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-β1. 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-β1, 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-β1, are able to induce androstenedione conversion.

The major in vivo effect of 17 beta-estradiol (17 beta E-2) on bone is the maintenance of bone mass by decreasing bone resorption, However, whether 17 beta E-2 affects osteoclast differentiation and/or function directly or through its action on osteoblasts is unclear. To investigate this question we examined a human pre-osteoclastic cell line (FLG 29.1) for evidence of functional estrogen receptors (ER), Binding of [H-3] 17 beta E-2 to nuclear ER was steroid-specific with approximately 400 saturable, high affinity (Kd similar to 1 nhl) binding sites per cell nucleus, Pretreatment with 17 beta E-2 induced Specific binding of progesterone to FGL 29.1 cells, In addition, stimulation of these cells with 10 nM and 100 nM of 17 beta E-2 significantly (p<0.005) reduced cell proliferation, These observations clearly indicate the presence of functional estrogen receptors in osteoclast precursors, Moreover using a cell-impermeant and fluorescent estrogen conjugate, 17 beta-estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate, we demonstrated the presence of specific plasma membrane binding sites for 17 beta E-2. Stimulation of FLG 29.1 with 17 beta E-2 induced different rapid responses, thus suggesting a nongenomic action of 17 beta E-2 on osteoclast precursors. LY 139478 is a member of the non-steroidal antiestrogens, described to be full estrogen agonists in bone acting via an estrogen receptor mediated mechanism, To investigate the in vitro properties of LY 139478 on osteoclast precursors, the human pre-osteoclastic cell line FLG 29.1 was examined for evidence of bioeffects of this compound, We demonstrated that its mechanism of action is mediated through the binding to the estrogen receptor.

Besides functional estrogen receptors, the presence of signalling cell surface binding sites for 17β-estradiol (17βE2) has been reported in osteoblast- and osteoclast-like cells, suggesting that 17βE2 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 preosteoclastic 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 17βE2, and whether 17βE2 could elicit cell signalling. Using a cell-impermeant and fluorescent estrogen conjugate, 17β-estradiol-6-carboxy-methyloxime-bovine serum albumin-fluorescein isothiocyanate, we demonstrated the presence of specific plasma membrane binding sites for 17βE2. Stimulation of FLG 29.1 cells with low (1 nM) and high (1 μM) doses of 17βE2 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 17βE2 with a dose-dependent response. Finally, a rapid increase of intracellular calcium ion concentration [Ca2+] was also induced by 1 nM 17βE2, as measured in single cells using an image analysis system. Our findings strongly suggest a non-genomic action of 17βE2 on osteoclast precursors.

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 aK d 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.

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.

LY 139478, the hydrochloride salt of LY 117018, is a member of the nonsteroidal antiestrogens, benzothiophene derivatives, described to be full estrogen agonists in bone acting via an estrogen receptor-mediated mechanism. However, the cellular actions of these compounds on bone remodelling need to be established. To investigate the "in vitro" properties of LY 139478 on osteoclast precursors, the human pre-osteoclastic cell line FLG 29.1 was examined for evidence of bioeffects of this compound. Binding studies with tritiated 17β-estradiol (17βE2) demonstrated that the relative potency of LY 139478 in inhibiting estrogen binding to its receptor was equal to that of 17βE2.Significant (p<0.05) dose-dependent inhibition of cell growth was induced by LY 139478 at 10 nM, 100 nM and 1 μM. Calcitonin-induced cAMP accumicrolation was significantly increased by low (1 pM) and high (1 μM) doses of both 17βE2 and the compound with a dose-dependent response. Differently than estrogen, LY 139478 at high dose significantly reduced IL-6 release by these cells. In addition, pharmacological doses of both 17βE2 and LY 139478 activated apoptotic cell death. These findings show that the benzothiophene-derived LY 139478 acts directly on the human pre-osteoclastic cell line FLG 29.1 as an estrogen agonist.

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.

Increasing evidence suggests that transforming growth factor‐β (TGF‐β) is involved in bone formation during remodeling. Using a recently cloned human leukemic cell line (FLG 29.1 cells) we demonstrate that these cells synthesize and secrete TGF‐β1 and that exogenous or autocrine TGF‐β1 can induce the same features of osteoclastic‐like cells, exerting its effects through the binding to TGF‐β specific receptors. Scatchard analysis of 125I‐labeled TGF‐β1 to FLG 29.1 cells revealed the presence of a single high affinity binding site with a Kd value of ∼25 pM and a binding capacity of ∼900 sites/cell. Affinity labeling experiments showed that FLG 29.1 cells express type I and type II TGF‐β receptors. Stimulation of FLG 29.1 cells with low TGF‐β1 doses reduced cell proliferation and increased cell adhesion and tartrate resistant acid phosphatase (TRAcP) activity. Pretreatment of FLG 29.1 cells with TGF‐β1 caused a significant and dose‐dependent response to calcitonin. Northern blot of total mRNA and analysis of the conditioned media (CM) showed that TGF‐β1 was synthesized by FLG 29.1 cells. TPA treatment, which induces partial differentiation of these cells, markedly increased TGF‐β1 mRNA expression and growth factor release. The majority of TGF‐β1 secreted by TPA‐treated cells was in its latent form. However, anti‐TGF‐β antibodies inhibited TGF‐β1 and TPA‐induced growth inhibition, calcitonin responsiveness, and TRAcP activity, suggesting that the TPA effect is mediated in part by autocrine TGF‐β1 and indicating that the cells can activate and respond to the TGF‐β that they secrete. These findings support a potential autocrine role for TGF‐β1 in osteoclast differentiation. © 1994 Wiley‐Liss, Inc.

Ipriflavone, a synthetic isoflavone derivative, reduces bone resorption by inhibiting osteoclasts activity. In order to evaluate the role of Ipriflavone on osteoclast growth and differentiation, we tested Ipriflavone and its four "in vivo" main metabolites (Metabolites I, II, III, and V) on a clonal population of human osteoclast precursor cells (FLG 29.1). Pharmacological doses of Ipriflavone and Metabolite III were able to inhibit cell proliferation and interleukin 6 release. In co-cultures of FLG 29.1 cells and osteoblastic (Saos-2) cells Ipriflavone at 1 μM dose inhibited the adhesion of FLG 29.1 cells to the osteoblastic monolayer and reduced the immunocytochemical reaction of the vitronectin receptor. Binding studies with tritiated Ipriflavone showed the presence of a single specific binding site, with a Kd of about 70 nM and a binding capacity of 8 fmol/106 cells. These results demonstrate a direct effect of Ipriflavone and of Metabolite III on the human osteoclast precursor cell line FLG 29.1.

[125I]Atrial natriuretic peptide (ANP) was used to identify ANP receptors on a clonal line of bovine bone endothelial (BBE) cells. Specific binding of [125I]ANP was saturable and of high affinity. Computer analysis of the equilibrium binding data indicated that the Scatchard plots are best fit by a straight line (Kd = 69.3 +/- 20.9 pM; binding capacity = 37.9 fmol/10(6) cells). The order of potency for competing with [125I]ANP binding was human ANP (hANP) > rat atriopeptin-1 (rAP-1) > porcine brain natriuretic peptide (pBNP) > porcine C-type natriuretic peptide. Affinity cross-linking studies indicated the presence of two major 130- and 70-kilodalton bands that specifically bound to hANP, rAP-1, pBNP, and porcine C-type natriuretic peptide. The binding of natriuretic peptides to BBE cells resulted in an increase in cGMP production and a significant decrease in Na+/K+/Cl- cotransport, without effects on cAMP intracellular accumulation. hANP, rAP-1, and pBNP at 100-nM concentrations, significantly inhibited PTH-induced cAMP production. Treatment with natriuretic hormones was also associated with an increase in 6-keto-prostaglandin F1 alpha levels in the culture medium of BBE cells and a higher cell growth rate. These studies demonstrate that bone endothelial cells bear receptors for natriuretic hormones associated with changes in PTH-induced cAMP production, prostaglandin production, and cell proliferation.

Although many studies have shown the relationship between therapy with high doses of heparin and osteoporosis, the possible role of heparin in the process of bone remodeling is still unknown. In order to evaluate if heparin directly acts on bone tissue, we studied the effects of various doses of calcium heparin on growth and differentiation of human osteoblastic cells in primary culture (PC), the human osteoblastic cell line Saos-2 and clonal bovine bone endothelial (BBE) cells. High doses of calcium heparin inhibited basal levels of alkaline phosphatase and collagen synthesis of Saos-2 cells and of human osteoblastic cells in primary culture. In contrast, calcium heparin did not affect collagen synthesis of BBE cells. Moreover, high doses of calcium heparin enhanced mitogenic effect of acidic Fibroblast Growth Factor (aFGF) on human osteoblastic cells in primary culture, on Saos-2 and BBE cells. The present data show that calcium heparin modulates bone endothelial and osteoblastic cell phenotype in in vitro studies. Further characterization of the effects of heparin on bone-derived cells other than osteoblasts and endothelial cells may explain the mechanism through which heparin influences bone remodeling.

Protective effects of ipriflavone, an isoflavone derivative, in osteoporosis are believed to be caused by the inhibitory action on bone resorption. A direct effect of ipriflavone on bone formation is as yet unknown. Ipriflavone and four of its metabolites (I, II, III, and V) were examined for their effects on parathyroid hormone response, collagen synthesis, alkaline phosphatase activity, and cell proliferation in a clonal cell population of rat osteoblastic cells. Pretreatment of osteoblasts with high concentrations of ipriflavone for 48 h significantly inhibited the cAMP response to parathyroid hormone, producing a shift in the dose-response curve; at lower concentrations metabolites II and III potentiated the cAMP accumulation induced by low doses of parathyroid hormone. The 48 h treatment with metabolite V at the 1 nM dose significantly stimulated collagen synthesis in osteoblastic cells. Ipriflavone and metabolite I showed a biphasic stimulatory action on the alkaline phosphatase activity of osteoblasts, with a maximal effect at the 0.1 and 1 nM doses, respectively. A similar biphasic response was observed with ipriflavone and metabolite I on osteoblastic cell growth, with a maximal effect at the 0.1 nM concentration. These results suggest a direct role of ipriflavone in modulating the synthetic and growth properties of osteoblast-like cells.

Although an autoimmune pathogenesis for non toxic goiter has been suggested, reports concerning circulating antibodies to TSH receptor structures have been conflicting. Intra thyroid lymphocytes, capable of secreting IgG, have been shown to be involved in the pathogenesis of Graves' and Hashimoto's diseases; therefore, the ability of conditioned media obtained from intra thyroid lymphocyte culture, and of IgG purified from these media, to stimulate cAMP accumulation and [3H]-Thymidine (TdR) uptake in FRTL-5 cells was investigated. The activity of IgG produced "in vitro" was compared with that of circulating IgG. Thyroid tissue samples were obtained at surgery from 21 patients with non toxic multinodular goiter (MNG), 5 patients with active Graves' disease (GD), and from 10 normal subjects, undergoing neck surgery for non-thyroidal pathology. IgG purified from media of GD lymphocyte cultures stimulated both cAMP accumulation and [3H]-TdR in 5 out of 5 cases: all of the IgG purified from control or MNG lymphocyte culture media was not active in either assay. Circulating IgG did not affect cAMP accumulation or [3H]-TdR in any of the non toxic MNG cases: controls showed no changed at all. However, both activities represented were increased by GD IgG. Conditioned media from intra thyroid lymphocyte cultures significantly inhibited basal cAMP accumulation in 7 out of the 21 non toxic MNG samples and totally abolished the response in all GD patients. [3H]-TdR was not affected by IgG of any of the controls, but it had an inhibitory effect on 8 out of 21 non toxic MNG patients, and significantly stimulated [3H]-TdR in all GD patients. In conclusion, present data demonstrate that intra thyroid lymphocytes from non toxic MNG do not produce antibodies capable of mimicking TSH actions through the adenylate cyclase cascade. Conversely, soluble factors interacting in TSH-mediated functions of FRTL-5 cells are present in conditioned media of intra thyroid lymphocytes of GD and MNG thyroid lymphocytes of GD and MNG thyroid cultures.