Effects of the selective estrogen receptor modulator ospemifene on bone in rats.

Ospemifene is a non-estrogen agent that exerts tissue-specific estrogen agonistic and weak antagonistic effects (i. e., is a selective estrogen receptor modulator [SERM]). The effects of various once-daily oral doses of ospemifene on bone are examined across 3 studies for 4 or 52 weeks after surgery in the ovariectomized (OVX) rat model of postmenopausal bone loss. Ospemifene treatment reduced the loss of bone mineral content and density observed in untreated OVX rats, significantly increased distal femur bone mineral content at 51 weeks at 25 mg/kg dose compared with untreated OVX rats (p<0.01), and significantly increased trabecular bone mineral density of the distal femur and proximal tibia with 1, 5, or 25 mg/kg doses after 52 weeks. Ospemifene 5 and 25 mg/kg preserved distal femur trabecular structure; trabecular number was significantly increased, whereas trabecular separation and eroded surface values were significantly decreased (all p<0.01). Structural changes associated with ospemifene were accompanied by increased mechanical strength of femurs and 4th lumbar vertebra compared with untreated OVX rats. Ospemifene 10 mg/kg prevented OVX-induced bone loss; trabecular bone volume of distal femurs was increased after 4 weeks. Further, histomorphometric measures revealed decreased bone resorption after 4 weeks of ospemifene treatment, with effects similar to other SERMs (raloxifene and droloxifene). Ospemifene 3 and 10 mg/kg significantly inhibited OVX-induced increases in osteoclast number, and doses ≥0.3 mg/kg dose-dependently reversed the OVX-induced increase in the double-labeled volume:bone volume ratio. These results demonstrate antiresorptive, selective agonist effects of ospemifene on bone that appear similar to raloxifene in this in vivo animal model of estrogen deficiency.

Effects of ospemifene on breast tissue morphology and proliferation: a comparative study versus other selective estrogen receptor modulators in ovariectomized rats.

Ospemifene is a tissue-selective estrogen agonist/antagonist that was recently approved for the treatment of dyspareunia associated with vulvar and vaginal atrophy, which occurs in up to approximately 50% of postmenopausal women. The current analyses were conducted to determine whether ospemifene exhibits estrogenic activity in the mammary glands of ovariectomized rats and to compare potential estrogenic activity with selective estrogen receptor modulators (tamoxifen, raloxifene, and toremifene). Three separate studies with differing durations (6, 9, and 28 days) were conducted using similar procedures in ovariectomized Sprague-Dawley rats. Estradiol treatment and sham-treated ovariectomized rats were used as positive and negative controls, respectively. Cell proliferation was examined using labeled 5-bromo-2-deoxyuridine; cytoplasmic prolactin was characterized with antibody staining. The morphology of the mammary gland was studied by histological staining of sections from the right fourth mammary glands, and the excised gland from the left side was used for counting the lobulus number. Neither ospemifene nor selective estrogen receptor modulators substantially induced 5-bromo-2-deoxyuridine staining, altered the morphology of the mammary glands, or changed prolactin immunostaining in ovariectomized rats compared with the ovariectomized controls. With the exception of toremifene, the selective estrogen receptor modulators did not cause a substantial induction in mammary gland lobuli. Estradiol had effects opposite to those of the selective estrogen receptor modulators in these studies. Ospemifene exhibited no substantial estrogenic activity in the mammary gland of ovariectomized rats. Activity in the mammary gland of ovariectomized rats with ospemifene was comparable to raloxifene and tamoxifen.

Effects of FGFR inhibitors TKI258, BGJ398 and AZD4547 on breast cancer cells in 2D, 3D and tissue explant cultures.

PURPOSE: Fibroblast growth factor receptors (FGFR) and pathways are important players in breast cancer (BC) development. They are commonly altered, and BCs exhibiting FGFR gene amplification are currently being studied for drug development. Here, we aimed to compare the effects of three FGFR inhibitors (FGFRis), i.e., non-selective TKI258 and selective BGJ398 and AZD4547, on different BC-derived cell lines (BCCs) and primary tissues. METHODS: The human BCCs MCF-7 and MDA-MB-231(SA) (wild-type FGFR) and MFM223 (amplified FGFR1 and FGFR2) were analyzed for FGFR expression using qRT-PCR, and the effects of FGFRis on FGFR signaling by Western blotting. The effects of FGFRis on proliferation, viability, migration and invasion of BCCs were assessed in 2D cultures using live-cell imaging, and in 3D cultures using phenotypic analysis of organoids. To study radio-sensitization, FGFRi treatment was combined with irradiation. Patient-derived BC samples were treated with FGFRis in explant cultures and immunostained for Ki67 and cleaved caspase 3. RESULTS: We found that all FGFRis tested decreased the growth and viability of BC cells in 2D and 3D cultures. BGJ398 and AZD4547 were found to be potent at low concentrations in FGFR-amplified MFM233 cells, whereas higher concentrations were required in non-amplified MCF7 and MDA-MB-231(SA) cells. TKI258 inhibited the migration and invasion, whereas BGJ398 and AZD4547 only inhibited the invasion of MDA-MB-231(SA) cells. FGFRi treatment of MCF7 and MFM223 cells enhanced the inhibitory effect of radiotherapy, but this effect was not observed in MDA-MB-231(SA) cells. FGFRi-treated primary BC explants with moderate FGFR levels showed a tendency towards decreased proliferation and increased apoptosis. CONCLUSIONS: Our results indicate that, besides targeting FGFR-amplified BCs with selective FGFRis, also BCs without FGFR amplification/activation may benefit from FGFRi-treatment. Combination with other treatment modalities, such as radiotherapy, may allow the use of FGFRis at relatively low concentrations and, thereby, contribute to better BC treatment outcomes.

Prevalence and associated factors of restless legs in a 57-year-old urban population in northern Finland.

OBJECTIVE: We examined the prevalence and associated factors of restless legs syndrome (RLS) in a 57-year-old unselected urban population in northern Finland. METHODS: A health survey was conducted in 2002 that targeted persons born in 1945 and residing in the city of Oulu on 31 December, 2001. Their history of RLS, coronary heart disease (CHD), daytime sleepiness, depressive symptoms and snoring was assessed by means of questionnaires. RESULTS: Altogether 995 of 1332 eligible subjects (74%) participated (556 women, 439 men). The overall prevalence of RLS > or = 1 per week was 20% in women and 15% in men. In the fitted multiple logistic regression model, RLS was found to be associated with female gender (OR 1.64, 95% CI 0.98-2.72), CHD (OR 2.92, 95% CI 1.18-7.23), daytime sleepiness (OR 2.12, 95% CI 1.32-3.41), moderately elevated (31-45) or high (46-65) Zung sum scores (OR 1.95, 95% CI 1.09-3.48 and OR 3.67, 95% CI 1.71-7.90, respectively), antidepressant medication (OR 2.10, 95% CI 1.06-4.19) and arthropathy (OR 1.69, 95% CI 1.04-2.72). Insufficient evidence was found of an association between RLS and type 2 diabetes or impaired glucose regulation. CONCLUSIONS: Restless legs syndrome is fairly common in subjects aged 57 years. A particularly strong positive association was observed between RLS and depressive symptoms and CHD.

Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes.

Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage.

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.

Differential effects of selective oestrogen receptor modulators (SERMs) tamoxifen, ospemifene and raloxifene on human osteoclasts in vitro.

BACKGROUND AND PURPOSE: Several selective oestrogen receptor modulators (SERMs) with oestrogen agonist effects in bone cells and without increased risk of breast and endometrial cancer have been developed. Here, we have investigated the effects of different types of SERMs on osteoclast differentiation, bone resorption and apoptosis in vitro. EXPERIMENTAL APPROACH: Human peripheral blood-derived CD14+ monocytes were cultured on bovine bone slices in the presence of RANKL, M-CSF, TNF-alpha and dexamethasone for seven days. Also, CD14+ monocytes were co-cultured either with human SaOS-2 or MG-63 osteosarcoma cells, in the presence of parathyroid hormone. Osteoclast cultures were treated with different SERMs. TRACP+ multinucleated cells and C-terminal telopeptide of type I collagen were used as markers for osteoclast formation and bone resorption, respectively. KEY RESULTS: In CD14+ monocyte cultures, tamoxifen directly inhibited human osteoclast formation and bone resorption, while raloxifene and ospemifene had no inhibitory effect. In the co-cultures either with SaOS-2 or MG-63 cells, ospemifene and raloxifene as well as tamoxifen inhibited osteoclast formation in a concentration-dependent manner. The inhibitory effect was associated with an increased production of osteoprotegerin. The anti-oestrogen ICI 182 780 completely reversed the effects of these SERMs. CONCLUSION AND IMPLICATIONS: Tamoxifen had an oestrogen receptor dependent, direct, inhibitory effect on human osteoclast differentiation and bone resorption, whereas ospemifene and raloxifene required osteoblastic cells to achieve a similar inhibition. The effects of ospemifene and raloxifene were mediated by oestrogen receptors by a mechanism involving paracrine induction of osteoprotegerin in cultures with osteoblast derived osteosarcoma cells.

Control of cell proliferation by steroids: the role of 17HSDs.

Sex steroid hormone signaling regulates the development, growth, and functioning of the breast and the prostate and plays a role in the development and progression of cancer in these organs. The intracellular concentration of active sex steroid hormones in target tissues is regulated by several enzymes, including 17beta-hydroxysteroid dehydrogenases (17HSDs). Changes in the expression patterns of these enzymes may play a pathophysiological role in malignant transformation. We recently analyzed the mRNA expressions of the 17HSD type 1, 2, and 5 enzymes in about 800 breast carcinoma specimens. Both 17HSD type 1 and 2 mRNAs were detected in normal breast tissue from premenopausal women but not in specimens from postmenopausal women. The patients with tumors expressing 17HSD type 1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients. The expression of 17HSD type 5 was significantly higher in breast tumor specimens than in normal tissue. Cox multivariate analyses showed that 17HSD type 1, tumor size, and estrogen receptor alpha (ERalpha) had independent prognostic significance. We developed, using a LNCaP prostate cancer cell line, a model to study the malignant transformation of prostate cancer and showed that androgen-sensitive LNCaP cells are transformed into neuroendocrine-like cells when cultured without androgens and, eventually into highly proliferating androgen-independent cells. We conducted Northern hybridizations and microarrays to analyze the gene expression during these processes. Substantial changes in the expressions of steroid metabolizing enzymes occurred during the transformation process. The variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of organs.

Apoptosis in toremifene-induced growth inhibition of human breast cancer cells in vivo and in vitro.

BACKGROUND: Antiestrogens inhibit the stimulative effects of estrogens on breast cancer growth, but the mechanism(s) by which they trigger tumor regression are not completely understood. Growth retardation and tumor regression can be achieved by enhanced cell death and/or arrested cell proliferation. PURPOSE: Our aim was to investigate the effect of a new antiestrogen, toremifene, on human breast cancer cells grown either in culture or as tumors in nude mice. METHODS: The growth and morphology of in vitro cultured cells of the human breast cancer cell line MCF-7 were monitored by time-lapse video. MCF-7 cells and ZR-75-1 human breast cancer cells were grown as tumors in nude mice and subsequently examined by electron microscopy. The integrity of DNA isolated from these cells was determined by standard gel electrophoretic techniques. Northern blot hybridization analysis was used to determine the steady-state levels of the mRNAs for testosterone-repressed prostatic message-2 (TRPM-2), tumor growth factor beta-1 (TGF beta 1), and pS2 (a small, cysteine-rich protein of unknown function). RESULTS: Time-lapse video microscopy of the cell cultures indicated that treatment with 7.5 microM toremifene for 3 days caused approximately 60% of the cells to exhibit morphologic characteristics typical of cells undergoing programmed death, or apoptosis. The number of mitoses gradually decreased to zero over a 3- to 4-day period. Estrogen withdrawal for the same length of time resulted in an approximately equal number of apoptoses and mitoses. These changes were not associated with the pattern of DNA fragmentation, detectable as ladders in agarose gels, that is characteristic of the DNA of cells undergoing apoptosis. Elevated levels of TRPM-2 and TGF beta 1 mRNAs were observed in in vitro or in vivo grown tumor cells treated with 5-10 microM toremifene. Elevated levels of TRPM-2, but not TGF beta 1, mRNA were observed in the tumor cells after estrogen withdrawal. The steady-state level of pS2 mRNA in the tumor cells dropped in response to either toremifene treatment or estrogen withdrawal. CONCLUSION: Toremifene causes growth inhibition of estrogen-sensitive breast cancer cells by inducing some cells to undergo apoptosis and by inhibiting other cells from entering mitosis. The higher than normal amounts of TRPM-2 and TGF beta 1 protein that would likely result from the elevated levels of TRPM-2 and TGF beta 1 mRNAs measured in these cells after toremifene treatment may have an important role in the growth inhibition process. IMPLICATION: Apoptosis as an active, targeted process provides a potential new therapeutic approach for treating breast cancer.

Loss of heterozygosity at 16q24.1-q24.2 is significantly associated with metastatic and aggressive behavior of prostate cancer.

Several studies have indicated that in prostate cancer, frequent aberrations take place in several genomic regions. In the present study, we have analyzed allelic losses in chromosome 16 region q in 50 prostate cancer specimens of various histological grades. The most frequently deleted region was located at 16q23-16q24.2 between loci D16S504 and D16S422. The highest percentage of loss of heterozygosity (LOH) at 16q was also found within this area at loci HSD17B2 and D16S422 located at 16q24.1-q24.2. The LOH at 16q24.1-q24.2 was significantly associated with clinically aggressive behavior of the disease, metastatic disease, and higher tumor grade. Of the metastatic diseases, 83% showed LOH, whereas only 40% of the nonmetastatic diseases were found to show it. Similarly, LOH was found in 76% of the clinically aggressive diseases and in 33% of the nonaggressive diseases. The data suggest that a potentially important gene associated with prostate cancer progression is located close to 16q24.1-q24.2.

Hormone regulation of human prostate in organ culture.

We have established organ cultures of human prostate for in vitro analysis of the hormone responsiveness of prostatic carcinoma. Tissue samples were obtained from total prostatectomies for localized cancer. Normal prostate tissues with age-related hyperplastic changes were obtained from cystoprostatectomies of bladder cancer patients representing the same age group, and they wer cultivated as controls. The explants of prostates were cultured for 7 days in basal medium containing 5% dextran charcoal-treated fetal calf serum, insulin (0.08 IU/ml), and dexamethasone (10(-7) M) with or without dihydrotestosterone (DHT) (10(-7) M) or estradiol (10(-9) M). Control prostates showed involutive changes of morphology when cultured in basal medium. These changes were prevented by DHT, which also maintained a strong epithelial immunostaining for PSA (prostate specific antigen), which was used as a marker for tissue-specific functions. The concentration of PSA in the medium was high. The rate of [3H]thymidine incorporation into DNA was stimulated by DHT in some cultures of control prostates, but no increase was seen in the others. Androgen stimulation of [3H]thymidine incorporation was consistently inhibited by the antihormone cyproterone acetate. The main morphological response of cultured control prostates to estradiol was induction of squamous metaplasia. This was associated with increased incorporation of [3H]thymidine, which was radioautographically localized to the basal layer of epithelium. Estradiol effects were counteracted by the antihormone toremifene. The expression of androgen receptor mRNA and protein in cultured control prostate was demonstrated by Northern blotting and immunohistochemistry, respectively. Also, the expression of estrogen receptor was demonstrated by the polymerase chain reaction analysis of total mRNA from cultured control and cancer prostate. The cultured explants of prostate cancer maintained the overall morphology of the original carcinoma. However, the presence of DHT improved the morphology of cancerous acini in all better differentiated carcinomas (3 grade I and 5 grade II), and corresponding responses to DHT were observed in the rate of DNA labeling with [3H]thymidine. In 2 of 3 grade I carcinomas, DHT increased DNA synthesis, but in grade II cancers the patterns of hormone responses were more variable. The poorly differentiated grade III prostatic carcinomas did not respond to either hormone as measured by [3H]thymidine uptake, and no hormone effects could be seen in morphology. Immunostaining for PSA differed from that in control prostates: besides cancerous acini, the surrounding stroma was also intensively stained, which suggests unpolarized and impaired secretion of PSA by the cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhanced invasion and tumor growth of fibroblast growth factor 8b-overexpressing MCF-7 human breast cancer cells.

Fibroblast growth factor 8 (FGF-8) is a secreted heparin-binding protein, which has mitogenic and transforming activity. Increased expression of FGF-8 has been found in human breast cancer, and it has a potential autocrine role in its progression. Human FGF-8 is alternatively spliced to generate four protein isoforms (a, b, e, and f). Isoform b has been shown to be the most transforming. In this work, we studied the role of FGF-8b in the growth (in vitro and in vivo) of MCF-7 human breast cancer cells, which proliferate in an estrogen-dependent manner. Constitutive overexpression of FGF-8b in MCF-7 cells down-regulated FGF-8b-binding receptors FGF receptor (FGFR) 1IIIc, FGFR2IIIc, and FGFR4 found to be expressed in these cells. FGF-8b overexpression led to an increase in the anchorage-independent proliferation rate in suspension culture and colony formation in soft agar, when MCF-7 cells were cultured with or without estradiol. FGF-8b also provided an additional growth advantage for cells stimulated with estradiol. In addition, FGF-8b-transfected cells invaded more actively through Matrigel than did control cells. This was possibly due to the increased secretion of matrix metalloproteinase 9. In vivo, FGF-8b-transfected MCF-7 cells formed faster growing tumors than vector-only-transfected cells when xenografted into nude mice. The tumors formed by FGF-8b-transfected cells were more vascular than the tumors formed by vector-only-transfected cells. In conclusion, FGF-8b expression confers a growth advantage to MCF-7 breast carcinoma cells, both in vitro and in vivo. In addition to stimulation of proliferation, this growth advantage probably arises from increased invasion and tumor vascularization induced by FGF-8b. The results suggest that FGF-8b signaling may be an important factor in the regulation of tumorigenesis and progression of human breast cancer.

FGF-8b increases angiogenic capacity and tumor growth of androgen-regulated S115 breast cancer cells.

Fibroblast growth factor 8 (FGF-8) is a secreted heparin-binding protein, which has transforming potential. Alternative splicing of the mouse Fgf-8 gene potentially codes for eight protein isoforms (a-h) which differ in their transforming capacity in transfected cells. S115 mouse mammary tumor cells express a transformed phenotype and secrete FGF-8 in an androgen-dependent manner. In order to study the role of FGF-8 isoforms in the induction of transformed phenotype of breast cancer cells, we over-expressed FGF-8 isoforms a, b and e in S115 cells. Over-expression of FGF-8b, but not FGF-8a or FGF-8e, induced androgen and anchorage independent growth of S115 cells. FGF-8b-transfected S115 cells formed rapidly growing tumors with increased vascularization when injected s.c. into nude mice. FGF-8a also slightly increased tumor growth and probably tumor vascularization but FGF-8e was not found to have any effects. The angiogenic activity of FGF-8b and heparin-binding growth factor fraction (HBGF) of S115 cell conditioned media was tested in in vitro and in vivo models for angiogenesis using immortomouse brain capillary endothelial cells (IBEC) and chorion allantoic membrane (CAM) assays. Recombinant FGF-8b protein was able to stimulate proliferation, migration, and vessel-like tube formation of IBECs. In addition, stimulatory effect of S115-HBGF on IBE cell proliferation was evident. A positive angiogenic response to FGF-8b was also seen in CAM assay. The results demonstrate that the expression of Fgf-8b is able to promote vessel formation. Angiogenic capacity probably markedly contributes to the ability of FGF-8b to increase tumor growth of androgen-regulated S115 mouse breast cancer cells.

Enzymes as modulators in malignant transformation.

Experimental data suggest that sex steroids have a role in the development of breast and prostate cancers. The biological activity of sex steroid hormones in target tissues is regulated by several enzymes, including 17beta-hydroxysteroid dehydrogenases (17HSD). Changes in the expression patterns of these enzymes may significantly modulate the intracellular steroid content and play a pathophysiological role in malignant transformation. To further clarify the role of 17HSDs in breast cancer, we analyzed the mRNA expressions of the 17HSD type 1, 2, and 5 enzymes in 794 breast carcinoma specimens. Both 17HSD type 1 and 2 mRNAs were detected in normal breast tissue from premenopausal women but not in specimens from postmenopausal women. Of the breast cancer specimens, 16% showed signals for 17HSD type 1 mRNA, 25% for type 2, and 65% for type 5. No association between the 17HSD type 1, 2, and 5 expressions was detected. The patients with tumors expressing 17HSD type 1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients. The expression of 17HSD type 5 was significantly higher in breast tumor specimens than in normal tissue. The group with 17HSD type 5 overexpression had a worse prognosis than the other patients. Cox multivariate analyses showed that 17HSD type 1 mRNA, tumor size, and ERalpha had independent prognostic significance. Using an LNCaP prostate cancer cell line, we developed a cell model to study the progression of prostate cancer. In this model, androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in oxidative 17HSD activity was seen, whereas reductive activity seemed to increase. Since local steroid metabolism controls the bioavailability of active steroid hormones of target tissues, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of organs.

Differential regulation of carbonic anhydrase II by androgen and estrogen in dorsal and lateral prostate of the rat.

Hormone regulation of carbonic anhydrase II (CA II) was studied in rat dorsal and lateral prostate. CA II is a major soluble protein in these accessory sex glands. The immunoelectronmicroscopy showed that CA II is expressed in their epithelial cells only. For studies on hormone regulation, adult male rats were castrated for 2 or 7 days. Groups of 7-day castrates and normal rats were treated daily either with testosterone or 17-beta-estradiol for 6 days and 2-day castrates for 1 day. CA II protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified by RIA. The levels of CA II mRNA were studied by Northern blotting and hybridization of total RNA with a 32P-labeled mouse CA II cDNA clone. Castration of the rats decreased the concentration of CA II in lateral prostate but increased in dorsal prostate. These changes were reversed in both prostatic lobes by testosterone treatment. Estrogen treatment of castrated rats enhanced CA II concentration in lateral prostate but no effects were seen in the dorsal prostate of the same animals. In normal rats estrogen increased CA II concentration of dorsal prostate but there was no change in lateral prostate. Corresponding changes were observed in the levels of CA II mRNA in both tissues. The morphometric analyses showed that the castration- and hormone-induced changes of the mRNA and protein levels of the exclusively epithelial CA II could not be explained by any alterations in the proportions of epithelial and stromal components of the glands after hormone manipulations. The results demonstrate the differential steroid regulation of CAII in two prostatic lobes. Androgen regulates the expression of CAII at messenger RNA level, but the responses of CAII to testosterone are opposite in dorsal and lateral prostate. Estrogen increases CA II expression in lateral prostate but in dorsal prostate the castration-like effects of estrogen on CAII expression are probably indirect.

Androgen and fibroblast growth factor (FGF) regulation of FGF receptors in S115 mouse mammary tumor cells.

We studied the androgen regulation of fibroblast growth factor (FGF) receptors (FGFRs) in the Shionogi 115 (S115) mouse mammary tumor cell line and its genetic variant Clone 22. In S115 cells, androgen maintains a transformed morphology, rate of proliferation, and serum and anchorage independence. Similar effects were induced by treatment of the cells with FGF-2 or a heparin-binding growth factor (HBGF) fraction prepared from the medium conditioned by the cells. The effects of androgen and FGF-2 could be partly reversed with a specific anti-FGF-2 immunoglobulin G or by suramin, which inhibits binding of FGFs to their high affinity receptors. Testosterone and FGF-2 increased the expression of FGFR-1 messenger RNA (mRNA) and, to a lesser extent, FGFR-3 mRNA, but down-regulated FGFR-2 mRNA in S115 cells. No FGFR-4 mRNA was detected. FGF-2 also down-regulated the expression of syndecan-1, a heparan sulfate proteoglycan that binds FGF with low affinity. The binding of radiolabeled FGF-2 to FGFRs was lower in the cells cultured with testosterone or in the presence of the HBGFs from androgen-treated cells, presumably because of the autocrine production of FGF-like factors. In Clone 22 cells, FGFRs and syndecan-1 responded to androgen as in S115 cells, but they were less sensitive to FGF-2. Androgen or FGF-2 could not induce morphological transformation, although both stimulated proliferation. Androgen-increased proliferation was not, however, decreased by anti-FGF-2 immunoglobulin G in Clone 22 cells. These data suggest that of the HBGFs produced, FGF-2 is required in androgen induction of morphological change, whereas the effect on proliferation involves other factors as well (perhaps mostly FGF-8). The results show that androgen differentially regulates the expression of the high and low affinity FGF receptors, which could mediate androgen induction of the transformed phenotype in S115 cells by an autocrine mechanism. The differential responses of the Clone 22 variant cells to androgen and FGF-2 suggest that the pathways of steroid induction of different parameters of the transformed phenotype, such as transition to fibroblastic morphology and stimulation of proliferation, are divergent.

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.

Estrogen reduces the depth of resorption pits by disturbing the organic bone matrix degradation activity of mature osteoclasts.

Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-terminal fragment, the C-terminal cross-linked peptide of type I collagen. E2 significantly inhibited the release of the C-terminal cross-linked peptide of type I collagen into the culture medium without affecting the release of cross-linked carboxyl-terminal telopeptide of type I collagen in osteoclast cultures. These results suggest that organic matrix degradation is initiated by MMPs and continued by cysteine proteases; the latter event is regulated by E2.

Prolactin is a survival factor for androgen-deprived rat dorsal and lateral prostate epithelium in organ culture.

PRL is one of several polypeptide factors that regulate growth and differentiation of prostate epithelium besides steroid hormones. This hormone may also participate in the development of pathologic changes of the prostate, as evidenced by marked prostate hyperplasia in hyperprolactinemic mice. We have previously demonstrated expression of PRL receptors and androgen-dependent local production of PRL in rat and human prostate epithelium, suggesting the existence of an autocrine loop. We now show that PRL acts as a survival factor for epithelial cells of rat dorsal and lateral prostate but not ventral prostate, using long-term organ cultures as an in vitro model. Culture of prostate explants in androgen-free medium was associated with a transient surge of apoptosis during the first 2-4 days of culture in rat ventral, dorsal, and lateral prostate tissues, as quantified by either nuclear morphology or in situ DNA fragmentation analysis. PRL significantly inhibited apoptosis in androgen-deprived dorsal and lateral prostate cultures, by 40-60%, as determined by the two methods. The present study has established conditions and methodology for analysis of apoptosis in organ cultures of rat prostate and suggests a physiological role for PRL as a survival factor for prostate epithelium.

Multihormonal control of synthesis and secretion of prostatein in cultured rat ventral prostate.

The synthesis and accumulation of prostatein, a major secretory protein of the rat ventral prostate, was examined in organ culture conditions. For the quantitation of this protein in the medium, a sensitive enzyme immunoassay was developed. The rat ventral prostate could be maintained in organ culture in defined medium for at least 2 weeks. Morphologically the changes in explants cultured without hormones resembled those of castration. These involutive changes could be postponed by testosterone and totally prevented by a combination of testosterone, corticosterone, and insulin in the culture medium. Newly synthesized prostatein, studied by fluorography of [35S] methionine-labeled proteins, accumulated only in the presence of testosterone. Its synthesis also took place in cultured prostate derived from castrated rats. Neither corticosterone nor insulin alone could sustain prostatein synthesis. Insulin increased the testosterone-dependent prostatein synthesis in the beginning of culture, but later, inhibition, rather than stimulation, could be noted. Corticosterone increased the testosterone-dependent synthesis of prostatein throughout the culture. The results show that organ culture of adult rat ventral prostate provides an in vitro model for studies of differentiated prostatic function.