Altered levels of Smad2 and Smad4 are associated with human prostate carcinogenesis.

Alterations have been demonstrated in ligand and cognate receptor system of the transforming growth factor beta (TGF-beta) pathway in prostate cancer (PC). Still, little is known about changes in the activity of the intracellular Smad cascade of TGF-beta signaling during prostate carcinogenesis. We used immunohistochemistry to analyze phosphorylated Smad2 (p-Smad2), nuclear Smad4 and inhibitory-Smad7 in epithelial cells of normal, hyperplastic and malignant prostate. Specimens comprised 49 tissue cores of PC, 10 benign prostate hypertrophies and three normal prostates. Nuclear p-Smad2 (P<0.001) and nuclear Smad4 (P=0.023) were significantly decreased in PC with remarkable variations in cytoplasmic Smad7 levels. Substantial decreases in p-Smad2 and Smad4 levels were found in specimens with primary Gleason grades 3 and 4, whereas in grade 5, levels were markedly higher. Our results provide the first evidence for changes and reversible attenuation in the Smad system of the TGF-beta pathway during prostate carcinogenesis.

Vitamin D fortification as public health policy: significant improvement in vitamin D status in young Finnish men.

BACKGROUND: Vitamin D insufficiency is common in northern countries during wintertime. In Finland, after the recommendation by the Ministry of Social Affairs and Health, vitamin D has been added to liquid milk products and margarines from February 2003. OBJECTIVE: We determined the effects of national policy on vitamin D fortification on vitamin D status among young Finnish men. DESIGN: A comparison before and after intervention with study population of 196 young Finnish men (18-28 years) was carried out. Serum 25-hydroxyvitamin D3 (25-OHD3) concentrations were determined with the OCTEIA enzymeimmunoassay by IDS (Immunodiagnostic Systems Limited, Bolden, UK) in January 2003 (n = 96) and in January 2004 (n = 100), nearly 1 year after national vitamin D fortification had started. RESULTS: The mean serum 25-OHD3 concentrations during the wintertime increased by 50% after implementation of the vitamin D fortification of dairy products. Correspondingly, the prevalence of vitamin D insufficiency (serum 25-OHD3 < 40 nmol/l) was decreased by 50% from 78% in January 2003 to 35% in January 2004. CONCLUSIONS: Our results demonstrate that national vitamin D fortification substantially improved the vitamin D status of young Finnish men. Still, a third remained vitamin D insufficient.

The effect of dihydrotestosterone on the estrogen-induced cytodifferentiation of the chick oviduct.

The androgenic effects on the estrogen-induced cytodifferentiation of the chick oviduct epithelium were investigated. Dihydrotestosterone was shown to have an effect on the organization of stromal cells. Since these cells contained androgen receptor (AR), it is reasonable to assume an involvement of androgens in the differentiation and functioning of these cells through a direct action. Immunohistochemical analysis revealed a wide distribution of AR. AR was shown to be expressed in both the endothelial and smooth muscle cells of blood vessels. In the immature oviduct AR was located in the epithelial, mesenchymal and mesothelial cells. In the differentiating oviduct, whether induced by exogenous estrogen or normally by endogenous hormones, AR was also expressed by the tubular gland cells. Dihydrotestosterone alone had no effect on the morphology of the immature oviduct, suggesting the involvement of the determinants of differentiation in the action of androgen together with estrogen.

Estrogen and prolactin regulation of rat dorsal and lateral prostate in organ culture.

Besides androgens, estrogen (E) and PRL are thought to have important roles in the regulation of the growth and function of the prostate. We have established organ cultures of rat dorsolateral prostate for the analysis of the multiple hormone actions. Explants of dorsal prostate (DP) and lateral prostate (LP) were cultured in a serum-free basal medium containing insulin and corticosterone with or without the hormones studied. The viability and overall integrity of the tissues were maintained for at least 14 days. The morphology of the explants showed castration-like changes in the basal medium, but the addition of testosterone (T) prevented them. Androgen receptors in the prostate cultured with T were demonstrated by immunohistochemistry. When the explants were grown with E the epithelium became stratified, and the cells were flat. The epithelium was also layered when the explants were grown with PRL, but the epithelial cells were hypersecretory and large. The glandular morphology of the cultured prostate was, however, best preserved if T was added along with E or PRL. The wet weights and DNA contents of the explants declined during the culture, but they were better maintained if T, E, or PRL were added to the medium. The rate of DNA labeling with [3H]thymidine was activated in the cultured explants, but it was higher in those grown with T, E, or PRL than in those grown in the basal medium. The tissue specific functions were evaluated by measuring the expression of the genes RWB and M-40.3 encoding androgen-regulated secretory proteins. The steady state levels of RWB and M-40.3 mRNA were low in the explants grown in the basal medium but in the presence of T they were high. E and PRL also increased the expression of RWB and M-40.3 messenger RNA, although the responses in DP and LP were somewhat different. The antihormones cyproterone and toremifene opposed the increase of M-40.3 messenger RNA by T and E, respectively. The results show that the cultured DP and LP of the rat maintain the androgen responsiveness and tissue-specific functions in vitro. In addition, E and PRL have androgen-independent, direct effects in them. Rat dorsolateral prostate in culture thus provides a useful model for the studies on the mechanisms of hormone regulation of the prostate.

Development and evaluation of an immunoenzymometric assay for the chicken progesterone receptor.

A specific and sensitive immunoenzymometric assay (IEMA) was developed for measuring the quantity of chicken progesterone receptor (PR) in tissue cytosol. The assay uses two monoclonal antibodies to the PR. One is used to capture the PR. The second (labelled with biotin) reacts first with the captured receptor and subsequently with avidin-labelled horseradish peroxidase to provide an enzymatic end-point. The method has a determination range from 0.3 to 60 pmol/l. Intra- and interassay coefficients of variation were 3.7% and 9.0% respectively. The assay can be performed with equal results as a rapid (3 h) or an overnight procedure. The IEMA is convenient, especially for signal measurement and the calculation of results. No ultracentrifugation of samples is needed, since the IEMA can be performed on low-speed cytosol samples. Assay results correlated well (r = 0.927) with those obtained by the conventional ligand-binding assay used in our laboratory. Similar results were obtained with the IEMA and the ligand-binding assay after exposure of cytosol samples to increased temperatures: at 20 degrees C the PR remained stable for the 4-h period examined, whereas at 37 degrees C almost complete degradation of the PR was observed in 30 min. Being more than 100 times as sensitive as the ligand-binding assay, the IEMA enabled the quantification of PR for the first time in such tissues as the bursa and small intestine even of immature animals.

Progesterone-induced avidin as a marker of cytodifferentiation in the oviduct: comparison to ovalbumin.

Immunohistochemical analysis of avidin and ovalbumin expression in the normally developing chick oviduct was compared to those changes induced by exogenous estrogen. Oviduct maturation was found to occur in two consecutive phases: slow proliferation and rapid differentiation. Mitosis was induced in the epithelium by estrogen, whereas it was inhibited by progesterone. Endogenous progesterone may retard the proliferation and prevent the differentiation, an effect that is overridden by increased estrogen concentration at the beginning of differentiation. Short secondary stimulation was shown to closely mimic normal maturation. When chicks treated with diethylstilbestrol (DES) for 1 month were allowed to mature, there were marked alterations in oviduct histology and laying behavior. The tubular glands were found to form from the surface epithelium as budlike invaginations, and these cells also contained avidin and ovalbumin. Ovalbumin production was stable in tubular glands. In contrast, the intensity of avidin staining was variable between gland cells even in the same sections. It was conspicuous that the number of avidin-expressing gland cells diminished markedly when estrogen treatment was prolonged over 1 week. After 2-week stimulation with DES, avidin was expressed predominantly by cells of the basal layer of pseudostratified surface epithelium, and ovalbumin mainly by tubular glands and cells of the luminal layer of surface epithelium. Neither of these proteins was expressed by goblet cells. Expression of progesterone receptor, characterized by two antibodies (polyclonal IgG-RB and monoclonal PR6), did not explain the heterogeneity of expression of avidin and ovalbumin, but probably reflects various differentiation stages of epithelial cells.

Ontogeny of the estrogen receptor in the chick oviduct.

The distribution of estrogen receptor (ER) in the chick oviduct was studied immunohistochemically with monoclonal antibody H222, known to recognize chick ER [1]. The ontogeny of ER appeared to be very dependent on cellular differentiation. In the undifferentiated oviduct ER was located in the epithelial, mesothelial, stromal and smooth muscle cells. During differentiation ER disappeared from the surface epithelium, mesothelium, stromal and smooth muscle cells. At the onset of differentiation the protodifferentiated gland cells invaginated into the underlying stroma; these cells expressed ER. In the fully differentiated chick oviduct ER was located only in the tubular gland cells, which correlates with the known transcriptional activity of estrogen-induced ovalbumin-gene. However, we have reported estrogen dependency of PR also in ER-negative stromal cells, the mechanism being so far unknown. It is possible that there are mechanisms other than ER regulating the expression of PR. Estrogen-induced differentiation did not differ from normal maturation in regard to the distribution of ER. Since stromal, epithelial, mesothelial and smooth muscle cells were ER-negative in the mature oviduct, the concentration of ER, i.e. ER binding sites/cell is underestimated when whole tissue homogenates are used.

Characterization of the estrogen-sensitive cells expressing progesterone receptor in the bursa of Fabricius.

Cells expressing the progesterone receptor (PR) in the bursa of Fabricius (BF) were studied with immunohistochemistry at light-microscopic level, with immunoelectron microscopy (immuno-EM) and with non-specific esterase histochemistry. The antibody (IgG-RB) directed to the B component of the chick oviduct progesterone receptor was shown by immunoblotting to be specific for the PR and to recognize the PR also in the bursa. Two cell types in the BF contain the PR: stromal cells in the interfollicular-subepithelial area and smooth muscle cells lining the BF. The PR was localized in the nuclei of these cells. The bursal epithelium and the cells inside the follicles were not stained for PR. Electron microscopically the immunoreaction precipitate was localized on condensed heterochromatin and on dispersed euchromatin. The cells expressing the PR resembled electron microscopically fibroblasts. Their cytoplasm was rich in rough endoplasmic reticulum indicating active protein synthesis. By non-specific esterase histochemistry we showed that the PR-containing cells were not macrophages, which are morphologically indistinguishable from stromal cells. In the bursae of young untreated chicks the PR was not seen, but was inducible by estradiol treatment and was spontaneously expressed after the onset of sexual maturation. It is concluded that both the stromal fibroblasts and the smooth muscle cells in the BF are estrogen and progesterone sensitive. The expression of PR after the onset of sexual maturation indicates that the BF is directly affected by sexual maturation-associated factors. We suggest that estrogen and progesterone participate in tissue remodelling during bursal involution via the stromal cells and may affect bursal functions via the smooth muscle cells.

Sexual maturation-associated and estrogen-induced progesterone receptor expression in the bursa of Fabricius.

The expression of the progesterone receptor (PR) was studied in the chicken bursa of Fabricius (BF) in both sexes from the time of hatching until the bursal involution. Steroid binding studies, immunohistochemistry, and autoradiography were used to characterize and localize the receptor. Three different polyclonal antibodies (IgG-RB, IgG-G3, and IgG-RB2) directed against the chick oviduct progesterone receptor were used for the studies. With immunohistochemistry, no receptor-positive cells were detected in the bursae of young chicks. The first receptor-positive cells were occasionally seen at the age of 10 wk in the frozen sections, not in the paraffin sections. In older female chicks, the staining became more abundant. In males, the PR was expressed only after estradiol treatment. The staining was located in the nuclei of the subepithelial and the interfollicular cells, which were probably mesenchymal in origin. The bursal epithelium and the lymphocytes were not stained. By using a combined technique of autoradiography and immunohistochemistry, we were able to demonstrate that the same cells also concentrated tritiated ORG 2058 (a specific synthetic progestin) in their nuclei. In steroid binding studies with tritiated ORG 2058, the receptor concentration after the age of 10 wk was 50 to 120 fmol/mg protein. Low-level ORG 2058 binding was also detected in young chicks of both sexes before the age of 10 wk. The progestin-binding molecule resembled the progesterone receptor of the chick oviduct in molecular size (studied with HPLC) and binding properties. The PR expression in the BF was preceded by the expression of PR in the oviduct stromal cells and by an increase in oviduct epithelial proliferation, indicating the BF is affected by factors associated with sexual maturation. It is concluded that the subepithelial and the interfollicular stromal cells in the BF, but not the epithelial or follicular cells, are estradiol-sensitive in both sexes immediately after hatching. The endogenous estrogens, however, are not able to induce PR until after the onset of sexual maturation, and only in females. This implies that estrogen and progesterone may affect the structural organization of the BF through the stromal cells, but probably not before the onset of puberty.

Avidin induction by theophylline in vivo and in vitro.

Avidin induction in chick tissues in vivo and in vitro was studied by a phosphodiesterase inhibitor, theophylline, and compared to progesterone-dependent induction. Theophylline (100 mg/kg, ip) caused a significant increase in avidin content only in the oviduct of diethylstilbestrol-treated chicks, but not in the lung, muscle, intestine, plasma, or in the bursa of Fabricius. Diethylstilbestrol priming was necessary for oviductal avidin induction in vivo by theophylline. In the oviduct culture, theophylline at a concentration between 100 and 500 micrograms/ml caused a dose-dependent increase in avidin production. Effects of theophylline and progesterone on avidin synthesis in oviduct culture were synergistic. Avidin production was dependent on protein and RNA synthesis, since induction was inhibited by cycloheximide and actinomycin D. Avidin induction by theophylline resembled progesterone-dependent induction, beginning 9 h after the injection in vivo and 12 h after administration of these drugs in vitro. Avidin induced by theophylline showed heat-induced biotin exchange identical to that of progesterone-induced avidin, indicating close similarity of these proteins. The results suggest that theophylline can mimic the action of progesterone on avidin production, and that cyclic nucleotides may have a role in the regulation of avidin synthesis.

Effect of the progesterone-inducible proteins human lactoferrin and chicken avidin on lymphocyte proliferation.

Lactoferrin and avidin are progesterone-inducible glycoproteins in the human and chicken reproductive tracts, respectively. The effect of these proteins on lymphocyte proliferation was studied. The results showed that both proteins suppressed Concanavalin A (Con A)-induced responses so that a 10(3)-fold higher concentration of Con A was required for an optimal response. In contrast, avidin and lactoferrin had no effect on lymphocyte proliferation induced by other mitogens, tuberculin PPD or allogeneic cells. The effect of lactoferrin and avidin on lymphocyte proliferation seem to be caused by their binding to Con A. The significance of these findings is discussed.

Similarity of biotin-binding activity and immunoreactivity in chicken oviduct and non-oviduct avidin.

Biotin-binding and immunological methods were employed to demonstrate the similarity of oviduct and non-oviduct avidin in the chicken. Oviduct avidin was induced after oestrogen pretreatment by progesterone and non-oviduct avidin by intestinal tissue injury or by intraperitoneal actinomycin D administration. Avidin in the intestine, lung, bursa of Fabricius, plasma, pectoral muscle and liver after injury had biotin-binding activity similar to that of progesterone-induced oviduct avidin: (1) a temperature of 79-83 degree C was required for 50% of the maximum [14C]biotin uptake, (2) maximal exchange occurred only at 90 or 100 degree C and (3) denaturation of protein, i.e., loss of biotin-binding activity, was not yet observed at 100 degree C. Avidin in the intestine, lung, bursa of Fabricius, plasma and pectoral muscle also showed an identical cross-reaction with oviduct avidin. Furthermore, the increase in avidin-like biotin binding in the oviduct and most non-oviduct tissues was significantly correlated with the increase in avidin-like antigen in the tissue. This indicates that avidin induced in chicken non-oviduct tissues by injury or inflammation caused by actinomycin D administration is similar to progesterone-dependent oviduct avidin.

Induction of an antimicrobial biotin-binding egg white protein (avidin) in chick tissues in septic Escherichia coli infection.

The induction of avidin in chick tissues was found in septic Escherichia coli infection. Avidin concentrations in the plasma roughly corresponded to those in the other tissues studied which suggests that avidin in chicks is a secretory protein.

Progesterone-independent avidin induction in chick tissues caused by tissue injury and inflammation.

Progesterone was administered to oestrogen-treated and untreated chicks, or inflammation in the abdominal cavity was caused by intestine and liver injury or intraperitoneal actinomycin D administration. Local injury to the pectoral muscle was also carried out. Chicks were killed 24--26 h after the treatment and the biotin-binding egg white protein, avidin, was assayed in a number of tissues using a [14C]biotin-binding method and radioimmunoassay. Ovalbumin was also assayed with a radioimmunoassay. Avidin was not found in the tissues of control chicks. Progesterone induced avidin only in the oviducts of oestrogen-treated chicks. After intestine and liver injury avidin was found, however, in all the tissues of oestrogen-treated and untreated chicks studied except for the brain. The concentrations were highest in the oviduct, lung, intestine and bursa of Fabricius. Actinomycin D (200 microgram/kg) caused ascites and subcutaneous oedema in 40--60% of the chicks, and avidin was found only in the tissues of these inflamed animals. Avidin production caused by the local muscular injury was restricted to the injured area. Tissue injury and inflammation did not induce ovalbumin in any tissue. The study shows that avidin can be induced besides the oviduct also in non-oviductal chick tissues, and it is proposed that there are both progesterone-dependent and -independent avidin induction mechanisms.

Avidin induction by tissue injury and inflammation in male and female chickens.

1. The occurrence and inducibility of the biotin-binding egg white protein (avidin) in the chicken is not restricted to the oviduct. 2. Inflammatory treatments (intestinal injury, actinomycin D) induced avidin in a number of tissues of young and adult hens and roosters, but not of female rats and mice. Highest avidin concentrations were found in the organs containing epithelial cells and serous membrane. 3. The expression of the avidin gene by tissue injury and inflammation suggests that avidin has a significant function in the injured and inflamed chicken tissues.