Transfection of Sertoli cells with androgen receptor alters gene expression without androgen stimulation

Abstract

BackgroundAndrogens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR.ResultsTransfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes.ConclusionWe conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0051-7) contains supplementary material, which is available to authorized users.

Importance of the Androgen Receptor Signaling in Gene Transactivation and Transrepression for Pubertal Maturation of the Testis

“…Although recent microarray studies have identified similar numbers of up-regulated and down-regulated genes in Sertoli cells during the process of postnatal maturation [76], and especially in response to androgens in Sertoli cells [6,77,78], most of the androgen-regulated genes thoroughly studied so far are positively regulated by androgens. Amongst those, Rhox5 (reproductive homeobox-5), formerly known as Pem , is perhaps one of the best characterized androgen-responsive genes [79].…”

Myofibroblast androgen receptor expression determines cell survival in co-cultures of myofibroblasts and prostate cancer cells in vitro

“…The stable transduction of AR into WPMY human prostate myofibroblasts significantly altered their gene expression pattern compared to those transduced with empty vector, in the absence of DHT [ 27 ]. Knockdown of AR by siRNA in an AR-positive cancer-associated fibroblast line produced significant differences in the expression of several growth factor genes, and the proliferation and migration of PC3 cells in transwell co-cultures [ 40 ], and the transfection of human AR into AR-deficient mouse Sertoli cells significantly altered the expression of 672 genes in the absence of androgen stimulation [ 41 ]. These latter two studies did not specify whether stripped medium was used.…”