Stimulation of FSHβ transcription by blockade of endogenous pituitary follistatin production: Efficacy of adenoviral-delivered antisense RNA in the rat

Author： Haisenleder Daniel Aylor Kevin Burger Laura Dalkin Alan Marshall John

Publisher： Humana Press, Inc

ISSN： 0969-711X

Source： Endocrine Journal, Vol.29, Iss.3, 2006-06, pp. : 399-404

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Abstract

This study investigated FSHβ 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—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 FSHβ primary transcript and FSH secretion. Treatment with testosterone alone stimulated FSHβ 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 FSHβ expression, which might otherwise involve the autocrine/paracrine actions of FST.