X-linked spinal and bulbar muscular atrophy (Kennedy's disease) is an adult-onset form of motorneuron disease which may be associated with signs of androgen insensitivity. We have now investigated whether the androgen receptor gene on the proximal long arm of the X chromosome is a candidate gene for this disease. In patient samples we found androgen receptor gene mutations with increased size of a polymorphic tandem CAG repeat in the coding region. These amplified repeats were absolutely associated with the disease, being present in 35 unrelated patients and none of 75 controls. They segregated with the disease in 15 families, with no recombination in 61 meioses (the maximum log likelihood ratio (lod score) is 13.2 at a recombination rate of 0). The association is unlikely to be due to linkage disequilibrium, because 11 different disease alleles were observed. We conclude that enlargement of the CAG repeat in the androgen receptor gene is probably the cause of this disorder.

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Purpose. Prostate cancer that recurs during androgen deprivation therapy is referred to as androgen-independent. High levels of expression of androgen receptor and androgen receptor-regulated genes in recurrent prostate cancer suggest a role for androgen receptor and its ligands in prostate cancer recurrence.Experimental Design. Recurrent prostate cancer specimens from 22 men whose prostate cancer recurred locally during androgen deprivation therapy and benign prostate specimens from 48 men who had received no prior treatment were studied. Androgen receptor expression was measured using monoclonal antibody and automated digital video image analysis. Tissue androgens were measured using radioimmunoassay.Results. Epithelial nuclei androgen receptor immunostaining in recurrent prostate cancer (mean optical density, 0.284 ؎ SD 0.115 and percentage positive nuclei, 83.7 ؎ 11.6) was similar to benign prostate (mean optical density, 0.315 ؎ 0.044 and percentage positive nuclei, 77.3 ؎ 13.0). Tissue levels of testosterone were similar in recurrent prostate cancer (2.78 ؎ 2.34 pmol/g tissue) and benign prostate (3.26 ؎ 2.66 pmol/g tissue). Tissue levels of dihydrotestosterone, dehydroepiandrosterone, and androstenedione were lower (Wilcoxon, P ‫؍‬ 0.0000068, 0.00093, and 0.0089, respectively) in recurrent prostate cancer than in benign prostate, and mean dihydrotestosterone levels, although reduced, remained 1.45 nM. Androgen receptor activation in recurrent prostate cancer was suggested by the androgenregulated gene product, prostate-specific antigen, at 8.80 ؎ 10.80 nmol/g tissue.Conclusions. Testosterone and dihydrotestosterone occur in recurrent prostate cancer tissue at levels sufficient to activate androgen receptor. Novel therapies for recurrent prostate cancer should target androgen receptor directly and prevent the formation of androgens within prostate cancer tissue.

Summary Adult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts and subsequently contribute to restoration of the hepatocyte mass.

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The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH 2 -terminal region which is not conserved. Recent biochemical and crystallographic studies revealed the molecular basis of AF2 is hormone-dependent recruitment of LXXLL motifcontaining coactivators, including the p160 family, to a hydrophobic cleft in the ligand binding domain. Our previous studies demonstrated that AF2 in the androgen receptor (AR) binds only weakly to LXXLL motif-containing coactivators and instead mediates an androgendependent interaction with the AR NH 2 -terminal domain required for its physiological function. Here we demonstrate in a mammalian two-hybrid assay, glutathione S-transferase fusion protein binding studies, and functional assays that two predicted ␣-helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, mediate the androgendependent, NH 2 -and carboxyl-terminal interaction. Nuclear receptors facilitate ligand-dependent increases of gene transcription by direct interactions with nuclear coactivators. p160 coactivators have histone acetyltransferase activity (1) and interact with nuclear receptors through their ligand binding and NH 2 -terminal regions (2-4). Additional acetyltransferases p300/cAMP response element binding protein and p300/cAMP response element binding protein associated factor interact with the p160 coactivators (5-7). Nuclear receptors also interact with multiprotein complexes referred to as thyroid hormone receptor-associated proteins (8), activator-recruited cofactor (9), or vitamin D receptor-interacting protein complex (10). The p160 coactivators and at least one of the thyroid hormone receptor-associated proteins/activator-recruited cofactor/vitamin D receptor-interacting protein subunit 205 interact in a ligand-dependent manner with activation function 2 (AF2) 1 in the ligand binding domain (LBD) of nuclear receptors through the consensus sequence LXXLL, where L is leucine and X is any amino acid (11-16). Crystal structures of nuclear receptor LBDs have shown that a hydrophobic cleft within a multilayered ␣-helical structure serves as the LXXLL coactivator binding surface AF2 (17, 18). In the estrogen receptor, agonist binding positions helix 12 over the binding cavity to complete the AF2 surface (19,20), whereas binding of an antagonist such as 4-hydroxytamoxifen displaces helix 12 (21) causing an LXXLL-like sequence in helix 12 to mimic and thereby block coactivator binding (22).It has become apparent that the AF2 region overlaps with regions that serve as the binding site for a variety of LXXLLrelated sequences as recently shown for corepressor binding (23)(24)(25). Furthermore, we demonstrated that the AR AF2 region mediates an androgen-dependent NH 2 -terminal/carboxylterminal (N/C) interaction (26). Therefore we investigated the possibility that AF2 in the ...

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.

Activation function 2 in the ligand binding domain of nuclear receptors forms a hydrophobic cleft that binds the LXXLL motif of p160 transcriptional coactivators. Here we provide evidence that activation function 2 in the androgen receptor serves as the contact site for the androgen dependent NH 2 -and carboxyl-terminal interaction of the androgen receptor and only weakly interacts with p160 coactivators in an LXXLL-dependent manner. Mutagenesis studies indicate that it is the NH 2 -/ carboxyl-terminal interaction that is required by activation function 2 to stabilize helix 12 and slow androgen dissociation critical for androgen receptor activity in vivo. The androgen receptor recruits p160 coactivators through its NH 2 -terminal and DNA binding domains in an LXXLL motif-independent manner. The results suggest a novel function for activation function 2 and a unique mechanism of nuclear receptor transactivation.Steroid receptors interact with coactivators during the recruitment of active transcription initiation complexes required for hormone-regulated gene transcription (1). Transcriptional activation domains in the steroid receptors that may mediate these interactions include activation function 1 in the NH 2 -terminal domain and activation function 2 (AF2) 1 in the ligand binding domain (LBD). Recent studies have focused on a family of p160 coactivators that interact with the AF2 region that include steroid receptor coactivator 1 (SRC1) (2) and the human transcriptional intermediary factor 2 (TIF2) (3). SRC1 and TIF2 contain distinct nuclear receptor interaction domains in the central and/or carboxyl-terminal regions (3, 4). Mutagenesis studies demonstrated a functional link between AF2 activity in the LBD and the binding of p160 coactivators (5, 6). The p160 coactivators interact with the AF2 hydrophobic surface of the LBD through conserved LXXLL motifs that form amphipathic ␣ helices (7, 8). Recent co-crystal structures of nuclear receptor LBDs and LXXLL motif fragments confirm that AF2 recruits TIF2 and SRC1 through their LXXLL motifs (6, 9 -11). A multistep mechanism for transcriptional activation by nuclear receptors involves hormone-dependent recruitment and association through these LXXLL binding motifs of histone acetyltransferase activity associated with the p160 coactivator family, CREB-binding protein/p300, and p300/CREB-binding protein-associated factor, resulting in chromatin remodeling (12, 13) and the formation of a transcriptionally competent Srb/mediator coactivator complex (thyroid hormone receptorassociated protein/vitamin D receptor-interacting protein) complex (14). However, androgen receptor (AR) AF2 activity is not detected in a variety of mammalian cell lines (15-18) despite homology of the region with other nuclear receptors. We therefore investigated the mechanism whereby AR recruits p160 coactivators and the role of AF2 in AR function. It is demonstrated that weak interactions between the AR LBD and SRC1 and TIF2 correspond with weak AR AF2 activity. The AF2 surface in the AR LBD ins...