c-Jun activation-dependent tumorigenic transformation induced paradoxically by overexpression or block of S-adenosylmethionine decarboxylase.

All mammalian cells absolutely require polyamines (putrescine, spermidine, and spermine) for growth. Here we show that the overexpression of cDNA for S-adenosylmethionine decarboxylase (AdoMetDC), the main regulatory enzyme in the biosynthesis of higher polyamines, induces transformation of rodent fibroblasts when expressed in the sense or the antisense orientation. Both transformants were able to induce invasive tumors in nude mice. Neither transformation was associated with activation of the mitogen-activated protein kinases Erk1 and Erk2. Instead, the AdoMet DC sense, but not antisense, transformants displayed constitutive activation of the c-Jun NH(2)-terminal kinase (JNK) pathway. However, both transformations converged on persistent phosphorylation of endogenous c-Jun at Ser73. The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis. Similarly, TAM-67 reverted the morphology of the AdoMetDC-antisense expressors. This report is the first demonstration of a protein whose overexpression or block of synthesis can induce cell transformation. In addition, we show that the polyamine biosynthetic enzymes require c-Jun activation for eliciting their biological effects.

Mutant strain of Chinese hamster ovary cells with no detectable ornithine decarboxylase activity.

We previously described an arginase-deficient, polyamine-dependent Chinese hamster ovary cell line which grows in serum-free medium. From this strain we isolated a new mutant strain that has no detectable catalytic ornithine decarboxylase activity. The mutant cells contain, however, immunoreactive ornithine decarboxylase-like protein roughly in the same quantity as the parent strain. The mutant and the parent cell line strains also contain similar amounts of ornithine decarboxylase-mRNA hybridizable to a specific cDNA. If polyamines are omitted from the medium, proliferation of the mutant cells is considerably retarded and ceases in 6 to 10 days. Addition of ornithine or alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, has no effect on these cells. Putrescine and spermidine decreased in the mutant cells to undetectable levels during polyamine starvation, whereas spermine was reduced to 1/5th of that found in the control cultures. Polyamines appear to be indispensable for the mutant strain, but this was obvious only after the amount of polyamines, found as impurities in bovine serum albumin used in the medium, was reduced by dialysis to 10(-12) M. Because sera contain polyamines, the ability of the mutant strain to grow in serum-free medium is a great advantage in elucidation of the mechanisms of polyamine function.

Identification of a novel RING finger protein as a coregulator in steroid receptor-mediated gene transcription.

Using the DNA-binding domain of androgen receptor (AR) as a bait in a yeast two-hybrid screening, we have identified a small nuclear RING finger protein, termed SNURF, that interacts with AR in a hormone-dependent fashion in both yeast and mammalian cells. Physical interaction between AR and SNURF was demonstrated by coimmunoprecipitation from cell extracts and by protein-protein affinity chromatography. Rat SNURF is a highly hydrophilic protein consisting of 194 amino acid residues and comprising a consensus C3HC4 zinc finger (RING) structure in the C-terminal region and a bipartite nuclear localization signal near the N terminus. Immunohistochemical experiments indicated that SNURF is a nuclear protein. SNURF mRNA is expressed in a variety of human and rat tissues. Overexpression of SNURF in cultured mammalian cells enhanced not only androgen, glucocorticoid, and progesterone receptor-dependent transactivation but also basal transcription from steroid-regulated promoters. Mutation of two of the potential Zn2+ coordinating cysteines to serines in the RING finger completely abolished the ability of SNURF to enhance basal transcription, whereas its ability to activate steroid receptor-dependent transcription was maintained, suggesting that there are separate domains in SNURF that mediate interactions with different regulatory factors. SNURF is capable of interacting in vitro with the TATA-binding protein, and the RING finger domain is needed for this interaction. Collectively, we have identified and characterized a ubiquitously expressed RING finger protein, SNURF, that may function as a bridging factor and regulate steroid receptor-dependent transcription by a mechanism different from those of previously identified coactivator or integrator proteins.

Activation of androgen receptor function by a novel nuclear protein kinase.

Androgen receptor (AR) belongs to the nuclear receptor superfamily and mediates the biological actions of male sex steroids. In this work, we have characterized a novel 130-kDa Ser/Thr protein kinase ANPK that interacts with the zinc finger region of AR in vivo and in vitro. The catalytic kinase domain of ANPK shares considerable sequence similarity with the minibrain gene product, a protein kinase suggested to contribute to learning defects associated with Down syndrome. However, the rest of ANPK sequence, including the AR-interacting interface, exhibits no apparent homology with other proteins. ANPK is a nuclear protein that is widely expressed in mammalian tissues. Its overexpression enhances AR-dependent transcription in various cell lines. In addition to the zinc finger region, ligand-binding domain and activation function AF1 of AR are needed, as the activity of AR mutants devoid of these domains was not influenced by ANPK. The receptor protein does not appear to be a substrate for ANPK in vitro, and overexpression of ANPK does not increase the extent of AR phosphorylation in vivo. In view of this, it is likely that ANPK-mediated activation of AR function is exerted through modification of AR-associated proteins, such as coregulatory factors, and/or through stabilization of the receptor protein against degradation.

Phosphorylated human keratinocyte ornithine decarboxylase is preferentially associated with insoluble cellular proteins.

Ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, is highly regulated by many trophic stimuli, and changes in its levels and organization correlate with cytoskeletal changes in normal human epidermal keratinocytes (NHEK). NHEK ODC exhibits a filamentous perinuclear/nuclear localization that becomes more diffuse under conditions that alter actin architecture. We have thus asked whether ODC colocalizes with a component of the NHEK cytoskeleton. Confocal immunofluorescence showed that ODC distribution in NHEK was primarily perinuclear; upon disruption of the actin cytoskeleton with cytochalasin D, ODC distribution was diffuse. The ODC distribution in untreated NHEK overlapped with that of keratin in the perinuclear but not cytoplasmic area; after treatment with cytochalasin D, overlap between staining for ODC and for keratin was extensive. No significant overlap with actin and minimal overlap with tubulin filament systems were observed. Subcellular fractionation by sequential homogenizations and centrifugations of NHEK lysates or detergent and salt extractions of NHEK in situ revealed that ODC protein and activity were detectable in both soluble and insoluble fractions, with mechanical disruption causing additional solubilization of ODC activity (three- to sevenfold above controls). Fractionation and ODC immunoprecipitation from [(32)P]orthophosphate-labeled NHEK lysates showed that a phosphorylated form of ODC was present in the insoluble fractions. Taken together, these data suggest that two pools of ODC exist in NHEK. The first is the previously described soluble pool, and the second is enriched in phospho-ODC and associated with insoluble cellular material that by immunohistochemistry appears to be organized in conjunction with the keratin cytoskeleton.

Regulation of ornithine decarboxylase gene expression in MCF-7 breast cancer cells by antiestrogens.

Ornithine decarboxylase (ODC) is an enzyme intimately related to cell growth regulation. The metabolic products of ODC, the polyamines, are known to play a vital role in the structure and function of biological macromolecules including nucleic acids and proteins. The activity of ODC is stimulated by estrogens in their target cells. In order to gain insight into the molecular mechanism of action of antiestrogens in human breast cancer, we studied the effect of tamoxifen and 4-hydroxytamoxifen on the concentration of ODC mRNA, ODC activity, and the polyamine levels in a hormone-responsive breast cancer cell line, MCF-7. ODC mRNA concentration was reduced to 40% of the controls after 6 h of treatment of the cells with 100 nM 4-hydroxytamoxifen, but tamoxifen had no significant effect on ODC mRNA after treating with even 1 microM concentration for 36 h. ODC activity was, however, reduced to 40 and 75% of the controls after 24 h of treatment with 4-hydroxytamoxifen and tamoxifen, respectively. There was a significant reduction in the concentration of putrescine to 63% of control in tamoxifen-treated cells, but spermidine and spermine levels were not affected. With 4-hydroxytamoxifen, putrescine, spermidine, and spermine levels were reduced to 41, 62, and 79% of the control, respectively. In addition, exogenous putrescine was able to reverse the growth inhibitory effects of 4-hydroxytamoxifen. Overall, these results indicate that ODC and polyamine levels in MCF-7 cells are controlled by antiestrogens, and that suppression of polyamine biosynthesis plays a critical role in the growth inhibitory effects of antiestrogens.

Effect of dexamethasone on the activity and expression of ornithine decarboxylase in rat liver and thymus.

A single intraperitoneal injection of the synthetic glucocorticoid dexamethasone into rats resulted in a marked stimulation (more than 60-fold) of hepatic ornithine decarboxylase (ODC) at 4 h after the injection, whereas the enzyme activity in thymus was almost totally (about 95%) depressed at the same time. The stimulation of ODC activity in liver was in all likelihood attributable to a greatly enhanced accumulation of mRNA species for the enzyme as revealed by Northern blot and dot-blot hybridization analyses. ODC activity in thymus, in response to dexamethasone, was only 5% of that found in control animals, but this decrease was apparently not accompanied by similar reductions of the levels of ODC message, which was in fact decreased only by 50% at the maximum. In addition to two mRNA species (2.1 and 2.6 kilobases; kb), typical to mouse cells, rat tissues seemed to contain a third hybridizable message for ODC, smaller (1.6 kb) than the above-mentioned species and not seen in samples obtained from mouse or human cells. Interestingly, these smaller poly(A)+ RNA sequences, hybridizable with cDNA complementary to mouse ODC mRNA, were apparently constitutively expressed, as the treatment with glucocorticoid altered the amount of these sequences only slightly.

Disruption of the murine PIASx gene results in reduced testis weight.

PIASx belongs to the PIAS protein family, the members of which modulate activities of several transcription factors and act as E3 ligases in the sumoylation pathway. The PIASx gene is highly expressed in testis, suggesting a role in spermatogenesis. To investigate the function of PIASx in vivo, we have disrupted the PIASx gene in mice. Interestingly, the knockout mice were viable and fertile. Despite the normal fertility, the testis weight of the mutant animals was reduced and their number of apoptotic testicular cells was increased. Also, the sperm count of mutant mice tended to be reduced, but the quality of their sperm cells was normal. No significant changes were observed in the serum levels of LH and FSH or in the intratesticular testosterone concentration between the knockout animals and their wild-type littermates. Compensatory increases in other PIAS protein mRNAs were not observed in the knockout mice. These results imply that PIASx is required quantitatively rather than qualitatively for normal spermatogenesis.

Progestins can mimic, inhibit and potentiate the actions of androgens.

There is an extensive background on the androgen responsiveness of the mouse kidney which can be demonstrated histologically by hypertrophy of the Bowman's capsule and the proximal convoluted tubule. Although androgens increase many renal proteins, beta-glucuronidase and ODC are distinguished by exquisite genetic regulation of the magnitude of the response induced by testosterone. Both the qualitative and quantitative expression of the genes for these enzymes are strain specific, and are dependent upon regulatory alleles. Ornithine decarboxylase is of particular interest since the response of this enzyme is rapid compared to that of beta-glucuronidase. Recent studies using a newly developed androgen receptor assay have demonstrated that the duration of retention of the androgen receptor complex in the nucleus correlates with the magnitude of the androgenic response. Progestins can mimic, inhibit, or potentiate the action of androgens. These responses have been termed the androgenic, antiandrogenic and synandrogenic actions of progestins, respectively. The androgenic and antiandrogenic action of this class of steroids are manifest on many tissues and on many endpoints within a given organ. These effects are believed to involve an early step(s) of androgen action which is common to all sensitive tissues. Results to date suggests that this early step involves the androgen receptor. By contrast, the synandrogenic action of progestins is limited in that it is not observed on all tissues, and not even on all endpoints within a single organ. In the mouse kidney, the synandrogenic actions of progestins have been most extensively studied on beta-glucuronidase. With this enzyme this unusual response to progestins can be demonstrated only in mice which carry the Gus-ra allele. This observation suggests that the potentiating action of progestins on beta-glucuronidase is manifest directly on the Gus gene complex. It is not certain at this time whether a similar mechanism is involved in the potentiation of androgen action on other organs such as the prostate. The androgenic action of progestins is believed to be similar to that of other androgens. Androgenic progestins such as MPA bind to the androgen receptors and translocate them to nuclei. This is followed by a dose dependent increase of proteins similar to what is observed after testosterone administration. In addition, the regulatory genes which modulate androgen action have the same effect on the androgenic effect of progestins. The fact that the potency of progestins such as MPA is less than that of testosterone is believed to relate in part to their lower affinity for the androgen receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Disrupted amino- and carboxyl-terminal interactions of the androgen receptor are linked to androgen insensitivity.

We have compared the functional consequences of seven single-point mutations in the ligand-binding domain (LBD) of the androgen receptor (AR). The mutations span helices 3 to 11 and are present in patients suffering from androgen insensitivity syndromes (AIS) and other male-specific disorders. The mutants, except M742V, bound to androgen response elements in vivo and in vitro and showed a testosterone-dependent conformational change. With regard to functional activity, the mutant M742V had severely blunted ability to transactivate or exhibit the androgen-dependent amino/carboxyl-terminal (N/C) interaction; mutants F725L, G743V, and F754L showed reduced transactivation potential and attenuated N/C interaction; and mutants V715M, R726L, and M886V had minor functional impairments. The mutants belonging to the first two groups also displayed reduced response to coexpressed GRIP1. In addition, mutations of amino acids M894 and A896 in the putative core activation domain 2 (AF2) in helix 12 confirmed that this helix is important for N/C interactions. Thus, amino acids located between helices 3 and 4 (F725 and R726), in helix 5 (M742, G743, and F754), and in helix 12 (M894 and A896) play critical roles in mediating the N/C interaction of AR. The data also show that disrupted N/C interaction is a potential molecular abnormality in AIS cases in which LBD mutations have not resulted in markedly impaired ability to bind androgen.

Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells.

The effects of androgen withdrawal and replacement on the concentrations of androgen receptor (AR) protein and AR mRNA were investigated in rat ventral prostate and seminal vesicles and in cultured human hepatoma (HepG2) cells. AR mRNA concentrations were determined by Northern blotting with single stranded AR cRNA as the hybridization probe, whereas antibodies raised against two synthetic 17-amino acid long peptides corresponding to the N-terminal and steroid-binding regions of the AR were employed in immunological receptor assays. AR mRNA levels in both prostate and seminal vesicles increased about 2-fold within 24 h after castration and continued to rise within the next 48 h to values that were 9- to 11-fold higher than those in intact controls. Administration of pharmacological doses of testosterone (400 micrograms steroid/day) to 1-day castrated animals for 24-48 h brought about a decrease in AR mRNA levels in accessory sex organs to levels in intact controls. Similar results were obtained in cultured HepG2 cells where a switch to serum- and steroid-free medium elicited a rapid increase (approximately 4-fold in 10 h) in the AR mRNA level, which was prevented by inclusion of 10(-7) M testosterone in culture medium. Similar, but quantitatively less marked, changes occurred in the AR protein concentration in prostate, seminal vesicles, and HepG2 cells, as determined by immunoblotting using antibodies against AR peptides. In addition, immunohistochemical studies showed that AR is a nuclear protein of the prostatic epithelial cells in both intact and castrated rats, and suggested that short term castration increases the concentration of nuclear AR in the prostate. Taken together, these data indicate that androgens down-regulate the concentration of AR protein and AR mRNA in a variety of target tissues.

Genetic variation in androgen regulation of ornithine decarboxylase gene expression in inbred strains of mice.

Previous studies have indicated that androgen regulation of certain gene products in murine kidney is genetically controlled. In the present work, the expression of renal ornithine decarboxylase (ODC) gene(s) was used as a biological marker to study androgen responsiveness of eight inbred strains of mice (A/J, C57BR/cdJ, 129/J, C57L/J, BALB/cJ, SM/J, RF/J, and C57BL/6J). Kidneys of untreated females from these strains did not have significantly different basal ODC activities or ODC mRNA concentrations. However, renal enzyme concentrations in intact male mice exhibited marked strain-dependent variation; three strains (RF/J, SM/J, and C57BR/cdJ) had 5- to 20-fold higher activities than the other five strains. Renal ODC mRNA content showed similar genetic variability in the male mice; animals with highest enzyme activity had higher mRNA levels than those with low activity. These results could not be explained by differences in either serum testosterone levels or renal nuclear androgen receptor content, suggesting that the animals were differentially sensitive to endogenous androgens. To evaluate further the androgen regulation of ODC gene expression, female mice were treated with testosterone-releasing implants for 5-7 days. The two strains (A/J and C57BL/6J) that had low enzyme activity in response to endogenous testosterone in male mice also showed blunted responses to exogenous androgen administration, as measured by the induction of ODC and its mRNA. The relative distribution of the two mRNA species coding for ODC (2.2 and 2.7 kb in size) exhibited strain-dependent variation that did not, however, correlate with the androgen responsiveness. Studies of the mRNA levels in reciprocal F1 hybrids of C57BR/cdJ and C57BL/6J mice suggested that androgen sensitivity of ODC gene expression, at least in these crosses, was inherited in an autosomal dominant manner.

Identification by hybridization histochemistry of human endometrial cells expressing mRNAs encoding a uterine beta-lactoglobulin homologue and insulin-like growth factor-binding protein-1.

A beta-lactoglobulin homologue (beta LG/PP14) and insulin-like growth factor-binding protein-1 (IGFBP-1) are two major secretory proteins of the human endometrium. In the present study, we have shown that beta LG/PP14 mRNA is expressed in the endometrium in a cyclic manner, being hardly detectable in midcycle and most abundant during the late secretory phase. IGFBP-1 mRNA is also expressed in endometrium, but in amounts smaller than those encoding beta LG/PP14 and with maximal accumulation earlier in the secretory phase. The expression of these two mRNAs occurs in different cell types of the endometrium, as revealed by in situ hybridization techniques using single-stranded RNA probes. The glandular epithelial cells accumulate beta LG/PP14 mRNA during the late secretory phase of the cycle, whereas only the stromal cells of the late secretory endometrium express IGFBP-1 mRNA. In contrast to the endometrium, the two mRNAs are present at very low abundance in the fallopian tubes where they are expressed in the epithelial cells of the mucosa.

Androgen receptor-mediated transcriptional regulation in the absence of direct interaction with a specific DNA element.

Androgen receptor (AR) brings about a ligand-dependent inhibition of low-affinity neurotrophin receptor (p75) promoter constructs in cultured cells, with the greatest inhibition being achieved with a reporter gene containing 1050 nucleotides (nt) of the promoter. The receptor domain critical for trans-repression localizes to the same region (amino acids 147-296) as that mandatory for transactivation. In contrast to trans-activation, AR does not interact directly with specific DNA elements to elicit trans-repression of p75 promoter constructs, although an intact DNA-binding domain of the receptor is required for both actions. In a search for interacting partners, both extensively purified full-length AR and AR-DNA binding domain were found to inhibit c-Jun/AP-1 site interaction without themselves binding to the AP-1 element. Prior binding of c-Jun to the AP-1 element protected the complex from the receptor's interference. Repression was not mutual, as c-Jun did not inhibit AR-androgen response element interaction or trans-activation through an androgen response element-containing promoter. The 1050-nt-long p75 promoter sequence does not contain an AP-1 element; an AP-1-like site in the vector backbone mediates the trans-repression by the AR in recipient cells. Intriguingly, an AR form with a large N-terminal deletion (the delta 46-408 mutant) behaved as a transcriptional activator of the p75 promoter through a mechanism that was also independent of specific DNA binding. Collectively, these data indicate that, in a proper context, AR is able to elicit both transrepression and trans-activation without interacting directly with specific DNA elements. Sequences responsible for the down-regulation of p75 mRNA by androgens in vivo are, however, not located in the proximal 1050 nt of the p75 promoter.

ARIP3 (androgen receptor-interacting protein 3) and other PIAS (protein inhibitor of activated STAT) proteins differ in their ability to modulate steroid receptor-dependent transcriptional activation.

Steroid receptors mediate their actions by using various coregulatory proteins. We have recently characterized ARIP3/PIASx alpha as an androgen receptor (AR)-interacting protein (ARIP) that belongs to the PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] protein family implicated in the inhibition of cytokine signaling. We have analyzed herein the roles that four different PIAS proteins (ARIP3/PIASx alpha, Miz1/PIASx beta, GBP/PIAS1, and PIAS3) play in the regulation of steroid receptor- or STAT-mediated transcriptional activation. All PIAS proteins are able to coactivate steroid receptor-dependent transcription but to a differential degree, depending on the receptor, the promoter, and the cell type. Miz1 and PIAS1 are more potent than ARIP3 in activating AR function on minimal promoters. With the natural probasin promoter, PIAS proteins influence AR function more divergently, in that ARIP3 represses, but Miz1 and PIAS1 activate it. Miz1 and PIAS1 possess inherent transcription activating function, whereas ARIP3 and PIAS3 are devoid of this feature. ARIP3 enhances glucocorticoid receptor-dependent transcription more efficiently than Miz1 or PIAS1, and all PIAS proteins also activate estrogen receptor- and progesterone receptor-dependent transcription but to a dissimilar degree. The same amounts of PIAS proteins that modulate steroid receptor-dependent transcription influence only marginally transactivation mediated by various STAT proteins. It remains to be established whether the PIAS proteins play a more significant physiological role in steroid receptor than in cytokine signaling.

Dominant negative regulation of trans-activation by the rat androgen receptor: roles of the N-terminal domain and heterodimer formation.

A series of deletion mutants was constructed for the rat androgen receptor (AR) to delineate sequences involved in transcriptional activation. Using transient expression conditions in CV-1 cells and in vitro DNA-binding studies, the amino-terminal domain of the receptor was shown to contain a region (residues 147-296) that is mandatory for trans-activation. Receptors with deletions (residues 147-408) in the N-terminal domain, but with intact DNA- and ligand-binding domains, interacted in vitro with androgen-responsive elements albeit with affinities lower than that of the wild type receptor. Coexpression of N-terminal deletion mutants (delta 46-408 and delta 38-296) with the wild type AR blunted trans-activation by the latter protein in a dominant fashion. By contrast, a hormone-binding-deficient receptor (delta 788-902) that had poor intrinsic activity potentiated the trans-activation by the native receptor. Mechanisms by which deletion mutants in the N-terminal region abolish the function of the wild type protein appear to involve heterodimer formation during interaction with DNA and direct competition for available binding sites on DNA, rather than squelching of accessory proteins. In contrast to impaired trans-activation, binding of the ligand to N-terminal deletion mutants brought about conformational changes that were comparable in wild type and mutant forms, as judged by electrophoretic mobility shift assays. Taken together, these data have specified a region in the N-terminal domain of the AR that plays a decisive role in transcriptional regulation.

Mutant and wild-type androgen receptors exhibit cross-talk on androgen-, glucocorticoid-, and progesterone-mediated transcription.

Androgen, glucocorticoid, and progesterone receptors (ARs, GRs, and PRs) often can regulate transcription via composite hormone response elements in target genes. We have used artificial and natural mutant ARs from patients with androgen resistance to study their effects on dominant negative activity on wild type AR, GR, and PR function on mouse mammary tumor virus (MMTV) and tyrosine aminotransferase (TAT) promoters. Artificial ARs that contained internal deletions within the amino-terminal region had minimal transcriptional activity but blocked ligand-mediated transcription by wild type AR. Mutants containing deletions of the DNA-binding and ligand-binding domains had minimal or weak dominant negative activity. We then tested the ability of wild type and mutant ARs to modulate GR- and PR-mediated transcriptional activity. The amino-terminal deletion mutants exerted dominant negative effects on GR- and PR-mediated activity, both in the absence and presence of testosterone. Surprisingly, wild type AR, which had approximately 20% of the maximal transcriptional activity of GR on the MMTV promoter, also had dominant negative activity on dexamethasone-regulated transcription mediated by GR. This dominant negative activity likely involves DNA binding because a point mutation in the DNA-binding domain abrogated such activity of an amino-terminal deletion mutant. Additionally, natural human AR mutants from patients with androgen resistance, which do not bind either DNA or ligand, did not block dexamethasone-mediated transcription. In summary, these studies suggest that mutant and wild type ARs can display dominant negative activity on other steroid hormone receptors that bind to a composite hormone response element This cross-regulation may be important in regulating maximal transcriptional activity in tissues where these receptors are coexpressed and may contribute to the phenotype of patients with steroid hormone resistance.

Cyclin D1 binds the androgen receptor and regulates hormone-dependent signaling in a p300/CBP-associated factor (P/CAF)-dependent manner.

The androgen receptor (AR) is a ligand-regulated member of the nuclear receptor superfamily. The cyclin D1 gene product, which encodes the regulatory subunit of holoenzymes that phosphorylate the retinoblastoma protein (pRB), promotes cellular proliferation and inhibits cellular differentiation in several different cell types. Herein the cyclin D1 gene product inhibited ligand-induced AR- enhancer function through a pRB-independent mechanism requiring the cyclin D1 carboxyl terminus. The histone acetyltransferase activity of P/CAF (p300/CBP associated factor) rescued cyclin D1-mediated AR trans-repression. Cyclin D1 and the AR both bound to similar domains of P/CAF, and cyclin D1 displaced binding of the AR to P/CAF in vitro. These studies suggest cyclin D1 binding to the AR may repress ligand-dependent AR activity by directly competing for P/CAF binding.

Effects of mitogens on androgen receptor-mediated transactivation.

The effects of mitogens and agents affecting tyrosine phosphorylation signaling on androgen-regulated transcription were investigated. CV-1 and HeLa cells were cotransfected with an androgen receptor (AR) expression vector and an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse mammary tumor virus promoter. Growth factors [epidermal growth factor (EGF) and insulin-like growth factor I] that activate receptor tyrosine kinases, an inhibitor of phosphotyrosine phosphatases (vanadate), or an inhibitor of tyrosine kinases (genistein) did not influence basal promoter activity or that of unliganded AR. However, EGF, insulin-like growth factor I, and vanadate enhanced AR-dependent transactivation by 1.5- to 2.5-fold, and genistein diminished it by two thirds in the presence of androgen. None of the treatments affected pRSV-CAT or pSV-beta-galactosidase expression, suggesting that gross activation of the transcription machinery was not involved. A reporter with two androgen response elements (AREs) in front of the thymidine kinase promoter (p delta ARE2tk-CAT) was used to examine promoter specificity. EGF activated this reporter even in the absence of androgen. However, when EGF was used concomitantly with testosterone, it augmented the action of androgen. Vanadate enhanced androgen-induced transactivation 2-fold without altering basal promoter activity. Neither EGF nor vanadate altered immunoreactive AR content or elicited changes in the receptor's DNA-binding properties. The intracellular content of hormone-binding AR was not influenced by EGF, but was decreased by vanadate and increased by genistein, as judged by [3H]mibolerone binding assays. An AR form lacking the hormone-binding domain (delta 641-902 mutant) transactivated p delta ARE2tk-CAT reporter similar to or better than the wild-type receptor in the presence of androgen. The transactivation by the delta 641-902 mutant was augmented by EGF and vanadate, but was attenuated by genistein, implying that the steroid-binding region is not critical for regulatory events initiated by tyrosine phosphorylation. Collectively, these data indicate that there is cross-talk between androgen-mediated signaling systems and growth factor/receptor tyrosine kinase pathways.

Nuclear androgen receptors in different stages of the seminiferous epithelial cycle and the interstitial tissue of rat testis.

Testicular androgen receptors were measured with a recently developed exchange assay using [3H]methyltrienolone from samples of interstitial tissue, whole seminiferous tubules, and segments of tubules in different stages of the cycle of the seminiferous epithelium. The hexylene glycol method was used to isolate nuclei and pyridoxal 5'-phosphate for extraction of the androgen receptors. This method proved superior to other techniques employed by our own and other laboratories. As a consequence, higher levels of androgen receptors were detected in seminiferous tubules than previously reported. Androgen receptors in seminiferous tubules and interstitial tissue had high affinity for methyltrienolone (Kd = approximately 3 nM) and steroid binding specificity similar to that of these receptors in other tissues. The concentration of nuclear androgen receptors in whole seminiferous tubules was 670 +/- 100 fmol/mg DNA (mean +/- SE), while that of the interstitial tissue was 1070 +/- 295 fmol/mg DNA. Tubules in stages IX-XII and XIII-I of the epithelial cycle contained significantly more nuclear androgen receptors (900 +/- 170 and 805 +/- 125 fmol/mg DNA, respectively) than those in stages II-VI and VII-VIII (485 +/- 95 and 485 +/- 65 fmol/mg DNA, respectively). These results suggest that there are local differences in androgen receptor concentration along the length of the seminiferous tubule. A high concentration of nuclear androgen receptors was also present in interstitial tissue. Androgen receptors were measurable in cytosol prepared from interstitial tissue, but such measurements were obscured in cytosol from tubules, because of a high capacity binding protein for the 3H-labeled ligand. We conclude that nuclear androgen receptors can be measured in various testicular compartments, including different stages of the seminiferous tubules, using an exchange assay that maximizes recovery. The concentration of nuclear androgen receptors in the tubules varies with the cycle of the seminiferous epithelium.