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The prostate and seminal vesicle (SV) are androgen-dependent secretory glands of the male genital tract. The epithelial cells of these glands produce the bulk of the seminal secretions. The objective of the present study was to examine the ontogeny of cytokeratin and androgen receptor (AR) expression in the rat SV, anterior prostate (AP) and ventral prostate (VP). The study utilized organ culture to examine the effects of androgens on the development of these markers and castration of adult rats to examine androgenic effects on their maintenance. Tissues were examined from 14 days of gestation to adulthood. The SV was a tubular organ from its inception while the prostate formed from solid epithelial cords. These prostatic buds canalized in a proximal to distal manner starting at day 1 postnatal in the VP and day 5 in the AP. The expression of cytokeratins and AR was visualized by immunocytochemistry. In all three glands keratins 5, 7, 8, 14, 18 and 19 were initially uniformly expressed in all epithelial cells. In the SV, segregation of cytokeratins between the luminal and basal cell types started at 4 days postnatally with keratin 7 localizing to basal cells. Five days after birth, keratins 5 and 14 were also localized to the basal epithelium, while keratins 8 and 18 were only expressed by luminal cells, Keratin 19 was expressed in all epithelial cells throughout development and into adulthood. In the VP and AP the same pattern of cytokeratin segregation occurred as in the SV. Epithelial differentiation occurred in a proximal to distal fashion in the prostate. In the proximal VP ducts keratins 7 and 14 were basally localized by 2 days postnatally, while keratin 5 did not clearly segregate to basal cells until day 9 after birth. In the AP keratin 14 was basally localized by 1 day postnatal but keratin 5 and 7 did not colocalize to the basal cells until days 9 and 12, respectively. AR were expressed in the epithelium of the urogenital sinus from 19 days of gestation. At 19 and 20 days of embryonic development AR-negative prostatic buds were seen emerging from the AR-positive urogenital sinus epithelium. By birth AR were detectable in the epithelium of both prostatic lobes and the SV. The role of androgens in the development of the prostatic and SV epithelium was investigated in a serum-free organ culture system. These experiments showed that differentiation of prostatic and SV luminal and basal epithelial cell types was accelerated as compared to the in vivo situation in the presence of androgens, and did not occur in their absence. Following castration of adult animals the prostate and SV regressed with preferential loss of luminal epithelium. The relative numbers of basal cells was increased, though some flattened cells expressing a luminal cell pattern of cytokeratins were still observed. AR were detected in the prostatic and SV epithelium of long-term castrated animals. In summary, the rat prostate was found to be derived from undifferentiated solid epithelial cords. Canalization occurred concurrent with the differentiation of clear epithelial subtypes. Epithelial AR were expressed from around the time of birth and expression levels increased with age. The SV was canalized from its inception but likewise was derived from an undifferentiated epithelial precursor.
We have shown that a polypeptide of M(r) 60,000 (60K) that shares N-terminal homology with a calcium-binding protein, calreticulin, can bind to an amino-acid sequence motif, KXGFFKR, found in the cytoplasmic domains of all integrin alpha-subunits. The homologous amino-acid sequence, KXFFKR (where X is either G, A or V), is also present in the DNA-binding domain of all known members of the steroid hormone receptor family; amino acids in this sequence make direct contact with nucleotides in their DNA-responsive elements and are crucial for DNA binding. Here we show that both the 60K protein (p60), purified on a KLGFFKR-Sepharose affinity matrix, and recombinant calreticulin can inhibit the binding of androgen receptor to its hormone-responsive DNA element in a KXFFKR-sequence-specific manner. Calreticulin can also inhibit androgen receptor and retinoic acid receptor transcriptional activities in vivo, as well as retinoic acid-induced neuronal differentiation. Our results indicate that calreticulin can act as an important modulator of the regulation of gene transcription by nuclear hormone receptors.
The functional and structural interactions of two androgen receptor-binding sites in the 5'-flanking DNA of the rat probasin gene were determined. Deletion mapping and DNase I footprinting analysis had previously identified two androgen receptor-binding sites (ARBS) necessary for androgen induction of the probasin gene: ARBS-1, which resembled a glucocorticoid-responsive element, and ARBS-2, which had a unique sequence. In this study, maximal androgen induction in transient transfection studies only occurred when both sites were present. Neither binding site functioned independently, and deletion of the DNA sequence between the sites resulted in a 60% loss of androgen inducibility. Moreover, point mutations in either ARBS-1 or ARBS-2 led to > 90% loss in activity. Scatchard analysis indicated that ARBS-1 and ARBS-2 bound a synthetic androgen receptor, AR2, with Kd values of 20.0 and 6.7 nM, respectively. Consistent with the higher affinity, ARBS-2 bound AR2 at half the threshold concentration (200 ng) of that required in reciprocal DNase I footprinting experiments with ARBS-1. By comparison, protection occurred at a much lower threshold concentration of AR2 (60 ng) and to the same extent over each site when both sites were present, suggesting a cooperative interaction between the two sites. The cooperative effect was further substantiated when a point mutation in ARBS-1 blocked AR2 binding not only to ARBS-1, but also to ARBS-2. Similarly, a point mutation in ARBS-2 also prevented receptor binding to both sites. Androgen-specific regulation of probasin gene transcription therefore required an androgen-responsive region (positions -286 and +28) containing two androgen receptor-binding sites, where the binding of the androgen receptor to both sites occurred in a cooperative, mutually dependent manner.
We studied the effect of retinoic acid on the growth and secretory activity of the androgen-responsive prostatic carcinoma cell line LNCaP. Our data showed that retinoic acid at 0.01 microM. stimulated the proliferation of LNCaP cells but inhibited their growth at 0.1 microM. under androgen-free conditions. In the presence of 0.1 nM. dihydrotestosterone (DHT), LNCaP cell proliferation was inhibited by 10 microM. retinoic acid but not by lower concentrations of retinoic acid. Retinoic acid reduced LNCaP cell growth at concentrations of 0.1 microM. in the presence of 10 nM. DHT. Retinoic acid (10 microM.) also reduced the growth response of LNCaP cells to epidermal growth factor and transforming growth factor alpha and potentiated the inhibitory effect of transforming growth factor beta. In additional studies, retinoic acid induced a dose-dependent increase in prostate specific antigen (PSA) secretion at concentrations of 0.1 to 1 microM. Dihydrotestosterone (10 nM.) also enhanced the secretion of PSA by LNCaP cells, and this effect was potentiated in a dose-dependent fashion by the addition of retinoic acid at 0.1-10 microM. Competitive binding studies showed that retinoic acid did not bind to androgen receptors. Overall, retinoic acid had a biphasic effect on LNCaP proliferation and promoted the secretion of PSA. The biphasic effect of retinoic acid on LNCaP growth should be considered in designing in vivo studies to determine the impact of retinoic acid on solid prostatic tumor growth. In addition, the ability of retinoic acid to increase PSA secretion may complicate the interpretation of serum PSA levels used for diagnostic and prognostic purposes.
This report describes the development and characterization of an epithelial cell line (BPH-1) from human prostate tissue obtained by transurethral resection. Primary epithelial cell cultures were immortalized with SV40 large T antigen. One of the isolated clones was designated BPH-1. These cells have a cobblestone appearance in monolayer culture and are non-tumorigenic in nude mice following subcutaneous injection or subrenal capsule grafting. They express the SV40 large T antigen and exhibit increased levels of p53, as determined by immunocytochemistry. Cytogenetic analysis by G-banding demonstrated an aneuploid karyotype with a modal chromosome number of 76 (range 71 to 79, n = 28) and 6 to 8 marker chromosomes. Some structurally rearranged chromosomes were observed, but the Y chromosome was normal. The expressed cytokeratin profile was consistent with a prostatic luminal epithelial cell. This profile was the same as that of primary prostatic epithelial cultures from which the BPH-1 cells were derived. In serum-free culture in plastic dishes epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, fibroblast growth factor (FGF) 1 (aFGF), and FGF 7 (KGF) induced increased proliferation in these cells whereas FGF 2 (bFGF), TGF-beta 1, and TGF-beta 2 inhibited proliferative activity. Testosterone had no direct effect on the proliferative rate of BPH-1 cells. 5 alpha-Reductase, 3 alpha-hydroxysteroid oxidoreductase, and 17 beta-hydroxy-steroid oxidoreductase activities were detected in BPH-1 cells. Expression of androgen receptors and the secretory markers, prostate specific antigen and prostatic acid phosphatase, were not detectable by immunocytochemistry, biochemical assay, or RT-PCR analysis.