Robert S. Chapkin

Protein kinase C ζ isoform is critical for mitogenic signal transduction

The requirement of protein kinase C zeta (zeta PKC) for maturation of X. laevis oocytes in response to insulin, p21ras, and phosphatidylcholine-hydrolyzing phospholipase C has recently been shown. Here we present experimental evidence demonstrating that activation of zeta PKC is not only necessary but also sufficient by itself to activate maturation in oocytes and to produce deregulation of growth control in mouse fibroblasts. Furthermore, by using a dominant kinase-defective mutant of zeta PKC, we confirm that this kinase is required for mitogenic activation in oocytes and fibroblasts. These results permit us to propose zeta PKC as a critical step downstream of p21ras in mitogenic signal transduction.

Dietary docosahexaenoic and eicosapentaenoic acid: Emerging mediators of inflammation

The inflammatory response is designed to help fight and clear infection, remove harmful chemicals, and repair damaged tissue and organ systems. Although this process, in general, is protective, the failure to resolve the inflammation and return the target tissue to homeostasis can result in disease, including the promotion of cancer. A plethora of published literature supports the contention that dietary n-3 polyunsaturated fatty acids (PUFA), and eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in particular, are important modulators of a hosts inflammatory/immune responses. The following review describes a mechanistic model that may explain, in part, the pleiotropic anti-inflammatory and immunosuppressive properties of EPA and DHA. In this review, we focus on salient studies that address three overarching mechanisms of n-3 PUFA action: (i) modulation of nuclear receptor activation, i.e., nuclear factor-kappaB (NF-kappaB) suppression; (ii) suppression of arachidonic acid-cyclooxygenase-derived eicosanoids, primarily prostaglandin E(2)-dependent signaling; and (iii) alteration of the plasma membrane micro-organization (lipid rafts), particularly as it relates to the function of Toll-like receptors (TLRs), and T-lymphocyte signaling molecule recruitment to the immunological synapse (IS). We propose that lipid rafts may be targets for the development of n-3 PUFA-containing dietary bioactive agents to down-modulate inflammatory and immune responses and for the treatment of autoimmune and chronic inflammatory diseases.

Dietary docosahexaenoic acid suppresses T cell protein kinase Cθ lipid raft recruitment and IL-2 production

To date, the proximal molecular targets through which dietary n-3 polyunsaturated fatty acids (PUFA) suppress the inflammatory process have not been elucidated. Because cholesterol and sphingolipid-enriched rafts have been proposed as platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane, we determined the in vivo effects of fish oil and highly purified docosahexaenoic acid (DHA; 22:6n-3) on T cell microdomain lipid composition and the membrane subdomain distribution of signal-transducing molecules (protein kinase C (PKC)θ, linker for activation of T cells, and Fas/CD95), before and after stimulation. Mice were fed diets containing 5 g/100 g corn oil (control), 4 g/100 g fish oil (contains a mixture of n-3 PUFA) plus 1 g/100 g corn oil, or 4 g/100 g corn oil plus 1 g/100 g DHA ethyl ester for 14 days. Dietary n-3 PUFA were incorporated into splenic T cell lipid raft and soluble membrane phospholipids, resulting in a 30% reduction in raft sphingomyelin content. In addition, polyclonal activation-induced colocalization of PKCθ with lipid rafts was reduced by n-3 PUFA feeding. With respect to PKCθ effector pathway signaling, both AP-1 and NF-κB activation, IL-2 secretion, and lymphoproliferation were inhibited by fish oil feeding. Similar results were obtained when purified DHA was fed. These data demonstrate for the first time that dietary DHA alters T cell membrane microdomain composition and suppresses the PKCθ signaling axis.

n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon

Caveolae, by virtue of their unique lipid environment, serve as signaling platforms that regulate cellular events. Perturbations in caveolae lipid composition have been shown in vitro to displace proteins from lipid microdomains, thereby altering their functionality and subsequent downstream signaling. Because membrane remodeling may not be accurately represented by using pharmacological treatments and in vitro models, we investigated the in vivo ability of dietary n‐3 polyunsaturated fatty acids (PUFA) to alter caveolae lipid environment and the compartmentalization of resident proteins in mouse colonic mucosa. n‐3 PUFA were examined for their chemoprotective, membrane lipid‐modifying properties. Colonic caveolae in mice fed n‐6 or n‐3 PUFA enriched diets were characteristically enriched in cholesterol, sphingomyelin, and caveolin‐1. n‐3 PUFA feeding, compared with n‐6 PUFA, significantly altered colonic caveolae microenvironment by increasing phospholipid n‐3 fatty acyl content and reducing both cholesterol (by 46%) and caveolin‐1 (by 53%), without altering total cellular levels. Concomitantly, localization of caveolae‐resident signaling proteins H‐Ras and eNOS in colonic caveolae was decreased by n‐3 PUFA, by 45 and 56%, respectively. The distribution of non‐caveolae proteins K‐Ras and clathrin was unaffected. Moreover, EGF‐stimulated H‐Ras, but not K‐Ras activation was significantly suppressed following n‐3 PUFA feeding, in parallel with the selective alterations in their microlocalization. These findings reveal a novel modality by which n‐3 PUFA remodel membrane microdomains in vivo and thereby alter caveolae protein localization and functionality.

Chemopreventive n-3 Polyunsaturated Fatty Acids Reprogram Genetic Signatures during Colon Cancer Initiation and Progression in the Rat

The mechanisms by which n-3 polyunsaturated fatty acids (PUFAs) decrease colon tumor formation have not been fully elucidated. Examination of genes up- or down-regulated at various stages of tumor development via the monitoring of gene expression relationships will help to determine the biological processes ultimately responsible for the protective effects of n-3 PUFA. Therefore, using a 3 × 2 × 2 factorial design, we used Codelink DNA microarrays containing ∼9000 genes to help decipher the global changes in colonocyte gene expression profiles in carcinogen-injected Sprague Dawley rats. Animals were assigned to three dietary treatments differing only in the type of fat (corn oil/n-6 PUFA, fish oil/n-3 PUFA, or olive oil/n-9 monounsaturated fatty acid), two treatments (injection with the carcinogen azoxymethane or with saline), and two time points (12 hours and 10 weeks after first injection). Only the consumption of n-3 PUFA exerted a protective effect at the initiation (DNA adduct formation) and promotional (aberrant crypt foci) stages. Importantly, microarray analysis of colonocyte gene expression profiles discerned fundamental differences among animals treated with n-3 PUFA at both the 12 hours and 10-week time points. Thus, in addition to demonstrating that dietary fat composition alters the molecular portrait of gene expression profiles in the colonic epithelium at both the initiation and promotional stages of tumor development, these findings indicate that the chemopreventive effect of fish oil is due to the direct action of n-3 PUFA and not to a reduction in the content of n-6 PUFA.

A metagenomic study of diet-dependent interaction between gut microbiota and host in infants reveals differences in immune response

BackgroundGut microbiota and the host exist in a mutualistic relationship, with the functional composition of the microbiota strongly affecting the health and well-being of the host. Thus, it is important to develop a synthetic approach to study the host transcriptome and the microbiome simultaneously. Early microbial colonization in infants is critically important for directing neonatal intestinal and immune development, and is especially attractive for studying the development of human-commensal interactions. Here we report the results from a simultaneous study of the gut microbiome and host epithelial transcriptome of three-month-old exclusively breast- and formula-fed infants.ResultsVariation in both host mRNA expression and the microbiome phylogenetic and functional profiles was observed between breast- and formula-fed infants. To examine the interdependent relationship between host epithelial cell gene expression and bacterial metagenomic-based profiles, the host transcriptome and functionally profiled microbiome data were subjected to novel multivariate statistical analyses. Gut microbiota metagenome virulence characteristics concurrently varied with immunity-related gene expression in epithelial cells between the formula-fed and the breast-fed infants.ConclusionsOur data provide insight into the integrated responses of the host transcriptome and microbiome to dietary substrates in the early neonatal period. We demonstrate that differences in diet can affect, via gut colonization, host expression of genes associated with the innate immune system. Furthermore, the methodology presented in this study can be adapted to assess other host-commensal and host-pathogen interactions using genomic and transcriptomic data, providing a synthetic genomics-based picture of host-commensal relationships.

n-3 Polyunsaturated Fatty Acids Suppress the Localization and Activation of Signaling Proteins at the Immunological Synapse in Murine CD4+ T Cells by Affecting Lipid Raft Formation

The molecular properties of immunosuppressive n-3 polyunsaturated fatty acids (PUFA) have not been fully elucidated. Using CD4+ T cells from wild-type control and fat-1 transgenic mice (enriched in n-3 PUFA), we show that membrane raft accumulation assessed by Laurdan (6-dodecanoyl-2-dimethyl aminonaphthalene) labeling was enhanced in fat-1 cells following immunological synapse (IS) formation by CD3-specific Ab expressing hybridoma cells. However, the localization of protein kinase Cθ, phospholipase Cγ-1, and F-actin into the IS was suppressed. In addition, both the phosphorylation status of phospholipase Cγ-1 at the IS and cell proliferation as assessed by CFSE labeling and [3H]thymidine incorporation were suppressed in fat-1 cells. These data imply that lipid rafts may be targets for the development of dietary agents for the treatment of autoimmune and chronic inflammatory diseases.

Immunomodulatory Effects of (n-3) Fatty Acids: Putative Link to Inflammation and Colon Cancer

Chronic inflammation and colorectal cancer are closely linked. Although the overall mechanisms of inflammation-associated gastrointestinal carcinogenesis are complex, it is clear that antiinflammatory therapy is efficacious against neoplastic progression and malignant conversion. From a dietary perspective, fish oil containing (n-3) polyunsaturated fatty acids (PUFAs) has antiinflammatory properties, but for years the mechanism has remained obscure. Of relevance to the immune system in the intestine, we showed that (n-3) PUFA feeding alters the balance between CD4+ T-helper (Th1 and Th2) subsets by directly suppressing Th1 cell development (i.e., clonal expansion). This is noteworthy because Th1 cells mediate inflammatory diseases and resistance to intracellular pathogens or allergic hypersensitivity, and Th2 cells mediate resistance to extracellular pathogens. Therefore, any changes induced by (n-3) PUFAs in T-cell subset balance and function are important because the outcome is expected to suppress the development of autoimmune diseases and possibly the occurrence of colon cancer. Precisely how the immunomodulatory effects of (n-3) PUFAs influence inflammation-associated colonic tumor development is the subject of an ongoing investigation.

n-3 Polyunsaturated fatty acids modulate carcinogen-directed non-coding microRNA signatures in rat colon

We have hypothesized that dietary modulation of intestinal non-coding RNA [microRNA (miRNA)] expression may contribute to the chemoprotective effects of nutritional bioactives (fish oil and pectin). To fully understand the effects of these agents on the expression of miRNAs, Sprague-Dawley rats were fed diets containing corn oil or fish oil with pectin or cellulose and injected with azoxymethane (AOM, a colon-specific carcinogen) or saline (control). Real-time polymerase chain reaction using miRNA-specific primers and Taq Man probes was carried out to quantify effects on miRNA expression in colonic mucosa. From 368 mature miRNAs assayed, at an early stage of cancer progression (10 week post AOM injection), let-7d, miR-15b, miR-107, miR-191 and miR-324-5p were significantly (P < 0.05) affected by diet x carcinogen interactions. Overall, fish oil fed animals exhibited the smallest number of differentially expressed miRNAs (AOM versus saline treatment). With respect to the tumor stage (34 week post AOM injection), 46 miRNAs were dysregulated in adenocarcinomas compared with normal mucosa from saline-injected animals. Of the 27 miRNAs expressed at higher (P < 0.05) levels in tumors, miR-34a, 132, 223 and 224 were overexpressed at >10-fold. In contrast, the expression levels of miR-192, 194, 215 and 375 were dramatically reduced (< or = 0.32-fold) in adenocarcinomas. These results demonstrate for the first time the utility of the rat AOM model and the novel role of fish oil in protecting the colon from carcinogen-induced miRNA dysregulation.

Reduced Colitis-Associated Colon Cancer in Fat-1 (n-3 Fatty Acid Desaturase) Transgenic Mice

Bioactive food components containing n-3 polyunsaturated fatty acids (PUFA) modulate multiple determinants that link inflammation to cancer initiation and progression. Therefore, in this study, fat-1 transgenic mice, which convert endogenous n-6 PUFA to n-3 PUFA in multiple tissues, were injected with azoxymethane followed by three cycles of dextran sodium sulfate (DSS) to induce colitis-associated cancer. Fat-1 mice exhibited a reduced number of colonic adenocarcinomas per mouse (1.05 +/- 0.29 versus 2.12 +/- 0.51, P = 0.033), elevated apoptosis (P = 0.03), and a decrease in n-6 PUFA-derived eicosanoids, compared with wild-type (wt) mice. To determine whether the chemoprotective effects of n-3 PUFA could be attributed to its pleiotropic anti-inflammatory properties, colonic inflammation and injury scores were evaluated 5 days after DSS exposure followed by either a 3-day or 2-week recovery period. There was no effect of n-3 PUFA at 3 days. However, following a 2-week recovery period, colonic inflammation and ulceration scores returned to pretreatment levels compared with 3-day recovery only in fat-1 mice. For the purpose of examining the specific reactivity of lymphoid elements in the intestine, CD3(+) T cells, CD4(+) T helper cells, and macrophages from colonic lamina propria were quantified. Comparison of 3-day versus 2-week recovery time points revealed that fat-1 mice exhibited decreased (P < 0.05) CD3(+), CD4(+) T helper, and macrophage cell numbers per colon as compared with wt mice. These results suggest that the antitumorigenic effect of n-3 PUFA may be mediated, in part, via its anti-inflammatory properties.