Abstract
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 β. In conclusion, HNF4α appears to play a key role in intestinal lipid metabolism as well as intestinal anti-oxidative and anti-inflammatory defense mechanisms.
Highlights
Metabolism of fatty acid (FA),2 cholesterol, glucose, and urea [4]
HNF4␣ expression was measured in the selected clones, cultured on filters, 13 days after their differentiation, and compared with noncoding shRNA vector control cells
A significant reduction in gene expression (ϳ80%) was noted in HNF4␣ shRNA-infected cells compared with Mock cells infected with empty vector or with untreated cells as measured by quantitative RTPCR (Fig. 1A)
Summary
Metabolism of fatty acid (FA), cholesterol, glucose, and urea [4]. Transcriptional activation by HNF4␣ is mediated by its binding as a homodimer to direct repeat-1 promoter sequences of target genes [5]. HNF4␣ has been shown to be necessary for hepatoblast differentiation into hepatocytes [14], epithelial morphology [15], gene-encoding proteins of cell junctions [16], and liver architecture, including the organization of the sinusoidal endothelium [15]. Did HNF4␣ regulate the expression of many genes such as apolipoproteins (apo) (20 – 25), but it has been found associated with susceptibility to abnormal intestinal permeability and inflammation [26]. These findings indicate that HNF4␣ is critical for intestinal tissue development and the maintenance of a number of metabolic pathways. To gain more insight into the physiological and biological significance of HNF4␣ in the intestine, molecular strategies were devised in the Caco-2 cell line, a reliable human intestinal model, to thoroughly examine the specific role of HNF4␣ in oxidative stress, inflammation, lipid transport and metabolism, apobiogenesis, and lipoprotein assembly
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