Elsevier

Bone

Volume 32, Issue 2, February 2003, Pages 136-141
Bone

Regular article
The effects of estrogen on osteoprotegerin, RANKL, and estrogen receptor expression in human osteoblasts

https://doi.org/10.1016/S8756-3282(02)00953-5Get rights and content

Abstract

Estrogen is essential for bone growth and development and for the maintenance of bone health in adulthood. The cellular responses of osteoblasts and osteoclasts to estrogen are initiated via two high-affinity receptors (ERs). Osteoblasts synthesize RANKL (receptor activator of NF-κB ligand), necessary for osteoclast formation and function, and osteoprotegerin (OPG), its decoy receptor. To investigate the effects of estrogen on the expression of OPG, RANKL, and ERs in human osteoblasts, cells were cultured with physiological (10−10 M) and high-dose (10−7 M) 17β-estradiol for 24 and 48 h. Proteins and corresponding mRNA levels were quantitatively determined by immunocytochemistry and RT–PCR. OPG expression was significantly increased three- and sevenfold at 24 h with 10−10 M (P < 0.05) and 10−7 M (P < 0.01) estradiol, respectively, compared to untreated cells. Similar but smaller increases were seen at 48 h (P < 0.05). Osteoblasts treated with estradiol demonstrated increased RANKL protein expression at 24 h (P < 0.05), but this was not maintained at 48 h. ERα expression was significantly increased by high-dose estradiol (P < 0.01) at 24 h and dose-dependently increased at 48 h (P < 0.01), while ERβ was only increased at 24 h (P < 0.01). The estrogen-induced protein expression of ER, OPG, and RANKL was abrogated when cells were cultured in the presence of the estrogen antagonist ICI 182,780. mRNA levels at 24 h demonstrated a significant suppression of RANKL with the low-dose but not the high dose. ERα mRNA but not ERβ expression was up-regulated by estrogen. Our results suggest that estrogen may exert its anti-resorptive effects on bone, at least in part, by stimulating ER and OPG expression in 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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