Transactivation properties of wild-type and mutant androgen receptors in transiently transfected primary human fibroblasts.
BACKGROUND
Stromal cells play key roles during androgen-mediated male sexual differentiation. Our objective was to establish a transient transfection method for primary human fibroblasts enabling functional characterization of wild-type (wt) and mutant androgen receptor (AR) plasmid constructs, corresponding to partial and complete androgen insensitivity syndrome (PAIS/CAIS).


METHODS
An AR-negative fibroblast strain (ARD842) was established from the gonads of a CAIS patient. Wt-AR or either mutants L712F (PAIS), R774C or V866M (CAIS) were transfected using a polyamine-based procedure. Alternatively, two AR-positive male foreskin fibroblast strains were investigated. Androgen-induced activation of two co-transfected reporter plasmids ((ARE)(2)TATA-, MMTV-luciferase) was measured.


RESULTS
All three fibroblast strains showed a ligand-dependent rise of luciferase activity after transfection of wt-AR. Mutant plasmids were assessed in AR-negative ARD842 cells. While L712F showed high partial activity, R774C and V866M were nearly inactive. The intrinsic AR of normal foreskin fibroblasts revealed no measurable ligand-inducible reporter gene activity.


CONCLUSIONS
Polyamine-based transfection of AR plasmids into cultured fibroblasts provides a promising tool for analysis of AR transactivation, thereby considering a stromal cellular background. This is supported by the mutant ARs which showed the expected levels of impaired transactivation with respect to the corresponding AIS phenotypes. The role of the intrinsic AR in normal male human foreskin fibroblasts needs further exploration.
Holterhus
P M
PM
Department of Pediatrics, University Hospital Schleswig Holstein, Campus Lubeck, Lubeck, Germany.
Salzburg
J
J
Werner
R
R
Hiort
O
O
D013734:Androgen-Insensitivity Syndrome / Q000378:metabolism / Q000503:physiopathology*; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology* / Q000502:physiology*; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D008297:Male; D016296:Mutagenesis; D010641:Phenotype; D010957:Plasmids; D011944:Receptors, Androgen / Q000235:genetics* / Q000378:metabolism*; D012867:Skin / Q000166:cytology; D014162:Transfection / Q000379:methods*
D011944:Receptors, Androgen