G. Fiorelli

Membrane binding sites and non-genomic effects of estrogen in cultured human preosteoclastic cells

Besides functional estrogen receptors, the presence of signalling cell surface binding sites for 17beta-estradiol (17betaE2) has been reported in osteoblast- and osteoclast-like cells, suggesting that 17betaE2 may influence bone remodelling by a dual mechanism of action: to affect gene expression mediated by the nuclear activity of the steroid-receptor complex, and to initiate rapid responses triggered by a signal-generating receptor on the cell surface. Recently, we demonstrated that the human pre-osteoclastic cell line FLG 29.1 bears functional estrogen receptors. In this study we examined FLG 29.1 cells for the presence of cell surface binding sites for 17betaE2, and whether 17betaE2 could elicit cell signalling. Using a cell-impermeant and fluorescent estrogen conjugate, 17beta-estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate, we demonstrated the presence of specific plasma membrane binding sites for 17betaE2. Stimulation of FLG 29.1 cells with low (1 nM) and high (1 microM) doses of 17betaE2 induced a prompt and significant (P < 0.05) increase of cellular pH, as measured in single cells using an image analysis system. In addition, both cAMP and cGMP were significantly increased by 17betaE2 with a dose-dependent response. Finally, a rapid increase of intracellular calcium ion concentration [Ca2+] was also induced by 1 nM 17betaE2, as measured in single cells using an image analysis system. Our findings strongly suggest a non-genomic action of 17betaE2 on osteoclast precursors.

Characterization and function of the receptor for IGF-I in human preosteoclastic cells

Using a coculture system, we have recently demonstrated that insulin-like growth factor I (IGF-I) is a mediator of preosteoclastic cell migration toward bone-derived endothelial cells. To better characterize the mechanisms of IGF-I action on preosteoclastic cells we evaluated the expression of type I IGFs receptor in the human leukemic cell line, FLG 29.1, which differentiates toward the osteoclastic phenotype following phorbol ester (TPA) treatment. Scatchard analysis of 125I-labeled IGF-I to FLG 29.1 cells revealed the presence of a single high affinity binding site in both untreated and TPA-treated cells with a similar Kd value (0.3 +/- 0.2 nmol/L and 0.4 +/- 0.1 nmol/L, respectively). In untreated cells, IGF-I binding capacity (1.43 +/- 0.41 fmol/10(6) cells) was significantly (p < 0.05) lower than in TPA-treated cells (2.62 +/- 0.87 fmol/10(6) cells). Competition analyses and crosslinking studies revealed the presence of type I IGF receptor both in untreated and TPA-treated cells. Northern analysis demonstrated that mRNA for IGF-I receptor was expressed by both untreated and TPA-treated FLG 29.1 cells. In addition, FLG 29.1 cells released in the conditioned medium IGFBP-2 and IGFBP-4, whose expression was increased by TPA treatment as demonstrated by ligand and immunoblot analyses. The previous observations of chemotactic action of IGF-I on FLG 29.1 cells was confirmed by ultrastructural observations. Indeed, these cells revealed a marked migratory activity in response to nanomolar concentrations of IGF-I. In addition, the IGF-I receptor alpha IR-3 antiserum inhibited the IGF-I-induced FLG 29.1 cells migratory activity. These findings clearly show that type IIGF receptor is expressed by osteoclast precursors and that IGF-I induces migration of these through the binding to type I IGF receptors. Binding proteins expressed by osteoclast precursors may play an autocrine role in modulating the IGF-I bioeffects.

Age-related secretion of androstenedione, testosterone and dihydrotestosterone by the human testis

To study the production of testosterone, dihydrotestosterone (DHT), and androstenedione by the human testis during advancing age, these substances were measured in the spermatic venous blood plasma of 38 17-80 year old men. Samples of blood from the spermatic veins were collected during the operative repair of inguinal hernias. Plasma concentrations were determined by radioimmunoassay after paper chromatography. Control experiments were done with added known amounts of these substances to steroid free plasma. It was found that in old age the testicular production of DHT decreases significantly as well as its concentration in peripheral venous plasma. Spermatic androstenedione is unchanged while testosterone is decreased in senesence. This finding suggests that the decreased Leydig cell function in old age may be partly due to an enzymatic defect in the testicular steroidogenesis pathway because androstenedione is a direct precursor of testosterone.

Estrogen synthesis in human colon cancer epithelial cells.

Epidemiological and experimental data suggest an involvement of estrogen in the development and progression of colorectal cancer. In order to determine whether local synthesis of estrogen occurred in human colonic cancer cells, two colorectal cancer cell lines, HCT8 and HCT116, were evaluated for gene expression and enzyme activity of cytochrome P450 aromatase. In addition, the effect on aromatase expression of charcoal-stripped fetal calf serum, of quercetin and genistein and of tamoxifen and raloxifene was investigated in both cell lines. RT-PCR analysis revealed that colorectal adenocarcinoma cell lines contain aromatase as a major component. The conversion of [(3)H]-androstenedione to estrone and labeled water was dose-dependently inhibited by 4-hydroxyandrostenedione and obeyed Michaelis-Menten kinetic with apparent Km values of approximately 20 nM and V(max) values of approx. 200 and 500 fmol/mg protein/h for HCT8 and HCT116 cells, respectively. After 24 h incubation, genistein (1 microM) significantly increased aromatase activity in HCT8 cells, with no effect on HCT116 cells. In accord with previous observation in reproductive tissues, quercetin (1 microM) significantly inhibited the enzyme activity in both cell lines. Also tamoxifen (100 nM) acted as inhibitor, while raloxifene (10 nM) decreased the enzyme activity only in HCT116 cells. The aromatase gene expression modulation by these effective agents was consistent with their effects on enzyme activity. These findings demonstrate for the first time that colorectal adenocarcinoma cell lines express aromatase. Interestingly, the enzyme activity was inhibited by quercetin, one major dietary flavonoid, by tamoxifen, a hormonal therapeutic agent for breast cancer, and by raloxifene, used in the prevention of postmenopausal osteoporosis.

Growth factors in the human prostate.

Recent studies have focused on the potential role of local polypeptide growth-regulating factors in the etiology of benign prostatic hyperplasia (BPH) and prostatic carcinoma. In our studies we confirmed the presence of specific receptors for epidermal growth factor (EGF) in prostatic tissues from patients affected by BPH. In addition, we demonstrated that specific receptors for insulin-like growth factor type I (IGF-I) are present in BPH tissues. In order to identify a possible interaction between androgens and these growth-regulating factors, we investigated the effect of testicular suppression-induced androgen withdrawal on both EGF and IGF-I receptor concentrations in prostatic tissue from patients affected by BPH treated with a long-acting luteinizing hormone-releasing hormone analog. Both EGF and IGF-I binding capacities were significantly increased after treatment. This finding suggests that in vivo IGF-I and EGF receptor levels may be under negative androgenic regulation, indicating a potential role for these growth-regulating factors in the mechanism of response to the castration-induced regression of androgen-dependent prostatic tissue. Moreover, preliminary studies indicate that in human BPH prostatic tissue multiple IGF-binding proteins (IGF-BP) are present. This finding suggests a possible role of IGF-BP in modulating IGFs biological activities at the prostate level.

Determination of plasma corticosteroids and urinary cortisol by a competitive protein-binding method using dextran-coated charcoal

Abstract Corticosteroid-binding globulin and dextran-coated charcoal have been used to measure the corticosteroids in plasma and the free cortisol in urine. 0.5 ml of plasma or 1.0 ml of urine was extracted with methylene chloride and the extract washed with sodium hydroxide and water. The extract was dried, dissolved in a standard plasma pool containing [1,2- 3 H]11-deoxycortisol. Dextran-coated charcoal was employed to separate protein-bound and unbound corticosteroid. The method has a high degree of precision and accuracy. The specificity was good in plasma; in urine the values of CBG-charcoal method were higher than the values found by a competitive binding method after purification of extracts with paper chromatography.

Simultaneous determination of androstenedione testosterone and 5α-dihydrotestosterone in human spermatic and peripheral venous plasma

Androstenedione (Δ), Testosterone (T) and 5α-Dihydrotestosterone (DHT) concentrations in spermatic and peripheral venous blood obtained from twenty four men have been measured by radioimmunoassay. The concentrations (mean ± S.D.) of 5α-dihydrotestosterone in the spermatic vein (0.42 ± 0–33 μg/100ml) were higher than those found in the cubital vein (0.030 ± 0.012 μg/100ml). The blood plasma levels of 5α-dihydrotestosterone correlated significantly with testosterone levels in spermatic (P < 0.05) as well as in peripheral venous blood plasma (P < 0.01); on the other hand, 5α-dihydrotestosterone did not correlate with androstenedione. The results confirm the production of 5α-dihydrotestosterone by the human testis. Since there is a significant correlation between testosterone and 5α-dihydrotestosterone both in peripheral and in spermatic venous blood and an insignificant correlation between 5α-dihydrotestosterone and androstenedione, testosterone has been confirmed to be the most important precursor of 5α-dihydrotestosterone in the testis as well as in the other androgen target tissues.

Interactions between ipriflavone and the estrogen receptor

Estrogen replacement therapy is effective in the prevention of postmenopausal osteoporosis, and a direct action of 17-β-estradiol (17βE2) on osteoblastic and osteoclastic cells has been demonstrated. The inhibition of bone resorption by ipriflavone (IP), an isoflavone derivative devoid of estrogenic properties but active in potentiating the effects of estroge on bone tissue, has been shown in in vitro and in vivo studies and confirmed by clinical data. To investigate the molecular mechanisms that underlie IP effect, we studied the possible interactions of IP and its four main in vivo metabolites (I, II, III, and V) with the estrogen receptor (ER) in the human preosteoclastic cell line FLG 29.1, whose growth and function are modulated by the compound. In parallel experiments, the human breast cancer cell line MCF7 was also analyzed. IP binding sites were demonstrated in the nuclear fraction of FLG 29.1 cells. 17βE2 and other steroid compounds failed to displace IP binding to intact FLG 29.1 cells. Similarly, IP and metabolites I, III, and V were not able to displace 17βE2 binding to intact MCF7 cells, whereas metabolite II showed an IC50 of 61 nM. 17βE2 binding to FLG 29.1 cells was increased after preincubation with metabolites I, III, and V. IP and its metabolites did not induce FR-dependent gene expression in FLG 29.1 and MCF7 cells transfected with a reporter gene and an estrogen response element (ERE). These results suggest that IP effects on osteoclast precursors are not mediated by a direct interaction with the ER, even if a crosstalk between the mechanisms of action of IP and 17βE2 cannot be excluded.

Human testicular secretion with increasing age.

Δ4 and Δ5 androgens were measured in spermatic venous blood of 25 subjects (age range 20–70) during surgical intervention for hernia repair. All androgens measured decreased significantly with age. However the androstenedione to testosterone ratio in spermatic venous blood of men aged between 60–70 was higher than that of subjects aged between 20–40. 17β-Oestradiol levels were unchanged in spermatic venous blood of the same subjects. These results seem to demonstrate that there is a general decrease with age of androgen secretion by the human testis and the increase of oestradiol in systemic blood in senescence is due to an increase in peripheral conversion of aromatizable androgens.

RADIOIMMUNOASSAY OF PLASMA TESTOSTERONE

A radioimmunoassay for the determination of testosterone in human plasma is described. After extraction from plasma, testosterone is separated by paper chromatography. The radioimmunoassay is performed using an antiserum to testosterone‐3‐oxime‐rabbit serum albumin and a saturated solution of ammonium sulphate is used for separation. The reliability criteria of the method in terms of precision, accuracy, sensitivity and specificity have been evaluated.