The role of androgen in determining differentiation and regulation of androgen receptor expression in the human prostatic epithelium transient amplifying population

The original article to which this erratum refers was published in J Cell Phys (2007) 212:572–578. J. Cell. Physiol. 215: 283–284, 2008. © 2007 Wiley‐Liss, Inc.     

The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes

Androgens and mesenchymal factors are essential extracellular signals for the development as well as the functional activity of the prostate epithelium. Little is known of the intraepithelial determinants that are involved in prostatic differentiation. Here we found that hepatocyte nuclear factor-3 alpha (HNF-3 alpha), an endoderm developmental factor, is essential for androgen receptor (AR)-mediated prostatic gene activation. Two HNF-3 cis-regulatory elements were identified in the rat probasin (PB) gene promoter, each immediately adjacent to an androgen response element. Remarkably, similar organization of HNF-3 and AR binding sites was observed in the prostate-specific antigen (PSA) gene core enhancer, suggesting a common functional mechanism. Mutations that disrupt these HNF-3 motifs significantly abolished the maximal androgen induction of PB and PSA activities. Overexpressing a mutant HNF-3 alpha deleted in the C-terminal region inhibited the androgen-induced promoter activity in LNCaP cells where endogenous HNF-3 alpha is expressed. Chromatin immunoprecipitation revealed in vivo that the occupancy of HNF-3 alpha on PSA enhancer can occur in an androgen-depleted condition, and before the recruitment of ligand-bound AR. A physical interaction of HNF-3 alpha and AR was detected through immunoprecipitation and confirmed by glutathione-S-transferase pull-down. This interaction is directly mediated through the DNA-binding domain/hinge region of AR and the forkhead domain of HNF-3 alpha. In addition, strong HNF-3 alpha expression, but not HNF-3 beta or HNF-3 gamma, is detected in both human and mouse prostatic epithelial cells where markers (PSA and PB) of differentiation are expressed. Taken together, these data support a model in which regulatory cues from the cell lineage and the extracellular environment coordinately establish the prostatic differentiated response.     

Concentration and cellular distribution of androgen receptor in human prostatic neoplasia: can estrogen treatment increase androgen receptor content?

The concentration of androgen receptor in cytosol (free and total sites) and nuclear fractions from benign (28 specimens) and malignant prostatic tissue from treated (16 specimens) and untreated patients (10 specimens) were assayed using [3H]methyltrienolone (3H R-1881) as ligand under conditions which stabilize AR and prevent binding of 3H R-1881 to progesterone receptor. It was found that optimum results were obtained when sodium molybdate (10 mM) was added after separation of the nuclear pellet rather than during tissue homogenization; when cytosol and nuclear exchange assays were carried out at 15 degrees C rather than at 0 degrees C; and when hydroxylapatite was used to separate free and bound steroid in the nuclear assay. Although AR values were variable in both BPH and carcinoma tissue, certain patterns of concentration, occupancy, and cellular distribution were observed in different patient groups. In BPH and untreated carcinoma tissue, the mean occupancy of cytosol AR by endogenous androgens was high, but the mean nuclear AR concentration was higher in BPH than in carcinoma tissue. Androgen receptor concentrations in tissue from orchiectomized patients were consistent with the effects of androgen deprivation: total cell AR was depleted, and a higher proportion was present as free cytosol AR. However, in tissue from most patients who had been treated with diethylstilbestrol (DES) on a long-term basis, total cell AR values were high. Although most of the AR was present as free cytosol AR, in three of four patients who had been treated with both orchiectomy and DES, the concentrations of bound cytosol AR and nuclear AR were similar to those in untreated patients.     

Vitamin D and androgen regulation of prostatic growth

Vitamin D has been reported to inhibit the growth of prostate cancer cells and model systems. In this study, we examined the interaction between 1,25-dihydroxyvitamin D(3) (1,25 D) in the presence or absence of endogenous testosterone on the growth and development of the adult rat prostate. Male Sprague-Dawley rats (165 days old) were either kept intact or castrated. Seven days after castration, the rats were treated with vehicle (control) or 1,25 D for 3 weeks and then sacrificed. Both ventral and dorsal lateral prostates were harvested; whole tissue lysates were collected and AR and VDR protein levels were analyzed by immunoblot analyses. Administration of 1,25 D in the intact animals decreased the prostatic size by 40%, compared to control animals, whereas 1,25 D did not influence the size of the prostate in castrated rats. 1,25 D administration in intact groups also increased both the AR and VDR protein levels by approximately twofold, whereas in castrated groups, 1,25 D only increased the AR protein level by 1.5-2.5-fold. 1,25 D in the presence of endogenous testosterone inhibits prostatic growth, whereas 1,25 D in the absence of endogenous testosterone does not affect prostatic growth. The growth inhibitory activity of 1,25 D in the presence of testosterone may be mediated through the ligand activated AR and VDR pathways. These studies may reveal important information about the potential efficacy of 1,25 D as well as hormonal status in understanding the development of prostate diseases.     

The role of human interleukin-6 in B-cell isotype regulation and differentiation

Human Interleukin-6 (IL-6) is a cytokine secreted by T cells, as well as a variety of other cell types, which exhibits B-cell differentiating activity. The recent cloning of the gene that codes for this molecule has allowed us the opportunity to study the function of this molecule alone and in conjunction with other lymphokines in human B-cell isotype-regulation and differentiation. Recombinant human IL-6 enhances immunoglobulin (Ig) M and G secretion by B-cells activated by Staphylococcal A Cowan strain (SAC) and enhances IgM, IgG, and IgA secretion by B-cells activated by pokeweed mitogen. IL-6 also augments immunoglobulin secretion of differing isotypes from various Epstein-Barr Virus transformed B-cell lines. However, IL-6 does not alter the secreted isotype of naive surface IgM-positive B-cells. As human T-cells secrete other lymphokines in association with IL-6 after activation we examined the interaction of Interleukin-2 (IL-2) gamma-interferon (IFN-gamma) and Interleukin-4 (IL-4) with IL-6 on B-cell immunoglobulin secretion. IL-2 and IL-4 synergized with IL-6 in augmenting immunoglobulin secretion by SAC-activated B-cells. IFN-gamma significantly inhibited the Ig secretion of SAC-activated B-cells cocultured with IL-6 alone or in combination with IL-2. These results demonstrate that human recombinant IL-6 augments immunoglobulin secretion of isotype-committed B-cells but it does not induce a change in the isotype secreted. In addition, this lymphokine synergizes with IL-2 and IL-4 in supporting Ig secretion. However, IFN-gamma significantly inhibits IL-6 induced Ig secretion.     

Tissue-specific expression and androgen regulation of different genes encoding rat prostatic 22-kilodalton glycoproteins homologous to human and rat cystatin

22-Kilodalton (kDa) protein cDNA clones were isolated from a rat prostatic library. Nucleotide sequence analysis revealed three different cDNA sequences encoding two somewhat different open reading frames of 176 amino acids. The N-terminal 24 amino acids of these sequences show the typical characteristics of signal peptides of secretory proteins. The C-terminal end of the derived protein sequences displays sequence similarity to a number of cysteine proteinase inhibitors, called cystatins, suggesting a common physiological function. Upon Northern blotting with a labeled cDNA fragment, three different 22-kDa protein mRNAs, i.e. 950 nucleotides (nt), 920 nt and 860 nt, could be detected in the rat ventral prostate and the lacrymal gland. In both tissues these messengers were regulated by androgens showing the most rapid androgen response for the 950 nt mRNA form. Administration of cycloheximide nearly completely abolished the observed androgen effect suggesting that a short-living protein is required for the full induction of the 22-kDa protein genes. Hybridization experiments with specific oligonucleotides which distinguish between the mRNAs encoding both 22-kDa protein variants indicate that one protein form is less androgen dependent in the ventral prostate and not expressed in the lacrymal gland.     

Steroid hormone regulation of prostatic acid phosphatase expression in cultured human prostatic carcinoma cells

We have investigated the modulation of prostatic acid phosphatase expression in the human prostatic cancer cell line LNCaP in response to the natural androgens testosterone and dihydrotestosterone, the female sex steroid estradiol and the synthetic androgen R1881 (methyltrienolone). Testosterone and dihydrotestosterone at 1 microgram/ml enhance the acid phosphatase synthesis by a factor of 3.5, while a hundred-fold lower concentration of the synthetic androgen R1881 induces an almost five-fold increase in the expression of this enzyme. The stimulation by all androgens tested and estradiol was dose-dependent. The synthetic glucocorticoid triamcinolone acetonide does not modulate the prostatic acid phosphatase expression in LNCaP cells, neither alone nor in combination with R1881.     

A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression

MDR1/P-glycoprotein is an efflux transporter determining the absorption and presystemic elimination of many xenobiotics in the gut. Thus, interindividual differences in MDR1 expression may affect the efficacy of drug treatment. The expression of MDR1 is partially controlled by the pregnane X receptor (PXR), which mediates induction by many xenobiotics. Since it has been described that the nuclear receptors PXR and constitutive androstane receptor (CAR) can bind to the same binding sites, we investigated the role of CAR in the regulation of MDR1 gene expression. We demonstrate here by gel shift and transfection experiments that CAR binds to distinct nuclear receptor response elements in the -7.8 kbp enhancer of MDR1 and transactivates MDR1 expression through DR4 motifs to which the receptor binds as a heterodimer with RXR or as a monomer, respectively. Expression of the endogenous MDR1 gene is elevated in cells stably expressing CAR, thus arguing for the functional relevance of CAR-dependent activation of MDR1 . The physiological relevance of the regulation of MDR1 by CAR is further suggested by correlation of the expression of CAR and MDR1 in the human small intestine. In summary, our data suggest that CAR plays a role in the regulation of intestinal MDR1 expression.     

Epigenetic regulation of cell type-specific expression patterns in the human mammary epithelium

Differentiation is an epigenetic program that involves the gradual loss of pluripotency and acquisition of cell type-specific features. Understanding these processes requires genome-wide analysis of epigenetic and gene expression profiles, which have been challenging in primary tissue samples due to limited numbers of cells available. Here we describe the application of high-throughput sequencing technology for profiling histone and DNA methylation, as well as gene expression patterns of normal human mammary progenitor-enriched and luminal lineage-committed cells. We observed significant differences in histone H3 lysine 27 tri-methylation (H3K27me3) enrichment and DNA methylation of genes expressed in a cell type-specific manner, suggesting their regulation by epigenetic mechanisms and a dynamic interplay between the two processes that together define developmental potential. The technologies we developed and the epigenetically regulated genes we identified will accelerate the characterization of primary cell epigenomes and the dissection of human mammary epithelial lineage-commitment and luminal differentiation.