-3 and occludin in IEC-6 crypt cells, while having no effect on transient receptor potential vanilloid family channels-5/6, plasma membrane Ca(2+)-ATPase (PMCA)-1b and Na(+)/Ca(2+) exchanger-1 expression. In conclusion, IEC-6 crypt cells did not respond to PRL by increasing proliferation or differentiation into villous cells. The present results thus supported the previous hypothesis that PRL enhanced mineral absorption predominantly by increasing transporter expression and activity in the absorptive villous cells.     

Compartmentalization of Fas and Fas ligand may prevent auto- or paracrine apoptosis in epithelial cells

Oligomerization of Fas receptor by its ligand, FasL, activates a signaling cascade that leads to apoptosis of Fas bearing cells. Interestingly, many epithelia coexpress Fas and FasL, yet FasL does not trigger Fas present on the same or neighboring cells to induce spontaneous apoptosis. Here, we show that Fas and FasL are segregated from each other to different cellular compartments in kidney epithelial MDCK cells. While Fas is restricted to the basolateral surface, FasL is sequestered to an intracellular compartment and, a lesser extent, the apical surface. This spatial segregation of Fas and FasL may explain how epithelial cells can constitutively express a functional Fas pathway but avoid auto- or paracrine cell death. Compromising this spatial segregation in physiological or pathological situations may play a so far underestimated role in initiating apoptosis of epithelial cells.     

Hyperoxia-induced apoptosis and Fas/FasL expression in lung epithelial cells

Alveolar epithelial apoptosis is an important feature of hyperoxia-induced lung injury in vivo and has been described in the early stages of bronchopulmonary dysplasia (chronic lung disease of preterm newborn). Molecular regulation of hyperoxia-induced alveolar epithelial cell death remains incompletely understood. In view of functional involvement of Fas/FasL system in physiological postcanalicular type II cell apoptosis, we speculated this system may also be a critical regulator of hyperoxia-induced apoptosis. The aim of this study was to investigate the effects of hyperoxia on apoptosis and apoptotic gene expression in alveolar epithelial cells. Apoptosis was studied by TUNEL, electron microscopy, DNA size analysis, and caspase assays. Fas/FasL expression was determined by Western blot analysis and RPA. We determined that in MLE-12 cells exposed to hyperoxia, caspase-mediated apoptosis was the first morphologically and biochemically recognizable mode of cell death, followed by necrosis of residual adherent cells. The apoptotic stage was associated with a threefold upregulation of Fas mRNA and protein expression and increased susceptibility to direct Fas receptor activation, concomitant with a threefold increase of FasL protein levels. Fas gene silencing by siRNAs significantly reduced hyperoxia-induced apoptosis. In murine fetal type II cells, hyperoxia similarly induced markedly increased Fas/FasL protein expression, confirming validity of results obtained in transformed MLE-12 cells. Our findings implicate the Fas/FasL system as an important regulator of hyperoxia-induced type II cell apoptosis. Elucidation of regulation of hyperoxia-induced lung apoptosis may lead to alternative therapeutic strategies for perinatal or adult pulmonary diseases characterized by dysregulated type II cell apoptosis.     

Down-regulation of MEK/ERK signaling by E-cadherin-dependent PI3K/Akt pathway in differentiating intestinal epithelial cells

In vitro experiments have shown that the establishment of cell-cell contacts in intestinal epithelial cell cultures is a critical step in initiating ERK inhibition, cell cycle arrest, and induction of the differentiation process. Herein, we determined the mechanisms through which E-cadherin-mediated cell-cell contacts modulate the ERK pathway in intestinal epithelial cells. We report that: (1) removal of calcium from the culture medium of newly confluent Caco-2/15 cells (30 min, 4 mM EGTA) results in the disruption of both adherens and tight junctions and clearly decreases Akt phosphorylation while increasing MEK and ERK activities. Akt, MEK, and ERK activation levels return to control levels 60 min after calcium restoration; (2) the use of E-cadherin blocking antibodies efficiently prevents Akt phosphorylation and MEK-ERK inhibition after 70 min of calcium restoration; (3) using the PI3K inhibitor LY294002 (15 microM) in calcium switch experiments, we demonstrate that the assembly of adherens junctions activates Akt activity and triggers the inhibition of ERK1/2 activities in a PI3K-dependent manner; (4) adenoviral infection of confluent Caco-2/15 cells with a constitutively active mutant of Akt1 strongly represses ERK1/2 activities; (5) inhibition of PI3K abolishes Akt activity but leads to a rapid and sustained activation of the MEK-ERK1/2 in confluent differentiating Caco-2/15 cells, but not in undifferentiated growing Caco-2/15 cells. Our data suggest that E-cadherin engagement leads to MEK/ERK inhibition in a PI3K/Akt-dependent pathway. This mechanism may account for the role of E-cadherin in proliferation/differentiation transition along the crypt-villus axis of the human intestinal epithelium.     

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-kappaB signaling in intestinal epithelial cells

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-B signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-B activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF- plus cycloheximide (TNF-/CHX). Specific inhibition of NF-B by the recombinant adenoviral vector containing the IB superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-B activity and increased the sensitivity to TNF-/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-B activity and resulted in increased resistance to TNF-/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-/CHX-induced apoptosis, at least partially, through the activation of NF-B signaling in IECs. polyamines; -difluoromethylornithine; X-linked inhibitor of apoptosis protein; IB     

Expression of sodium-linked nucleoside transport activity in monolayer cultures of IEC-6 intestinal epithelial cells

Mature, confluent monolayer cultures of IEC-6 rat intestinal epithelial cells in conventional growth media express both Na(+)-linked, concentrative nucleoside transport (NT) activity and equilibrative, inhibitor-sensitive NT activity, but do not show morphologic differentiation. Na(+)-dependent fluxes of Ado and formycin B were minor in early subconfluent IEC-6 monolayers, but increased severalfold to become the major component of influx of these agents in confluent monolayers grown in medium containing Nu-Serum, a commercial medium supplement with a low serum content. In monolayers cultured in medium with fetal bovine serum, cell proliferation rates were similar to those in medium supplemented with Nu-Serum, but expression of Na(+)-linked NT activity was 6-8-fold lower than in monolayers grown in the latter medium. Inclusion of hydrocortisone in growth medium with Nu-Serum caused a 2-fold increase in the expression of Na(+)-linked NT activity. Experiments in which components of medium supplementation were withheld showed that insulin and epidermal growth factor were important in expression of the Na(+)-linked NT activity. Because the Na(+)-linked NT system has a brush border location in fresh intestinal epithelium, it is concluded that the regulated expression of this activity in the IEC-6 monolayers is a differentiative change.