Antioxidative properties of paraoxonase 2 in intestinal epithelial cells.

Paraoxonase (PON) family members seem central to a wide variety of human illnesses, but appreciation of their antioxidative function in the gastrointestinal tract is in its infancy. The major objective of the present work is to highlight the role of the ubiquitously expressed PON2 in the small intestine. With use of pLKO lentiviral vector containing short hairpin RNA (shRNA) lentivirus, PON2 expression was knocked down in intestinal Caco-2/15 cells, where antioxidative status, lipid peroxidation, and degree of inflammation were evaluated. As a consequence of PON2 inactivation in the epithelial cells, we observed 1) imbalanced primary and secondary antioxidative responses, characterized by increased superoxide dismutases and decreased catalase, 2) high concentrations of H(2)O(2) and malondialdehyde, along with low glutathione-to-glutathione disulfide ratio, 3) upregulation of TNF-α, IL-6, and monocyte chemoattractant protein-1 gene expression after induction of oxidative stress, and 4) raised level of the activation of transcription factor NF-κB, which was likely implicated in exacerbation of the inflammatory activation. These results suggest that PON2 is involved in the antioxidative and anti-inflammatory response in intestinal epithelial cells.

The three-gene paraoxonase family: physiologic roles, actions and regulation.

The paraoxonase (PON) gene family is composed of three members (PON1, PON2, PON3) that share considerable structural homology and are located adjacently on chromosome 7 in humans. By far the most-studied member is PON1, a high-density lipoprotein-associated esterase/lactonase, also endowed with the capacity to hydrolyze organophosphates, but all the three proteins prevent oxidative stress and fight inflammation. They therefore seem central to a wide variety of human illnesses, including atherosclerosis, diabetes mellitus, mental disorders and inflammatory bowel disease. The major goal of this review is to highlight the regulation of each of the paraoxonase components by diverse nutritional molecules and pharmacological agents as well as a number of pathophysiological events, such as oxidative stress and inflammation. Considerable and detailed cell-based studies and animal model experiments have been provided to allow a thorough scrutiny of PON modulation, which will increase our understanding and ability to target these genes in order to efficiently increase their transcriptional activity and decrease the risks of developing different disorders.

Modification in oxidative stress, inflammation, and lipoprotein assembly in response to hepatocyte nuclear factor 4alpha knockdown in intestinal epithelial cells.

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor mainly expressed in the liver, intestine, kidney, and pancreas. Many of its hepatic and pancreatic functions have been described, but limited information is available on its role in the gastrointestinal tract. The objectives of this study were to evaluate the anti-inflammatory and antioxidant functions of HNF4α as well as its implication in intestinal lipid transport and metabolism. To this end, the HNF4A gene was knocked down by transfecting Caco-2 cells with a pGFP-V-RS lentiviral vector containing an shRNA against HNF4α. Inactivation of HNF4α in Caco-2 cells resulted in the following: (a) an increase in oxidative stress as demonstrated by the levels of malondialdehyde and conjugated dienes; (b) a reduction in secondary endogenous antioxidants (catalase, glutathione peroxidase, and heme oxygenase-1); (c) a lower protein expression of nuclear factor erythroid 2-related factor that controls the antioxidant response elements-regulated antioxidant enzymes; (d) an accentuation of cellular inflammatory activation as shown by levels of nuclear factor-κB, interleukin-6, interleukin-8, and leukotriene B4; (e) a decrease in the output of high density lipoproteins and of their anti-inflammatory and anti-oxidative components apolipoproteins (apo) A-I and A-IV; (f) a diminution in cellular lipid transport revealed by a lower cellular secretion of chylomicrons and their apoB-48 moiety; and (g) alterations in the transcription factors sterol regulatory element-binding protein 2, peroxisome proliferator-activated receptor α, and liver X receptor α and ß. In conclusion, HNF4α appears to play a key role in intestinal lipid metabolism as well as intestinal anti-oxidative and anti-inflammatory defense mechanisms.

Comparative expression analysis reveals differences in the regulation of intestinal paraoxonase family members.

The paraoxonase (PON) gene cluster contains three members (PON1, PON2, and PON3), located on chromosome 7q21.3-22.1. Until now there has been little insight into their regulation in human intestine. This study was designed to determine the regulation of PONs by oxidative stress and inflammatory factors. Differentiated Caco-2/15 cells, cultured on polycarbonate Transwell filter inserts, exhibited transcripts of the 3 PONs whereas Western blot revealed the protein expression of PON2 and PON3 only. Iron-ascorbate-mediated lipid peroxidation, lipopolysaccharides (LPS), tumor necrosis factor-alpha and interferon-gamma induced differential effects on the gene expression and protein mass of PONs. In particular, LPS down-regulated PON2 protein expression, which was accompanied with decreased levels of IkappaBalpha, the inhibitor of the proinflammatory transcription factor nuclear factor-kappa B (NF-kappaB). Selective inactivation of NF-kappaB by the action of caffeic acid phenethyl ester (CAPE) partially attenuated but did not abolish LPS-triggered decline of PON2. However, the combination of CAPE and antioxidants completely abrogated the negative impact of LPS on PON2. Therefore, our data indicate that oxidative stress and proinflammatory agents selectively affect the expression of PONs. Our findings also suggest that both NF-kappaB pathway and lipid peroxidation are implicated in LPS-dependent diminution of PON2.

Regulation of the proprotein convertase subtilisin/kexin type 9 in intestinal epithelial cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally promotes the degradation of the low-density lipoprotein receptor (LDLr) in hepatocytes and increases plasma LDL cholesterol. It is not clear, however, whether PCSK9 plays a role in the small intestine. Here, we characterized the patterns of variations of PCSK9 and LDLr in fully differentiated Caco-2/15 cells as a function of various potential effectors. Cholesterol (100 microM) solubilized in albumin or micelles significantly downregulated PCSK9 gene (30%, P<0.05) and protein expression (50%, P<0.05), surprisingly in concert with a decrease in LDLr protein levels (45%, P<0.05). Cells treated with 25-hydroxycholesterol (50 microM) also displayed significant reduction in PCSK9 gene (37%, P<0.01) and protein (75% P<0.001) expression, whereas LDLr showed a decrease at the gene (30%, P<0.05) and protein (57%, P<0.01) levels, respectively. The amounts of PCSK9 mRNA and protein in Caco-2/15 cells were associated to the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein-2 (SREBP-2) that can transcriptionally activate PCSK9 via sterol-regulatory elements located in its proximal promoter region. On the other hand, depletion of cholesterol content by hydroxypropyl-beta-cyclodextrin upregulated PCSK9 transcripts (20%, P<0.05) and protein mass (540%, P<0.001), in parallel with SREBP-2 protein levels. The addition of bile acids (BA) taurocholate and deoxycholate to the apical culture medium lowered PCSK9 gene expression (25%, P<0.01) and raised PCSK9 protein expression (30%, P<0.01), respectively, probably via the modulation of farnesoid X receptor. Furthermore, unconjugated and conjugated BA exhibited different effects on PCSK9 and LDLr. Altogether, these data indicate that intestinal PCSK9 is highly modulated by sterols and emphasize the distinct effects of BA species.

Biological role, protein expression, subcellular localization, and oxidative stress response of paraoxonase 2 in the intestine of humans and rats.

Oxidative stress is a cardinal manifestation of various intestinal disorders. However, very little knowledge is available on the intestine's inherent defense mechanisms against free radicals. This study was designed to determine the protein expression, subcellular localization and oxidative stress response of paraoxonase 2 (PON2), a member of a powerful antioxidant family in human and rat intestine. Biochemical and ultrastructural experiments all showed a substantial expression of PON2 in human and rat intestine. Western blot analysis disclosed higher levels of PON2 in the jejunum than in the duodenum, ileum, and colon. Cell fractionation revealed a predominant PON2 association with microsomes and lysosomes in the human jejunum, which differed from that in rats. PON2 was detected in the intestine as early as week 15 of gestation and was significantly increased by week 20. Iron ascorbate-mediated lipid peroxidation induced a marked decrease in PON2 expression in intestinal specimens coincidental to an abundant rise in malondialdehyde (MDA). On the other hand, preincubation with potent antioxidants, such as butylated hydroxytoluene, Trolox, and N-acetylcysteine, prevented iron-ascorbate-generating PON2 reduction in parallel with MDA suppression. Finally, the preincubation of permeabilized Caco-2 cells with purified PON2 led to a protection against iron-ascorbate-induced lipid peroxidation. These observations demonstrate that the human intestine is preferentially endowed with a marked PON2 expression compared with the rat intestine and this expression shows a developmental and intracellular pattern of distribution. Furthermore, our observations suggest PON2 protective effects against prooxidant stimuli in the small intestine.