The current study provides the first evidence for an AR regulatory pathway controlled by mammalian SIRT1. Endogenous androgen-regulated gene expression was induced by the SIRT1 inhibitor nicotinamide. hSIRT1 colocalized with the AR by confocal microscopy and physically associated with the AR in vitro and in vivo. AR function was repressed by hSIRT1, requiring the NAD-dependent enzymatic activity of hSIRT1. SIRT1 inhibited androgen-dependent gene expression in a ligand-dependent, AR and DNA sequence-specific manner. Acetylation of the AR at a conserved motif in the AR hinge region enhanced the physical association between the AR and coactivators in vitro and in cultured cells (17
). SIRT1 regulated the acetylation levels of the AR in LNCaP cells, and an 18-amino-acid peptide encoding the AR hinge region acetylation site was sufficient to interact with SIRT1. SIRT1 deacetylated the AR with Km
values similar to those of p53 and p300. SIRT1 expression inhibited contact-independent cellular growth of AR-expressing prostate cancer cells. Thus, the NAD-dependent histone deacetylase SIRT1 represses androgen receptor signaling and function.
hSIRT1 inhibited AR-induced reporter gene activity. Repression of AR activity was abrogated by point mutation of the acetylated AR lysine resides (ARK63OT). hSIRT1 inhibition of AR activity involved the deacetylation function of SIRT1. hSIRT1 inhibited DHT-induced AR activity in a DNA sequence-dependent manner, repressing expression of AR and activities of the PSA and MMTV promoters. An ARE site was sufficient for hSIRT1 repression. Point mutation of the hSIRT1 core domain histidine residue (H363Y), which abolishes the deacetylase activity of SIRT1, abrogated the repression of DHT-induced androgen signaling. The conserved lysine motifs of the AR, which are acetylated by p300 and other histone acetylases, were deacetylated by Sir2 in vitro. The murine orthologue of hSirT1, mSir2α, deacetylated an 18-mer peptide containing the three acetyl lysines (630, 632, and 633) corresponding to the native AR sequence. Enzymes derived from Archaea (Archaeoglobus fulgius AF2SIR2) and yeast (Saccharomyces cerevisiae SIRT) sources, closely related to hSir2, also deacetylated these residues. The deacetylation of the AR at 0.5 nM NAD+ and 202 nM SIRT1 enzyme demonstrated a Km value of 174 μM and a Kcat of 0.0395−1. These association and catalysis constants are comparable to those obtained for p300 and p53 peptide substrates.
The current findings extend previous observations that both histones and nonhistone proteins are targets for deacetylation by hSIRT1 proteins (57
) and establish a role for SIRT1 in regulating cellular growth through the AR. SIRT1 inhibited DHT-induced prostate cancer cellular proliferation and contact-independent cellular growth. The endogenous AR was required for both DHT-induced proliferation and the effect of SIRT1. The ability of SIRT1 to deacetylate AR and repress AR activity mechanistically resembles the deacetylation of p53, RelA/p65, and FOXO but contrasts with the effects of SIRT1 on MyoD. SIRT1 directly bound and deacetylated the AR, whereas the effect of Sir2 on MyoD is indirect, as Sir2 deacetylates P/CAF (21
) to reduce MyoD function (21
). SIRT1 bound the AR both in vitro and in vivo in cultured prostate cancer cells and repressed and deacetylated the AR directly. The repression of AR coactivator proteins (P/CAF and p300) by SIRT1 (9
) may also contribute to repression of AR activity. When coexpressed in cultured cells, p300 and the p160 coactivator proteins are recruited to AREs in the context of local chromatin. In addition, arginine methyltransferases form part of the complex that coordinates cyclical recruitment of AR nuclear receptor complexes (33
). It is known that the AR-acetylated lysine residues serve as key docking sites for the association with p300 versus NCoR-HDAC-Smad3 complexes of the AR (18
) (Fig. ). The role that SIRT1-dependent deacetylation plays in coordinating recruitment of specific complexes to the AR remains to be determined.
The current study provides evidence for functional antagonism between SIRT1 and AR activities at the site acetylated by p300. The acetylated motif residues of the AR are predicted to reside in proximity to the superior aspect of the AR hydrophobic pocket. Previous studies have suggested functional antagonism between p300 and histone deacetylase. In Caenorhabditis elegans
, inactivation of the cbp1
gene, an orthologue of p300/CBP, causes developmental arrest of C. elegans
embryos except for neuronal differentiation. Coinactivation of genes encoding components of the HDAC complex, such as hda-1
, and rba-2
, which antagonize HAT activity, rescues some of the cbp1
null phenotype (52
), suggesting that CBP specifies differentiation by functioning along with the early cell fate-determining transcription factors, and the conserved histone deacetylase complex blocks CBP-mediated cellular differentiation by antagonizing the functions of CBP (52
). SIRT1-dependent repression of AR activity was reduced by a point mutation (K630T) found in prostate cancer cells at the lysine residue acetylated by p300 but was not affected by mutations within the carboxyl terminus of AR. Here, SIRT1 repressed the ligand-augmented AR amino- and carboxyl-terminal intramolecular interaction. The N- and C-terminal interaction within the AR is enhanced by CBP/p300 (28
). The ligand-binding domain of the AR recruits its AF-1 function via an N-terminal FXXXLF motif. The hydrophobic cleft of the AR ligand-binding domain created by ligand binding in the C-terminal domain interacts strongly with the AR N-terminal motif. As p300 acetylates the AR, the independent role of p300 repression by SIRT1 versus the direct repression of AR by SIRT1 cannot be dissociated in p300−/−
cells or using small interfering RNA.
What might be the significance of the finding that AR expression and DHT signaling are regulated by nicotinamide and NAD-dependent deacetylation? The requirement for NAD in Sir2 enzymatic activity has led to suggestions that Sir2 activity may be regulated by the intracellular concentration of NAD, by the NAD/NADH ratio, or by the intracellular concentration of nicotinamide (2
). Endogenous levels of nicotinamide may limit Sir2 activity (49
), suggesting that the concentration of nicotinamide in response to physiological changes could affect Sir2 function. Metabolic changes in muscle induced by pyruvate, for example, increase the NAD+
/NADH ratio and inhibit muscle gene expression, whereas lactate reduces the NAD/NADH ratio and stimulates muscle gene expression (21
). Androgens maintain male muscle mass (10a
) and induce muscle cellular gene expression. During prostate cancer progression, metabolism shifts toward cytosolic glycolysis (1
). The increased production of lactate that occurs during prostate cancer progression (5
) is predicted to inhibit SIRT1 and thereby enhance AR function. A recent study also suggested that global histone modification in prostate tumor tissues, including acetylation of H3K18 and H4 K12, dimethylation of H3K4 and H4R3, and acetylation of H3K9, a target of SirT1 (54
), predict a risk of prostate cancer recurrence (51
Here, SIRT1 inhibited androgen-dependent prostate cancer cellular growth and repressed the endogenous androgen-responsive target gene, AR. AR
is an androgen-responsive gene, and sirtinol, a Sir2-specific inhibitor, increased the abundance of acetylated-AR acetylation. Nicotinamide, a noncompetitive inhibitor of Sir2, induced expression of the endogenous androgen-responsive AR
gene. The selective chemical inhibitor of SIRT activity, splitomycin, enhanced AR-dependent gene expression, suggesting that endogenous SirT1 contributes to maintenance of the AR in a repressed state. The androgen receptor's function is a critical determinant of human prostate cancer pathogenesis and progression (11
). Androgen ablation therapy remains a major therapeutic intervention in metastatic disease. Reexpression of androgen-responsive genes is a characteristic of progression of human prostate cancer, as monitored clinically by the androgen-regulated PSA gene. Although AR
gene amplification or mutation contributes to reexpression of androgen-responsive genes in a subset of patients, in the majority of patients, the mechanism remains poorly defined. Multiple mechanisms, including AR overexpression and mutations, activation of growth factor signaling, and altered cointegrator expression and function, have been implicated in therapy resistance (13
). Increased AR activity through loss of AR corepressors has been implicated in prostate cancer growth, and acetylation mimic mutants of the AR promote prostate cancer cellular growth (17
). The current study provides evidence for an important role for SIRT1 as a regulator of AR expression and function.