The β1 isoform of protein kinase C mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells (original) (raw)
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Using intestinal monolayers, we showed that F-actin cytoskeletal stabilization and Ca 2ϩ normalization contribute to epidermal growth factor (EGF)-mediated protection against oxidant injury. However, the intracellular mediator responsible for these protective effects remains unknown. Since the protein kinase C-1 (PKC-1) isoform is abundant in our naive (N) cells, we hypothesized that PKC-1 is essential to EGF protection. Monolayers of N Caco-2 cells were exposed to H 2 O 2 Ϯ EGF, PKC, or Ca 2ϩ modulators. Other cells were transfected to over-express PKC-1 or to inhibit its expression and then pretreated with low or high doses of EGF or a PKC activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), before H 2 O 2 . In N monolayers exposed to oxidant, pretreatment with EGF or PKC activators activated PKC-1, enhanced 45 Ca 2ϩ efflux, normalized Ca 2ϩ , decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of the actin. PKC inhibitors prevented these protective effects. Transfected cells stably overexpressing PKC-1 (ϩ3.1-fold) but not N cell monolayers were protected from injury by even lower doses of EGF or OAG. EGF or OAG rapidly activated the over-expressed PKC-1. Antisense inhibition of PKC-1 expression (Ϫ90%) prevented all measures of EGF protection. Inhibitors of Ca 2ϩ -ATPase prevented EGF protection in N cells as well as protective synergism in transfected cells. EGF protects the assembly of the F-actin cytoskeleton in intestinal monolayers against oxidants in large part through the activation of PKC-1. EGF normalizes Ca 2ϩ by enhancing Ca 2ϩ efflux through PKC-1. We have identified novel biologic functions, protection of actin and Ca 2ϩ homeostasis, among the classical isoforms of PKC.
Journal of Pharmacology and Experimental Therapeutics, 2002
Using intestinal monolayers, we showed that F-actin cytoskeletal stabilization and Ca 2ϩ normalization contribute to epidermal growth factor (EGF)-mediated protection against oxidant injury. However, the intracellular mediator responsible for these protective effects remains unknown. Since the protein kinase C-1 (PKC-1) isoform is abundant in our naive (N) cells, we hypothesized that PKC-1 is essential to EGF protection. Monolayers of N Caco-2 cells were exposed to H 2 O 2 Ϯ EGF, PKC, or Ca 2ϩ modulators. Other cells were transfected to over-express PKC-1 or to inhibit its expression and then pretreated with low or high doses of EGF or a PKC activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), before H 2 O 2 . In N monolayers exposed to oxidant, pretreatment with EGF or PKC activators activated PKC-1, enhanced 45 Ca 2ϩ efflux, normalized Ca 2ϩ , decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of the actin. PKC inhibitors prevented these protective effects. Transfected cells stably overexpressing PKC-1 (ϩ3.1-fold) but not N cell monolayers were protected from injury by even lower doses of EGF or OAG. EGF or OAG rapidly activated the over-expressed PKC-1. Antisense inhibition of PKC-1 expression (Ϫ90%) prevented all measures of EGF protection. Inhibitors of Ca 2ϩ -ATPase prevented EGF protection in N cells as well as protective synergism in transfected cells. EGF protects the assembly of the F-actin cytoskeleton in intestinal monolayers against oxidants in large part through the activation of PKC-1. EGF normalizes Ca 2ϩ by enhancing Ca 2ϩ efflux through PKC-1. We have identified novel biologic functions, protection of actin and Ca 2ϩ homeostasis, among the classical isoforms of PKC.
Key role of PKC and Ca2+in EGF protection of microtubules and intestinal barrier against oxidants
American Journal of Physiology-gastrointestinal and Liver Physiology, 2001
Using monolayers of human intestinal (Caco-2) cells, we showed that growth factors (GFs) protect microtubules and barrier integrity against oxidative injury. Studies in nongastrointestinal cell models suggest that protein kinase C (PKC) signaling is key in GF-induced effects and that cytosolic calcium concentration ([Ca 2ϩ ]i) is essential in cell integrity. We hypothesized that GF protection involves activating PKC and maintaining normal [Ca 2ϩ ]i. Monolayers were pretreated with epidermal growth factor (EGF) or PKC or Ca 2ϩ modulators before exposure to oxidants (H 2O2 or HOCl). Oxidants disrupted microtubules and barrier integrity, and EGF protected from this damage. EGF caused rapid distribution of PKC-␣, PKC-I, and PKC-isoforms to cell membranes, enhancing PKC activity of membrane fractions while reducing PKC activity of cytosolic fractions. EGF enhanced 45 Ca 2ϩ efflux and prevented oxidant-induced (sustained) rises in [Ca 2ϩ ]i. PKC inhibitors abolished and PKC activators mimicked EGF protection. Oxidant damage was mimicked by and potentiated by a Ca 2ϩ ionophore (A-23187), exacerbated by high-Ca 2ϩ media, and prevented by calcium removal or chelation or by Ca 2ϩ channel antagonists. PKC activators mimicked EGF on both 45 Ca 2ϩ efflux and [Ca 2ϩ ]i. Membrane Ca 2ϩ-ATPase pump inhibitors prevented protection by EGF or PKC activators. In conclusion, EGF protection of microtubules and the intestinal epithelial barrier requires activation of PKC signal transduction and normalization of [Ca 2ϩ ]i.
PKC-β1 mediates EGF protection of microtubules and barrier of intestinal monolayers against oxidants
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2001
Using monolayers of human intestinal (Caco-2) cells, we found that oxidants and ethanol damage the cytoskeleton and disrupt barrier integrity; epidermal growth factor (EGF) prevents damage by enhancement of protein kinase C (PKC) activity and translocation of the PKC-β1 isoform. To see if PKC-β1 mediates EGF protection, cells were transfected to stably over- or underexpress PKC-β1. Transfected monolayers were preincubated with low or high doses of EGF (1 or 10 ng/ml) or 1-oleoyl-2-acetyl- sn-glycerol [OAG; a PKC activator (0.01 or 50 μM)] before treatment with oxidant (0.5 mM H2O2). Only in monolayers overexpressing PKC-β1 (3.1-fold) did low doses of EGF or OAG initiate protection, increase tubulin polymerization (assessed by quantitative immunoblotting) and microtubule architectural integrity (laser scanning confocal microscopy), maintain normal barrier permeability (fluorescein sulfonic acid clearance), and cause redistribution of PKC-β1 from cytosolic pools into membrane and/or c...
American Journal of Physiology-gastrointestinal and Liver Physiology, 2002
Using monolayers of human intestinal (Caco-2) cells, we showed that epidermal growth factor (EGF) protects intestinal barrier integrity against oxidant injury by protecting the microtubules and that protein kinase C (PKC) is required. Because atypical PKC-isoform is abundant in wild-type (WT) Caco-2 cells, we hypothesized that PKCmediates, at least in part, EGF protection. Intestinal cells (Caco-2 or HT-29) were transfected to stably over-or underexpress PKC-. These clones were preincubated with low or high doses of EGF or a PKC activator [1-oleoyl-2-acetyl-snglycerol (OAG)] before oxidant (0.5 mM H2O2). Relative to WT cells exposed to oxidant, only monolayers of transfected cells overexpressing PKC-(2.9-fold) were protected against oxidant injury as indicated by increases in polymerized tubulin and decreases in monomeric tubulin, enhancement of architectural stability of the microtubule cytoskeleton, and increases in monolayer barrier integrity toward control levels (62% less leakiness). Overexpression-induced protection was OAG independent and even EGF independent, but EGF significantly potentiated PKC-protection. Most overexpressed PKC-(92%) resided in membrane and cytoskeletal fractions, indicating constitutive activation of PKC-. Stably inhibiting PKC-expression (95%) with antisense transfection substantially attenuated EGF protection as demonstrated by reduced tubulin assembly and increased microtubule disassembly, disruption of the microtubule cytoskeleton, and loss of monolayer barrier integrity. We conclude that 1) activation of PKC-is necessary for EGF-induced protection, 2) PKC-appears to be an endogenous stabilizer of the microtubule cytoskeleton and of intestinal barrier function against oxidative injury, and 3) we have identified a novel biological function (protection) among the atypical isoforms of PKC. cytoskeleton; growth factors; epidermal growth factor; Caco-2 cells; gut barrier; protection; transfection; protein kinase C isoforms; inflammatory bowel disease A FUNDAMENTAL PROPERTY OF epithelial cells of the gastrointestinal (GI) tract is to function as a highly selective permeability barrier, permitting the absorption
Journal of Pharmacology and Experimental Therapeutics, 2003
Oxidant damage and gut barrier disruption contribute to the pathogenesis of a variety of inflammatory gastrointestinal disorders, including inflammatory bowel disease (IBD). In our studies using a model of the gastrointestinal (GI) epithelial barrier, monolayers of intestinal (Caco-2) cells, we investigated damage to and protection of the monolayer barrier. We reported that activation of nuclear factor-B (NF-B) via degradation of its endogenous inhibitor I-B␣ is key to oxidant-induced disruption of barrier integrity and that growth factor (epidermal growth factor, EGF) protects against this injury by stabilizing the cytoskeletal filaments. Protein kinase C (PKC) activation seems to be required for monolayer maintenance, especially activation of the atypical isoform of PKC. In an attempt to investigate, at the molecular level, the fundamental events underlying EGF protection against oxidant disruption, we tested the intriguing hypothesis that EGF-induced activation of PKC-prevents oxidant-induced activation of NF-B and the consequences of NF-B activation, namely, cytoskeletal and barrier disruption. Monolayers of wild-type (WT) Caco-2 cells were incubated with oxidant (H 2 O 2) with or without EGF or modulators. In other studies, we used the first gastrointestinal cell clones created by stable transfection of varying levels (1-5 g) of cDNA to either overexpress PKC-or to inhibit its expression. Transfected cell clones were then pretreated with EGF or a PKC activator (diacylglycerol analog 1-oleoyl-2-acetyl-glycerol, OAG) before oxidant. We monitored the following endpoints: monolayer barrier integrity, stability of the microtubule cytoskeleton, subcellular distribution and activity of the PKC-isoform, intracellular levels and phosphorylation of the NF-B inhibitor I-B␣, and nuclear translocation and activity of NF-B subunits p65 and p50. Monolayers were also fractionated and processed to assess alterations in the structural protein of the microtubules, polymerized tubulin (S2), and monomeric tubulin (S1). Our data indicated that relative to WT monolayers exposed only to oxidant, pretreatment with EGF protected cell monolayers by 1) increasing native PKC
Gut, 2000
Background-Intestinal barrier dysfunction concomitant with high levels of reactive oxygen metabolites (ROM) in the inflamed mucosa have been observed in inflammatory bowel disease (IBD). The cytoskeletal network has been suggested to be involved in the regulation of barrier function. Growth factors (epidermal growth factor (EGF) and transforming growth factor (TGF-)) protect gastrointestinal barrier integrity against a variety of noxious agents. However, the underlying mechanisms of oxidant induced disruption and growth factor mediated protection remain elusive. Aims-To determine: (1) if oxidation and disassembly of actin (a key cytoskeletal component) plays a major role in ROM induced epithelial monolayer barrier dysfunction; and (2) if growth factor mediated protection involves prevention of theses alterations. Methods-Caco-2 monolayers were preincubated with EGF, TGF-, or vehicle before incubation with ROM (H 2 O 2 or HOCl). EVects on cell integrity, barrier function, and G-and F-actin (oxidation, disassembly, and assembly) were determined. Results-ROM dose dependently and significantly increased F-and G-actin oxidation (carbonylation), decreased the stable F-actin fraction (index of stability), and increased the monomeric G-actin fraction (index of disassembly). Concomitant with these changes were disruption of the actin cytoskeleton and loss of the monolayer barrier function. In contrast, growth factor pretreatment decreased actin oxidation and enhanced the stable F-actin, while in concert prevented actin disruption and restored normal barrier function of monolayers exposed to ROM. Cytochalasin-D, an inhibitor of actin assembly, not only caused actin disassembly and barrier dysfunction but also abolished the protective action of growth factors. Moreover, an actin stabilising agent, phalloidin, mimicked the protective actions of the growth factors. Conclusions-Oxidation, disassembly, and instability of the actin cytoskeleton appears to play a key role in the mechanism of oxidant induced loss of intestinal barrier integrity. In contrast, organisation and stabilisation of actin through promotion of its assembly plays a critical role in the mechanism of growth factor mediated protection.
AJP Gastrointestinal and Liver Physiology
PKC-prevents oxidantinduced iNOS upregulation and protects the microtubules and gut barrier integrity. Using intestinal (Caco-2) monolayers, we reported that inducible nitric oxide synthase (iNOS) activation is key to oxidant-induced barrier disruption and that EGF protects against this injury. PKC-was required for protection. We thus hypothesized that PKCactivation and iNOS inactivation are key in EGF protection. Wild-type (WT) Caco-2 cells were exposed to H2O2 (0.5 mM) Ϯ EGF or PKC modulators. Other cells were transfected to overexpress PKC-or to inhibit it and then pretreated with EGF or a PKC activator (OAG) before oxidant. Relative to WT cells exposed to oxidant, pretreatment with EGF protected monolayers by 1) increasing PKC-activity; 2) decreasing iNOS activity and protein, NO levels, oxidative stress, tubulin oxidation, and nitration); 3) increasing polymerized tubulin; 4) maintaining the cytoarchitecture of microtubules; and 5) enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected cells overexpressing PKC-(ϩ2.9-fold) were protected as indicated by decreases in all measures of iNOS-driven pathways and enhanced stability of microtubules and barrier function. Overexpressioninduced inhibition of iNOS was OAG independent, but EGF potentiated this protection. Antisense inhibition of PKC-(Ϫ95%) prevented all measures of EGF protection against iNOS upregulation. Thus EGF protects against oxidative disruption of the intestinal barrier by stabilizing the cytoskeleton in large part through the activation of PKC-and downregulation of iNOS. Activation of PKC-is by itself required for cellular protection against oxidative stress of iNOS. We have thus discovered novel biologic functions, suppression of the iNOS-driven reactions and cytoskeletal oxidation, among the atypical PKC isoforms.
Journal of Pharmacology and Experimental Therapeutics, 2002
Using monolayers of intestinal (Caco-2) cells, we showed that oxidants disassemble the microtubule cytoskeleton and disrupt barrier integrity (permeability) (Banan et al., 2000a). Because exposure of our parental cells to oxidants causes protein kinase C (PKC)-␦ to be translocated to particulate fractions, we hypothesized that PKC-␦ activation is required for these oxidant effects. Monolayers of parental Caco-2 cells were incubated with oxidant (H 2 O 2) Ϯ modulators. Other cells were transfected with an inducible plasmid to stably overexpress PKC-␦ or with a dominant negative plasmid to stably inhibit the activity of native PKC-␦. In parental cells, oxidants caused translocation of PKC-␦ to the particulate (membrane ϩ cytoskeletal) fractions, activation of PKC-␦ isoform, increases in monomeric (S1) tubulin and decreases in polymerized (S2) tubulin, disruption of the microtubule cytoarchitecture, and loss of barrier integrity