Ali Banan

Oxidant injury to epithelial cells and gut barrier disruption are key factors in the pathogenesis of inflammatory bowel disease. Studying monolayers of intestinal (Caco-2) cells, we reported that oxidants disrupt the cytoskeleton and cause barrier dysfunction (hyperpermeability). Because the λ isoform of protein kinase C (PKC-λ), an atypical diacylglycerol-independent isozyme, is abundant in parental (wild type) Caco-2 cells and is translocated to the particulate fractions upon oxidant exposure, we hypothesized that PKC-λ is critical to oxidative injury to the assembly and architecture of cytoskeleton and the intestinal barrier function. To this end, Caco-2 cells were transfected with an inducible plasmid, a tetracycline-responsive system, to create novel clones stably overexpressing native PKC-λ. Other cells were transfected with a dominant-negative plasmid to stably inhibit the activity of native PKC-λ. Cells were exposed to oxidant (H₂O₂) ± modulators. Parental Caco-2 cells were treated similarly. We then monitored barrier function (fluorescein sulfonic acid clearance), microtubule cytoskeletal stability (confocal microscopy, immunoblotting), subcellular distribution of PKC-λ (immunofluorescence, immunoblotting, immunoprecipitation), and PKC-λ isoform activity (in vitro kinase assay). Monolayers were also processed to assess alterations in tubulin assembly, polymerized tubulin (S2, an index of cytoskeletal integrity), and monomeric tubulin (S1, an index of cytoskeletal disassembly) (polyacrylamide gel electrophoresis fractionation and immunoblotting. In parental cells, oxidant caused: 1) translocation of PKC-λ from the cytosol to the particulate (membrane + cytoskeletal) fractions, 2) activation of native PKC-λ, 3) tubulin pool instability (increased monomeric S1 and decreased polymerized S2), 4) disruption of cytoskeletal architecture, and 5) barrier dysfunction (hyperpermeability). In transfected clones, overexpression of the atypical (74 kDa) PKC-λ isoform by itself (∼3.2-fold increase) led to oxidant-like disruptive effects, including cytoskeletal and barrier hyperpermeability. Overexpressed PKC-λ was mostly found in particulate cell fractions (with a smaller cytosolic distribution) indicating its activation. Disruption by PKC-λ overexpression was also potentiated by oxidant challenge. Stable inactivation of endogenous PKC-λ (∼99.6%) by a dominant-negative protected against all measures of oxidant-induced disruption. We conclude that: 1) oxidant induces disruption of epithelial barrier integrity by disassembling the cytoskeleton, in large part, through the activation of PKC-λ isoform; and 2) activation of PKC-λ by itself appears to be sufficient for disruption of cellular cytoskeleton and monolayer barrier permeability. The unique ability to mediate an oxidant-like injury and cytoskeletal depolymerization and instability is a novel mechanism not previously attributed to the atypical subfamily of PKC isoforms.

Using monolayers of intestinal Caco-2 cells, we discovered that the θ isoform of protein kinase C (PKC), a member of the “novel” subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remain largely unknown. Here, we sought to determine whether the mechanism by which PKC-θ disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC-θ-induced alterations in claudin isotypes. We used cell clones that we recently developed, clones that were transfected with varying levels of plasmid to either stably suppress endogenous PKC-θ activity (antisense, dominant-negative constructs) or to ectopically express PKC-θ activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC-θ subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC-θ led to monolayer instability as shown by reduced 1) endogenous PKC-θ activity, 2) claudin isotypes in the membrane and cytoskeletal pools (↓claud-1, ↓claud-4 assembly), 3) claudin isotype phosphorylation (↓ phospho-serine, ↓ phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC-θ activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC-θ (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-θ/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC-θ caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 and -4 hypophosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a third series of studies, sense overexpression of the PKC-θ caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC-θ inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC-θ activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC-θ are mediated at the molecular level by changes in phosphorylation, membrane assembly, and/or organization of the subunit components of two barrier function proteins: claudin-1 and claudin-4 isotypes. The ability of PKC-θ to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.

Using monolayers of intestinal cells, we reported that upregulation of inducible nitric oxide synthase (iNOS) is required for oxidative injury and that activation of NF-κB is key to cytoskeletal instability. In the present study, we hypothesized that NF-κB activation is crucial to oxidant-induced iNOS upregulation and its injurious consequences: cytoskeletal oxidation and nitration and monolayer dysfunction. Wild-type (WT) cells were pretreated with inhibitors of NF-κB, with or without exposure to oxidant (H₂O₂). Other cells were transfected with an IκBα mutant (an inhibitor of NF-κB). Relative to WT cells exposed to vehicle, oxidant exposure caused increases in IκBα instability, NF-κB subunit activation, iNOS-related activity (NO, oxidative stress, tubulin nitration), microtubule disassembly and instability (increased monomeric and decreased polymeric tubulin), and monolayer disruption. Monolayers pretreated with NF-κB inhibitors (MG-132, lactacystin) were protected against oxidation, showing decreases in all measures of the NF-κB → iNOS → NO pathway. Dominant mutant stabilization of IκBα to inactivate NF-κB suppressed all measures of the iNOS/NO upregulation while protecting monolayers against oxidant insult. In these mutants, we found prevention of tubulin nitration and oxidation and enhancement of cytoskeletal and monolayer stability. We concluded that 1) NF-κB is required for oxidant-induced iNOS upregulation and for the consequent nitration and oxidation of cytoskeleton; 2) NF-κB activation causes cytoskeletal injury following upregulation of NO-driven processes; and 3) the molecular event underlying the destabilizing effects of NF-κB appears to be increases in carbonylation and nitrotyrosination of the subunit components of cytoskeleton. The ability to promote NO overproduction and cytoskeletal nitration/oxidation is a novel mechanism not previously attributed to NF-κB in cells.

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the θ-isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC-θ is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC-θ (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC-θ showed monolayer injury as indicated by decreased native PKC-θ activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC-θ was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC-θ was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-θ/tubulin complex. Second, DN transfection to inhibit the endogenous PKC-θ led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC-θ led to a mostly cytosolic distribution of θ-isoform (

Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-κB (NF-κB) activation is required for oxidant-induced disruption of cytoskeletal and barrier integrity. Epidermal growth factor (EGF) stabilizes the F-actin cytoskeleton and protects against oxidant damage, but the mechanism remains unclear. We hypothesized that the mechanism involves activation of phospholipase C-γ (PLC-γ), which prevents NF-κB activation and the consequences of this activation, namely, cytoskeletal and barrier disruption. We studied wild-type and transfected cells. The latter were transfected with varying levels (1-5 μg) of cDNA to either stably overexpress PLC-γ or to inhibit its activation. Cells were pretreated with EGF before exposure to oxidant (H₂O₂). Stably overexpressing PLC-γ (+2.0-fold) or preincubating with EGF protected against oxidant injury as indicated by 1) decreases in several NF-κB-related variables [NF-κB (p50/p65 subunit) nuclear translocation, NF-κB subunit activity, inhibitory-κBα (I-κBα) phosphorylation and degradation]; 2) increases in F-actin and decreases in G-actin; 3) stabilization of the actin cytoskeletal architecture; and 4) enhancement of barrier function. Overexpression induced inactivation of NF-κB was potentiated by EGF. PLC-γ was found mostly in membrane and cytoskeletal fractions (<9% in the cytosolic fractions), indicating its activation. Dominant negative inhibition of endogenous PLC-γ (-99%) substantially prevented all measures of EGF protection against NF-κB activation. We concluded 1) EGF protects against oxidant-induced barrier disruption through PLC-γ activation, which inactivates NF-κB; 2) Activation of PLC-γ by itself is protective against NF-κB activation; 3) the ability to modulate the dynamics of NF-κB/I-κ Bα is a novel mechanism not previously attributed to the PLC family of isoforms in cells; and 4) development of PLC-γ mimetics represents a possible new therapeutic strategy for IBD.

Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-κB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-β1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-β1 prevents oxidant-induced activation of NF-κB and the consequences of NF-κB activation, F-actin, and barrier dysfunction. We used wild-type (WT) and transfected cells. The latter were transfected with varying levels of cDNA to overexpress or underexpress PKC-β1. Cells were pretreated with EGF or PKC modulators ± oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-β1 activity, decreasing IκBα phosphorylation/degradation, suppressing NF-κB activation (p50/p65 subunit nuclear translocation/activity), enhancing stable actin (increased F-actin-to-G-actin ratio), increasing stability of actin cytoskeleton, and reducing barrier hyperpermeability. Cells stably overexpressing PKC-β1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IκBα stabilization, NF-κB inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-β1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-β1 substantially prevented all measures of EGF's protection against NF-κB activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-β1 activation and NF-κB suppression. The molecular event underlying this unique effect of PKC-β1 involves inhibition of phosphorylation and increases in stabilization of IκBα. The ability to inhibit the dynamics of NF-κB/IκBα and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.

Upregulation of inducible nitric oxide synthase (iNOS) is key to oxidant-induced disruption of intestinal (Caco-2) monolayer barrier, and EGF protects against this disruption by stabilizing the cytoskeleton. PLC-γ appears to be essential for monolayer integrity. We thus hypothesized that PLC-γ activation is essential in EGF protection against iNOS upregulation and the consequent cytoskeletal oxidation and disarray and monolayer disruption. Intestinal cells were transfected to stably overexpress PLC-γ or to inhibit its activation and were then pretreated with EGF ± oxidant (H₂O₂). Wild-type (WT) intestinal cells were treated similarly. Relative to WT monolayers exposed to oxidant, pretreatment with EGF protected monolayers by: increasing native PLC-γ activity; decreasing six iNOS-related variables (iNOS activity/protein, NO levels, oxidative stress, actin oxidation/nitration); increasing stable F-actin; maintaining actin stability; and enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected monolayers overexpressing PLC-γ (+2.3-fold) were protected, as indicated by decreases in all measures of iNOS-driven pathway and enhanced actin and barrier integrity. Overexpression-induced inhibition of iNOS was potentiated by low doses of EGF. Stable inhibition of PLC-γ prevented all measures of EGF protection against iNOS upregulation. We conclude that 1) EGF protects against oxidative stress disruption of intestinal barrier by stabilizing F-Actin, largely through the activation of PLC-γ and downregulation of iNOS pathway; 2) activation of PLC-γ is by itself essential for cellular protection against oxidative stress of iNOS; and 3) the ability to suppress iNOS-driven reactions and cytoskeletal oxidation and disassembly is a novel mechanism not previously attributed to the PLC family of isoforms.

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-κB (NF-κB) via degradation of its endogenous inhibitor I-κBα 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 ζ 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-ζ prevents oxidant-induced activation of NF-κB and the consequences of NF-κB activation, namely, cytoskeletal and barrier disruption. Monolayers of wild-type (WT) Caco-2 cells were incubated with oxidant (H₂O₂) 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 μg) of cDNA to either overexpress PKC-ζ 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-ζ isoform, intracellular levels and phosphorylation of the NF-κB inhibitor I-κBα, and nuclear translocation and activity of NF-κB 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-ζ activity; 2) decreasing several variables related to NF-κB activation [NF-κB (both p50 and p65 subunits) nuclear translocation, NF-κB subunits activity, I-κBα degradation, and phosphorylation]; 3) increasing stable tubulin (increased polymerized S2 tubulin and decreased monomeric S1 tubulin); 4) maintaining the cytoarchitectural integrity of microtubules; and 5) preventing hyperpermeability (barrier disruption). In addition, relative to WT cells exposed to oxidant, monolayers of transfected cells stably overexpressing PKC-ζ (∼3.0-fold increase) were protected as indicated by decreases in all measures of NF-κB activation as well as enhanced stability of microtubule cytoarchitecture and barrier function. Overexpression induced stabilization of I-κBα and inactivation of NF-κB was OAG-independent, although EGF potentiated this protection. Approximately 90% of the overexpressed PKC-ζ resided in particulate (membrane + cytoskeletal) fractions (with less than 10% in cytosolic fractions), indicating constitutive activation of the ζ isoform of PKC. Furthermore, antisense transfection to stably inhibit native PKC-ζ expression (–95%) and activation (–99%) prevented all measures of EGF-induced protection against NF-κB activation and monolayer disruption. We conclude the following: 1) EGF protects against oxidant disruption of the intestinal barrier integrity, in large part, through the activation of PKC-ζ and inactivation of NF-κB (an inflammatory mediator); 2) activation of PKC-ζ is by itself required for monolayer protection against oxidant stress of NF-κB activation; 3) the mechanism underlying this novel biological effect of the atypical PKC isoform ζ seems to involve suppression of phosphorylation and enhancement of stabilization of I-κBα; and 4) development of agents that can mimic or enhance PKC-ζ-induced suppression of NF-κB activation may be a useful therapeutic strategy for preventing oxidant damage to GI mucosal epithelium in disorders such as IBD. To our knowledge, this is the first report that PKC-ζ can inhibit the dynamics of NF-κB and cytoskeletal disassembly in cells.

Using intestinal (Caco-2) cells, we found that oxidant-induced disruption of barrier integrity requires microtubule disassembly. Protein kinase C (PKC)-δ isoform seems to be essential for disruption, but the mechanism is unknown. Because inducible nitric-oxide synthase (iNOS) is key to oxidant stress, we hypothesized that PKC-δ activation is essential in oxidant-induced iNOS up-regulation and the consequent cytoskeletal oxidation and disarray and monolayer barrier dysfunction. Cells were transfected with an inducible plasmid to overexpress native PKC-δ or with a dominant-negative to inhibit the activity of native PKC-δ. Clones were then incubated with oxidant (H₂O₂) ± modulators. Parental cells were treated similarly. Exposure to oxidant-disrupted monolayers by increasing native PKC-δ activity, increasing six iNOS-related variables (iNOS activity and protein, nitric oxide, oxidative stress, tubulin oxidation and nitration), decreasing polymerized tubulin, disrupting the cytoarchitecture of microtubules, and causing monolayer dysfunction. Induction of PKC-δ overexpression by itself (3.5-fold) led to oxidant-like disruptive effects, including activation of the iNOS-driven pathway. Overexpression-induced up-regulation of iNOS was potentiated by oxidants. iNOS inhibitors or oxidant scavengers were protective. Dominant inhibition of native PKC-δ activity (99.5%) prevented all measures of oxidant-induced iNOS up-regulation and protected the monolayer barrier. The conclusions are as follows. 1) Oxidants induce loss of epithelial barrier integrity by oxidizing and disassembling the cytoskeleton, in part, through the activation of PKC-δ and up-regulation of iNOS. 2) Overexpression and activation of PKC-δ are by themselves key for cellular injury by oxidative stress of iNOS. 3) We thus report a pathophysiological mechanism, activation of iNOS pathway and its injurious consequences to the cytoskeleton, including oxidation and nitration, among the “novel” subfamily of PKC isoforms.