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.     

Human homolog of disc-large is required for adherens junction assembly and differentiation of human intestinal epithelial cells

We and others have shown that phosphatidylinositol 3-kinase (PI3K) is recruited to and activated by E-cadherin engagement. This PI3K activation is essential for adherens junction integrity and intestinal epithelial cell differentiation. Here we provide evidence that hDlg, the homolog of disc-large tumor suppressor, is another key regulator of adherens junction integrity and differentiation in mammalian epithelial cells. We report the following. 1) hDlg co-localizes with E-cadherin, but not with ZO-1, at the sites of cell-cell contact in intestinal epithelial cells. 2) Reduction of hDlg expression levels by RNA(i) in intestinal cells not only severely alters adherens junction integrity but also prevents the recruitment of p85/PI3K to E-cadherin-mediated cell-cell contact and inhibits sucrase-isomaltase gene expression. 3) PI3K and hDlg are associated with E-cadherin in a common macromolecular complex in living differentiating intestinal cells. 4) This interaction requires the association of hDlg with E-cadherin and with Src homology domain 2 domains of the p85/PI3K subunit. 5) Phosphorylation of hDlg on serine and threonine residues prevents its interaction with the p85 Src homology domain 2 in subconfluent cells, whereas phosphorylation of hDlg on tyrosine residues is essential. We conclude that hDlg may be a determinant in E-cadherin-mediated adhesion and signaling in mammalian epithelial cells.     

Uric acid induced epithelial−mesenchymal transition of renal tubular cells through PI3K/p-Akt signaling pathway

The phenotypic changes of tubular epithelial cell are hallmark features of renal diseases caused by abnormal uric acid levels. We hereby intend to investigate whether PI3K/p-Akt signaling plays a role in uric-acid induced epithelial−mesenchymal transition process. The normal rat kidney cell line (NRK-52E) was used as a proximal tubular cell model in this study. NRK-52E cells were exposed to different concentrations of uric acid, or PI3K inhibitor LY294002, or both, respectively. The effects of uric acid on cell morphology were examined by phase contrast microscopy, while molecular alternations were assessed by western blot analysis and immunofluorescence staining. We found that uric acid induced visible morphological alterations in NRK-52E cells accompanied by increased expression of α-smooth muscle actin and reduced expression of E-cadherin. Moreover, phosphorylation of Akt protein was obviously increased, whereas Akt level remained stable. Furthermore, the above effects were abolished when PI3K/p-Akt pathway was blocked by the PI3K inhibitor. These findings demonstrated that high uric acid could induce phenotypic transition of cultured renal tubular cells, which was probably via activating PI3K/p-Akt signaling pathway.     

Cardiotrophin-1 stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through PI3K/Akt-dependent signaling pathways

Cardiotrophin-1 (CT1) plays an important role in the differentiation, development, and survival of neural stem cells. In this study, we analyzed its effects on the stimulation of human umbilical cord blood-derived mesenchymal stem cells in terms of their potential to differentiate into neuron-like cells, their survival characteristics, and the molecular mechanisms involved. The treatment of cells with neural induction medium (NIM) and CT1 generated more cells that were neuron-like and produced stronger expression of neural-lineage markers than cells treated with NIM and without CT1. Bcl-2 and Akt phosphorylation (p-Akt) expression levels increased significantly in cells treated with both NIM and CT1. This treatment also effectively blocked cell death following neural induction and decreased Bax, Bak and cleaved-caspase 3 expression compared with cells treated with NIM without CT1. In addition, the inhibition of phosphatidylinositol 3-kinase (PI3K) abrogated p-Akt and Bcl-2 expression. Thus, PI3K/Akt contribute to CT1-stimulated neural differentiation and to the survival of differentiated cells.     

Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation.

The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brush-border formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation.     

Inducible expression of p120Cas1B isoform corroborates the role for p120-catenin as a positive regulator of E-cadherin function in intestinal cancer cells

Over the past decade, the exact function of p120-catenin in regulation of E-cadherin/catenins complex has remained particularly controversial. We have previously reported that E-cadherin-mediated adhesion is tightly regulated by tyrosine phosphorylation of catenins. However, this effect is not observed in human colon carcinoma cell line Caco-2. Here, we have generated inducible Caco-2 clones that display p120Cas1B, a p120-catenin isoform poorly expressed by these cells. As a result, neither expression of the transgene nor tyrosine phosphorylation of catenins induces redistribution of E-cadherin to the cytosol and disassembly of adherens and tight junctions. In contrast, E-cadherin appears markedly increased reinforcing cell-cell adhesion. Interestingly, a substantial decrease in p120-catenin levels is found in MDCK cells expressing Snail, in which E-cadherin expression is strongly inhibited. Additionally, we show that the specific depletion of p120-catenin decreases cell-cell contacts, and increases cell motility and scattering of colonies established by HT-29 M6 cells. Together our results corroborate that p120-catenin plays an essential role in the maintenance of the required E-cadherin protein levels that prevent the loss of epithelial characteristics occurred during tumorigenesis.     

Human intestinal epithelial cell survival: differentiation state-specific control mechanisms

To investigate whether human intestinal epithelial cell survival involves distinct control mechanisms depending on the state of differentiation, we analyzed the in vitro effects of insulin, pharmacological inhibitors of Fak, MEK/Erk, and PI3-K/Akt, and integrin (beta1, beta4)-blocking antibodies on the survival of the well-established human Caco-2 enterocyte-like and HIEC-6 cryptlike cell models. In addition, relative expression levels of six Bcl-2 homologs (Bcl-2, Bcl-X(L), Mcl-1, Bax, Bak, and Bad) and activation levels of Fak, Erk-2, and Akt were analyzed. Herein, we report that 1) the enterocytic differentiation process results in the establishment of distinct profiles of Bcl-2 homolog expression levels, as well as p125(Fak), p42(Erk-2), and p57(Akt) activated levels; 2) the inhibition of Fak, of the MEK/Erk pathway, or of PI3-K, have distinct impacts on enterocytic cell survival in undifferentiated (subconfluent Caco-2, confluent HIEC-6) and differentiated (30 days postconfluent Caco-2) cells; 3) exposure to insulin and the inhibition of Fak, MEK, and PI3-K resulted in differentiation state-distinct modulations in the expression of each Bcl-2 homolog analyzed; and 4) Fak, beta1 and beta4 integrins, as well as the MEK/Erk and PI3-K/Akt pathways, are distinctively involved in cell survival depending on the state of cell differentiation. Taken together, these data indicate that human intestinal epithelial cell survival is regulated according to differentiation state-specific control mechanisms.     

Activation of autophagy and Akt/CREB signaling play an equivalent role in the neuroprotective effect of rapamycin in neonatal hypoxia-ischemia

We have previously shown that in neonatal rats subjected to hypoxia-ischemia (HI) rapamycin administration increases autophagy, decreases apoptosis and significantly reduces brain damage. After HI, when autophagy is blocked neuronal cells rapidly progress toward necrotic cell death. The present study was undertaken to assess the potential role of activation of autophagic and phosphatidylinositol 3-kinase (PI3K)/Akt kinase pathways in the neuroprotective effect of rapamycin. Rapamycin administration caused a significant reduction of 70 kDa S6 kinase (p70S6K) phosphorylation and a significant increase of the autophagic proteins beclin 1 and microtubule-associated protein 1 light chain 3 (LC3), as of monodansylcadaverine (MDC) labelling in the lesioned side. The phosphorylation of Akt and cAMP response element binding protein (CREB) was increased in neuronal cells, and both p-Akt and p-CREB co-localized with beclin 1. Wortmannin (WT) administration significantly reduced Akt and CREB phosphorylation as well as the neuroprotective effect of rapamycin but did not affect the phosphorylation of p70S6K, the expression of beclin 1 and LC3, and MDC labelling. In contrast, 3-methyladenine (3MA) reduced the increased beclin 1 expression, the MDC labelling and the neuroprotective effect of rapamycin without affecting Akt phosphorylation. However, both compounds significantly increased necrotic cell death. Taken together, these data indicate that in neonatal HI autophagy can be part of an integrated pro-survival signalling which includes the PI3K-Akt-mammalian target of rapamycin (mTOR) axis. When the autophagic or the PI3K-Akt-mTOR pathways are interrupted cells undergo necrotic cell death.     

Rifampicin Prevents SH-SY5Y Cells from Rotenone-Induced Apoptosis via the PI3K/Akt/GSK-3β/CREB Signaling Pathway

In addition to its original application for treating tuberculosis, rifampicin has multiple potential neuroprotective effects in chronic neurodegenerative diseases including Parkinson’s disease (PD) and Alzheimer’s disease. Inflammatory reactions and the PI3K/Akt pathway are strongly implicated in dopaminergic neuronal death in PD. This study aims to investigate whether rifampicin protects rotenone-lesioned SH-SY5Y cells via regulating PI3K/Akt/GSK-3β/CREB pathway. Rotenone-treated SH-SY5Y cells were used as the cell model to investigate the neuroprotective effects of rifampicin. Cell viability and apoptosis of SH-SY5Y cells were determined by CCK-8 assay and flow cytometry, respectively. The expression of Akt, p-Akt, GSK-3β, p-GSK-3β, CREB and p-CREB were measured by Western blot. Our results showed that the cell viability and level of phospho-CREB significantly decreased in SH-SY5Y cells exposed to rotenone when compared to the control group. Both the cell viability and the expression of phospho-CREB in cells pretreated with rifampicin were higher than those of cells exposed to rotenone alone. Moreover, pretreatment of SH-SY5Y cells with rifampicin enhanced phosphorylation of Akt and suppressed activity of GSK-3β. The addition of LY294002, a PI3K inhibitor, could suppress phosphorylation of Akt and CREB and activate GSK-3β, resulting in abolishment of neuroprotective effects of rifampicin on cells exposed to rotenone. Rifampicin provides neuroprotection against dopaminergic degeneration, partially via the PI3K/Akt/GSK-3β/CREB signaling pathway. These findings suggest that rifampicin could be an effective and promising neuroprotective candidate for treating PD.     

PGE2 inhibits apoptosis in human adenocarcinoma Caco-2 cell line through Ras-PI3K association and cAMP-dependent kinase A activation

PGE2 plays a critical role in colorectal carcinogenesis. We have previously shown that COX-2 expression and PGE2 synthesis are mediated by IGF-II/IGF-I receptor signaling in the Caco-2 cell line and that the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt protects the cell from apoptosis. In the present study, we demonstrate that PGE2 has the ability to increase Ras and PI3K association and decrease the level of apoptosis in the same experimental system. The effect of PGE2 on PI3K/Ras association is dependent on the activation of EP4 receptor, the increase of cAMP levels, and the activation of PKA. In fact, treatment of cells with the PKA inhibitor H89 decreases the association of Ras and PI3K and Ras-associated PI3K activity. PKA inhibitor H89 is able to decrease threonine phosphorylation of Akt and to increase serine phosphorylation of Akt by p38 MAPK and counteracts the cytoprotective effect induced by PGE2. In addition, PGE2 is able to activate p38 MAPK and the inhibition of p38 MAPK, with SB203580 specific inhibitor or with dominant negative MKK6 kinase, is able to revert the apoptotic effect of H89 and serine phosphorylation of Akt. The effect of PGE2 on Caco-2 cell survival through PKA activation is mediated and regulated by the balance of threonine/serine phosphorylation of Akt by p38 kinase and PI3K. In conclusion, our data elucidate a novel mechanism for regulation of colon cancer cell survival and provide evidences for new combinatory treatments of colon cancer.     

结肠腺瘤息肉蛋白突变经PI3K/AKT 信号通路影响犬肾细胞MDCK的细胞粘附

结肠腺瘤息肉蛋白（APC）是一个肿瘤抑制因子,它不仅参与Wnt信号通路的传导,而且对细胞粘附、细胞骨架的组织和迁移等都有影响.APC突变发生于大多数结肠癌中.为了探讨APC突变对细胞粘附的影响及机制,本研究利用细胞粘附实验分析了MDCK-APC-N1和对照MDCK-GFP稳定表达细胞株系的细胞粘附情况.实验结果显示,在MDCK细胞中过表达APC-N1导致细胞-细胞间的粘附减少,细胞-基质间的粘附增加.荧光定量PCR和Western印迹实验表明,在MDCK-APC+N1细胞中,E-cadherin表达水平降低,CD29、P-FAK (Y397)、β-catenin和 P-AKT (T308)表达水平升高. 在MDCK-APC-N1细胞中,敲减β-catenin导致E-cadherin表达量升高,而CD29表达没有明显变化.进一步利用PI3K抑制剂LY294002处理MDCK-APC-N1细胞,结果发现,E-cadherin表达量明显升高,CD29表达量明显降低.这些结果揭示,APC-N1可活化 PI3K/AKT 信号通路,进而改变粘附蛋白E-cadherin和CD29影响细胞粘附.     

臭椿酮诱导人黑色素瘤A375细胞凋亡及其机制研究

为研究臭椿酮(Ailanthone,AIL)诱导人黑色素瘤A375细胞凋亡的作用及作用机制,以人黑色素瘤A375细胞为研究对象,采用MTT法测定AIL对人黑色素瘤A375细胞生长增殖的抑制作用。用倒置相差显微镜观察AIL对A375细胞形态的影响,用荧光倒置显微镜观察Hoechst33258染色后AIL对A375细胞核的影响,用AnnexinV-FITC/PI双染法检测AIL诱导A375细胞凋亡的作用,用分光光度法检测caspase-3和caspase-9的活性,Westernblot检测p-PI3Kβ(Ser1070),PI3Kβ,p-Akt(Ser473)和Akt蛋白表达水平的变化,接着用PI3K抑制剂LY294002进行干预,进一步验证AIL对PI3K/Akt信号通路及细胞凋亡的影响。实验结果表明,AIL能够明显抑制A375细胞增殖,使A375细胞数目变少、附着力和透光性减弱,AIL能够诱导A375细胞凋亡,使其细胞核染色质发生固缩并呈现高亮,且使A375细胞早期及晚期凋亡率均增加,AIL作用后能够使caspase-3和caspase-9活性增加,AIL能够抑制PI3K和Akt蛋白磷酸化,从而使PI3K/Akt信号通路失活。较AIL单独作用,AIL和LY294002共同作用后对PI3K和Akt蛋白磷酸化的抑制作用增强且诱导凋亡作用增加,进一步说明AIL通过失活PI3K/Akt信号通路来诱导A375细胞凋亡。     

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration

Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3-5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury.     

Formation of E-cadherin-mediated cell-cell adhesion activates AKT and mitogen activated protein kinase via phosphatidylinositol 3 kinase and ligand-independent activation of epidermal growth factor receptor in ovarian cancer cells

E-cadherin is a well characterized adhesion molecule that plays a major role in epithelial cell adhesion. Based on findings that expression of E-cadherin is frequently lost in human epithelial cancers, it has been implicated as a tumor suppressor in carcinogenesis of most human epithelial cancers. However, in ovarian cancer development, our data from the current study showed that E-cadherin expression is uniquely elevated in 86.5% of benign, borderline, and malignant ovarian carcinomas irrespective of the degree of differentiation, whereas normal ovarian samples do not express E-cadherin. Thus, we hypothesize that E-cadherin may play a distinct role in the development of ovarian epithelial cancers. Using an E-cadherin-expressing ovarian cancer cell line OVCAR-3, we have demonstrated for the first time that the establishment of E-cadherin mediated cell-cell adhesions leads to the activation of Akt and MAPK. Akt activation is mediated through the activation of phosphatidylinositol 3 kinase, and both Akt and MAPK activation are mediated by an E-cadherin adhesion-induced ligand-independent activation of epidermal growth factor receptor. We have also demonstrated that suppression of E-cadherin function leads to retarded cell proliferation and reduced viability. We therefore suggest that the concurrent formation of E-cadherin adhesion and activation of downstream proliferation signals may enhance the proliferation and survival of ovarian cancer cells. Our data partly explain why E-cadherin is always expressed during ovarian tumor development and progression.     

Poorly Differentiated Colon Carcinoma Cell Lines Deficient in α-Catenin Expression Express High Levels of Surface E-cadherin but Lack Ca2+-Dependent Cell-Cell Adhesion

Studies on several different types of carcinomas, with the notable exception of colon carcinoma, have shown that poorly differentiated tumors are frequently deficient in E-cadherin dependent cell-cell adhesion. In this study, we examined Ca²⁺-dependent cell-cell adhesion in colon carcinoma cell lines. Five poorly differentiated (Clone A, MIP 101, RKO, CCL 222, CCL 228) and four moderately-well differentiated (CX-1, CCL 235, DLD-2, CCL 187) colon carcinoma cell lines were assayed for their ability to form cell-cell aggregates and for their levels of E-cadherin expression. All of the poorly differentiated cell lines exhibited low levels of Ca²⁺-dependent cell-cell aggregation, in contrast to the moderately-well differentiated cell lines. Contrary to most previous studies, however, we observed that three of the five poorly differentiated cell lines examined expressed E-cadherin by FACS analysis and immunoprecipitation using an E-cadherin mAb. In fact, two of these cell lines expressed a 3- to 4-fold higher level of E-cadherin than that found in the moderately-well differentiated cell lines. mRNA levels for E-cadherin, as evaluated by both RT-PCR and Northern hybridization, corresponded to the levels of protein expression in each of the cell lines. Immunoprecipitation with an E-cadherin mAb, which is known to co-precipitate the catenins, demonstrated that the three poorly differentiated cell lines expressing E-cadherin did not co-precipitate α-catenin, although all of the moderately-well differentiated cell lines expressed both α- and β-catenin. RT-PCR confirmed the absence of the α-catenin mRNA from two of these cell lines. Stable expression of an α-catenin cDNA in one of the poorly differentiated cell lines lacking α-catenin expression resulted in a 5-fold increase in its level of Ca²⁺-dependent cell-cell aggregation, providing evidence that α-catenin is directly responsible for the loss of cell-cell adhesion in some cell lines. The α-catenin transfectants also exhibited a marked reduction in migration on collagen I. These data indicate that loss of α-catenin expression, as well as E-cadherin expression, can lead to a phenotype associated with poorly differentiated colon carcinomas.     

Activation of the protein kinase Akt/PKB by the formation of E-cadherin-mediated cell-cell junctions. Evidence for the association of phosphatidylinositol 3-kinase with the E-cadherin adhesion complex.

E-cadherins are surface adhesion molecules localized at the level of adherens junctions, which play a major role in cell adhesiveness by mediating calcium-dependent homophylic interactions at sites of cell-cell contacts. Recently, E-cadherins have been also implicated in a number of biological processes, including cell growth and differentiation, cell recognition, and sorting during developmental morphogenesis, as well as in aggregation-dependent cell survival. As phosphatidylinositol (PI) 3-kinase and Akt play a critical role in survival pathways in response to both growth factors and extracellular stimuli, these observations prompted us to explore whether E-cadherins could affect intracellular molecules regulating the activity of the PI 3-kinase/Akt signaling cascade. Using Madin-Darby canine kidney cells as a model system, we show here that engagement of E-cadherins in homophylic calcium-dependent cell-cell interactions results in a rapid PI 3-kinase-dependent activation of Akt and the subsequent translocation of Akt to the nucleus. Moreover, we demonstrate that the activation of PI 3-kinase in response to cell-cell contact formation involves the phosphorylation of PI 3-kinase in tyrosine residues, and the concomitant recruitment of PI 3-kinase to E-cadherin-containing protein complexes. These findings indicate that E-cadherins can initiate outside-in signal transducing pathways that regulate the activity of PI 3-kinase and Akt, thus providing a novel molecular mechanism whereby the interaction among neighboring cells and their adhesion status may ultimately control the fate of epithelial cells.