Michael J. McPhaul

Neuropathogenic Forms of Huntingtin and Androgen Receptor Inhibit Fast Axonal Transport

Huntingtons and Kennedys disease are autosomal dominant neurodegenerative diseases caused by pathogenic expansion of polyglutamine tracts. Expansion of glutamine repeats must in some way confer a gain of pathological function that disrupts an essential cellular process and leads to loss of affected neurons. Association of huntingtin with vesicular structures raised the possibility that axonal transport might be altered. Here we show that polypeptides containing expanded polyglutamine tracts, but not normal N-terminal huntingtin or androgen receptor, directly inhibit both fast axonal transport in isolated axoplasm and elongation of neuritic processes in intact cells. Effects were greater with truncated polypeptides and occurred without detectable morphological aggregates.

A and B forms of the androgen receptor are expressed in a variety of human tissues.

Human genital skin fibroblasts contain both the full-length 110 K androgen receptor protein (AR-B, apparent M(r) approximately 110,000) and an 87 K N-terminally truncated AR isoform (AR-A, apparent M(r) approximately 87,000). These two AR species are structurally analogous to the A- and B-isoforms of the progesterone receptor (PR). We examined the distribution pattern of human AR isoforms in a variety of fetal and adult tissues by Western blot analysis. Relative levels of immunoreactive AR proteins in high salt tissue extracts were estimated by densitometry in comparison to a standard normal genital skin fibroblast preparation. High AR levels (AR-A + AR-B = 0.8-7.7) were present in male and female reproductive tissues from mid-trimester fetuses, including penis, prostate, testis, epididymis, scrotal skin, labial skin, uterus/cervix, and ovary. AR-A and AR-B (0.08-0.9) also were found in 14 non-genital fetal tissues (bladder, fat, lung, great vessel, trachea, muscle, scalp skin, kidney, thyroid, intestine, thymus, ureter, stomach and rectum). AR-A accounted for 4-26% of the AR protein detected in these tissues. Ten other fetal tissues had low levels of AR-B (0.02-0.3) and little or no detectable AR-A. AR-B also was the predominant or only immunoreactive AR species found in 17 adult human tissues. AR levels in adult reproductive tissues (prostate, endometrium, ovary, uterus, fallopian tube, testis, seminal vesicle, myometrium, and ejaculatory duct) ranged from 0.1 to 2.2. Immunoreactive AR (0.4-0.8) also was present in specimens of prostate carcinoma, endometrial carcinoma, thyroid carcinoma and kidney. Lower levels of AR (0.03-0.1) were detected in adult breast, colon, lung and adrenal gland specimens. This study demonstrates that immunoreactive AR protein is present in a wide variety of human fetal and adult tissues and that two AR isoforms are expressed in many tissues.

Changes in Androgen Receptor Nongenotropic Signaling Correlate with Transition of LNCaP Cells to Androgen Independence

A cure for prostate cancer (CaP) will be possible only after a complete understanding of the mechanisms causing this disease to progress from androgen dependence to androgen independence. To carry on a careful characterization of the phenotypes of CaP cell lines before and after acquisition of androgen independence, we used two human CaP LNCaP sublines: LNCaPnan, which is androgen dependent (AD), and LNCaP-HP, which is androgen independent (AI). In AD LNCaPnan cells, dihydrotestosterone (DHT) stimulated in an androgen receptor (AR)-dependent way a phosphorylation signaling pathway involving steroid receptor coactivator (Src)–mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)-1/2–ERK-1/2–cAMP-response element binding-protein (CREB). Activation of this pathway was associated with increased [3H]thymidine incorporation and resistance to apoptosis. Use of dominant-negative forms of MEK-1/2 and CREB demonstrated in LNCaPnan cells that DHT induced [3H]thymidiine incorporation through a thus far unidentified molecule activated downstream of MEK-1/2, and antiapoptosis through phosphorylation of the transcription factor CREB. In contrast, in AI LNCaP-HP cells, the Src–MEK-1/2–ERK-1/2–CREB pathway was constitutively active. Because it was not further stimulated by addition of DHT, no increase of [3H]thymidine incorporation or apoptosis resistance was demonstrated in LNCaP-HP cells. Additional experiments showed that Src and the scaffold protein MNAR coimmunoprecipitated with AR, indicating a role for Src as an apical molecule in the Src–MEK-1/2–ERK-1/2–CREB pathway. Interestingly, differences between the two cell lines were that in LNCaP-HP cells presence of an AI phenotype and lack of response to DHT were associated with constitutive activation of the protein kinase Src and interaction among Src, AR, and MNAR. In contrast, in LNCaPnan cells, presence of an AD phenotype and ability to respond to DHT were associated with DHT-dependent activation of Src kinase activity and interaction among Src, AR, and MNAR. Intriguingly, in LNCaPnan cells, we found that transcription through the prototypical CREB-responsive promoter c-fos could be induced in a DHT-dependent way, and this action was inhibited by the AR antagonist Casodex and MEK-1 inhibitor PD98059. In contrast, transcription through the PSA P/E promoter, a prototypical AR-dependent promoter directly activated by agonist, was obliterated only by Casodex. Additional experiments with genital skin fibroblasts derived from patients with a variety of AR abnormalities indicated that nongenotropic AR signaling does not depend on an intact DNA-binding domain or on the ability of AR to translocate to the nucleus. The results suggest the following: (1) Constitutive activation of the Src–MEK-1/2–ERK-1/2–CREB pathway is associated with the AI phenotype observed in LNCaP-HP cells. (2) Activation of the Src–MEK-1/2–ERK-1/2–CREB pathway is DHT dependent in AD LNCaPnan cells. (3) DHT activation of this pathway is associated with induction of [3H]thymidine incorporation by a molecule activated downstream of MEK-1/2 and of antiapoptosis through activation of the transcription factor CREB in AD LNCaPnan cells. (4) AR regulates transcription either directly upon ligand binding and nuclear translocation or indirectly through kinase pathways leading to activation of downstream transcription factors. (5) Nuclear translocation and ability of the DNA-binding domain of AR to interact with DNA are not prerequisites for nongenotropic AR activity.

Testosterone and 5α-dihydrotestosterone interact differently with the androgen receptor to enhance transcription of the MMTV-CAT reporter gene ☆

Testosterone and its 5 alpha-reduced derivative 5 alpha-dihydrotestosterone exert different actions in the male during embryogenesis and in postnatal life. Nevertheless the two hormones bind to the same intracellular androgen receptor, and genetic and endocrinological studies in the Tfm mouse suggest that the actions of both hormones are mediated by this single receptor. Previous studies indicate that dihydrotestosterone binds more tightly to the androgen receptor but that the Bmax of binding of the two hormones is the same. To determine whether these differences in binding parameters could explain the mechanism by which the two hormones exert different physiological actions via the same receptor, we introduced a plasmid encoding the androgen receptor cDNA and a reporter plasmid encoding MMTV-CAT into Chinese hamster ovary cells. These cells do not express endogenous androgen receptor and do not convert testosterone to dihydrotestosterone. Therefore, it was possible to examine the relation between the concentration of each of the steroids and reporter gene expression. Both hormones enhanced CAT activity, but dihydrotestosterone was approximately 10 times as potent (half maximal of 0.018 nM) as testosterone (half maximal of 0.2 nM); the maximal activity achieved was the same for the two androgens. These findings are nearly identical to the apparent Kd values for the interaction of the two hormones with the androgen receptor. Although testosterone and dihydrotestosterone may influence the expression of other genes differently, these findings are compatible with a model system in which the differential effects can be explained as a consequence of different binding affinities to the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen increases androgen receptor protein while decreasing receptor mRNA in LNCaP cells

We have examined the effect of androgen treatment on androgen receptor mRNA and protein expression in the LNCaP human prostate carcinoma cell line. Incubation with androgen caused a decrease in cellular androgen receptor mRNA content that was concentration and time dependent. Maximal suppression to approximately 35% of control level was observed after 49 h of exposure to androgen. By contrast, incubation of LNCaP cells with androgen resulted in a 2-fold increase in the cellular content of androgen receptor protein at 24 h. At 49 h androgen receptor protein increased 30% as assayed by immunoblots and 79% as assayed by ligand binding. These results suggest that ligand-induced changes in androgen receptor stability and/or the translational efficiency of androgen receptor mRNA account for the phenomenon of androgen receptor upregulation observed in cultured LNCaP cells. Furthermore, the suppression of androgen mRNA and protein that is caused by prolonged incubation with androgen is incomplete and is reversible upon removal of ligand.

Transcriptional activation and transient expression of the human androgen receptor

A series of cDNAs containing deletions within the open-reading frame of the human androgen receptor (AR) were constructed and transiently expressed in CV1 cells to investigate the effects of these alterations on the level of expression of the protein and on its capacity to activate a model reporter gene (MMTV-luciferase). The levels of AR expression were assayed using immunoblots made using an antibody directed at an epitope (amino acids 1-21) preserved in all of the deletions. Treatment of the transfected cells with androgen increased the level of normal or mutant AR approximately five-fold in all constructs in which the hormone-binding domain was intact. This finding indicates that an intact hormone-binding domain is necessary and sufficient for the androgen-dependent increase in AR levels. Contraction of expansion or the glutamine repeat or deletion of the glycine repeat in the amino terminus diminished the capacity of the mutant ARs to activate the MMTV luciferase gene. The presence of a large-scale deletion within the amino terminus (amino acid residues 96-483), abolished receptor function, and two smaller deletions (bounded by residues 80-93 and 245-485) within the amino terminus substantially impaired receptor function. As previously described, deletion of the hormone-binding domain (amino acids 708-917) resulted in a constitutively active receptor. Unexpectedly, the large-scale deletion within the amino terminus (amino acids 96-483), in combination with deletion of the carboxy terminus also produced a constitutively active receptor that was almost as active as ligand-activated normal AR. None of the alterations in AR function could be explained by changes in the level of AR expression and the function of some mutant receptors was even more defective when the relative levels of mutant ARs expressed was considered. These findings imply that interaction of the sequences within the amino- and carboxy-terminal portions of the AR, or proteins that interact with these segments, is critical for regulation of transcription by the AR.

Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene

We have analyzed the nucleotide sequence of the androgen receptor from 22 unrelated subjects with substitution mutations of the hormone-binding domain. Eleven had the phenotype of complete testicular feminization, four had incomplete testicular feminization, and seven had Reifenstein syndrome. The underlying functional defect in cultured skin fibroblasts included individuals with absent, qualitative, or quantitative defects in ligand binding. 19 of the 21 substitution mutations (90%) cluster in two regions that account for approximately 35% of the hormone-binding domain, namely, between amino acids 726 and 772 and between amino acids 826 and 864. The fact that one of these regions is homologous to a region of the human thyroid hormone receptor (hTR-beta) which is a known cluster site for mutations that cause thyroid hormone resistance implies that this localization of mutations is not a coincidence. These regions of the androgen receptor may be of particular importance for the formation and function of the hormone-receptor complex.

Molecular basis of androgen resistance in a family with a qualitative abnormality of the androgen receptor and responsive to high-dose androgen therapy.

We have examined the nature of the mutant androgen receptor in a family with a severe defect in virilization associated with a qualitative defect in receptor function. The androgen receptor gene in this family contains two structural alterations: a single nucleotide substitution at position 2444 in exon 5 (adenosine----guanosine) that converts tyrosine 761 to a cysteine residue and a shortened glutamine homopolymeric segment in exon 1 that encodes 12 rather than the usual 20-22 glutamines. A family study was performed using polymerase chain reaction amplification of the glutamine-rich segment, and it was shown that the sister of the proband does not carry the mutant allele. The effects of these two mutations on the function of the androgen receptor were studied by introducing the changes, individually and in combination, into cDNAs encoding the normal human androgen receptor and analyzing the receptor protein produced after transfection of the cDNAs into eukaryotic cells. The presence of a cysteine residue at position 761 causes rapid dissociation of dihydrotestosterone from the receptor protein. Marked thermolability of the transfected receptor protein, however, was demonstrable only upon introduction of an androgen receptor cDNA containing both the partial deletion of the glutamine homopolymeric segment and a cysteine residue at position 761. Likewise, the ability of the receptor to stimulate a reporter gene is strikingly diminished only when both alterations are present, suggesting that the shortened glutamine homopolymeric segment amplifies the impairment of receptor function caused by the tyrosine to cysteine substitution.

Brain aromatase cytochrome P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat

Aromatase cytochrome P-450 (P-450AROM) enzyme activity catalyzes the conversion of androgens to estrogens in specific brain areas. During development local estrogen formation is thought to influence the sexual differentiation of neural structures (i.e. increase neurite growth and establish neural circuitry) and modulate reproductive functions. This study was undertaken to investigate the ontogeny of the (P-450AROM) enzyme and its messenger RNA (mRNA) in medial basal hypothalamic (MBH) and preoptic area (POA) tissue during late fetal and perinatal development of the rat. Aromatase activity in the MBH-POA was negligible before gestational day (GD) 16 (< 0.1 pmol/h/mg protein), increased over 10-fold at GD 17 and continued to increase (over 5-fold) to peak levels at GD 19 (> 5.0 pmol/h/mg protein), and then declined to low levels at GD 22 and 2 days post-birth (approximately 1 pmol/h/mg protein). The profile of P-450AROM mRNA in the MBH-POA tissue was characterized by a predominant 2.7 kilobase (kb) mRNA species, similar in size to the largest functional P-450AROM mRNA observed in adult rat ovarian tissue. At GD 15, the P-450AROM mRNA was undetectable; low but detectable levels were seen at GD 17, the abundance increased at later time points and remained at peak levels on GDs 18 through 20, decreased slightly by GD 22, and then declined further by 2 days post-birth. The developmental increase in P-450AROM mRNA levels correlated with the ascending pattern of enzyme activity before GD 19, but the marked decrease in enzyme activity seen after GD 19 was not accompanied by a corresponding decline in mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete testicular feminization caused by an amino-terminal truncation of the androgen receptor with downstream initiation.

We have characterized the molecular defect causing androgen resistance in two 46,XY siblings with complete testicular feminization. Although binding studies in genital skin fibroblasts showed a reduced Bmax, an increased dissociation rate of ligand, and an 8S peak of dihydrotestosterone binding on sucrose density gradient centrifugation, no immunoreactive androgen receptor (AR) was detected in immunoblots using anti-NH2-terminal antibodies, suggesting an abnormal amino terminus. Sequence analysis of the AR gene revealed a point mutation CAG-->TAG (Gln-->Stop) at nucleotide 340. In vitro mutagenesis studies suggest the synthesis of the mutant AR is initiated downstream of the termination codon at reduced levels and that each molecule is functionally impaired. These results define a novel mechanism causing androgen resistance: the combination of decreased amount and functional impairment of AR caused by an abnormality within the amino terminus of the receptor. These findings suggest that domains important to the in vivo function of the receptor reside within the amino terminus and that disruption of these domains can occur with only subtle effects on receptor binding. Identification of this mutation made it possible to identify the mutant allele within the family and to ascertain antenatally that it was not present in a 46,XY fetal sibling of the proband at 9 wk gestation.