Phosphorylation In Epithelial Cells Research Articles

Epithelial loss of mitochondrial oxidative phosphorylation leads to disturbed enamel and impaired dentin matrix formation in postnatal developed mouse incisor

The formation of dentin and enamel matrix depends on reciprocal interactions between epithelial-mesenchymal cells. To assess the role of mitochondrial function in amelogenesis and dentinogenesis, we studied postnatal incisor development in K320E-TwinkleEpi mice. In these mice, a loss of mitochondrial DNA (mtDNA), followed by a severe defect in the oxidative phosphorylation system is induced specifically in Keratin 14 (K14+) expressing epithelial cells. Histochemical staining showed severe reduction of cytochrome c oxidase activity only in K14+ epithelial cells. In mutant incisors, H&E staining showed severe defects in the ameloblasts, in the epithelial cells of the stratum intermedium and the papillary cell layer, but also a disturbed odontoblast layer. The lack of amelogenin in the enamel matrix of K320E-TwinkleEpi mice indicated that defective ameloblasts are not able to form extracellular enamel matrix proteins. In comparison to control incisors, von Kossa staining showed enamel biomineralization defects and dentin matrix impairment. In mutant incisor, TUNEL staining and ultrastructural analyses revealed differentiation defects, while in hair follicle cells apoptosis is prevalent. We concluded that mitochondrial oxidative phosphorylation in epithelial cells of the developed incisor is required for Ca2+ homeostasis to regulate the formation of enamel matrix and induce the differentiation of ectomesenchymal cells into odontoblasts.

Ras inhibits TGF-β-induced KLF5 acetylation and transcriptional complex assembly via regulating SMAD2/3 phosphorylation in epithelial cells.

Acetylated Kruppel-like factor 5 (KLF5) is essential for transforming growth factor-β (TGF-β) to properly regulate gene transcription in the inhibition of cell proliferation and tumor growth. Ras oncogenic signaling can convert TGF-β from a tumor suppressor to a tumor promoter; however, its ability to utilize the KLF5 transcription factor to modulate TGF-β functions is still unknown. Therefore, in this study, we sought to determine whether Ras signaling altered TGF-β-induced KLF5 acetylation and the assembly of the p300-KLF5-SMADs transcriptional complex in gene regulation. Not only did we determine that Ras signaling inhibited TGF-β-induced KLF5 acetylation and interfered with TGF-β function in p15 induction and Myc repression, but also TGF-β-induced SMAD3 C-terminal region phosphorylation was necessary for TGF-β to induce KLF5 acetylation. Moreover, Ras activation further interrupted the interactions amongst p300, KLF5, and SMAD4, as well as the binding of p300-KLF5-SMADs complex onto the TGF-β-responsive promoter elements for both p15 and Myc. These findings suggested that KLF5 mediated the crosstalk between TGF-β and Ras signaling, and that suppression of TGF-β-induced KLF5 acetylation by Ras activation; this altered TGF-β-induced assembly of p300-KLF5-SMADs complex onto gene promoters to convert the function of TGF-β in gene regulation.

Peyer's patch myeloid cells infection by Listeria signals through gp38+ stromal cells and locks intestinal villus invasion.

The foodborne pathogen Listeria monocytogenes (Lm) crosses the intestinal villus epithelium via goblet cells (GCs) upon the interaction of Lm surface protein InlA with its receptor E-cadherin. Here, we show that Lm infection accelerates intestinal villus epithelium renewal while decreasing the number of GCs expressing luminally accessible E-cadherin, thereby locking Lm portal of entry. This novel innate immune response to an enteropathogen is triggered by the infection of Peyer's patch CX3CR1+ cells and the ensuing production of IL-23. It requires STAT3 phosphorylation in epithelial cells in response to IL-22 and IL-11 expressed by lamina propria gp38+ stromal cells. Lm-induced IFN-γ signaling and STAT1 phosphorylation in epithelial cells is also critical for Lm-associated intestinal epithelium response. GC depletion also leads to a decrease in colon mucus barrier thickness, thereby increasing host susceptibility to colitis. This study unveils a novel innate immune response to an enteropathogen, which implicates gp38+ stromal cells and locks intestinal villus invasion, but favors colitis.

The Effects of Retinoic Acid and MAPK Inhibitors on Phosphorylation of Smad2/3 Induced by Transforming Growth Factor β1.

BackgroundTransforming growth factor β (TGF-β), retinoic acid (RA), p38 mitogen-activated protein kinase (MAPK), and MEK signaling play critical roles in cell differentiation, proliferation, and apoptosis. We investigated the effect of RA and the role of these signaling molecules on the phosphorylation of Smad2/3 (p-Smad2/3) induced by TGF-β1.MethodsA549 epithelial cells and CCD-11Lu fibroblasts were incubated and stimulated with or without all-trans RA (ATRA) and TGF-β1 and with MAPK or MEK inhibitors. The levels of p-Smad2/3 were analyzed by western blotting. For animal models, we studied three experimental mouse groups: control, bleomycin, and bleomycin+ATRA group. Changes in histopathology, lung injury score, and levels of TGF-β1 and Smad3 were evaluated at 1 and 3 weeks.ResultsWhen A549 cells were pre-stimulated with TGF-β1 prior to RA treatment, RA completely inhibited the p-Smad2/3. However, when A549 cells were pre-treated with RA prior to TGF-β1 stimulation, RA did not completely suppress the p-Smad2/3. When A549 cells were pre-treated with MAPK inhibitor, TGF-β1 failed to phosphorylate Smad2/3. In fibroblasts, p38 MAPK inhibitor suppressed TGF-β1-induced p-Smad2. In a bleomycin-induced lung injury mouse model, RA decreased the expression of TGF-β1 and Smad3 at 1 and 3 weeks.ConclusionRA had inhibitory effects on the phosphorylation of Smad induced by TGF-β1 in vitro, and RA also decreased the expression of TGF-β1 at 1 and 3 weeks in vivo. Furthermore, pre-treatment with a MAPK inhibitor showed a preventative effect on TGF-β1/Smad phosphorylation in epithelial cells. As a result, a combination of RA and MAPK inhibitors may suppress the TGF-β1-induced lung injury and fibrosis.

Transforming Growth Factor β-Induced Smad1/5 Phosphorylation in Epithelial Cells Is Mediated by Novel Receptor Complexes and Is Essential for Anchorage-Independent Growth

Transforming growth factor beta (TGF-beta) signals predominantly through a receptor complex comprising ALK5 and TbetaRII to activate receptor-regulated Smads (R-Smads) Smad2 and Smad3. In endothelial cells, however, TGF-beta can additionally activate Smad1 and Smad5. Here, we report that TGF-beta also strongly induces phosphorylation of Smad1/5 in many different normal epithelial cells, epithelium-derived tumor cells, and fibroblasts. We demonstrate that TbetaRII and ALK5, as well as ALK2 and/or ALK3, are required for TGF-beta-induced Smad1/5 phosphorylation. We show that the simultaneous activation of the R-Smads Smad2/3 and Smad1/5 by TGF-beta results in the formation of mixed R-Smad complexes, containing, for example, phosphorylated Smad1 and Smad2. The prevalence of these mixed R-Smad complexes explains why TGF-beta-induced Smad1/5 phosphorylation does not result in transcriptional activation via bone morphogenetic protein (BMP)-responsive elements, which bind activated Smad1/5-Smad4 complexes that are induced by BMP stimulation. Thus, TGF-beta induces two parallel pathways: one signaling via Smad2-Smad4 or Smad3-Smad4 complexes and the other signaling via mixed R-Smad complexes. Finally, we assess the function of the novel arm of TGF-beta signaling and show that TGF-beta-induced Smad1/5 activation is not required for the growth-inhibitory effects of TGF-beta but is specifically required for TGF-beta-induced anchorage-independent growth.

Preferential Phosphorylation of Focal Adhesion Kinase Tyrosine 861 Is Critical for Mediating an Anti-apoptotic Response to Hyperosmotic Stress

The results presented here demonstrate that focal adhesion kinase (FAK) Tyr-861 is the predominant tyrosine phosphorylation site stimulated by hyperosmotic stress in a variety of cell types, including epithelial cell lines (ileum-derived IEC-18, colon-derived Caco2, and stomach-derived NCI-N87), FAK null fibroblasts re-expressing FAK, and Src family kinase triple null fibroblasts (SYF cells) in which c-Src has been restored (YF cells). We show that hyperosmotic stress-stimulated FAK phosphorylation in epithelial cells is inhibited by Src family kinase inhibitors PP2 and SU6656 and that it does not occur in SYF cells. Unexpectedly, hyperosmotic stress-induced phosphorylation of FAK at Tyr-397, Tyr-576, and most dramatically at Tyr-861 was completely insensitive to the F-actin-disrupting agents, latrunculin A and cytochalasin D. Finally, we show that in FAK null cells exposed to hyperosmotic stress or growth factor withdrawal, re-expression of wild type FAK restored cell survival, whereas re-expression of FAK mutated from tyrosine to phenylalanine at position 861 (FAKY861F) did not. Our results indicate that FAK Tyr-861 phosphorylation is required for mammalian cell survival of hyperosmotic stress. Furthermore, the results suggest that FAK is an upstream regulator (rather than downstream effector) of F-actin reorganization in response to hyperosmotic stress. We propose that FAK/c-Src bipartite enzyme is a sensor of cytoplasmic shrinkage, and that the phosphorylation on FAK Tyr-861 by Src and subsequent reorganization of F-actin can initiate an anti-apoptotic signaling pathway that protects cells from hyperosmotic stress.

Nitric oxide attenuates hydrogen peroxide-induced barrier disruption and protein tyrosine phosphorylation in monolayers of intestinal epithelial cell

The intestinal epithelium provides a barrier to the transport of harmful luminal molecules into the systemic circulation. A dysfunctional epithelial barrier is closely associated with the pathogenesis of a variety of intestinal and systemic disorders. We investigated here the effects of nitric oxide (NO) and hydrogen peroxide (H2O2) on the barrier function of a human intestinal epithelial cell line, Caco-2. When treated with H2O2, Caco-2 cell monolayers grown on permeable supports exhibited several remarkable features of barrier dysfunction as follows: a decrease in transepithelial electrical resistance, an increase in paracellular permeability to dextran, and a disruption of the intercellular junctional localization of the scaffolding protein ZO-1. In addition, an induction of tyrosine phosphorylation of numerous cellular proteins including ZO-1, E-cadherin, and β-catenin, components of tight and adherens junctions, was observed. On the other hand, combined treatment of Caco-2 monolayers with H2O2 and an NO donor (NOC5 or NOC12) relieved the damage to the barrier function and suppressed the protein tyrosine phosphorylation induced by H2O2 alone. These results suggest that NO protects the barrier function of intestinal epithelia from oxidative stress by modulating some intracellular signaling pathways of protein tyrosine phosphorylation in epithelial cells.

Epithelial Cells Are Sensitive Detectors of Bacterial Pore-forming Toxins

Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins (including pneumolysin from Streptococcus pneumoniae, alpha-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis) at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial poreforming toxins, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by the insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response, while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.

Depolarization induces Rho-Rho kinase-mediated myosin light chain phosphorylation in kidney tubular cells

Myosin-based contractility plays important roles in the regulation of epithelial functions, particularly paracellular permeability. However, the triggering factors and the signaling pathways that control epithelial myosin light chain (MLC) phosphorylation have not been elucidated. Herein we show that plasma membrane depolarization provoked by distinct means, including high extracellular K(+), the lipophilic cation tetraphenylphosphonium, or the ionophore nystatin, induced strong diphosphorylation of MLC in kidney epithelial cells. In sharp contrast to smooth muscle, depolarization of epithelial cells did not provoke a Ca(2+) signal, and removal of external Ca(2+) promoted rather than inhibited MLC phosphorylation. Moreover, elevation of intracellular Ca(2+) did not induce significant MLC phosphorylation, and the myosin light chain kinase (MLCK) inhibitor ML-7 did not prevent the depolarization-induced MLC response, suggesting that MLCK is not a regulated element in this process. Instead, the Rho-Rho kinase (ROK) pathway is the key mediator because 1) depolarization stimulated Rho and induced its peripheral translocation, 2) inhibition of Rho by Clostridium difficile toxin B or C3 transferase abolished MLC phosphorylation, and 3) the ROK inhibitor Y-27632 suppressed the effect. Importantly, physiological depolarizing stimuli were able to activate the same pathway: L-alanine, the substrate of the electrogenic Na(+)-alanine cotransporter, stimulated Rho and induced Y-27632-sensitive MLC phosphorylation in a Na(+)-dependent manner. Together, our results define a novel mode of the regulation of MLC phosphorylation in epithelial cells, which is depolarization triggered and Rho-ROK-mediated but Ca(2+) signal independent. This pathway may be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and thereby regulate paracellular transport.

Determinants and consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter pylori

Background & Aims: The Helicobacter pylori cag pathogenicity island encodes a secretory system that translocates CagA into epithelial cells, where it becomes tyrosine phosphorylated and induces cytoskeletal rearrangements. Strains with more CagA tyrosine phosphorylation motifs are most closely associated with gastric cancer. Here we assess whether clinical strains can deliver CagA, whether strains with different numbers of CagA phosphorylation motifs have CagA phosphorylated to different degrees, and whether this induces different amounts of epithelial cytoskeletal change. Methods: Forty-four H. pylori strains from South African patients, all cagA gene positive, were cocultured with the gastric adenocarcinoma cell line AGS. CagA expression and phosphorylation were determined by Western blot and interleukin-8 secretion by enzyme-linked immunosorbent assay. The cagA 3′ variable regions of 22 strains were sequenced and shown to possess 3–6 phosphorylation motifs. These strains were used to quantify CagA phosphorylation and cytoskeletal rearrangements. Results:cagA genotype and typing of cag pathogenicity island genes were poorly predictive of phenotype. Thirty-four of 44 strains expressed CagA protein that could be delivered to and phosphorylated within AGS cells. Only these 34 strains induced interleukin-8 secretion from AGS cells. Among those strains, the number of CagA tyrosine phosphorylation motifs determined the degree of CagA phosphorylation and the level of biologic activity in terms of degree and extent of AGS cell elongation. Conclusions:H. pylori strains that deliver CagA with more phosphorylation motifs induce higher levels of CagA phosphorylation in epithelial cells, induce more cytoskeletal changes, and are more likely to be associated with gastric cancer.

Activation of extracellular regulated kinases is required for the increase in airway epithelial permeability during leukocyte transmigration.

The goal of this study was to determine whether the extracellular regulated kinases (ERK1/2) are involved in leukocyte transmigration across airway epithelium and the associated changes in epithelial permeability. In vitro, we used formyl-methionyl-leucyl-phenylalanine (fMLP) to induce migration of HL-60 cells (a human leukocyte cell line) across sheets of polarized Calu-3 airway epithelial cells and also to induce migration of human neutrophils across primary cultures of cow tracheal epithelial cells. In both systems, leukocyte migration decreased transepithelial electrical resistance (R(te)), increased epithelial permeability to albumin (P(alb)), and increased ERK1/2 phosphorylation in epithelial cells. Leukocyte migration and the associated changes in R(te), P(alb), and ERK1/2 phosphorylation were inhibited by calphostin C, a blocker of protein kinase C (PKC), and by PD98059 (a blocker of ERK1/2). Leukocyte transmigration in rat tracheas in vivo was induced with fMLP, and was associated with increased P(alb) and phosphorylation of epithelial ERK1/2. Again, migration and the associated changes were inhibited by luminal PD98059 or calphostin C though neither agent affected rat leukocyte migration in Boyden chambers in vitro. We conclude that PKC and ERK1/2 pathways are activated in airway epithelial cells during migration of leukocytes and are important regulators of airway epithelial permeability.

PPARγ activation inhibits interferon-γ stimulated STATI and JNK phosphorylation in epithelial cells

PPARγ activation inhibits interferon-γ stimulated STATI and JNK phosphorylation in epithelial cells

Epithelial cell adhesion to extracellular matrix proteins induces tyrosine phosphorylation of the Epstein-Barr virus latent membrane protein 2: a role for C-terminal Src kinase.

The Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) is expressed in latently EBV-infected B cells, where it forms patches in the plasma membrane and interferes with B-cell receptor signal transduction through dominant-negative effects on protein kinases. LMP2 transcripts are detected in nasopharyngeal carcinoma, an epithelial-cell malignancy. In this study the function of LMP2A in epithelial cells was investigated. LMP2A was found to coprecipitate with protein kinase activities and to become phosphorylated in in vitro kinase assays. Analysis of LMP2A deletion mutants demonstrated that tyrosines implicated in interacting with Src family kinase SH2 domains and the SH2 domain of Csk, as well as the LMP2A immunoreceptor tyrosine-based activation motif, are important for its phosphorylation in epithelial cells. LMP2A tyrosine phosphorylation was triggered by cell adhesion to extracellular-matrix (ECM) proteins. Src family kinases, whose involvement in cell-ECM signaling and LMP2A phosphorylation in B lymphocytes has been well established, were found not to be responsible for LMP2A phosphorylation in epithelial cells. Instead, coexpression of Csk, a negative Src regulator, and LMP2A led to an increase in LMP2A phosphorylation both in nonadherent cells and upon cell adhesion. Csk also phosphorylated LMP2A in vitro. These results suggest that LMP2A has a different role in epithelial cells, where it interacts with cell adhesion-initiated signaling pathways. Although tyrosine phosphorylation of LMP2A occurs in both cell types, different protein kinases seem to be used: Src family kinases in B lymphocytes and Csk in epithelial cells.

Evidence for autocrine basis of transformation in NIH-3T3 cells transfected with met/HGF receptor gene.

NIH-3T3 cells transformed with met/HGF receptor gene proliferate in the absence of serum and growth factors. Immunocytochemical staining with anti-HGF antibody revealed intense HGF staining in the transfected cells. Additionally, these cells secrete bioactive HGF as evidenced by the ability of the conditioned media to stimulate met/HGF receptor phosphorylation in epithelial cells, and to promote migration of bovine adrenal capillary endothelial cells in a modified Boyden chamber assay. The migration of endothelial cells could be specifically inhibited by anti-HGF antibody but not by an irrelevant antibody. Suramin, a drug known to disrupt ligand-receptor interactions, inhibits the serum and growth-factor free proliferation, and the endogenous phosphorylation of met/HGF receptor in the transformed cells. Taken together, our data suggests an autocrine mode of transformation in NIH-3T3 cells transfected with met/HGF receptor gene.

Protein kinase C mediates cholinergically regulated protein phosphorylation in a Cl(-)-secreting epithelium

T84 cell monolayers were used to study the cholinergic regulation of protein phosphorylation in epithelial cells. When T84 cell monolayers are labeled with 32Pi and stimulated with carbachol, six proteins exhibit altered phosphorylation. The most prominent response is a fivefold increase in labeling of p83, an acidic protein of Mr 83,000. Increasing labeling of p83 parallels stimulated secretion with respect to the onset of agonist action, agonist potency, and antagonism by atropine. However, the p83 and secretory responses differ in that the p83 response is more sustained. When T84 cell fractions are incubated with [gamma-32P]ATP, Ca2(+)-phospholipid stimulates p83 labeling. Phosphorylation of p83 also occurs when a T84 cell extract is incubated with purified protein kinase C and when intact cells are exposed to phorbol myristate acetate. p83 does not become phosphorylated in cell fractions incubated with adenosine 3',5'-cyclic monophosphate (cAMP) or in monolayers stimulated with agonists acting via cAMP. Thus carbachol stimulates the phosphorylation of an endogenous substrate for protein kinase C in T84 cells. The duration of this phosphorylation response suggests that protein kinase C may mediate a sustained response to carbachol, possibly acting to limit the duration of stimulated secretion.