Changes in peripheral blood mononuclear cells' mRNA expression of TLRs and CD14 during puerperal metritis in dairy cattle.

BACKGROUND: Peripheral blood mononuclear cells (PBMCs), commonly referred to as lymphocytes and monocytes, representing cells of the innate and adaptive immune systems. AIMS: To find out whether changes in PBMCs' mRNA expression of pattern recognition receptors (PRRs) are associated with puerperal metritis in Holstein cows. METHODS : Peripheral blood mononuclear cells were collected from 20 cows with puerperal metritis and 20 cows without metritis at 10 days postpartum. Expression of toll-like receptors 2 and 4 (TLR2 and TLR4), and cluster of differentiation 14 (CD14) genes were assessed in PBMCs using a quantitative real time-polymerase chain reaction (qRT-PCR) technique. The data was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference gene, and 2-∆∆Ct methodology was used for relative quantification. RESULTS: The results of the present study demonstrated that the expression of TLR4 (P=0.04) and CD14 (P=0.008) was significantly greater in cows with puerperal metritis compared to the control group. However, the expression of TLR2 (P=0.06) was not significantly different between cows with puerperal metritis and healthy cows. CONCLUSION: This study suggests that puerperal metritis significantly increases the expression of TLR4 and CD14 genes in the PBMCs which contributes to the proper stimulation of inflammation and uterine clearance of bacteria soon after calving.

Impact of Symmetry on Anisotropic Magnetoresistance in Textured Ferromagnetic Thin Films.

We report on the magnetoresistance of textured films consisting of 3d-ferromagnetic layers sandwiched by Pt. While the conventional cos^{2}φ behavior of the anisotropic magnetoresistance (AMR) is found when the magnetization M is varied in the film plane, cos^{2n}θ contributions (2n≤6) exist for rotating M in the plane perpendicular to the current. This finding is explained by the symmetry-adapted modeling of AMR of textured films demonstrating that the cos^{2}θ behavior cannot be used as a fingerprint for the presence of spin Hall magnetoresistance (SMR). Further, the interfacial MR contributions for Pt/Ni/Pt contradict the SMR behavior confirming the dominant role of AMR in all-metallic systems.

Allele specific-PCR and melting curve analysis showed relatively high frequency of ß-casein gene A1 allele in Iranian Holstein, Simmental and native cows.

There are two allelic forms of A1 and A2 of ß-casein gene in dairy cattle. Proteolytic digestion of bovine ß-casein A1 type produces bioactive peptide of ß-casomorphin-7 known as milk devil. ß-casomorphin-7 causes many diseases, including type 1 diabetes, cardiovascular disease syndrome, sudden death and madness. The aim of the present study was to determine the different allelic forms of ß-casein gene in Iranian Holstein, Simmental and native cattle in order to identify A1 and A2 variants. The blood samples were collected randomly and DNA was extracted using modified salting out method. An 854 bp fragment including part of exon 7 and part of intron 6 of ß-casein gene was amplified by allele specific polymerase chain reaction (AS-PCR). Also, the accuracy of AS-PCR genotyping has been confirmed by melting temperature curve analysis using Real-time PCR machinery. The comparison of observed allele and genotype frequency among the studied breeds was performed using the Fisher exact and Chi-squared test, respectively by SAS program. Obtained results showed the A1 allele frequencies of 50, 51.57, 54.5, 49.4 and 46.6% in Holstein, Simmental, Sistani, Taleshi and Mazandarani cattle populations, respectively. The chi-square test was shown that no any populations were in Hardy-Weinberg equilibrium for studied marker locus. Comparison and analysis of the test results for allelic frequency showed no any significant differences between breeds (P>0.05). The frequency of observed genotypes only differs significantly between Holstein and Taleshi breeds but no any statistically significant differences were found for other breeds (P>0.05). A relatively high frequency of ß-casein A1 allele was observed in Iranian native cattle. Therefore, determine the genotypes and preference alleles A2 in these native and commercial cattle is recommended.

Construction of bacterial ghosts for transfer and expression of a chimeric hepatitis C virus gene in macrophages.

The bacterial ghost (BG) production is a field of biotechnology for applications in vaccine and drug delivery. We assessed the capacity of BG for delivery of a recombinant gene encoded for both cell mediated and antibody dependent epitopes of hepatitis C virus (HCV) into murine macrophages. Escherichia coli (E. coli) cells were transformed with the lysis plasmid (pHH43). To produce chimeric gene, NS3 (non-structural protein 3) and core regions of HCV genome were fused together by splicing by overlap extension (SOEing) PCR and were cloned into plasmid pEGFP-C1. Bacterial ghosts were loaded with recombinant pEGFP-C1 and then were transferred to murine macrophages (RAW 264.7). To investigate plasmid transfection and chimeric mRNA transcription, fluorescent microscopy and RT-PCR were used. In vitro studies indicated that bacterial ghosts loaded with pEGFP-C1 plasmid were efficiently taken up by murine macrophages and indicated a high transfection rate (62%), as shown by fluorescent microscopy. RT-PCR from extracted intracellular mRNAs for chimeric Core-NS3 gene showed a specific 607 bp fragment of the gene. The sequence analysis of purified PCR products demonstrated the expected unique mRNA sequence. We constructed a chimeric HCV gene containing both cell mediated and antibody dependent epitopes with a significant expression in murine macrophages delivered by bacterial ghost.

GENETIC DIVERSITY, PARENTAGE VERIFICATION AND GENETIC BOTTLENECKS EVALUATION IN IRANIAN TURKMEN HORSE BREED.

The present study was undertaken to genetically evaluate Turkmen horses for genetic diversity and to evaluate whether they have experienced any recent genetic bottlenecks. A total of 565 individuals from Turkmen horses were characterized for within breed diversity using 12 microsatellite markers. The estimated mean allelic diversity was (9.42 ± 1.78) per locus, with a total of 131 alleles in genotyped samples. A high level of genetic variability within this breed was observed in terms of high values of effective number of alleles (4.70 ± 1.36), observed heterozygosity (0.757 ± 0.19), expected Nei's heterozygosity (0.765 ± 0.13), and polymorphism information content (0.776 ± 0.17). The estimated cumulative probability of exclusion of wrongly named parents (PE) was high, with an average value of 99.96% that indicates the effectiveness of applied markers in resolving of parentage typing in Turkmen horse population. The paternity testing results did not show any misidentification and all selected animals were qualified based on genotypic information using a likelihood-based method. Low values of Wright's fixation index, F(IS) (0.012) indicated low levels of inbreeding. A significant heterozygote excess on the basis of different models, as revealed from Sign and Wilcoxon sign rank test suggested that Turkmen horse population is not in mutation-drift equilibrium. But, the Mode-shift indicator test showed a normal 'L' shaped distribution for allelic class and proportion of alleles, thus indicating the absence of bottleneck events in the recent past history of this breed. Further research work should be carrying out to clarify the cause of discrepancy observed forbottleneck results in this breed. In conclusion, despite unplanned breeding in Turkmen horse population, this breed still has sufficient genetic variability and could provide a valuable source of genetic material that may use for meeting the demands of future breeding programs.

Inbreeding depression for economically important traits of Mazandaran native fowls.

1. The objective was to investigate inbreeding depression for some economic traits of Mazandaran native fowls using data collected from 1992 to 2012 (21 generations) using a REML 2. The mean inbreeding coefficient (F) for the whole population and dams was 4.67% and 4.12%, respectively, and most of the inbred birds (75.79%) and inbred dams (72.58%) had F < 12.5%. 3. Individual and dam inbreeding trends were 0.55% and 0.53% per year. 4. Inbreeding depression for body weight at hatch, at 8 weeks and 12 weeks of age, age at sexual maturity, weight at sexual maturity, egg weight at 1st d of laying and average egg weight at 28, 30 and 32 weeks of laying due to a 1% increase in individual inbreeding were -0.11 g, -3.1 g, -1.3 g, 0.15 d, 0.59 g, -0.05 g and -0.03 g, respectively. 5. A 1% increase in maternal inbreeding resulted in a reduction of 0.06, 0.6 and 3.6 g in body weight at hatch, 8 weeks and 12 weeks of age.

Incidence of human herpes virus-6 and human cytomegalovirus infections in donated bone marrow and umbilical cord blood hematopoietic stem cells.

This study examined the incidence of human herpes virus-6 (HHV-6) and human cytomegalovirus (HCMV) infections that are potentially transmitted to haematopoietic stem cells (HSC) transplant recipients via bone marrow (BM) or umbilical cord blood (UCB). Bone marrow progenitor cells were collected from 30 allogenic BM donors. UCB HSC were collected from 34 subjects. The extracted DNA was then processed using nested polymerase chain reaction (nPCR) technique. HCMV and HHV-6 serological status were determined by enzyme immunoassay (EIA). Nested PCR identified HCMV in 22 (73%) of 30 samples of BM progenitor cells but in only eight (23.5%) of 34 samples of UBC HSC ( P = 0.001). HHV-6 DNA was detected in 11 (36.6%) of 30 BM progenitor cells and in only one (2.9%) of 34 UBC cells ( P = 0.002). Both HHV-6 and HCMV infections were determined in nine (26.5%) of 34 bone marrow samples. The results indicate that, the risk of HCMV and HHV-6 via BM progenitor cells is higher than transmission by UCB cells ( P= 0.04).

The relevance of sleep abnormalities to chronic inflammatory conditions.

Sleep is vital to health and quality of life while sleep abnormalities are associated with adverse health consequences. Nevertheless, sleep problems are not generally considered by clinicians in the management of chronic inflammatory conditions (CIC) such as asthma, RA, SLE and IBD. To determine whether this practice is justified, we reviewed the literature on sleep and chronic inflammatory diseases, including effects of sleep on immune system and inflammation. We found that a change in the sleep-wake cycle is often one of the first responses to acute inflammation and infection and that the reciprocal effect of sleep on the immune system in acute states is often protective and restorative. For example, slow wave sleep can attenuate proinflammatory immune responses while sleep deprivation can aggravate those responses. The role of sleep in CIC is not well explored. We found a substantial body of published evidence that sleep disturbances can worsen the course of CIC, aggravate disease symptoms such as pain and fatigue, and increase disease activity and lower quality of life. The mechanism underlying these effects probably involves dysregulation of the immune system. All this suggests that managing sleep disturbances should be considered as an important factor in the overall management of CIC.

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.

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.