Transcriptional activation by the androgen receptor in X-linked spinal and bulbar muscular atrophy

233

146

The action of androgens is essential for the development of benign prostatic hyperplasia and carcinoma of the prostate. The androgen receptor is a ligand-dependent nuclear transcription factor. The transcriptional activation domain of the androgen receptor gene contains a polymorphic CAG repeat sequence. A shorter CAG repeat sequence within the normal range has been reported to be associated with increased risk of prostate cancer and symptomatic benign prostatic hyperplasia. Here, we examine the in vitro transcriptional activity of the androgen receptor (AR) with different numbers of CAG repeats within the normal range in a number of different cell lines of prostatic (LNCaP, PC3) and non-prostatic (COS-1, MCF7) origin. We utilize a luciferase reporter driven by the rat probasin promoter ( 286/+28) containing two androgen receptor binding sites. Transcriptional activation of the androgen responsive reporter was observed to be greater with the AR containing 15 vs 31 CAG repeats in COS-1 cells (123·2 16·6 vs 78·2 10·9, P value 0·01) and the well differentiated prostate cancer cell line LNCaP (103·4 17·7 vs 81·4 7·7, P value 0·045). No difference was observed in the poorly differentiated prostate cancer cell line, PC3 (106·9 21·9 vs 109·6 21·4, P value >0·5) or the breast cancer cell line MCF7 (120·4 39·4 vs 103·1 23·1, P value >0·5). Dose-response experiments with varying quantities of ligand (0·01, 0·1, 1 and 10 nM dihydrotestosterone) or AR cDNA did not demonstrate significant differences in transactivation of the androgen responsive reporter in PC3 cells by the different AR constructs. This suggests that the lack of influence of CAG number in this prostatic cell line is not related to dose of ligand or quantity of androgen receptor. Western immunoblot analysis of androgen receptor protein in transiently transfected COS-1 cells did not demonstrate a difference in the expression of the androgen receptor protein with different numbers of CAG repeats following incubation in the presence or absence of androgen. Gel shift assay did not demonstrate increased DNA binding by androgen receptor with a shorter CAG repeat sequence. These experiments using a relatively androgen-and prostate-specific reporter provide evidence for an inverse relationship between androgen receptor transcriptional activity and the number of CAG repeats in the transcriptional activation domain. The effect of CAG repeat number was cell specific suggesting the involvement of accessory factors expressed differentially between different cell lines.

Section: Discussionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Effect of the androgen receptor CAG repeat polymorphism on transcriptional activity: specificity in prostate and non-prostate cell lines

Beilin¹,

Ball²,

Favaloro³

et al. 2000

233

146

“…The stabilization of structure or at least a reduction in local structural plasticity may also result from the removal of the poly-Q repeat, with concomitant impact on protein-protein interactions. Expansion of the poly-Q repeat in the AR is proposed to lead to a receptor protein that is compromised for transcriptional activity (Mhatre et al 1993, Chamberlain et al 1994, Jenster et al 1994, Nakajima et al 1996, Tut et al 1997, Irvine et al 2000, Callewaert et al 2003, Wang et al 2004. Conversely, a reduction in poly-Q length has been associated with a more active AR (Chamberlain et al 1994, Irvine et al 2000, Callewaert et al 2003, Wang et al 2004.…”

Section: Discussionmentioning

confidence: 99%

Consequences of poly-glutamine repeat length for the conformation and folding of the androgen receptor amino-terminal domain

Davies

Watt²,

Kelly

et al. 2008

Poly-amino acid repeats, especially long stretches of glutamine (Q), are common features of transcription factors and cell-signalling proteins and are prone to expansion, resulting in neurodegenerative diseases. The amino-terminal domain of the androgen receptor (AR-NTD) has a poly-Q repeat between 9 and 36 residues, which when it expands above 40 residues results in spinal bulbar muscular atrophy. We have used spectroscopy and biochemical analysis to investigate the structural consequences of an expanded repeat (Q45) or removal of the repeat (DQ) on the folding of the AR-NTD. Circular dichroism spectroscopy revealed that in aqueous solution, the AR-NTD has a relatively limited amount of stable secondary structure. Expansion of the poly-Q repeat resulted in a modest increase in a-helix structure, while deletion of the repeat resulted in a small loss of a-helix structure. These effects were more pronounced in the presence of the structure-promoting solvent trifluoroethanol or the natural osmolyte trimethylamine N-oxide. Fluorescence spectroscopy showed that the microenvironments of four tryptophan residues were also altered after the deletion of the Q stretch. Other structural changes were observed for the AR-NTDQ45 polypeptide after limited proteolysis; in addition, this polypeptide not only showed enhanced binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonic acid but was more sensitive to urea-induced unfolding. Taken together, these findings support the view that the presence and length of the poly-Q repeat modulate the folding and structure of the AR-NTD.

“…Since the CAG repeat also affects AR function (Mhatre et al 1993, Chamberlain et al 1994, Kazemi-Esfarjani et al 1995, Gao et al 1996, Nakajima et al 1996, Buchanan et al 2004, Wang et al 2004, the combination of certain CAG and GGN alleles might have additive effects as suggested by the detection of functional differences between five analyzed CAG/GGN combinations (Gao et al 1996). In whites, there is no significant linkage disequilibrium between the two repeats (Kittles et al 2001), resulting in a variety of haplotypes with potential differences in AR function.…”

Section: Discussionmentioning

confidence: 99%

“…The effect of repeat lengths on receptor function has been investigated for both of these repeats. A large body of evidence suggests that a long CAG repeat results in reduced AR transactivity (Mhatre et al 1993, Chamberlain et al 1994, Kazemi-Esfarjani et al 1995, Gao et al 1996, Nakajima et al 1996, Buchanan et al 2004, Wang et al 2004. Fewer studies have been performed for the GGN repeat.…”

Section: Introductionmentioning

confidence: 99%

The two most common alleles of the coding GGN repeat in the androgen receptor gene cause differences in protein function

Brockschmidt¹,

Nöthen²,

Hillmer³

2007

Genetic studies have reported association of a polyglycine-encoding GGN repeat in exon 1 of the androgen receptor (AR) gene with common human traits. The polyglycine tract is located in the transactivating domain of the AR protein, suggesting an effect of repeat length on receptor function. Here, we compare the functional characteristics of the two most common alleles (23 and 24 repeats) and two extreme alleles (10 and 27 repeats) in a reporter gene assay in HeLa cells. A correlation between the repeat length and AR activity was observed. This is attributable to both a higher protein concentration, determined by ELISA, and a higher per-protein activity of long repeat alleles. Interestingly, protein concentration does not correlate with transcript quantity, determined by real-time PCR assays, and no influence of repeat length on protein stability could be detected in translation inhibition assays. This may suggest that repeat length affects translation efficiency. In conclusion, our data provide evidence of functional differences between the two most common alleles of the AR GGN repeat, supporting its potential role in the development of human traits.