Stimulation of FSHbeta transcription by blockade of endogenous pituitary follistatin production: Efficacy of adenoviral-delivered antisense RNA in the rat.
This study investigated FSHbeta transcriptional responses to the suppression of endogenous follistatin (FST) production using FST antisense RNA (FST-AS) expressing adenovirus constructs in female rat pituitary cells in vitro. Adenoviral delivery systems were characterized and optimized using an adenovirus-green fluorescent protein construct, and maximal infection (85-90% of cells) was seen 48 h post adenovirus treatment. A 424 bp fragment, which included the translational start site and exons 1-3 of the rat FST gene, was subcloned in the reverse orientation into an adenovirus vector. Construct efficacy was tested using cultured rat pituitary cells infected with the adenovirus-FST-AS construct. Infection with adenovirus-FST-AS increased FST-AS mRNA expression in a dose-dependent manner, reduced FST protein expression to undetectable levels, and stimulated increases in FSHbeta primary transcript and FSH secretion. Treatment with testosterone alone stimulated FSHbeta primary transcript and FSH release, and responses were doubled in the presence of adenovirus- FST-AS. These results demonstrate the effectiveness of adenovirus FST-AS in suppressing pituitary FST protein expression and enhancing FSH biological responses at the transcriptional level. Thus, the FST-deficient rat gonadotrope cell is a model that allows for the investigation of factors regulating FSHbeta expression, which might otherwise involve the autocrine/paracrine actions of FST.
Haisenleder
Daniel J
DJ
Division of Endocrinology and Metabolism, Department of Medicine, Center for Research in Reproduction, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA. djh2q@virginia.ed
Aylor
Kevin W
KW
Burger
Laura L
LL
Dalkin
Alan C
AC
Marshall
John C
JC
D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D037201:Follicle Stimulating Hormone, beta Subunit / Q000378:metabolism*; D038681:Follistatin / Q000096:biosynthesis* / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes
D037201:Follicle Stimulating Hormone, beta Subunit; D038681:Follistatin; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone