Use of dominant negative nuclear receptors to study xenobiotic-inducible gene expression in primary cultured hepatocytes.
BACKGROUND
To determine the feasibility of using dominant negative nuclear receptors to dissect the regulation of inducible gene expression in primary cultured hepatocytes, a series of dominant negative nuclear receptor expression plasmids were designed with truncated AF-2 subdomains.


METHODS
Plasmids expressing dominant negative or wild-type constitutive androstane receptor (CAR), pregnane X receptor (PXR), farnesoid X receptor (FXR), liver X receptor (LXR), or peroxisome proliferator-activated receptor alpha (PPARalpha) were transiently cotransfected into primary cultured rat hepatocytes, together with an appropriate reporter plasmid.


RESULTS
Treatment with prototypic inducers, 10(-4) M phenobarbital (CAR activator), 10(-5) M pregnenolone 16alpha-carbonitrile (PXR activator), 3x10(-5) M chenodeoxycholate (FXR activator), or 10(-4) M ciprofibrate (PPARalpha activator), significantly activated expression from the corresponding reporter plasmid. Treatment with 22(R)-hydroxycholesterol (LXR activator) only weakly activated the LXR-responsive reporter, while pregnenolone 16alpha-carbonitrile treatment significantly activated this reporter. Cotransfection with wild-type LXRalpha strongly enhanced 22(R)-hydroxycholesterol-inducible expression from the LXR-responsive reporter. Cotransfection of hepatocyte cultures with each of the dominant negative nuclear receptor plasmids significantly inhibited inducible expression of the corresponding reporter while, with one exception (LXRalpha), cotransfection with the wild-type receptor moderately enhanced or had little effect on reporter expression. When each dominant negative nuclear receptor was cross-examined against all inducer-reporter pairs, effects on multiple inducer-reporter pairs were frequently observed. However, in general, only cotransfection with the appropriate dominant negative inhibited inducible reporter expression to a greater extent than did cotransfection with the corresponding wild-type receptor.


CONCLUSIONS
We suggest that the application of dominant negative nuclear receptors has utility in transient transfection studies aimed at discerning the regulatory role of individual nuclear receptor transcription factors in inducible hepatic gene expression, provided that appropriate controls are employed.
Kocarek
Thomas A
TA
Institute of Environmental Health Sciences, Wayne State University, Room 4000, 2727 Second Avenue, Detroit, MI 48201, USA. t.kocarek@wayne.edu
Shenoy
Sarita D
SD
Mercer-Haines
Nancy A
NA
Runge-Morris
Melissa
M
D000818:Animals; D002478:Cells, Cultured; D002635:Chenodeoxycholic Acid / Q000494:pharmacology; D002995:Clofibric Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D000090702:Constitutive Androstane Receptor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D058607:Fibric Acids; D015870:Gene Expression / Q000187:drug effects*; D005799:Genes, Dominant; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D010634:Phenobarbital / Q000494:pharmacology; D000077297:Pregnane X Receptor; D011285:Pregnenolone Carbonitrile / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D015262:Xenobiotics / Q000494:pharmacology*; D000098825:Oxysterol Binding Proteins; D000099150:Receptor, Farnesoid X-Activated
D002635:Chenodeoxycholic Acid; D002995:Clofibric Acid; D000090702:Constitutive Androstane Receptor; D004268:DNA-Binding Proteins; D058607:Fibric Acids; D010634:Phenobarbital; D000077297:Pregnane X Receptor; D011285:Pregnenolone Carbonitrile; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors; D015262:Xenobiotics; D000098825:Oxysterol Binding Proteins; D000099150:Receptor, Farnesoid X-Activated; C019304:ciprofibrate