Diane Stroup

Regulation of cholesterol 7α-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXRα)

Abstract The cholesterol 7α-hydroxylase gene (CYP7A1) plays an important role in regulation of bile acid biosynthesis and cholesterol homeostasis. Oxysterol receptor, LXR, stimulates, whereas the bile acid receptor, FXR, inhibits CYP7A1 transcription. The goal of this study was to investigate the role of LXRα on the regulation of rat, human and hamster CYP7A1 transcription in its native promoter and cellular context. Cotransfection with LXRα and RXRα expression plasmids strongly stimulated rat CYP7A1/luciferase reporter activity in HepG2 cells and oxysterol was not required. However, LXRα had much less effect on hamster and no significant effect on human CYP7A1 promoter activity in HepG2 cells. In Chinese hamster ovary cells, cotransfection with LXRα stimulated reporter activity by less than 2-fold and addition of 22(R)-hydroxycholesterol caused a small but significant stimulation of rat, human and hamster CYP7A1 promoter activity. At least two direct repeats of AGGTCA-like sequences with 4-base spacing (DR4) and five-base spacing (DR5), in previously identified bile acid response elements of the rat CYP7A1 were able to bind LXRα/RXRα and confer LXRα stimulation. However, LXRα did not bind to the corresponding sequences of the human gene and bound weakly to hamster and mouse DR4 sequences. Therefore, rats and mice have the unusual capacity to convert cholesterol to bile acids by LXRα-mediated stimulation of CYP7A1 transcription, whereas other species do not respond to cholesterol and develop hypercholesterolemia on a diet high in cholesterol.

Synthesis of the major inner capsid protein VP6 of the human rotavirus Wa in Escherichia coli.

The gene for the major inner capsid protein VP6 of human rotavirus strain Wa has been cloned and placed into a bacterial expression vector under the control of the inducible hybrid trp-lac (tac) promoter. Recombinant VP6 was produced at low levels in a cell-free Escherichia coli transcription-translation system programmed with this expression plasmid. The yield of VP6 synthesized in the extract could be increased several-fold by introduction of point mutations upstream and downstream from the start codon. Upon induction with IPTG, E. coli JM105 cells harboring the mutated expression plasmid produced VP6 as shown by immunoblotting of proteins from bacterial lysates with anti-Wa antiserum. Recombinant VP6 appeared to inhibit the growth of E. coli and did not accumulate in the cells to high levels. Conformational analysis with a monoclonal antibody suggested that bacterially produced VP6 adopted an oligomeric structure characteristic for native VP6.