Abstract 1333: Amphipathic Helical Peptides Increase ApoA-I Synthesis and Secretion from Caco2 Cells Via Transcription Factor Regulation
Objective- We have previously shown that amphipathic peptides having either a class A amphipathic helix (D-4F) or a class G* amphipathic helix (D- [113–122] apoJ) function, in part, by enhancing intestinal synthesis and secretion of apolipoprotein (apo)A-I, the major protein component of high density lipoprotein (HDL). Recent studies implicate two members of the steroid receptor superfamily of transcription factors, HNF-4 and ARP-1, in the regulation of apoA1 transcription. HNF-4 activates while ARP-1 represses apoA-I gene expression in both HepG2 and Caco2 cells. ApoA-I transcription has also been shown to be upregulated by the orphan nuclear receptor ROR-a, suggesting that transcription of the apoA-I gene may be dependent on the intracellular balance of these positive and negative regulatory factors. The objective of the present study was to determine the role of ROR-a, HNF-4 and ARP-1 in amphipathic peptide mediated-regulation of apoA-I synthesis in a human intestinal cell line, Caco2.
Methods and Results- Microarray analysis of Caco2 cells treated with D-4F or D-[113–122]apoJ was carried out and confirmed by RT-PCR. Compared to controls, treatment of Caco2 cells with 0.25–250 micro g/ml of either D-4F or D-[113–122]apoJ cells resulted in 2.5–3-fold increases in both apoA-I mRNA expression by RT-PCR and in protein secretion into the media by ELISA by 48 hr after treatment (p<0.05). Both peptides reduced ARP-1 expression 3-fold but increased HNF-4 expression 150-fold by 8 hr post-treatment. Treatment with either peptide resulted in a significant 2–3-fold increase in ROR-a expression by 24 hr (p<0.05). Caco2 cells were also transfected with a luciferase-based apoA-I reporter construct (−1000/+500,) containing a binding site for ROR-a. Compared to control cells, treatment with peptides showed more than a 3-fold induction in luciferase activity. Conversely, knock-down of ROR-a by an antisense oligonucleotide completely inhibited induced luciferase activity following treatment with the amphipathic peptides.
Conclusions- The amphipathic peptides D-4F and D-[113–122]apoJ increase apoA-I synthesis and secretion from Caco2 cells and the mechanism of action appears to involve ROR-a and possibly the transcription factors, HNF-4 and ARP-1.