Abstract
In the present study, the length of 360, 1848 and 367 bp sequences of promoters from three subtypes of PI3K family (PI3KCa, PI3KC2b and PI3KC3) of yellow catfish Pelteobagrus fulvidraco were cloned and characterized. Bioinformatics analysis revealed that PI3KCa, PI3KC2b and PI3KC3 had different structures in their core promoter regions. The promoter regions of PI3KCa and PI3KC2b had CpG islands but no CAAT and TATA box. In contrast, the promoter of PI3KC3 had the canonical TATA and CAAT box but no CpG island. The binding sites of several transcription factors, such as HNF1, STAT and NF-κB, were predicted on PI3KCa promoter. The binding sites of transcription factors, such as FOXO1, PPAR-RXR, STAT, IK1, HNF6 and HNF3, were predicted on PI3KC2b promoter and the binding sites of FOXO1 and STAT transcription factors were predicted on PI3KC3 promoter. Deletion analysis indicated that these transcriptional factors were the potential regulators to mediate the activities of their promoters. Subsequent mutation analysis and electrophoretic mobility-shift assay (EMSA) demonstrated that HNF1 and IK1 directly bound with PI3KCa and PI3KC2b promoters and negatively regulated the activities of PI3KCa and PI3KC2b promoters, respectively. Conversely, FOXO1 directly bound with the PI3KC2b and PI3KC3 promoters and positively regulated their promoter activities. In addition, AS1842856 (AS, a potential FOXO1 inhibitor) incubation significantly reduced the relative luciferase activities of several plasmids of PI3KC2b and PI3KC3 but did not significantly influence the relative luciferase activities of the PI3KCa plasmids. Moreover, by using primary hepatocytes from yellow catfish, AS incubation significantly down-regulated the mRNA levels of PI3KCa, PI3KC2b and PI3KC3 and reduced triacylglyceride (TG) accumulation and insulin-induced TG accumulation, as well as the activities and the mRNA levels of several genes involved in lipid metabolism. Thus, the present study offers new insights into the mechanisms for transcriptional regulation of PI3Ks and for PI3Ks-mediated regulation of lipid metabolism by insulin in fish.
Highlights
Phosphatidylinositol-3 kinase (PI3K) is an intracellular transducer with lipid substrate specificity and implicates a wide range of signaling pathways involved in cell survival, proliferation and nutrient metabolism [1]
The characterization and tissue expression profile of seven PI3K members from yellow catfish were determined in our previous study [3], the underlying transcriptional mechanisms of PI3K member were still unknown
As a continuation of our series of studies involved in the structure and functional analysis of PI3K in yellow catfish, the present study cloned and characterized the sequences of PI3Ks promoter and explored theirs functions
Summary
Phosphatidylinositol-3 kinase (PI3K) is an intracellular transducer with lipid substrate specificity and implicates a wide range of signaling pathways involved in cell survival, proliferation and nutrient metabolism [1]. The regulation of gene expression can occur at different steps ranging from DNA–RNA transcription to post-translational modification of protein [4]. It involves the interaction of transcription factors with the transcription machinery as well as changes in DNA structure (epigenetic process including CpG dinucleotide methylation) which influences accessibility of promoter sequences [5]. Studies suggested that the transcription factor STAT, which can bind with the region of PI3K promoters, differed between fish and mammals [12,13]. To our best knowledge, information involved in the transcriptional regulation of PI3K members was very scarce in fish
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