Prolactin and prolactin receptors are expressed and functioning in human prostate.

Prolactin is widely expressed in different tissues, and it is presumed to have both local and systemic actions. In males it is known to influence reproductive functions but the significance and mechanisms of prolactin action in male accessory reproductive tissues are poorly understood. Here we show that prolactin acts as a direct growth and differentiation factor for human prostate, as measured by changes in DNA synthesis and epithelial morphology of organ cultures. Furthermore, we report the expression in human prostate of a short prolactin receptor form in addition to the long form, based upon ligand cross-linking studies and RT-PCR analysis of mRNA expression. The highest density of prolactin receptors was detected in the secretory epithelial cells by immunohistochemistry. Finally, we report that prolactin is locally produced in human prostate epithelium, as evidenced by marked prolactin immunoreactivity in a significant portion of prostate epithelial cells, with parallel expression of prolactin mRNA in human prostate. Collectively, these data provide significant support for the existence of an autocrine/paracrine loop of prolactin in the human prostate and may shed new light on the involvement of prolactin in the etiology and progression of neoplastic growth of the prostate.

Distribution and functions of parathyroid hormone-related protein in vertebrate cells.

Parathyroid hormone-related protein (PTHrP) was isolated from tumors and identified as the agent of humoral hypercalcemia of malignancy (HHM) in 1987. Since then its gene structure in several mammalian and an avian species has been analyzed and its gene expression demonstrated in many adult and embryonic tissues derived from all three germ layers. The composition and structure of PTHrP peptide depends on both differential gene splicing and posttranslational processing, which result in a range of peptides of potentially diverse functions. This chapter describes the distribution of PTHrP in both normal and neoplastic adult and embryonic tissues. PTHrP is of fundamental importance to cell survival because the absence of the gene is fatal; this aspect of PTHrP function in cell physiology becomes overwhelmingly important in neoplasia. Intracrine or paracrine actions for PTHrP seem to be most likely in mammalian and avian physiology, but in fishes high circulating levels suggest classic endocrine functions as well. Much remains to be learned of the biology of this fascinating protein.

Detection of prolactin receptor gene expression in the sheep pituitary gland and visualization of the specific translation of the signal in gonadotrophs.

In sheep, as in other mammalian species, the pronounced reduction in GnRH and gonadotropin secretion that characterizes stages of infertility is normally associated with a conspicuous increase in the secretion of PRL. A possible role of PRL in modulating gonadotropin release implies the presence and activation of specific receptors in target tissues (i.e. pituitary, hypothalamus). In this study, we investigated the expression of PRL receptor (PRL-R) messenger RNA (mRNA) in the sheep pituitary and the distribution of the translated product in specific pituitary cell types. Using primers designed to flank different regions of the extracellular and cytoplasmic domains of the PRL-R, two complementary DNA (cDNA) fragments, one of which was specific for the long-form PRL-R, were amplified by reverse transcriptase-PCR. Sequencing revealed more than 95% identity with nucleotides 267-1272 of the bovine PRL-R cDNA. When these cDNA fragments were used as probes for the detection of PRL-R mRNA expression by Northern analysis, three major transcripts of approximately 13, 10, and 3.5 kb were identified in the pituitary. Both probes detected identical transcripts, suggesting that primarily the long form of PRL-R is expressed in the sheep pituitary gland. No difference in the abundance of pituitary PRL-R mRNA transcripts was observed between anestrous and breeding season ewes (P > 0.05). Additional RT-PCR studies revealed the existence of a cDNA variant bearing a 39-bp insert with a premature stop codon. Translation of the PRL-R mRNA was confirmed by Western blot analysis. The identification of PRL-R in specific pituitary cell types was carried out by immunocytochemistry. Double immunofluorescent staining, using antibodies to the rat liver PRL-R and specific monoclonal antibodies to the LHbeta-subunit, FSHbeta-subunit, free alpha-subunit, PRL, or GH, revealed that in both the pars distalis and pars tuberalis, all pituitary cells expressing PRL-R immunoreactivity were positive for LHbeta, although only 53% of LHbeta-positive cells expressed PRL-R. A small proportion (2%) of gonadotrophs expressing PRL-R immunoreactivity were negative for FSHbeta, indicating the specific localization of PRL-R in LH (or LH/FSH) secreting cells. Further, a selective cytological association was detected in the pars distalis where LH gonadotrophs appeared surrounded by lactotrophs. In contrast to these observations, PRL-R immunoreactivity was completely absent in lactotrophs and in the vast majority (>98%) of somatotrophs. In conclusion, here we show the expression of PRL-R mRNA in the sheep pituitary and the specific translation of the signal in LH (or LH/FSH) gonadotrophs. These results support the hypothesis that PRL may be involved in the regulation of gonadotropin secretion through a paracrine mechanism within the pituitary gland and that this action does not seem to be mediated by changes in PRL-R mRNA expression.

Expression and hormone regulation of prolactin receptors in rat dorsal and lateral prostate.

We have studied the receptors that presumably mediate the biological effects of PRL in rat dorsal (DP) and lateral (LP) prostate. The PRL receptor proteins were localized to the glandular secretory epithelium of prostatic tissue by immunohistochemistry. Both the short and the long PRL receptor proteins were detected in DP and LP by Western blot analysis and cross-linking of [125I]human PRL to membrane preparations of DP and LP. Three messenger RNAs (mRNAs) for the long [1.3-1.7, 2.5, and 9.5-10 kilobases (kb)] and short (0.6-0.7, 3.0-4.6, and 10-12 kb) PRL receptors were expressed in dorsal and lateral lobes of rat prostate. Testosterone (T), estrogen (E), and PRL regulation of PRL receptor expression in rat DP and LP was studied in organ culture, which has been shown to be a suitable model to study hormone responses of prostatic tissue in vitro. The mRNAs of the short and long PRL receptors were differentially regulated in rat dorsolateral prostate. T, E, and PRL regulated the level of the long PRL receptor mRNAs in a tissue-specific manner, whereas hormone regulation of the short PRL receptor mRNAs was only modest. Furthermore, the hormonal responses of the different mRNA splicing variants of the long PRL receptor were not all similar; T, E, and PRL each increased the expression of 1.3- to 1.7-kb and 9.5- to 10-kb transcripts in DP, but only T did so in LP, whereas no clear regulation for the 2.5-kb mRNA could be observed in either tissue. This suggests that the hormonal regulation occurs at least at the posttranscriptional level. The effects of T and E were counteracted by the antihormones cyproterone and toremifene, respectively, indicating a specific receptor-mediated manner of steroid action.

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes.

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT-PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.

Calcitonin: characterisation and expression in a teleost fish, Fugu rubripes.

The present report describes the structure and expression of the calcitonin gene in Fugu rubripes. It is composed of 4 exons and 3 introns. Splicing of exons 1, 2 and 3 generates the calcitonin pre-proprotein, while splicing of exons 1, 2 and 4 generates calcitonin gene-related protein (CGRP). Exons 1 and 2 encoding the signal sequence and the N-terminal peptide are common in both the gene products and this gene organisation has been conserved in human, rat, chicken and salmon. The gene environment around calcitonin in Fugu has been poorly conserved when compared with human, apart from a small gene cluster. The calcitonin gene in Fugu has a widespread tissue distribution but it is most highly expressed in the brain. The abundance of gene expression in the ultimobranchial gland and the pituitary indicates that these are important sites of production and that the peptide is probably secreted into the circulation and/or acts as a paracrine or autocrine controlling factor. Whilst the function of calcitonin in fish is still largely unknown, the distribution described here suggests that one of the potential functions may be as a neuropeptide.

Sex-steroid receptors in the diethylnitrosamine model of hepatocarcinogenesis: modifications by gonadal ablation and steroid replacement therapy.

The results of this study confirm our previous report of increased androgen receptor expression in livers of female SUAH Wistar rats during development of liver tumours induced by diethylnitrosamine (DENA). In adult female rats not treated with DENA, removal of the ovary increased liver androgen receptor levels but testosterone did not further enhance the androgen receptor status of ovariectomized rats. In normal adult males the testis and/or testosterone maintained high levels of androgen receptors but oestrogen reduced them in castrated rats. Oestrogen receptor levels were not significantly changed in either males or females by gonadectomy. Treatment of female rats with DENA for 10 and 16 weeks increased liver androgen receptors but oestrogen receptors were only reduced by 16 weeks of DENA treatment, whether the rats were intact or ovariectomized. Concentrations of liver androgen receptors were increased in intact and castrated male rats by 10 and 16 weeks of DENA treatment, an increase not seen in the previous experiments. Oestrogen appeared to inhibit both the increases in liver androgen receptor expression and liver tumour development in rats treated with the weakly carcinogenic dose of 10 weeks of DENA. However, the full carcinogenic dose of 16 weeks of DENA increased liver androgen receptors and decreased oestrogen receptors in female rats regardless of sex-steroid status. Development of malignant hepatocellular carcinoma (HCC) was associated with both an increase in liver androgen receptors and a decrease in oestrogen receptors. Maintenance of relatively high levels of liver oestrogen receptors appeared to protect the liver against development of HCC.

In situ hybridization of parathyroid hormone-related protein in normal skin, skin tumors, and gynecological cancers using digoxigenin-labeled probes and antibody enhancement.

We describe a novel procedure for in situ hybridization that combines the use of digoxigenin-labeled oligonucleotide probes with an antibody enhancement step that can be performed on formalin-fixed, paraffin-embedded tissues. Addition of a second antibody enhances the visibility of parathyroid hormone-related protein (PTHrP) mRNA expression from barely to highly discernible and interpretable, with virtually no nonspecific background expression. This technique has allowed visualization of PTHrP mRNA in normal human skin and epithelium-derived tumors. PTHrP mRNA expression was confined to the basal and spinous keratinocyte layers of skin. There was strong hybridization in the spinous keratinocyte layer and a low level of hybridization in the basal layer. An extensive panel of positive and negative controls included poly d(T) probe to indicate total mRNA present in the sections. Squamous cell carcinomas and basal cell carcinomas of the skin, from pathology archives, were examined for the presence of PTHrP mRNA. The results reflected previous immunohistochemical studies, with every squamous cell carcinoma hybridizing strongly with the PTHrP probes. The basal cell carcinomas showed no expression of PTHrP mRNA, although the total mRNA signal was very strong. The localization of PTHrP mRNA in the tumors of the gynecological tract also reflected the immunohistochemical findings, with expression found in the squamous cell carcinomas but not in the adenocarcinomas. In situ hybridization with digoxigenin-labeled oligonucleotide probes and antibody enhancement has provided a sensitive, highly specific procedure for detection of PTHrP mRNA in tumors and normal tissue.

Effect of maternal dexamethasone treatment and ambient temperature on prolactin receptor abundance in Brown adipose and hepatic tissue in the foetus and new-born lamb.

We investigated the influence of maternal dexamethasone treatment and ambient temperature on prolactin receptor (PRLR) abundance in brown adipose tissue (BAT) and hepatic tissue from foetuses and 6-h-old lambs delivered by caesarean section. Lambs were either delivered into a warm (30 degrees C; WD) or cool (15 degrees C; CD) ambient temperature at 140 days gestation, 2 days after dexamethasone treatment, or at 146 days gestation for controls. Uncoupling protein-1 (UCP1) content of BAT was higher in dexamethasone-treated groups compared to controls. A range of tissue-specific PRLR isoforms was detected. For the long form of PRLR in BAT these isoforms had molecular weights of 66, 54, 34 and 19 kD compared with 88, 76, 66, 58, 54 and 48 kD in liver. In BAT, isoforms of the short form of PRLR had molecular weights of 66, 62, 54, 48, 33 and 31 kD compared with 82, 66, 56, 54, 48, 40 and 33 kD in liver. Dexamethasone treatment in CD lambs resulted in higher abundance of the 54 kD isoform of the short form of PRLR in liver, whilst in BAT dexamethasone resulted in a greater abundance of the 48 kD isoform of the short form, and lower abundance of the 66 kD isoform of the long form of PRLR, compared to controls. A negative correlation (r2 = 0.52) was observed between abundance of 66 kD isoform for the long form of PRLR and UCP1, compared with positive correlations (r2 = 0.58-0.60) for the abundance of the 54 and 48 kD isoforms for the short form of PRLR and UCP1. In conclusion, maternal dexamethasone treatment 1 week before term alters the abundance of PRLR isoforms in a tissue-specific manner. This response is dependent on ambient temperature after birth and may provide a critical endocrine signal for maximising non-shivering thermogenesis.

Microassay for prostatic androgen receptors correlated with quantitative histological assessment.

A new microassay in which cryostat sections of prostate tissue were used to provide the source of soluble androgen receptor for biochemical assay, was devised using an isoelectric focusing method, with [3H]-mibolerone as the androgenic radioligand. Adjacent cryostat sections from the same tissue block were stained for diagnostic and quantitative histological assessment. The assay was used to illustrate variations in tissue androgen receptor concentration for correlation with epithelial cell content in benign prostate hyperplasia and prostatic cancer, and to show the effects of androgen receptor concentration of resection of prostatic tissue by electroresection. The results indicate that the heat in electroresection renders prostatic tissue unsuitable for androgen receptor assays, and suggest that knowledge of the cellular composition of carcinomatous prostates may be of importance in the full assessment of androgen receptor assay results. This method incorporates both a biochemical assay and histological assessment of the assayed tissue on near-facsimile sections, an advantage over conventional biochemical assays.

Parathyroid hormone-related protein in lower vertebrates.

The genes for parathyroid hormone-related protein (PTHrP) have been cloned in two teleost fishes, cDNA of sea bream (Sparus aurata) and genomic DNA of puffer fish (Fugu rubripes). The gene sequences show that there is significant conservation of amino acid identity, with specific domains most highly conserved. The N-terminus, responsible for bone matrix lysis in mammals and chickens, is present in the fish genes with 52% sequence identity to higher vertebrate PTHrP peptides; the nuclear transporter region shares 73% identity, and the RNA-binding sequence is 65% identical. However, the peptides are shorter then mammalian PTHrP, lacking the C-terminus responsible for inhibition of osteoclast lytic activity, but they have an additional inserted sequence between amino acids 38 and 54 that is not present in higher vertebrate PTHrPs. The N-terminus 1-38 Fugu PTHrP proved to be hypercalcaemic in larval Sparus, suggesting that it may be a physiological regulator of calcium homeostasis in fish. Using homologous nucleotide probes for in situ hybridisation and reverse-transcription polymerase chain reaction (RT-PCR) of extracted RNA, PTHrP gene expression has been widely found in both developing and adult fish. Antiserum to the fish insert sequence demonstrated transcription of PTHrP in all stages of Sparus development, and also detected the same epitope in tissues of developing frog (Rana temporaria), indicating that this has been retained during evolution of the amphibia.