Phosphatidylinositol 3-kinase is required for adherens junction-dependent mammary epithelial cell spheroid formation

Adherens junctions facilitate and maintain epithelial cell-cell adhesion. This is true of mammary epithelial cells, both in two dimensional monolayers and in three-dimensional basement membrane cultures. Using the immortalized, functional mouse mammary epithelial scp2 cell line, we found that pharmacological inhibition of phosphatidylinositol 3-kinase (PI3-kinase) disrupted adherens junctions. In monolayers, this disruption was associated with decreased E-cadherin and beta-catenin at sites of cell-cell contact and decreased association of both proteins with the cytoskeleton. Changes in the distribution of f-actin after PI3-kinase inhibition suggest that this disruption of adherens junctions may be mediated by alterations to the cytoskeleton. In basement membrane cultures, PI3-kinase inhibition reversibly prevented adherens junction-dependent spheroid formation and differentiative milk protein gene expression, both in scp2 cells and in a second mouse mammary epithelial cell line, EpH4. Decreasing the calcium concentration in the culture medium produced similar, although less dramatic, phenotypic effects. These data indicate that adherens junctions contribute, at least in part, to the efficient induction of basement membrane-dependent differentiation of mammary epithelial cells.     

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.     

Oxidative stress-induced intestinal epithelial cell apoptosis is mediated by p38 MAPK

Free oxygen radicals are involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. The stress-activated p38 mitogen-activated protein kinase (MAPK) has been implicated in gut injury. Here, we found that phosphorylated p38 was detected primarily in the villus tips of normal intestine, whereas it was expressed in the entire mucosa in NEC. H(2)O(2) treatment resulted in a rapid phosphorylation of p38 MAPK and subsequent apoptosis of rat intestinal epithelial (RIE)-1 cells; this induction was attenuated by treatment with SB203580, a selective p38 MAPK inhibitor, or transfection with p38alpha siRNA. Moreover, SB203580 also blocked H(2)O(2)-induced PKC activation. In contrast, the PKC inhibitor (GF109203x) did not affect p38 activation, indicating that p38 MAPK activation occurs upstream of PKC activation in H(2)O(2)-induced apoptosis. H(2)O(2) treatment also decreased mitochondrial membrane potential; pretreatment with SB203580 attenuated this response. Our study demonstrates that the p38 MAPK/PKC pathway plays an important role as a pro-apoptotic cellular signaling during oxidative stress-induced intestinal epithelial cell injury.     

The tumor suppressor CYLD controls epithelial morphogenesis and homeostasis by regulating mitotic spindle behavior and adherens junction assembly

Epithelial morphogenesis and homeostasis are essential for animal development and tissue regeneration,and epithelial disorganization is associated with developmental disorders and tumorigenesis.However,the molecular mechanisms that contribute to the morphogenesis and homeostasis of the epithelium remain elusive.Herein,we report a novel role for the cylindromatosis(CYLD)tumor suppressor in these events.Our results show that CYLD depletion disrupts epithelial organization in both Drosophila egg chambers and mouse skin and intestinal epithelia.Microscopic analysis of proliferating cells in mouse epithelial tissues and cultured organoids reveals that loss of CYLD synergizes with tumor-promoting agents to cause the misorientation of the mitotic spindle.Mechanistic studies show that CYLD accu?mulates at the cell cortex in epithelial tissues and cultured cells,where it promotes the formation of epithelial adherens junctions through the modulation of microtubule dynamics.These data suggest that CYLD controls epithelial morphogenesis and homeostasis by modulating the assembly of adherens junctions and ensuring proper orientation of the mitotic spindle.Our findings thus provide novel insight into the role of CYLD in development,tissue homeostasis,and tumorigenesis.     

Epidermal growth factor-stimulated intestinal epithelial cell migration requires Src family kinase-dependent p38 MAPK signaling

Members of the epidermal growth factor (EGF) family of ligands and their receptors regulate migration and growth of intestinal epithelial cells. However, our understanding of the signal transduction pathways determining these responses is incomplete. In this study we tested the hypothesis that p38 is required for EGF-stimulated intestinal epithelial monolayer restitution. EGF-stimulated migration in a wound closure model required continuous presence of ligand for several hours for maximal response, suggesting a requirement for sustained signal transduction pathway activation. In this regard, prolonged exposure of cells to EGF activated p38 for up to 5 h. Furthermore genetic or pharmacological blockade of p38 signaling inhibited the ability of EGF to accelerate wound closure. Interestingly p38 inhibition was associated with increased EGF-stimulated ERK1/ERK2 phosphorylation and cell proliferation, suggesting that p38 regulates the balance of proliferation/migration signaling in response to EGF receptor activity. Activation of p38 in intestinal epithelial cells through EGF receptor was abolished by blockade of Src family tyrosine kinase signaling but not inhibition of phosphatidylinositol 3-kinase or protein kinase C. Taken together, these data suggest that Src family kinase-dependent p38 activation is a key component of a signaling switch routing EGF-stimulated responses to epithelial cell migration/restitution rather than proliferation during wound closure.     

p38 MAPK controls prothrombin expression by regulated RNA 3' end processing

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Highlights? External signals regulate 3′ end mRNA processing through p38 MAPK activation ? The mechanism is controlled by a competitive mRNP formation in the 3′UTR ? The mechanism is activated by acute phase stress and inflammation ? The prototypic thrombin links the mechanism to blood coagulation and tumor spread     

Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate.

Vascular endothelial cells undergo morphogenesis into capillary networks in response to angiogenic factors. We show here that sphingosine-1-phosphate (SPP), a platelet-derived bioactive lipid, activates the EDG-1 and -3 subtypes of G protein-coupled receptors on endothelial cells to regulate angiogenesis. SPP induces the Gi/mitogen-activated protein kinase/cell survival pathway and the small GTPase Rho- and Raccoupled adherens junction assembly. Both EDG-1-and EDG-3-regulated signaling pathways are required for endothelial cell morphogenesis into capillary-like networks. Indeed, SPP synergized with polypeptide angiogenic growth factors in the formation of mature neovessels in vivo. These data define SPP as a novel regulator of angiogenesis.     

Differential activation of p38MAPK isoforms by MKK6 and MKK3

All four members of the mammalian p38 mitogen-activated protein kinase (MAPK) family (p38α, p38β, p38γ and p38δ) are activated by dual phosphorylation in the TGY motif in the activation loop. This phosphorylation is mediated by three kinases, MKK3, MKK6 and MKK4, at least in vitro. The role of these MKK in the activation of p38α has been demonstrated in studies using fibroblasts that lack MKK3 and/or MKK6. Nonetheless, the physiological upstream activators of the other p38MAPK isoforms have not yet been reported using MKK knockout cells. In this study, we examined p38β, γ and δ activation by MKK3 and MKK6, in cells lacking MKK3, MKK6 or both. We show that MKK3 and MKK6 are both essential for the activation of p38γ and p38β induced by environmental stress, whereas MKK6 is the major p38γ activator in response to TNFα. In contrast, p38δ activation by ultraviolet radiation, hyperosmotic shock, anisomycin or by TNFα is mediated by MKK3. Moreover, in response to osmotic stress, MKK3 and MKK6 are crucial in regulating the phosphorylation of the p38γ substrate hDlg and its activity as scaffold protein. These data indicate that activation of distinct p38MAPK isoforms is regulated by the selective and synchronized action of two kinases, MKK3 and MKK6, in response to cell stress.     

p38 MAPK interacts with actin and modulates filament assembly during skeletal muscle differentiation

Skeletal muscle differentiation is marked by enhanced myotube formation and increased cytoskeletal rearrangement. Actin, a cytoskeletal protein is involved in various cellular functions such as glucose transport, intracellular trafficking, cell shape, and coordinated cell movement in response to various extracellular signals. The present study reveals an association between actin and p38 MAPK only in differentiated myotubes, not in proliferating myoblasts. Actin filament disassembly caused by cytochalasinD can be reversed using the potent activator of p38 MAPK, anisomycin. Pretreatment of myotubes with anisomycin partially resisted the effect of cytochalasinD. However, inhibition of p38 MAPK completely abolished the anisomycin-mediated actin remodeling. Data suggests that p38 MAPK interacts with actin and modulates actin filament rearrangement in differentiated L6E9 skeletal muscle cells.     

APPL1 mediates adiponectin-stimulated p38 MAPK activation by scaffolding the TAK1-MKK3-p38 MAPK pathway

The adaptor protein APPL1 mediates the stimulatory effect of adiponectin on p38 mitogen-activated protein kinase (MAPK) signaling, yet the underlying mechanism remains unclear. Here we show that, in C(2)C(12) cells, overexpression or suppression of APPL1 enhanced or suppressed, respectively, adiponectin-stimulated p38 MAPK upstream kinase cascade, consisting of transforming growth factor-β-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase 3 (MKK3). In vitro affinity binding and coimmunoprecipitation experiments revealed that TAK1 and MKK3 bind to different regions of APPL1, suggesting that APPL1 functions as a scaffolding protein to facilitate adiponectin-stimulated p38 MAPK activation. Interestingly, suppressing APPL1 had no effect on TNFα-stimulated p38 MAPK phosphorylation in C(2)C(12) myotubes, indicating that the stimulatory effect of APPL1 on p38 MAPK activation is selective. Taken together, our study demonstrated that the TAK1-MKK3 cascade mediates adiponectin signaling and uncovers a scaffolding role of APPL1 in regulating the TAK1-MKK3-p38 MAPK pathway, specifically in response to adiponectin stimulation.     

Amelogenin promotes odontoblast-like MDPC-23 cell differentiation via activation of ERK1/2 and p38 MAPK

Amelogenin (AMG) is a highly conserved protein secreted by ameloblasts. Some research indicates that AMG might induce the differentiation and maturation of odontoblasts. The aim of this study was to clarify the function of AMG during the differentiation of odontoblast-like MDPC-23 cells. The results revealed that the alkaline phosphatase activity and the number of mineralized nodules were significantly enhanced in AMG-overexpressing MDPC-23 cells during the mineralization process. Tissue-specific markers such as dentin matrix protein 1 and dentin sialophosphoprotein also elevated significantly, indicating the cell differentiation and maturation process. Furthermore, AMG could upregulate the phosphorylation levels of ERK1/2 and p38 MAPK. However, JNK, another MAPK pathway molecule, didn't change the activity at all. And the differentiation induced by AMG was abrogated when the MDPC-23 cells were treated with U0126 and SB203580, the inhibitors of ERK1/2 and p38, respectively. Taken together, our present results showed that AMG could promote the differentiation of odontoblast-like MDPC-23 cells via ERK1/2 and p38 MAPK pathways.     

Negative regulation of EGFR-Vav2 signaling axis by Cbl ubiquitin ligase controls EGF receptor-mediated epithelial cell adherens junction dynamics and cell migration

The E3 ubiquitin ligase Casitas B lymphoma protein (Cbl) controls the ubiquitin-dependent degradation of EGF receptor (EGFR), but its role in regulating downstream signaling elements with which it associates and its impact on biological outcomes of EGFR signaling are less clear. Here, we demonstrate that stimulation of EGFR on human mammary epithelial cells disrupts adherens junctions (AJs) through Vav2 and Rac1/Cdc42 activation. In EGF-stimulated cells, Cbl regulates the levels of phosphorylated Vav2 thereby attenuating Rac1/Cdc42 activity. Knockdown of Cbl and Cbl-b enhanced the EGF-induced disruption of AJs and cell motility. Overexpression of constitutively active Vav2 activated Rac1/Cdc42 and reorganized junctional actin cytoskeleton; these effects were suppressed by WT Cbl and enhanced by a ubiquitin ligase-deficient Cbl mutant. Cbl forms a complex with phospho-EGFR and phospho-Vav2 and facilitates phospho-Vav2 ubiquitinylation. Cbl can also interact with Vav2 directly in a Cbl Tyr-700-dependent manner. A ubiquitin ligase-deficient Cbl mutant enhanced the morphological transformation of mammary epithelial cells induced by constitutively active Vav2; this effect requires an intact Cbl Tyr-700. These results indicate that Cbl ubiquitin ligase plays a critical role in the maintenance of AJs and suppression of cell migration through down-regulation of EGFR-Vav2 signaling.     

Gas1 cooperates with Cdo and promotes myogenic differentiation via activation of p38MAPK

Skeletal myogenesis is a multistep process that involves cell cycle exit, expression of muscle-specific genes and formation of multinucleated myotubes. Growth arrest specific gene 1 (Gas1) is a GPI-linked membrane protein and originally identified as a growth arrest-linked gene in fibroblasts. Promyogenic cell surface protein, Cdo functions as a component of multiprotein complexes that include other cell adhesion molecules, like Cadherins to mediate cell contact signaling. Here we report that Gas1 and Cdo are coexpressed in muscle cells and form a complex in differentiating myoblasts. Interestingly, Cdo^−/− myoblasts display defects in Gas1 induction during differentiation. Overexpression or depletion of Gas1 enhances or decreases myogenic differentiation, respectively. During myoblast differentiation, Gas1 depletion causes defects in downregulation of Cdk2 and Cyclin D1 and up-regulation of miR-322, a negative regulator of Cdk2 activities. Furthermore overexpression or knockdown of Gas1 either enhances or decreases activation of p38MAPK that functions downstream of Cdo. Additionally, Gas1 overexpression in Cdo-depleted C2C12 cells restores p38MAPK activities and differentiation abilities. These data suggest that Gas1 promotes myogenic differentiation through regulation of cell cycle arrest and is critical to activate p38MAPK, most likely via association with Cdo/Cadherin multiprotein complexes.     

Myogenic differentiation requires signalling through both phosphatidylinositol 3-kinase and p38 MAP kinase

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) or of Akt induces myoblast differentiation. Activation of p38 MAP kinase also triggers myogenic differentiation. The current paper shows that PI 3-kinase and p38 MAP kinase signalling are activated by two separate pathways during myogenic differentiation; both are required for muscle differentiation. p38-induced myogenic differentiation can be inhibited by the PI 3-kinase inhibitor LY294002 without affecting p38 activity. Similarly, a constitutively active form of Akt, myristylated c-Akt (Myr-Akt), induces myogenic differentiation that is inhibited by the p38 inhibitor SB203580. An analysis of the two forms of p38, p38 and p38beta, shows that the activity of both is required for myogenic differentiation. These data suggest that PI 3-kinase and p38 signalling are essential and parallel pathways for myogenic differentiation. They may either affect different downstream targets required for myogenesis or they may converge on shared targets that require input from both signalling pathways.     

Unc119 regulates myofibroblast differentiation through the activation of Fyn and the p38 MAPK pathway

Unc119 is an adaptor protein that is involved in the development of the vertebrate nervous system. We have shown that Unc119 stimulates the induction of alpha-smooth muscle actin (alpha-SMA) and myofibroblast differentiation by TGF-beta in human lung fibroblasts. Unc119 increases the kinase activity of Fyn and associates with it in coprecipitation and colocalization studies. Phosphorylation and activation of Fyn in response to TGF-beta and platelet-derived growth factor is delayed in Unc119-deficient cells. This delay translates into suppressed cell proliferation. In Src family kinase-deficient (SYF) cells, Unc119 knockdown does not affect cell proliferation. The result suggests that Unc119 interacts with Fyn in the early stages of signal generation and its presence is essential for conducive signal transduction. Unc119 overexpression does not stimulate alpha-SMA in SYF cells and this defect is restored upon reconstitution with Fyn indicating that Unc119 stimulation of alpha-SMA requires at least Fyn. Unc119 overexpression stimulated p38, but not JNK, phosphorylation. Blocking p38 MAPK resulted in reduced alpha-SMA expression by Unc119 suggesting that the p38 pathway regulates Unc119-induced myofibroblast differentiation. Unc119 stimulates the production of TGF-beta and IL-6, known inducers of myofibroblast differentiation. Thus, Unc119 regulates receptor-mediated signal transduction and myofibroblast differentiation by activating Fyn and the p38 MAPK pathway. Using primary lung fibroblasts from patients with fibrotic lung diseases and control subjects, we show that the expression of alpha-smooth muscle actin is highly correlated with that of Unc119. Taken together, our results suggest that Unc119 plays an important role in fibrotic processes through myofibroblast differentiation.     

Sphingosine-1-phosphate stimulates human Caco-2 intestinal epithelial proliferation via p38 activation and activates ERK by an independent mechanism

Sphingosine-1-phosphate (S-1-P) has been identified as an extracellular mediator and an intracellular second messenger that may modulate cell motility, adhesion, proliferation, and differentiation and cancer cell invasion. Widely distributed, S-1-P is most abundant in the intestine. Although S-1-P is likely to modulate various intracellular pathways, activation of the mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase 1 (ERK1), ERK2, and p38 is among the best-characterized S-1-P effects. Because the MAPKs regulate proliferation, we hypothesized that S-1-P might stimulate intestinal epithelial cell proliferation by MAPK activation. Human Caco-2 intestinal epithelial cells were cultured on a fibronectin matrix because fibronectin is an important constituent of the gut mucosal basement membrane. We assessed ERK1, ERK2, and p38 activation by Western blotting with antibodies specific for their active forms and proliferation by Coulter counting at 24 h. Specific MAP kinase kinase (MEK) and p38 inhibitors PD98059 (20 microM) and SB202190 and SB203580 (10 and 20 microM) were used to probe the role of ERK and p38 in S-1-P-mediated proliferation. Three or more similar studies were pooled for the analysis. S-1-P stimulated Caco-2 proliferation and dose-responsively activated ERK1, ERK2, and p38. Proliferation peaked at 5 microM, yielding a cell number 166.3 +/- 2.7% of the vehicle control (n = 6, P < 0.05). S-1-P also maximally stimulated ERK1, ERK2, and p38 at 5 microM, to 164.4 +/- 19.9%, 232.2 +/- 38.5%, and 169.2 +/- 20.5% of the control, respectively. Although MEK inhibition prevented S-1-P activation of ERK1 and ERK2 and slightly but significantly inhibited basal Caco-2 proliferation, MEK inhibition did not block the S-1-P mitogenic effect. However, pretreatment with 10 microM SB202190 or SB203580 (putative p38 inhibitors) attenuated the stimulation of proliferation by S-1-P. Twenty micromolars of SB202190 or SB203580 completely blocked the mitogenic effect of S-1-P. Ten to twenty micromolars of SB202190 and SB203580 also dose-dependently ablated the effects of 5 microM S-1-P on heat shock protein 27 accumulation, a downstream consequence of p38 MAPK activation. Consistent with the reports in some other cell types, S-1-P appears to activate ERK1, ERK2, and p38 and to stimulate proliferation. However, in contrast to the mediation of the S-1-P effects in some other cell types, S-1-P appears to stimulate human intestinal epithelial proliferation by activating p38. ERK activation by S-1-P is not required for its mitogenic effect.