Regulation of apolipoprotein A-I gene expression in Hep G2 cells depleted of Cu by cupruretic tetramine.
Studies were designed to examine the regulation of apolipoprotein (apo) A-I gene expression in Cu-depleted Hep G2 cells. The cupruretic chelator N,N'-bis(2-aminoethyl)-1,3-propanediamine 4 HCl (2,3,2-tetramine or TETA) was used to maintain a 77% reduction in cellular Cu in Hep G2 cells. After two passages of TETA treatment, the relative abundance of apoA-I mRNA was elevated 52%. In TETA-treated cells, the rate of apoA-I mRNA decay measured by an actinomycin D chase study was accelerated 108%, and the synthesis of apoA-I mRNA determined by a nuclear runoff assay was enhanced 2.5-fold in TETA-treated cells. All of those changes could be reverted toward the control values with Cu supplementation for only 2 days. In transient transfection assays, a 26.7% increase in chloramphenicol O-acetyltransferase (CAT) activity for the reporter construct -256AI-CAT was observed in the treated cells. However, the ability of apoA-I regulatory protein 1 (ARP-1) to repress the CAT activity was not affected by the depressed Cu status. In addition, gel retardation experiments demonstrated that Cu depletion enhanced the binding of hepatocyte nuclear factor 4 (HNF-4) and other undefined nuclear factors to oligonucleotides containing site A, one of three regulatory sites of the apoA-I gene promoter. Moreover, the relative abundance of HNF-4 mRNA was increased 58% in the Cu-depleted cells. Thus the observed increase in apoA-I gene transcription may be mediated mostly by an elevated level of the regulatory factor, HNF-4. In summary, the present findings established the mechanism by which a depressed cellular Cu status can enhance apoA-I mRNA production and subsequently increase apoA-I synthesis.
Wu
J Y
JY
Department of Nutritional Sciences, University of Arizona, Tucson 85721, USA.
Zhang
J J
JJ
Wang
Y
Y
Reaves
S K
SK
Wang
Y R
YR
Lei
P P
PP
Lei
K Y
KY
D016632:Apolipoprotein A-I / Q000096:biosynthesis* / Q000235:genetics; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002467:Cell Nucleus / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology*; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003300:Copper / Q000378:metabolism*; D004268:DNA-Binding Proteins; D003609:Dactinomycin / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D007700:Kinetics; D010750:Phosphoproteins / Q000096:biosynthesis; D011073:Polyamines / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014157:Transcription Factors / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002614:Chelating Agents; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D011073:Polyamines; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C064279:N,N-bis(2-aminoethyl)-1,3-propanediamine; D003609:Dactinomycin; D003300:Copper; D015500:Chloramphenicol O-Acetyltransferase