Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.

Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.

Growth hormone therapy in hypophosphatemic rickets.

The effects of growth hormone therapy on the biochemical measures of bone metabolism were studied in 11 children aged 3.5 to 17 years who had familial hypophosphatemic rickets; five were male. Subjects were maintained on a regimen of stable doses of conventional therapy (calcitriol and phosphate). Subjects were studied at baseline receiving conventional therapy and during three sequential treatment periods: no therapy (4 weeks), growth hormone only (0.05 mg/kg per day for 4 weeks), and conventional therapy plus growth hormone (2 weeks). The nine youngest subjects were continued on a regimen of triple therapy for an additional 24 weeks. Serum phosphate averaged 0.93 +/- 0.13 mmol/L (mean +/- SD) at entry and decreased when the subjects were not receiving any therapy. During the 4 weeks of growth hormone only treatment, phosphate rose in all 11 subjects (0.70 +/- 0.08 mmol/L to 0.83 +/- 0.08 mmol/L). With triple therapy, phosphate remained higher than with no therapy. Calcitriol, osteocalcin, and parathyroid hormone increased as the subjects received growth hormone alone. Insulinlike growth factor I z scores rose significantly in response to growth hormone therapy alone. All nine subjects receiving 6 months of triple therapy increased their growth rate z scores. Exogenous growth hormone therapy may be useful in familial hypophosphatemic rickets.

Expression of recombinant androgen receptor in cultured mammalian cells.

Full-length rat and human androgen receptor (AR) cDNA clones were expressed in COS-7 and CV1 monkey kidney cells to analyze the AR protein using immunological and cotransfection techniques. The studies were aided by the development of two rabbit polyclonal antibodies, designated AR32 and AR52, directed against epitopes within the N-terminal region of AR. Each antibody recognizes native AR by sucrose gradient analysis and detects a 114-kilodalton protein in COS cells transfected with human or rat AR cDNA. Covalent binding of the synthetic androgen [3H]methyltrienolone (R1881) to the 114-kDa protein was saturable. The endogenous native AR was similarly 114 kDa on immunoblots of a human prostate adenocarcinoma cell line, LNCaP, and rat sex accessory gland extracts. AR was localized in nuclei of transfected COS cells and in LNCaP cells by immunocytochemical staining. Androgen induction of CAT activity was dose dependent in CV1 cells cotransfected with the AR expression vector and a reporter plasmid containing the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. It is concluded that antipeptide antibodies are useful reagents in characterizing both native and denatured forms of the AR protein. The 114-kDa protein expressed transiently in cultured cells represents the full-length AR protein, has a molecular size equivalent to that of endogenous AR, and mediates androgen-dependent transcriptional activation in CV1 cells.

A single base mutation in the androgen receptor gene causes androgen insensitivity in the testicular feminized rat.

The complete form of androgen insensitivity is an inherited X-linked syndrome in which genetic males fail to undergo masculinization in utero due to defective functioning of the androgen receptor (AR). The molecular basis of androgen insensitivity was investigated in the testicular feminized (Tfm) rat with this syndrome. AR mRNA size and amount, as well as nuclear AR protein revealed by immunocytochemistry, suggested normal expression of the AR gene in the Tfm rat. Sequence analysis of the AR coding region from Tfm and wild-type littermate male rats revealed a single transition mutation, guanine to adenine, within exon E, changing arginine 734 to glutamine within the steroid-binding domain of the AR. This arginine is highly conserved among the family of nuclear receptors and may be part of a phosphorylation recognition site. A recreated mutant AR (Arg734----Gln) expressed in COS cells had only 10-15% of the androgen-binding capacity of wild-type AR; the reduced androgen-binding capacity was similar to that of AR in tissue extracts of the Tfm rat. Stimulation of transcriptional activity by the recreated mutant AR was reduced relative to wild-type AR in cotransfection assays in CV1 cells using as reporter plasmid the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. Thus, arginine 734 appears essential for normal AR function both in androgen binding and transcriptional activation. Absence of these functions results in androgen insensitivity and lack of male sexual development.

Expression and localization of androgen receptor in the R-3327 Dunning rat prostatic adenocarcinoma.

The Dunning R-3327 rat prostatic adenocarcinoma and its sublines have been developed as a model system to study prostate tumor progression. We have used this system to study the changes in androgen receptor (AR) and AR mRNA expression which occur during tumor progression from androgen dependent to androgen independent growth. Dorsal prostate and all tumor sublines contained a 10-kilobase AR mRNA on Northern blot analysis. The levels of AR mRNA in each subline compared to dorsal prostate (100%) were: H (75%) greater than G (48%) greater than HI (25%) greater than HI-F = AT-1 = AT-3 = MAT-Lu = MAT-Ly-Lu = less than 5%. Immunocytochemistry showed AR predominantly in acinar epithelial cells of dorsal prostate and the androgen sensitive H subline. In the H subline, both acinar epithelial cells and locally invasive adenocarcinoma cells within the stroma showed positive immunostaining. The androgen responsive, anaplastic G subline also showed strong positive immunostaining. The androgen resistant AT-1 and MAT-Lu sublines lacked immunostaining for the AR. Steroid autoradiography revealed a similar cellular distribution of AR. These data suggest that in the Dunning system the loss of androgen binding and responsiveness is primarily due to selective changes in gene expression and not to gene rearrangements or posttranscriptional or translational modification of the AR mRNA or protein.

Autologous down-regulation of androgen receptor messenger ribonucleic acid.

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.

The rat androgen receptor: primary structure, autoregulation of its messenger ribonucleic acid, and immunocytochemical localization of the receptor protein.

A composite androgen receptor DNA sequence 4,181 base pairs in length was determined from three cDNA clones isolated from a rat epididymal bacteriophage lambda gt11 library. An open reading frame of 902 amino acids encodes a protein of 98,227 mol wt. Structural domains characteristic of the steroid receptor family include an amino-terminal region with five repeated amino acid motifs, a central DNA-binding domain homologous with other steroid receptors, and a carboxyl-terminal steroid-binding region. A receptor cDNA probe used in Northern blot analysis hybridized with a predominant 10-kilobase androgen receptor mRNA in male reproductive tissues of the rat. Autoregulation of androgen receptor mRNA was indicated in rat ventral prostate by an increase in the level of 10-kilobase mRNA after castration and suppression of receptor mRNA upon androgen restimulation. A 15 amino acid peptide with sequence derived from the deduced androgen receptor sequence was synthesized and used as immunogen in raising receptor antibodies in rabbits. Antisera reacted with high titer against the synthetic peptide by enzyme-linked immunosorbent assay and against the native [3H]dihydrotestosterone-labeled androgen receptor as evidenced by an increase in receptor sedimentation rate determined by sucrose gradient centrifugation. Immunocytochemical staining localized the androgen receptor to epithelial cell nuclei in rat ventral prostate.

Regulation of the beta 2-adrenergic receptor and its mRNA in the rat ventral prostate by testosterone.

To investigate the regulation of beta 2AR expression in rat ventral prostate the effects of castration and testosterone replacement on the beta 2AR were studied by ligand binding and Northern blot analysis. Orchidectomy depressed beta 2AR number by 50% within 4 days and testosterone administration to 4-day castrates produced a rapid and complete recovery of beta 2AR number within 24 h. In contrast to receptor number, beta 2AR mRNA levels did not change relative to total RNA following castration. However, during the testosterone replacement period beta 2AR mRNA levels rose transiently, reaching a maximum (3.5-fold) between 8 and 12 h, and this increase in mRNA preceded the recovery in beta 2AR number in the membrane. Regulation of beta 2AR gene expression by testosterone in the ventral prostate is thus complex and probably involves both transcriptional and post-transcriptional components.

Response of the mouse uterus to nafoxidine stimulation: agonism and antagonism.

Nafoxidine (NAF) acts as an estrogen agonist or antagonist depending on the animal model used. In the CD-1 mouse uterus, a three-day uterine bioassay of NAF produced a bell-shaped dose response curve with a maximal uterine wet weight increase at 200 micrograms/kg; this dose produced only a fractional increase in uterine dry weight. Combination treatment with NAF and estradiol antagonized estradiol stimulation of both wet and dry weight parameters. The time course of uterine wet weight stimulation following a single injection of NAF had an early pattern (0-10 h) similar to that of estradiol. However, at later times after stimulation, the patterns changed dramatically: the low NAF dose (200 micrograms/kg) returned to control levels by 24 h; estradiol and the high dose NAF (1.7 mg/kg) showed sustained stimulation, which peaked at 36 h with NAF compared to 24 h for estradiol. Nuclear estrogen receptor (ER) levels were measured after a single injection of 1.7 mg/kg NAF and showed a bimodal pattern similar to that seen with estradiol, with increases at 1 h and 8 h, although the overall ER levels were elevated above those seen with estradiol. Cytosolic ER levels with NAF decreased by 1 h and remained low up to 48 h. NAF treatment did stimulate uterine DNA and RNA synthesis, with a delayed time course compared to estradiol. DNA synthesis following a single 1.7 mg/kg dose of NAF was 2.5 times higher than that produced by 20 micrograms/kg estradiol. NAF treatment resulted in hypertrophy and hyperplasia in the luminal epithelium but not in the glandular epithelium. Long-term exposure to estradiol for 5 wk resulted in development of uterine cystic glandular hyperplasia and increased secretory activity; long-term exposure to NAF produced a more significant tissue hyperplasia but no secretions. These studies show that NAF stimulates some of the receptor-mediated responses attributed to an estrogen agonist in the mouse uterus; but, when co-administered with estradiol, NAF antagonizes some aspects of estrogen action.

Oncogene expression in prostate cancer: Dunning R3327 rat dorsal prostatic adenocarcinoma system.

Steady-state levels of myc, fos, p53, sis, and neu mRNAs were measured in eight variants derived from the Dunning R3327 rat prostate adenocarcinoma and compared to levels in normal dorsal prostate. Expression of the myb and erbB oncogenes in the Dunning tumors was below the limits of detection. Myc, p53, and sis mRNA levels in all tumors were at or above control levels. Fos mRNA levels were below control levels in four of five anaplastic tumors and were above control levels in the remaining tumors. A comparison of mRNA levels along the two Dunning lineages revealed that increased expression of these oncogenes did not correlate with tumor progression.

Expression of ras proto-oncogenes in the Dunning R3327 rat prostatic adenocarcinoma system.

Steady-state levels of c-Ha-ras mRNA were measured in eight sublines of the Dunning R3327 rat prostatic adenocarcinoma. As a control, normal dorsal prostate tissue was studied. Increased expression of c-Ha-ras is associated with tumor progression in one lineage of the Dunning R3327 system (H to AT1 to MAT-Lu and MAT-Ly-Lu). Here ras mRNA increases as the tumor advances from androgen dependence and a high degree of differentiation to an anaplastic aneuploid phenotype with high metastatic potential. However, in the other Dunning lineage (H to HI to HI-F to AT3), expression of c-Ha-ras is variable and does not correlate with tumor progression. Immunocytochemistry showed that levels of the c-Ha-ras p21 protein paralleled steady-state mRNA levels in variants. Transfection assays, using NIH/3T3 cells, suggested that the ras loci were not activated in the R3327 tumors. Levels of c-Ki-ras mRNA were also measured in the Dunning tumors; these did not correlate with tumor progression in either lineage. Expression of N-ras mRNA was not detected in the Dunning tumors.

Purification and characterization of mouse uterine estrogen receptor under conditions of varying hormonal status.

Mouse uterine estrogen receptor (ER) was purified about 11,000-fold from normal mouse uteri by affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified ER demonstrated a major component of 65,000 mol wt with minor fragments of the 54,000- and 37,000-dalton species, as judged by affinity labeling with [3H]tamoxifen aziridine and immunodetection with an ER monoclonal antibody (H-222). The minor fragments were not detected with additional monoclonals (H-226 or D-547), which recognize different domains on the ER molecule. Two-dimensional gel electrophoresis revealed that the major component with a mol wt of 65,000 had a pI of about 6.5. The 54,000- and 37,000-dalton components had similar pI values. Saturation binding and Scatchard plot analysis of purified ER yielded one class of binding sites with an apparent dissociation constant of about 1.4 nM. Changes in the hormonal status resulted in changes in the size of the ER even in the presence of molybdate and leupeptin.

Androgen regulation of c-myc messenger ribonucleic acid levels in rat ventral prostate.

Androgens regulate growth of the rat ventral prostate gland. In a search for possible mediators of androgen stimulated growth we have studied c-myc proto-oncogene expression in ventral prostate after androgen withdrawal and replacement. Steady state levels of c-myc mRNA were determined by Northern hybridization and compared with mRNA levels for prostatein, the major androgen dependent protein of ventral prostate. C-myc mRNA in ventral prostate increased nearly 4-fold within 1 day and 6- to 7-fold within 2 days after castration. Administration of androgen at the time of castration prevented this increase in c-myc mRNA levels. Androgen treatment of 4-day castrate rats caused c-myc mRNA levels to decrease within 4 h. Cycloheximide increased c-myc mRNA severalfold within 2 h. The net increase in c-myc mRNA after cycloheximide treatment was greater in the castrate than in the noncastrate or in androgen-treated castrate rats. These results suggest that androgen may influence both transcription and turnover of c-myc mRNA. Prostatein C3 mRNA decreased rapidly after castration and increased after androgen treatment of the castrate but was only slightly influenced by cycloheximide. Steady state levels of c-myc mRNA were higher in the 10-day-old rat and decreased with age while prostatein C3 mRNA increased with age. In situ hybridization demonstrated that both c-myc and prostatein mRNAs are expressed in the epithelial cells of ventral prostate acinar glands. These data indicate that androgens regulate the expression of c-myc in the ventral prostate.

Diethylstilbestrol metabolites and analogs. Biochemical probes for differential stimulation of uterine estrogen responses.

Diethylstilbestrol (DES) and certain chemically structural derivatives and analogs, indenestrol A (IA), indenestrol B (IB), indanestrol (IN), and pseudo-DES (PD), have been used as probes to examine various estrogenic responses previously considered interrelated and obligatory to the stimulation of uterine growth. All the analogs had poor uterotropic activity in vivo which ranged from 10-200 times less than that of estradiol or DES. The poor uterotropic activity was not due to poor binding affinity for the receptor. All compounds except IN interacted with the mouse uterine estrogen receptor with high affinity (approximately Ka 1.5-2.2 X 10(10) M-1). In addition, the compounds were able to translocate similar levels of receptor to the nucleus in vivo. Nuclear retention and occupancy of the estrogen receptor by the compounds was comparable to the patterns produced by DES or estradiol. The activity of uterine tissue responses was investigated during treatment with the compounds. Only IA stimulated uterine glucose-6-phosphate dehydrogenase to significant levels similar to DES or estradiol. Uterine progesterone receptor was induced to varying degrees by all compounds; the indenestrol isomers (IA and IB) were the most active. Uterine DNA synthesis was marginally stimulated by the derivatives and analogs except for IB which showed a response increase comparable to DES or estradiol. Because of the differential stimulation, these data suggest that in uterine tissue estrogen receptor stimulates certain biochemical responses independently and not in concert. The ability of a particular response to be increased may depend on the chemical nature of the ligand receptor complex and its interaction at genomic sites.

Differential regulation of protein synthesis by estradiol in uterine component tissues.

High resolution two-dimensional gel electrophoresis was used to examine [35S]methionine incorporation into intracellular proteins of the stromal, myometrial, and epithelial tissue fractions of the mouse uterus and into uterine secretory products. This procedure revealed that estradiol promotes preferential synthesis of different proteins in the myometrium, stroma, and epithelium. Conversely, synthesis of some proteins in the myometrial and stromal fractions was depressed by estradiol treatment; estrogen did not appear to depress synthesis of specific proteins in the epithelium. Thus, estradiol treatment influences protein synthesis in each uterine compartment in a different manner. This system provides a useful model to examine hormonal regulation of protein synthesis within different tissue compartments of a target organ.

The influence of 17 beta-estradiol on patterns of cell division in the uterus.

Uteri from immature (21-day-old) and adult mice show different patterns of cell division in response to a physiological dose of 17 beta-estradiol. In the immature mouse uterus, estradiol increased stromal and epithelial cell proliferation by shortening the cell generation time (Tc). The epithelial Tc was reduced from 100 to 14.5 h; the stromal Tc was reduced to 16 h. In both cell types, the reduction in Tc was primarily due to a reduction of the G1 phase of the cell cycle. In the adult mouse uterus, estradiol stimulated epithelial but not stromal cell proliferation. Here, estradiol reduced the epithelial Tc from 86 to 13 h, mostly by reducing the G1 phase of the cell cycle. Therefore, estrogens stimulate uterine hyperplasia by selectively decreasing the G1 phase of the cell cycle in specific cell populations. At some point during reproductive tract maturation, uterine stromal cells lose their ability to divide in response to estrogen stimulation. A developmental study showed that in the intact mouse, the stromal cell population gradually lost its ability to divide in response to estrogen stimulation during days 22-52 after birth. In prepubertally ovariectomized mice, the stromal cell population showed a very low mitotic response to estrogen stimulation at all ages. Thus, an ovarian mechanism may regulate the change in stromal responsiveness to estrogen stimulation.

Estrogen action in the mouse uterus: the influence of the neuroendocrine-adrenal axis.

Estrogen receptor levels were measured in uteri from ovariectomized-hypophysectomized mice. Cytosolic estrogen receptor levels had a range of 1.65-3.83 X 10(-13) mol/100 micrograms DNA and varied significantly with time after surgery. Nuclear receptor levels also varied with time and had a range of 0.26-1.19 X 10(-13) mol/100 micrograms DNA. Microsomal receptor levels varied from 0.24-0.72 X 10(-13) mol/100 micrograms DNA. Concurrent temporal changes in uterine or vaginal epithelial histology were not observed. Binding properties of the estrogen receptor from the ovariectomized-hypophysectomized mouse uterus were determined; Scatchard analysis of saturation binding data showed one class of binding sites (Kd = 1.32 nM; n = 276 fmol/mg protein) with values similar to those obtained from ovariectomized-adrenalectomized mouse uterus. The ability of the ovariectomized-hypophysectomized mouse uterus to respond to estrogen stimulation was determined in uterine wet weight and DNA synthesis assays; uteri from ovariectomized-hypophysectomized mice responded to estrogen in the same way as uteri from ovariectomized mice. Uteri from ovariectomized-adrenalectomized mice showed a similar growth response to estrogen stimulation. The temporal pattern of estrogen receptor distribution was determined in uteri from ovariectomized-adrenalectomized mice; there were two peaks of nuclear receptor translocation at 1 and 8 h, but the rate of cytosol receptor replenishment was slower than that previously seen in ovariectomized mouse uteri. Circulating estrogen and progesterone levels were measured in ovariectomized, ovariectomized-hypophysectomized, and ovariectomized-adrenalectomized mice at different times after surgery. There was no significant difference in systemic estradiol levels among the groups. However, there were significant differences in progesterone levels; mean values from ovariectomized, ovariectomized-hypophysectomized, and ovariectomized-adrenalectomized mice were 6.5, 2.1, and 0.029 ng/ml, respectively. The differences in progesterone levels correlate with previously reported differences in uterine estrogen receptor levels. Thus, data from this system suggest that adrenal-derived progesterone may play a greater role in regulating estrogen receptor levels in target tissues than was previously thought.

An ovarian independent population of uterine estrogen receptors.

Estrogen receptor levels in uteri from ovariectomized mice varied markedly with time after surgery; these changes were not due to nuclear translocation. Receptor cyclicity was abolished in ovariectomized-adrenalectomized mice, and receptor levels remained low. This indicates that the adrenals significantly influenced uterine estrogen receptor levels. However, these adrenal-mediated changes did not alter uterine responsiveness ot estrogen stimulation.

Effects of progesterone on uptake and metabolism of 17 beta-estradiol by mouse uterine luminal epithelium.

The influence of progesterone (P4) pretreatment on the uptake and retention of [3H]estradiol-17 beta ([3H]E2) in whole uterus and luminal epithelium was examined in ovariectomized mice. After s.c. injection of 50 ng [3H]E2 uterine, epithelial and epithelial nuclear levels of radioactivity were maximal 3 h post-injection: maximum epithelial levels were sustained from 3 to 6 h in Pa-treated, but not control tissue. P4 treatment did not significantly alter the proportion of epithelial or uterine radioactivity recovered as [3H]E2, or the intracellular distribution of the hormone. Preparation of epithelial suspensions in buffer containing excess 17 beta-estradiol (E2) did not significantly alter their [3H]E2 content. After intraluminal injection of 100 pg [3H]E2 rates of loss from uterine, epithelial and epithelial nuclear preparations did not differ significantly between control and P4-treated tissues. We conclude that P4 does not inhibit E2-induced luminal epithelial mitosis by preventing E2 reaching the epithelial cells and nuclei, by changing its distribution in the tissue, or by increasing its rate of loss from the tissue or its metabolism to less active estrogens like estrone.