Regular articleThe effects of estrogen on osteoprotegerin, RANKL, and estrogen receptor expression in human osteoblasts
Introduction
It is well established that estrogen has multifunctional roles influencing growth, differentiation, and function in many tissues. It is an important factor in the maintenance of bone health, estrogen deficiency at the time of menopause being associated with bone loss. Administration of conventional doses of hormone replacement therapy prevents menopausal bone loss by reducing bone turnover and inhibiting osteoclast activity [1], whereas high doses of estrogen have been shown to exert anabolic skeletal effects in rodents and postmenopausal women [2], [3], [4], [5]. Estrogens diffuse in and out of cells, but are retained in target cell nuclei by the estrogen receptor protein (ER). The ER is a nuclear hormone receptor and a member of a family of activated transcription factors that can initiate or enhance the transcription of genes. Two ER subtypes have been identified, α and β. They are distinct proteins encoded by separate genes and located on different chromosomes.
RANKL (receptor activator of NF-κB ligand), a membrane-bound molecule, is a newly identified member of the tumor necrosis factor (TNF) ligand family and has been shown to be crucial for osteoclast formation [6]. Two receptors for RANKL have been identified, RANK, a membrane-bound signaling receptor expressed on the cell surface of osteoclast progenitors, and osteoprotegerin (OPG), a secreted cytokine receptor. OPG, a member of the tumor necrosis factor receptor (TNF-R) superfamily [8], [9] acts as a decoy receptor by blocking the interaction of RANKL with its functional receptor RANK [10], thereby inhibiting osteoclastogenesis.
In vitro studies have demonstrated the synthesis of OPG by stromal cell lines [11] and osteoblasts [12] and its stimulation by estrogen [13]. Udagawa et al. [14] reported that osteoblasts constitutively express RANKL mRNA, the bone resorbing capacity of osteoclasts being enhanced when cocultured with osteoblasts.
The aim of this study was to investigate the relative effects of estrogen on ER, OPG, and RANKL mRNA and protein expression in human osteoblasts.
Section snippets
Cell culture and immunolocalization
Primary human osteoblasts from two young female donors (1 day and 1 year) were supplied by Clontech Laboratories (Basingstoke, UK), with each grown to confluence and expanded to yield cells for three replica experiments. Cells were seeded into eight-well chamber slides (NUNC) at 104 cells/well in McCoys 5A medium supplemented with 10% heat-inactivated FBS (Life Technologies), penicillin/streptomycin (Life Technologies), and ascorbic acid (100 mM, Wako, Alpha Labs, Eastleigh, UK). Cultures were
Protein expression
The result for untreated cells in each group was given a value of 1, and data for treated cells in each group were expressed as a relative ratio. OPG protein expression measured at 24 h demonstrated a significant 3-fold increase with low-dose E2 compared to untreated cells (3.00 ± 1.05, P < 0.05) and a 7-fold increase with high-dose E2 (6.9 ± 1.80, P < 0.01). At 48 h there was a more modest but still significant increase in OPG expression in the estrogen-treated cells (low dose, 1.2 ± 0.09,
Discussion
This in vitro study demonstrated that estrogen elicits changes in cellular protein and mRNA expression in human osteoblastic cells with OPG and ER protein expression significantly increased. The estrogen-induced changes in ER expression and up-regulation of the decoy receptor OPG suggest that osteoblasts may be involved in the mediation of the estrogen-induced anabolic skeletal effects in bone. We and other groups have demonstrated the presence of ERs in osteoblasts [16], [17], [18] and Udagawa
Acknowledgements
This work was funded by the Wellcome Trust.
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