Implementation of the fit body and soul, a church-based life style program for diabetes prevention in high-risk African Americans: a feasibility study.

PURPOSE: The purpose of this study was to determine the effects of a behavioral faith-based diabetes prevention program called the Fit Body and Soul program in a semi-urban African-American church using a community-based participatory approach. METHODS: The 12-session Fit Body and Soul program was modified from the group lifestyle balance intervention that was modified from the successful National Institute of Health (NIH) funded Diabetes Prevention Program. The Fit Body and Soul program was implemented in a semi-urban African-American church community. Based on the results of physical examinations and increased body mass index (BMI > or = 25), 40 adult members of the church were identified as being at high risk for diabetes. Four church ministers, after receiving Fit Body and Soul program training for 2 days, served as study interventionists. The primary objective was weight loss of at least 5% by the end of the 12-session Fit Body and Soul intervention. RESULTS: Screening of church participants was conducted at the Gospel Water Branch Baptist Church in Augusta, Georgia. A total of 40 individuals having a BMI > or = 25 were selected. Of the 40, a total of 35 (87.5%) attended at least 10 sessions and provided information required for the study. Of the 35, a total of 48% lost at least 5% of baseline weight, 26% lost 7% or more, and 14% lost >10% of baseline weight. CONCLUSIONS: This pilot trial suggests that carrying out a larger Fit Body and Soul study in a faith-based setting, using behavioral lifestyle interventions, in the context of a diabetes prevention program for African American communities is feasible, as is the possibility that subjects in that larger study will achieve a clinically significant degree of weight loss.

NF-kappaB activation as a key mechanism in ethanol-induced disruption of the F-actin cytoskeleton and monolayer barrier integrity in intestinal epithelium.

Intestinal barrier disruption has been implicated in several intestinal and systemic disorders including alcoholic liver disease (ALD). Using monolayers of intestinal (Caco-2) cells, we showed that ethanol (EtOH) disrupts the barrier integrity via destabilization of the cytoskeleton. Because proinflammatory conditions are associated with activation of NF-kappa B (NF-kappaB), we hypothesized that EtOH induces disruption of cytoskeletal assembly and barrier integrity by activating NF-kappaB. Parental cells were pretreated with pharmacological modulators of NF-kappaB. Other cells were stably transfected with a dominant negative mutant for the NF-kappaB inhibitor, I-kappaBalpha. Monolayers of each cell type were exposed to EtOH and we then monitored monolayer barrier integrity (permeability); cytoskeletal stability and molecular dynamics (confocal microscopy and immunoblotting); intracellular levels of the I-kappaBalpha (immunoblotting); subcellular distribution and activity of NF-kappaB (immunoblotting and sensitive ELISA); and intracellular alterations in the 43kDa protein of the actin cytoskeleton, polymerized F-actin, and monomeric G-actin (SDS-PAGE fractionation). EtOH caused destabilizing alterations, including I-kappaBalpha degradation, NF-kappaB nuclear translocation, NF-kappaB subunit (p50 and p65) activation, actin disassembly (upward arrow G-, downward arrow F-), actin cytoskeleton instability, and barrier disruption. Inhibitors of NF-kappaB and stabilizers of I-kappaBalpha (e.g., MG-132, lactacystin, etc) prevented NF-kappaB activation while protecting against EtOH-induced injury. In transfected I-kappaBalpha mutant clones, stabilization of I-kappaBalpha to inactivate NF-kappaB protected against all measures of EtOH-induced injury. Our data support several novel mechanisms where NF-kappaB can affect the molecular dynamics of the F-actin cytoskeleton and intestinal barrier integrity under conditions of EtOH injury. (1) EtOH induces disruption of the F-actin cytoskeleton and of intestinal barrier integrity, in part, through I-kappaBalpha degradation and NF-kappaB activation; (2) The mechanism underlying this pathophysiological effect of the NF-kappaB appears to involve instability of the assembly of the subunit components of actin network.

Symmetric division of cancer stem cells--a key mechanism in tumor growth that should be targeted in future therapeutic approaches.

As cancer stem cells (SCs) drive tumor growth, it is only through the elimination of those cancer SCs that a pharmacologic cure can be attained. To study ways to develop drugs that target cancer SC, we investigated changes in cellular mechanisms and kinetics that occur in SC populations during colorectal cancer (CRC) development. We used computer modeling to determine which changes could give rise to exponential increases in both SC and non-SC populations in CRC. Our results show that the only mechanism that can explain how these subpopulations increase exponentially in CRC development involves an increase in symmetric SC cell division. This finding suggests that any systemic therapies designed to effectively treat CRC and other cancers must act to control or eliminate symmetrical cancer SC division in tumors, while minimally affecting normal SC division in non-tumor tissues.

Regulation of oxidant-induced intestinal permeability by metalloprotease-dependent epidermal growth factor receptor signaling.

Inflammatory bowel disease (IBD) affects more than 1 million Americans with more than 30,000 new cases diagnosed each year. IBD increases patient morbidity and susceptibility to colorectal cancer, yet its etiology remains unknown. Current models identify two key determinants of IBD pathogenesis: hyperpermeability of the gut epithelial barrier to bacterial products and an abnormal immune response to these products. Two factors seem critical for hyperpermeability: oxidant-induced stress and proinflammatory cytokines (e.g., tumor necrosis factor-alpha). The aim of this study was to investigate the role of oxidant stress-mediated transactivation of the epidermal growth factor receptor (EGFR) in intestinal hyperpermeability. This study used the Caco-2 human colonic epithelial cell in vitro model of intestinal epithelium. Cells were grown on inserts for permeability and signaling studies and glass coverslips for microscopy studies. show that oxidant-induced intestinal hyperpermeability can be blocked by specific inhibitors of the EGFR, tumor necrosis factor convertase (TACE) metalloprotease, transforming growth factor (TGF)-alpha, and mitogen-activated protein kinases, especially extracellular signal-regulated kinase 1/2. We also show that oxidant initiates these signaling events, in part by causing translocation of TACE to cell-cell contact zones. In this study, our data identify a novel mechanism for oxidant-induced intestinal hyperpermeability relevant to IBD. We propose a new intestinal permeability model in which oxidant transactivates EGFR signaling by activation of TACE and cleavage of precursor TGF-alpha. These data could have a significant effect on our view of IBD pathogenesis and provide new therapeutic targets for IBD treatment.

The role of protein kinase C isoforms in modulating injury and repair of the intestinal barrier.

Gastrointestinal cells express a diverse group of protein kinase C (PKC) isoforms that play critical roles in a number of cell functions, including intracellular signaling and barrier integrity. PKC isoforms expressed by gastrointestinal epithelial cells consist of three major PKC subfamilies: conventional isoforms (alpha, beta1, beta2, and gamma), novel isoforms (delta, epsilon, theta, eta, and mu), and atypical isoforms (lambda, tau, and zeta). This review highlights recent discoveries, including our own, that some PKC isoforms in gastrointestinal epithelia monolayer cell culture are involved in injury to, whereas others are involved in protection of, intestinal barrier integrity. For example, certain PKC isoforms aggravate oxidative damage, whereas others protect against it. These findings suggest that the development of agents that selectively activate or inhibit specific PKC isoforms may lead to new therapeutic modalities for important gastrointestinal disorders such as cancer and inflammatory bowel disease.

theta Isoform of protein kinase C alters barrier function in intestinal epithelium through modulation of distinct claudin isotypes: a novel mechanism for regulation of permeability.

Using monolayers of intestinal Caco-2 cells, we discovered that the 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- disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC--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- activity (antisense, dominant-negative constructs) or to ectopically express PKC- activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC- subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC- led to monolayer instability as shown by reduced 1) endogenous PKC- activity, 2) claudin isotypes in the membrane and cytoskeletal pools ( downward arrowclaud-1, downward arrowclaud-4 assembly), 3) claudin isotype phosphorylation ( downward arrow phospho-serine, downward arrow phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC- activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC- (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC- 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- caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC- inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC- activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC- 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- to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.

Critical role of the atypical {lambda} isoform of protein kinase C (PKC-{lambda}) in oxidant-induced disruption of the microtubule cytoskeleton and barrier function of intestinal epithelium.

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 lambda isoform of protein kinase C (PKC-lambda), 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-lambda 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-lambda. Other cells were transfected with a dominant-negative plasmid to stably inhibit the activity of native PKC-lambda. Cells were exposed to oxidant (H(2)O(2)) +/- 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-lambda (immunofluorescence, immunoblotting, immunoprecipitation), and PKC-lambda 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-lambda from the cytosol to the particulate (membrane + cytoskeletal) fractions, 2) activation of native PKC-lambda, 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-lambda isoform by itself ( approximately 3.2-fold increase) led to oxidant-like disruptive effects, including cytoskeletal and barrier hyperpermeability. Overexpressed PKC-lambda was mostly found in particulate cell fractions (with a smaller cytosolic distribution) indicating its activation. Disruption by PKC-lambda overexpression was also potentiated by oxidant challenge. Stable inactivation of endogenous PKC-lambda ( approximately 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-lambda isoform; and 2) activation of PKC-lambda 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.

Novel effect of NF-kappaB activation: carbonylation and nitration injury to cytoskeleton and disruption of monolayer barrier in intestinal epithelium.

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-kappaB is key to cytoskeletal instability. In the present study, we hypothesized that NF-kappaB 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-kappaB, with or without exposure to oxidant (H(2)O(2)). Other cells were transfected with an IkappaBalpha mutant (an inhibitor of NF-kappaB). Relative to WT cells exposed to vehicle, oxidant exposure caused increases in IkappaBalpha instability, NF-kappaB 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-kappaB inhibitors (MG-132, lactacystin) were protected against oxidation, showing decreases in all measures of the NF-kappaB --> iNOS --> NO pathway. Dominant mutant stabilization of IkappaBalpha to inactivate NF-kappaB 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-kappaB is required for oxidant-induced iNOS upregulation and for the consequent nitration and oxidation of cytoskeleton; 2) NF-kappaB activation causes cytoskeletal injury following upregulation of NO-driven processes; and 3) the molecular event underlying the destabilizing effects of NF-kappaB 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-kappaB in cells.

Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta.

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 theta-isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC-theta 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-theta (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-theta showed monolayer injury as indicated by decreased native PKC-theta 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-theta was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC-theta was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-theta/tubulin complex. Second, DN transfection to inhibit the endogenous PKC-theta led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC-theta led to a mostly cytosolic distribution of theta-isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1). PKC-theta isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2). the molecular event underlying this novel biological effect of PKC-theta involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-theta.

Inhibition of oxidant-induced nuclear factor-kappaB activation and inhibitory-kappaBalpha degradation and instability of F-actin cytoskeletal dynamics and barrier function by epidermal growth factor: key role of phospholipase-gamma isoform.

Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-kappaB (NF-kappaB) 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-gamma (PLC-gamma), which prevents NF-kappaB 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 microg) of cDNA to either stably overexpress PLC-gamma or to inhibit its activation. Cells were pretreated with EGF before exposure to oxidant (H(2)O(2)). Stably overexpressing PLC-gamma (+2.0-fold) or preincubating with EGF protected against oxidant injury as indicated by 1) decreases in several NF-kappaB-related variables [NF-kappaB (p50/p65 subunit) nuclear translocation, NF-kappaB subunit activity, inhibitory-kappaBalpha (I-kappaBalpha) 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-kappaB was potentiated by EGF. PLC-gamma was found mostly in membrane and cytoskeletal fractions (<9% in the cytosolic fractions), indicating its activation. Dominant negative inhibition of endogenous PLC-gamma (-99%) substantially prevented all measures of EGF protection against NF-kappaB activation. We concluded 1) EGF protects against oxidant-induced barrier disruption through PLC-gamma activation, which inactivates NF-kappaB; 2) Activation of PLC-gamma by itself is protective against NF-kappaB activation; 3) the ability to modulate the dynamics of NF-kappaB/I-kappa Balpha is a novel mechanism not previously attributed to the PLC family of isoforms in cells; and 4) development of PLC-gamma mimetics represents a possible new therapeutic strategy for IBD.

PKC-beta1 isoform activation is required for EGF-induced NF-kappaB inactivation and IkappaBalpha stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers.

Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-kappaB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-beta1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-beta1 prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB 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-beta1. Cells were pretreated with EGF or PKC modulators +/- oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-beta1 activity, decreasing IkappaBalpha phosphorylation/degradation, suppressing NF-kappaB 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-beta1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IkappaBalpha stabilization, NF-kappaB inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-beta1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-beta1 substantially prevented all measures of EGF's protection against NF-kappaB activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-beta1 activation and NF-kappaB suppression. The molecular event underlying this unique effect of PKC-beta1 involves inhibition of phosphorylation and increases in stabilization of IkappaBalpha. The ability to inhibit the dynamics of NF-kappaB/IkappaBalpha and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.

Zeta isoform of protein kinase C prevents oxidant-induced nuclear factor-kappaB activation and I-kappaBalpha degradation: a fundamental mechanism for epidermal growth factor protection of the microtubule cytoskeleton and intestinal barrier integrity.

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-kappaB (NF-kappaB) via degradation of its endogenous inhibitor I-kappaBalpha 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 zeta 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-zeta prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB 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 microg) of cDNA to either overexpress PKC-zeta 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-zeta isoform, intracellular levels and phosphorylation of the NF-kappaB inhibitor I-kappaBalpha, and nuclear translocation and activity of NF-kappaB 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-zeta activity; 2) decreasing several variables related to NF-kappaB activation [NF-kappaB (both p50 and p65 subunits) nuclear translocation, NF-kappaB subunits activity, I-kappaBalpha 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-zeta (approximately 3.0-fold increase) were protected as indicated by decreases in all measures of NF-kappaB activation as well as enhanced stability of microtubule cytoarchitecture and barrier function. Overexpression induced stabilization of I-kappaBalpha and inactivation of NF-kappaB was OAG-independent, although EGF potentiated this protection. Approximately 90% of the overexpressed PKC-zeta resided in particulate (membrane + cytoskeletal) fractions (with less than 10% in cytosolic fractions), indicating constitutive activation of the zeta isoform of PKC. Furthermore, antisense transfection to stably inhibit native PKC-zeta expression (-95%) and activation (-99%) prevented all measures of EGF-induced protection against NF-kappaB 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-zeta and inactivation of NF-kappaB (an inflammatory mediator); 2) activation of PKC-zeta is by itself required for monolayer protection against oxidant stress of NF-kappaB activation; 3) the mechanism underlying this novel biological effect of the atypical PKC isoform zeta seems to involve suppression of phosphorylation and enhancement of stabilization of I-kappaBalpha; and 4) development of agents that can mimic or enhance PKC-zeta-induced suppression of NF-kappaB 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-zeta can inhibit the dynamics of NF-kappaB and cytoskeletal disassembly in cells.

Evidence that nuclear factor-kappa B activation is critical in oxidant-induced disruption of the microtubule cytoskeleton and barrier integrity and that its inactivation is essential in epidermal growth factor-mediated protection of the monolayers of intestinal epithelia.

Using monolayers of intestinal (Caco-2) cells, we showed that oxidants disrupt the microtubule cytoskeleton and barrier integrity; epidermal growth factor (EGF) was protective via stabilization of the microtubules. Because proinflammatory conditions activate nuclear factor-kappaB (NF-kappaB), we hypothesized that oxidants disrupt barrier integrity through activation of NF-kappaB and that EGF protects by suppressing NF-kappaB. Parental cells were pretreated with EGF or NF-kappaB or inhibitory kappaBalpha (I-kappaBalpha) modulators. Other cells were stably transfected with varying levels of a dominant negative mutant for the NF-kappaB inhibitor I-kappaBalpha. Both types of cells were grown as monolayers and then exposed to oxidant (H2O2). We then monitored monolayer barrier integrity (permeability), stability of the microtubule cytoskeleton (confocal microscopy, immunoblotting), intracellular levels of the I-kappaBalpha (immunoblotting), translocation, and activity of NF-kappaB (immunoblotting, sensitive enzyme-linked immunosorbent assay). Monolayers were also fractionated and processed to assess alterations in 1) polymerized tubulin (S2; an index of cytoskeletal integrity) and 2) monomeric tubulin (S1; an index of disassembly) (polyacrylamide gel electrophoresis fractionation and immunoblotting). We found the following: 1) Oxidants caused I-kappaBalpha degradation, NF-kappaB translocation, NF-kappaB (p50 and p65 subunits) activation, tubulin disassembly ( upward arrow S1, downward arrow S2), microtubule architectural instability, and barrier disruption. I-kappaBalpha stabilizers and NF-kappaB inhibitors [e.g., carbobenzyloxy-leuleu-leucinol (MG-132), lactacystin] suppressed oxidants injurious effects. 2) EGF (10 ng/ml) stabilized I-kappaBalpha and prevented both NF-kappaB translocation and activation while protecting monolayers against oxidants. 3) In stably transfected cells, transfection-induced stabilization of I-kappaBalpha by itself led to EGF-like protective effects. In these mutant cells, protection was not potentiated by EGF (10 ng/ml). Conclusions are 1) oxidants induce disruption of the cytoskeleton and intestinal barrier integrity, in part, through I-kappaBalpha degradation and subsequent NF-kappaB activation, 2) I-kappaBalpha stabilization is by itself protective, mimicking EGF, and 3) EGF protects cell monolayers through I-kappaBalpha stabilization and NF-kappaB inactivation. To our knowledge, this is the first report that NF-kappaB can affect the dynamics of cytoskeletal assembly and intestinal barrier integrity.

Key role of PLC-gamma in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly.

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-gamma appears to be essential for monolayer integrity. We thus hypothesized that PLC-gamma 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-gamma 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-gamma 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-gamma (+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-gamma 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-gamma and downregulation of iNOS pathway; 2) activation of PLC-gamma 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.

Increases in free radicals and cytoskeletal protein oxidation and nitration in the colon of patients with inflammatory bowel disease.

BACKGROUND: Overproduction of colonic oxidants contributes to mucosal injury in inflammatory bowel disease (IBD) but the mechanisms are unclear. Our recent findings using monolayers of intestinal cells suggest that the mechanism could be oxidant induced damage to cytoskeletal proteins. However, oxidants and oxidative damage have not been well characterised in IBD mucosa. AIMS: To determine whether there are increases in oxidants and in tissue and cytoskeletal protein oxidation in IBD mucosa. METHODS: We measured nitric oxide (NO) and markers of oxidative injury (carbonylation and nitrotyrosination) to tissue and cytoskeletal proteins in colonic mucosa from IBD patients (ulcerative colitis, Crohn's disease, specific colitis) and controls. Outcomes were correlated with IBD severity score. RESULTS: Inflamed mucosa showed the greatest increases in oxidants and oxidative damage. Smaller but still significant increases were seen in normal appearing mucosa of patients with active and inactive IBD. Tissue NO levels correlated with oxidative damage. Actin was markedly (>50%) carbonylated and nitrated in inflamed tissues of active IBD, less so in normal appearing tissues. Tubulin carbonylation occurred in parallel; tubulin nitration was not observed. NO and all measures of oxidative damage in tissue and cytoskeletal proteins in the mucosa correlated with IBD severity. Disruption of the actin cytoarchitecture was primarily within the epithelial cells and paracellular area. CONCLUSIONS: Oxidant levels increase in IBD along with oxidation of tissue and cytoskeletal proteins. Oxidative injury correlated with disease severity but is also present in substantial amounts in normal appearing mucosa of IBD patients, suggesting that oxidative injury does not necessarily lead to tissue injury and is not entirely a consequence of tissue injury. Marked actin oxidation (>50%)-which appears to result from cumulative oxidative damage-was only seen in inflamed mucosa, suggesting that oxidant induced cytoskeletal disruption is required for tissue injury, mucosal disruption, and IBD flare up.

The delta-isoform of protein kinase C causes inducible nitric-oxide synthase and nitric oxide up-regulation: key mechanism for oxidant-induced carbonylation, nitration, and disassembly of the microtubule cytoskeleton and hyperpermeability of barrier of intestinal epithelia.

Using intestinal (Caco-2) cells, we found that oxidant-induced disruption of barrier integrity requires microtubule disassembly. Protein kinase C (PKC)-delta 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-delta 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-delta or with a dominant-negative to inhibit the activity of native PKC-delta. Clones were then incubated with oxidant (H(2)O(2)) +/- modulators. Parental cells were treated similarly. Exposure to oxidant-disrupted monolayers by increasing native PKC-delta 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-delta 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-delta 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-delta and up-regulation of iNOS. 2) Overexpression and activation of PKC-delta 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.

PKC-zeta prevents oxidant-induced 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-zeta was required for protection. We thus hypothesized that PKC-zeta activation and iNOS inactivation are key in EGF protection. Wild-type (WT) Caco-2 cells were exposed to H(2)O(2) (0.5 mM) +/- EGF or PKC modulators. Other cells were transfected to overexpress PKC-zeta 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-zeta 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-zeta (+2.9-fold) were protected as indicated by decreases in all measures of iNOS-driven pathways and enhanced stability of microtubules and barrier function. Overexpression-induced inhibition of iNOS was OAG independent, but EGF potentiated this protection. Antisense inhibition of PKC-zeta (-95%) 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-zeta and downregulation of iNOS. Activation of PKC-zeta 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.

Activation of delta-isoform of protein kinase C is required for oxidant-induced disruption of both the microtubule cytoskeleton and permeability barrier of intestinal epithelia.

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)-delta to be translocated to particulate fractions, we hypothesized that PKC-delta activation is required for these oxidant effects. Monolayers of parental Caco-2 cells were incubated with oxidant (H(2)O(2)) +/- modulators. Other cells were transfected with an inducible plasmid to stably overexpress PKC-delta or with a dominant negative plasmid to stably inhibit the activity of native PKC-delta. In parental cells, oxidants caused translocation of PKC-delta to the particulate (membrane + cytoskeletal) fractions, activation of PKC-delta isoform, increases in monomeric (S1) tubulin and decreases in polymerized (S2) tubulin, disruption of the microtubule cytoarchitecture, and loss of barrier integrity (hyperpermeability). In transfected cells, induction of PKC-delta overexpression by itself (3.5-fold over its basal level) led to oxidant-like disruptive effects. Disruption induced by PKC-delta overexpression was potentiated by oxidants. Overexpressed PKC-delta resided in particulate fractions, indicating its activation. Stable inhibition of native PKC-delta activity (98%) by dominant negative transfection substantially protected against all measures of oxidative disruption. We conclude that 1) oxidants induce loss of intestinal epithelial barrier integrity by disassembling the microtubules in large part through the activation of the PKC-delta isoform; and 2) overexpression and activation of PKC-delta is by itself a sufficient condition for disruption of these cytoskeleton and permeation pathways. Thus, PKC-delta activation may play a key role in intestinal dysfunction in oxidant-induced diseases such as inflammatory bowel disease.

Development of cross-resistance to tamoxifen in raloxifene-treated breast carcinoma cells.

Selective estrogen receptor modifiers (SERMs) are used chronically in the treatment of breast cancer and osteoporosis but some patients become resistant, at which point second-line SERMs are considered as options. Because the use of SERMs is increasing and breast cancer is so common, we tested the hypothesis that treatment with SERMs can induce cross-resistance to other SERMs. We used three cultured breast carcinoma cell lines (MCF-7, ZR-75-1, and T47D) which are estrogen-receptor-positive (ER+) and are prone to developing resistance to hormonal treatment. Cell lines were exposed to increasing doses of raloxifene. Raloxifene-resistant clones were selected and tested for cross-resistance to tamoxifen. Compared to untreated cells, raloxifene-resistant clones showed an increased IC50 (reduced potency) of about 15,000-fold with no apparent change in maximal inhibition of cell growth. These same raloxifene-resistant clones were also about 15-fold more resistant to the growth-inhibiting effects of tamoxifen. While the resistance to tamoxifen is considerably less marked (1000-fold less), it is large enough to raise the question as to whether patients who become resistant to raloxifene will benefit by switching to tamoxifen or vice versa.

The beta 1 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.

Using intestinal monolayers, we showed that F-actin cytoskeletal stabilization and Ca(2+) 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-beta1 (PKC-beta1) isoform is abundant in our naive (N) cells, we hypothesized that PKC-beta1 is essential to EGF protection. Monolayers of N Caco-2 cells were exposed to H(2)O(2) +/- EGF, PKC, or Ca(2+) modulators. Other cells were transfected to over-express PKC-beta1 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-beta1, enhanced (45)Ca(2+) efflux, normalized Ca(2+), decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of the actin. PKC inhibitors prevented these protective effects. Transfected cells stably over-expressing PKC-beta1 (+3.1-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-beta1. Antisense inhibition of PKC-beta1 expression (-90%) prevented all measures of EGF protection. Inhibitors of Ca(2+)-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-beta1. EGF normalizes Ca(2+) by enhancing Ca(2+) efflux through PKC-beta1. We have identified novel biologic functions, protection of actin and Ca(2+) homeostasis, among the classical isoforms of PKC.