Chronic ethanol downregulates PKA activation and ciliary beating in bovine bronchial epithelial cells

Previously, we reported that ethanol (EtOH) stimulates a rapid increase in ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBEC). Agents activating cAMP-dependent protein kinase (PKA) also stimulate CBF. EtOH stimulates BBEC CBF through cyclic nucleotide kinase activation. However, EtOH-stimulated CBF is maximal by 1 h and subsides by 6 h, returning to baseline by 24 h. We hypothesized that the loss of EtOH-stimulated CBF was a result of downregulation of PKA activity. To determine the PKA activation state in response to EtOH, ciliated BBEC were stimulated for 0--72 h with various concentrations of EtOH and assayed for PKA. EtOH (100 mM) treatment of the cells for 1 h increased PKA activity threefold over unstimulated controls. PKA activity decreased with increasing time from 6 to 24 h. When BBEC were preincubated with 100 mM EtOH for 24 h, the stimulation of PKA by isoproterenol or 8-bromo-cAMP was abrogated. EtOH desensitizes BBEC to PKA-activating agents, suggesting that EtOH rapidly stimulates, whereas long-term EtOH downregulates, CBF via PKA in BBEC.     

Morphine stimulates cell migration of oral epithelial cells by delta-opioid receptor activation

Oral mucositis is one of the most common side effects of chemoradiation regimens and manifestation can be dose-limiting for the therapy, can impair the patient''s nutritional condition and quality of life due to severe pain. The therapeutic options are limited; often only an alleviation of the symptoms such as pain reduction by using systemic opioids is possible. Stimulating opioid receptors on peripheral neurons and dermal tissue, potent analgesic effects are induced e.g. in skin grafted patients. Advantageous effects on the cell migration and, thus, on the wound healing process are described, too. In this study, we investigated whether opioid receptors are also expressed on oral epithelial cells and if morphine can modulate their cell migration behavior. The expression of the opioid receptors MOR, DOR and KOR on primary human oral epithelial cells was verified. Furthermore, a significantly accelerated cell migration was observed following incubation with morphine. The effect even slightly exceeded the cell migration stimulating effect of TGF-ß: After 14 h of morphine treatment about 86% of the wound area was closed, whereas TGF-ß application resulted in a closed wound area of 80%. With respect to morphine stimulated cell migration we demonstrate that DOR plays a key role and we show the involvement of the MAPK members Erk 1/2 and p38 using Western blot analysis.Further studies in more complex systems in vitro and in vivo are required. Nevertheless, these findings might open up a new therapeutic option for the treatment of oral mucositis.     

uPA/plasmin system-mediated MMP-9 activation is implicated in bronchial epithelial cell migration

To examine the effects of the uPA/plasmin system on cell migration in relation to the activation of MMP-9, we used ex vivo and in vitro wound-repair models of human bronchial epithelial cells and videomicroscopy techniques that make possible cell tracking and quantification of cell migration speeds. We observed that uPA was only detected in migrating cells at the wound edges and located at crucial sites for cell/extracellular matrix interactions. The implication of uPA in human bronchial epithelial cell migration was studied by incubating cultures with a monoclonal antibody raised against uPA and these experiments led to a 70% reduction in cell velocity. To examine the effects of the plasmin system on cell migration, we incubated cultures with increasing concentrations of plasmin or activated MMP-9. We observed a significant dose-dependent increase in cell migration velocity with plasmin (P < 0.001) and MMP-9 (P < 0.001). Moreover, addition of exogenous plasmin led to a twofold increase of activated MMP-9 in migrating cells. We also demonstrated that the addition of anti-uPA IgG led to an inhibition of 43% of activated MMP-9. In conclusion, these results show that uPA is involved in human bronchial epithelial cells migration. This action is mediated by the generation of plasmin, which in turn activates MMP-9, thus making possible cell migration.     

Purified scatter factor stimulates epithelial and vascular endothelial cell migration

Fibroblasts and smooth muscle cells release a protein activity which causes epithelial sheets to "scatter" into isolated cells. Purification of scatter factor (SF) activity from ras-transformed 3T3 cells was reported recently. We purified ras-3T3 SF by a slightly different method with essentially similar findings. Purified factor showed a single band at 77 +/- 3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Scatter activity was eluted from gel slices at this molecular size. Reduction with mercaptoethanol caused the loss of activity and the appearance of two bands (58 and 31 kDa). We report the amino acid composition of ras-3T3 SF and sequences of several tryptic peptides. These sequences were not similar to the known proteins in the Protein Database. We have shown previously that partially purified ras-3T3 scatter activity stimulates migration of epithelial and vascular endothelial cells in a new migration assay utilizing microcarrier beads. We now demonstrate that the same purified ras-3T3 protein scatters epithelial cells and stimulates epithelial and endothelial migration in microcarrier bead and Boyden chamber assays. Partially purified human smooth muscle scatter activity shares these activities, but the protein(s) responsible has not been isolated. Migration-stimulating activity was maximal at ras-3T3 protein concentrations less than 10 ng/ml (0.13 nM). ras-3T3 SF had no collagenolytic activity and did not stimulate DNA synthesis in fibroblast growth factor-responsive human melanocytes. ras-3T3 SF appears to be a new protein which regulates endothelial and epithelial mobility; and, therefore, it may be involved in vascular repair and wound healing.     

Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration

Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.     

Helicobacter pylori stimulates epithelial cell migration via CagA-mediated perturbation of host cell signaling

Helicobacter pylori CagA is delivered into gastric epithelial cells, where undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif to interact with Src homology 2-containing protein tyrosine phosphatase-2 (SHP2) oncoprotein. CagA also binds to partitioning-defective 1 (PAR1) polarity-regulating kinase via the CagA multimerization (CM) sequence. To investigate pathophysiological role of CagA-SHP2 and/or CagA-PAR1 interaction in H. pylori infection, we generated H. pylori isogenic strains producing a phosphorylation-resistant CagA and a CagA without CM sequence. Infection studies revealed that deregulation of epithelial cell motility was more prominent in the wild-type strain than in the mutant strains. Thus, both CagA-SHP2 and CagA-PAR1 interactions are involved in the pathogenicity of cagA-positive H. pylori.     

Adenosine stimulates Cl- channels of nonpigmented ciliary epithelial cells

Ciliaryepithelial cells possess multiple purinergic receptors, and occupancyof A₁ andA₂ adenosine receptors isassociated with opposing effects on intraocular pressure. Aqueousadenosine produced increases in short-circuit current across rabbitciliary epithelium, blocked by removingCl and enhanced by aqueousBa²⁺. Adenosine's actions werefurther studied with nonpigmented ciliary epithelial (NPE) cells fromcontinuous human HCE and ODM lines and freshly dissected bovine cells.With gramicidin present, adenosine (3 µM) triggered isosmoticshrinkage of the human NPE cells, which was inhibited by theCl channel blockers5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) and niflumic acid. At 10 µM, the nonmetabolizable analog 2-chloroadenosine and AMP alsoproduced shrinkage, but not inosine, UTP, or ATP. 2-Chloroadenosine(1 µM) triggered increases of whole cell currents in HCE cells,which were partially reversible,Cl dependent, andreversibly inhibited by NPPB. Adenosine (10 µM) also stimulatedwhole cell currents in bovine NPE cells. We conclude that occupancy ofadenosine receptors stimulatesCl secretion in mammalianNPE cells.

     

Activation of protein kinase A accelerates bovine bronchial epithelial cell migration

Bronchial epithelial cell migration is required for the repair of damaged airway epithelium. We hypothesized that bronchial epithelial cell migration during wound repair is influenced by cAMP and the activity of its cyclic nucleotide-dependent protein kinase, protein kinase A (PKA). We found that, when confluent monolayers of bronchial epithelial cells are wounded, an increase in PKA activity occurs. Augmentation of PKA activity with a cell-permeable analog of cAMP, dibutyryl adenosine 3',5'-cyclic monophosphate, isoproterenol, or a phosphodiesterase inhibitor accelerated migration of normal bronchial epithelial cells in in vitro wound closure assays and Boyden chamber migration assays. A role for PKA activity was also confirmed with a PKA inhibitor, KT-5720, which reduced stimulated migration. Augmentation of PKA activity reduced the levels of active Rho and the formation of focal adhesions. These studies suggest that PKA activation modulates Rho activity, migration mechanisms, and thus bronchial epithelial repair mechanisms.     

Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility.     

Relaxin stimulates leukocyte adhesion and migration through a relaxin receptor LGR7-dependent mechanism

Leukocytes are critical effectors of inflammation and tumor biology. Chemokine-like factors produced by such inflammatory sites are key mediators of tumor growth that activate leukocytic recruitment and tumor infiltration and suppress immune surveillance. Here we report that the endocrine peptide hormone, relaxin, is a regulator of leukocyte biology with properties important in recruitment to sites of inflammation. This study uses the human monocytic cell line THP-1 and normal human peripheral blood mononuclear cells to define a novel role for relaxin in regulation of leukocyte adhesion and migration. Our studies indicate that relaxin promotes adenylate cyclase activation, substrate adhesion, and migratory capacity of mononuclear leukocytes through a relaxin receptor LGR7-dependent mechanism. Relaxin-stimulated cAMP accumulation was observed to occur primarily in non-adherent cells. Relaxin stimulation results in increased substrate adhesion and increased migratory activity of leukocytes. In addition, relaxin-stimulated substrate adhesion resulted in enhanced chemotaxis to monocyte chemoattractant protein-1. These responses in THP-1 and peripheral blood mononuclear cells are relaxin dose-dependent and proportional to cAMP accumulation. We further demonstrate that LGR7 is critical for mediating these biological responses by use of RNA interference lentiviral short hairpin constructs. In summary, we provide evidence that relaxin is a novel leukocyte stimulatory agent with properties affecting adhesion and chemomigration.     

Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration

Ras proteins control the signalling pathways that are responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins that initiate the mitogen-activated protein kinase (MAPK) cascade, which mediates diverse biological functions such as cell growth, survival and differentiation. Here we show that prohibitin, a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf-MEK-ERK pathway by Ras. The membrane targeting and activation of C-Raf by Ras needs prohibitin in vivo. In addition, direct interaction with prohibitin is required for C-Raf activation. C-Raf kinase fails to interact with the active Ras induced by epidermal growth factor in the absence of prohibitin. Moreover, in prohibitin-deficient cells the adhesion complex proteins cadherin and beta-catenin relocalize to the plasma membrane and thereby stabilize adherens junctions. Our data show an unexpected role of prohibitin in the activation of the Ras-Raf signalling pathway and in modulating epithelial cell adhesion and migration.     

ANG II stimulates PKC-dependent ERK activation,DNA synthesis,and cell division in intestinal epithelial cells

PKC, a major target for the tumor-promoting phorbol esters, has been implicated in the signal transduction pathways that mediate important functions in intestinal epithelial cells, including proliferation and carcinogenesis. With the use of IEC-18 cells arrested in G0/G1, addition of phorbol esters resulted in a modest increase in [3H]thymidine incorporation and a slight shift toward the S and G2/M phases of the cell cycle, whereas the combination of EGF and phorbol 12,13-dibutyrate (PDB) synergistically stimulated DNA synthesis. To investigate the effects of receptor-mediated PKC activation on mitogenesis, we demonstrated that ANG II induced ERK activation, a response completely blocked by pretreatment with mitogen/extracellular signal-regulated kinase inhibitors or specific PKC inhibitors. Furthermore, ANG II stimulated an over threefold increase in [3H]thymidine incorporation that was corroborated by flow cytometric analysis of the cell cycle to levels comparable to that achieved by the combination of EGF and PDB. Taken together, our results indicate that receptor-mediated PKC activation, as induced by ANG II, transduces mitogenic signals leading to DNA synthesis and cell proliferation in IEC-18 cells.     

Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration.

Cell responses to soluble regulatory factors may be strongly influenced by the mode of presentation of the factor, as in matrix-bound versus diffusible modes. The possibly diverse effect of presenting a growth factor in autocrine as opposed to exogenous (or paracrine) mode is an especially important issue in cell biology. We demonstrate here that migration behavior of human mammary epithelial cells in response to stimulation by epidermal growth factor (EGF) is qualitatively different for EGF presented in exogenous (paracrine), autocrine, and intracrine modes. When EGF is added as an exogenous factor to the medium of cells that express EGF receptor (EGFR) but not EGF, cell migration speed increases while directional persistence decreases. When these EGFR-expressing cells are made to also express via retroviral transfection EGF in protease-cleaveable transmembrane form on the plasma membrane, migration speed similarly increases, but directional persistence increases as well. Addition of exogenous EGF to these cells abrogates their enhanced directional persistence, reducing their directionality to a level similar to wild-type cells. If the EGFR-expressing cells are instead transduced with a gene encoding EGF in a soluble form, migration speed and directional persistence were unaffected. Thus, autocrine presentation of EGF at the plasma membrane in a protease-cleavable form provides these cells with an enhanced ability to migrate persistently in a given direction, consistent with their increased capability for organizing into gland-like structures. In contrast, an exogenous/paracrine mode of EGF presentation generates a "scattering" response by the cells. These findings emphasize the functional importance of spatial restriction of EGFR signaling, and suggest critical implications for growth factor-based therapeutic treatments.     

Planar cell polarity induces local microtubule bundling for coordinated ciliary beating

Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)–dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.     

Flagellar and ciliary beating in trypanosome motility

The single flagellum of Leishmania and Trypanosoma parasites is becoming an increasingly attractive model for the analysis of flagellar function-driven largely by the abundance of genomic and proteomic information available for the organelle, the genetic manipulability of the organisms and the importance of motility for the parasite lifecycle. However, as yet, there is a paucity of published data on the beating of any genetically malleable trypanosomatid species. Here we undertook an in-depth analysis using high-speed videomicroscopy of the beating of free-swimming Leishmania major cells in comparison to Crithidia species (for which there is some existing literature). In so doing, we describe a simple and generally-applicable technique to facilitate the quantitative analysis of free-swimming cells. Our analysis thoroughly defines the parameters of the expected tip-to-base symmetrical flagellar beat in these species. It also describes beat initiation from points other than the flagellum tip and a completely different, base-to-tip highly-asymmetric beat that represents a ciliary beat of trypanosomatid flagella. Moreover, detailed analysis of parameter interrelationships revealed an unexpected dependency of wavelength on oscillator length that may be the result of reversible constraint of doublet sliding at the tip or resonance of the flagellar beat.     

Helicobacter pylori stimulates gastric epithelial cell MMP-1 secretion via CagA-dependent and -independent ERK activation

Because the mechanisms of Helicobacter pylori-induced gastric injury are incompletely understood, we examined the hypothesis that H. pylori induces matrix metalloproteinase-1 (MMP-1) secretion, with potential to disrupt gastric stroma. We further tested the role of CagA, an H. pylori virulence factor, in MMP-1 secretion. Co-incubation of AGS cells with Tx30a, an H. pylori strain lacking the cagA virulence gene, stimulated MMP-1 secretion, confirming cagA-independent secretion. Co-incubation with strain 147C (cagA(+)) resulted in CagA translocation into AGS cells and increased MMP-1 secretion relative to Tx30a. Transfection of cells with the recombinant 147C cagA gene also induced MMP-1 secretion, indicating that CagA can independently stimulate MMP-1 secretion. Co-incubation with strain 147A, containing a cagA gene that lacks an EPIYA tyrosine phosphorylation motif, as well as transfection with 147A cagA, yielded an MMP-1 secretion intermediate between no treatment and 147C, indicating that CagA tyrosine phosphorylation regulates cellular signaling in this model system. H. pylori induced activation of the MAP kinase ERK, with CagA-independent (early) and dependent (later) components. MEK inhibitors UO126 and PD98059 inhibited both CagA-independent and -dependent MMP-1 secretion, whereas p38 inhibition enhanced MMP-1 secretion and ERK activation, suggesting p38 negative regulation of MMP-1 and ERK. These data indicate H. pylori effects on host epithelial MMP-1 expression via ERK, with p38 playing a potential regulatory role.