U. Frediani

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.

Bone endothelial cells as estrogen targets

SummaryIn the present study, we investigated the effects of estrogens on bone endothelial cell metabolism and the presence of estrogen binding sites in the same cells. For these studies, we have used a continuous cell line of clonal bovine bone endothelial cells for evidence of a direct response to estrogensin vitro. Receptor analysis to intact viable cells was steroid specific and saturable, with an apparent dissociation constant of 17.2 nM and a Bmax of 3.2 × 104 sites/cell. Northern blot analysis revealed a 6.5-kilobase mRNA that hybridized with a cDNA to human estrogen receptor. The 6.5-kilobase size is in close agreement with the reported size of the human estrogen receptor mRNA.In vitro estrogen responses of bone endothelial cells included a stimulation of cell proliferation as well as an inhibition of parathyroid hormone responsiveness. These findings clearly demonstrate the presence of functional estrogen receptors in bone endothelial cellsin vitro, suggesting a role of estrogens in bone angiogenesis and in the entire process of bone remodeling.

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.

Catecholamines modulate growth and differentiation of human preosteoclastic cells

Using a clonal cell line of human osteoclast precursors (FLG 29.1 cells), that after treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) show many functional characteristics of osteoclasts, we demonstrated that catecholamines act as inducers of osteoclast maturation in vitro and as stimulators of osteoclast activity via the binding to β2 adrenergic receptors. Scatchard analysis of125I-labelled iodocyano-pindolol to untreated (undifferentiated) or TPA-treated (differentiated) FLG 29.1 cells revealed the presence of a single high-affinity site with aKd value around 24 pM and 8 pM respectively and with superimposable binding capacity (1.18 fmol/mg protein). Catecholamines increased in a dose-dependent fashion the intracellular cyclic AMP (cAMP) accumulation in both undifferentiated and TPA-treated FLG 29.1 cells. Pretreatment of untreated and TPA-treated FLG 29.1 cells with propranolol inhibited the catecholamine effect on cAMP accumulation, while pretreatment with clonidine had no effect. Catecholamines also reduced cell proliferation, increased tartrate-resistant acid phosphatase (TRAcP) activity, interleukin 6 (IL-6) production, multinuclearity and response to salmon calcitonin (sCT) in undifferentiated FLG 29.1 cells. In differentiated FLG 29.1 cells only IL-6 release was induced by catecholamine treatment. These findings support a potential role for catecholamines in modulating osteoclast differentiation and mature osteoclast activity.

Aromatase expression and activity in the human leukaemic cell line FLG 29.1

The recent observation that estrogen synthesis occurs in osteoblast-like cells has suggested the aromatase activity as a possible local modulator of bone remodeling in post-menopausal women. To provide further insights into the androstenedione conversion to estrogen in bone-derived cells, we examined the human leukaemic cell line FLG 29.1, which is induced to differentiate toward the osteoclastic phenotype by TPA and TGF-beta1. Southern blot of RT-PCR products with a 32P-labeled cDNA probe for the human aromatase demonstrated that FLG 29.1 cells express aromatase mRNA. The enzyme activity, determined by measuring [3H]H2O release from [3H]androstenedione, obeyed Michaelis-Menten kinetic with apparent Km and Vmax values ranging from 5 to 10 nM and from 200 to 400 fmol/mg protein/6 h. Gene expression, enzyme activity and protein immunoreactivity, evaluated by immunocytochemistry, were stimulated in a time-dependent fashion by 5% charcoal-stripped FCS and by either 1-100 nM TPA or 0.01-0.5 ng/ml TGF-beta1, with maximal responses after 2-3 h exposure. After 24 h incubation of FLG 29.1 cells in the absence of these stimuli the aromatase mRNA and the protein were barely detectable. These findings demonstrate that cells of the osteoclastic lineage synthesize estrogen in vitro and that local cytokines, such as TGF-beta1, are able to induce androstenedione conversion.

Histamine receptors and bioeffects on clonal parathyroid endothelial cells

Using a clonal line of bovine parathyroid endothelial cells (BPE-1) we defined the presence on these cells of a histamine H2 receptor and characterized its pharmacological properties. Interaction of histamine with its receptor induced an increase of cAMP accumulation in a dose- and time-dependent fashion. This effect appears unique for parathyroid endothelial cells, in fact, clonal parathyroid epithelial cells did not exhibit a similar response. No effect of histamine was observed on BPE-1 cell proliferation.