Deborah C. Ireland

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

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-kappaB 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) 17beta-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. ERalpha 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 ERbeta 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 182780. mRNA levels at 24 h demonstrated a significant suppression of RANKL with the low-dose but not the high dose. ERalpha mRNA but not ERbeta 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.

Colocalization of Glucocorticoid and Mineralocorticoid Receptors in Human Bone

Osteoporosis is a poorly understood but common complication of glucocorticoid therapy. The actions of glucocorticoids are mediated via glucocorticoid receptors (GRs), but in vitro, glucocorticoids also can bind to mineralocorticoid receptors (MRs). It is not known if MR protein is present in human bone and little is known of GR isoform expression (GRα and GRβ). GR and MR protein expression and possible sites of action were investigated in neonatal rib and adult iliac crest biopsy specimens using antibodies specific for MR, GRα, and GRαβ. Colocalization [MR GRα] [MR GRαβ] was performed using fluorescent‐conjugated secondary antibodies. GRα, GRβ, and MR show distinct but overlapping patterns of expression, suggesting important functions for each receptor type. Osteoclasts showed no staining for GRα but strong staining for GRαβ, indicating expression of GRβ and a specific role in addition to antagonizing the transcriptional activity of GRα. MR also was observed in osteoclasts and colocalized with GRαβ. Coexpression of MR, GRα, and GRαβ was seen in osteoblasts. Reverse‐transcription‐polymerase chain reaction (RT‐PCR) of cultured osteoblast RNA confirmed expression of both GRα and GRβ. Osteocytes stained with MR, GRα, and GRαβ antibodies but to a lesser degree than osteoblasts. In the neonatal rib cartilage, staining for GRα, GRαβ, and MR was present in approximately one‐half of the resting and hypertrophic chondrocytes and in most of proliferating chondrocytes and chondrocytes within the mineralizing matrix. Identification of MR raises the possibility that the physiological and pharmacologic effects of glucocorticoids on bone may be mediated via MR as well as GR and that GRα, GRβ, and MR synergize to influence corticosteroid metabolism in human bone.

Vacuolar H+-ATPase d2 subunit: molecular characterization, developmental regulation, and localization to specialized proton pumps in kidney and bone.

The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pumps constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henles loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target.

The role of surface wettability and surface charge of electrosprayed nanoapatites on the behaviour of osteoblasts

A new deposition method is presented, based on electrospraying, that can build bioceramic structures with desirable surface properties. This technology allows nanoapatite crystals, including hydroxyapatite (nHA), carbonate-substituted HA (nCHA) and silicon-substituted HA (nSiHA), to be electrosprayed on glass substrates. Human osteoblast cells cultured on nSiHA showed enhanced cell attachment, proliferation and protein expression, namely alkaline phosphatase, type 1 collagen and osteocalcin, as compared to nHA and nCHA. The modification of nanoapatite by the addition of silicon into the HA lattice structure renders the electrosprayed surface more hydrophilic and electronegatively charged.

Mechanisms by which high-dose estrogen therapy produces anabolic skeletal effects in postmenopausal women: role of locally produced growth factors

Conventional hormone replacement therapy acts primarily by preserving bone, but cannot restore lost bone in women with established osteoporosis. Studies in rodents have shown that high doses of estrogens have anabolic skeletal effects, and recent observations in a group of women treated long term with high doses of estrogen indicated that similar effects occur in humans. This study examines the hypothesis that locally produced growth factors, including transforming growth factor-beta (TGF-beta) and platelet-derived growth factors (PDGFs), are involved in mediating the anabolic effects of high-dose estrogen. Transiliac-crest bone biopsies were taken from ten women, aged 52-67 years (mean 58 years), who had been treated with high-dose estrogen for 15 years. Control samples were obtained from four age-matched postmenopausal women not receiving estrogen therapy. TGF-betas and PDGFs were analyzed for mRNA and protein expression by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Results showed both TGF-beta1 and -beta2 mRNA, expressed as a ratio to GAPDH, were increased in the estrogen-treated group with an eightfold increase for TGF-beta1 (0.258 +/- 0.246 [mean +/- SD] vs. 0.032 +/- 0.053 in the control group, p = 0.02) and a twofold increase for TGF-beta2 (p = n.s.). TGF-beta3 analysis showed only negligible amounts in both groups. Protein expression levels for TGF-beta1, -beta2, -betaRI and -RII were higher in the estrogen-treated group than in controls, the most marked effects being seen for TGF-beta1. PDGF-A protein expression was also significantly higher in osteoblasts and osteocytes in women treated with estrogen, whereas PDGF-B showed only modest differences. The percentage of bone surface occupied by osteoclasts, as determined by tartrate-resistant acid phosphatase (TRAP) staining, was significantly reduced in the estrogen-treated group (p = 0.001). These results demonstrate that high-dose estrogen therapy is associated with increased TGF-beta, TGF-betaR, and PDGF synthesis and decreased osteoclast activity, consistent with the hypothesis that these growth factors may mediate the actions of estrogen in bone.

The role of electrosprayed apatite nanocrystals in guiding osteoblast behaviour.

Apatite nanocrystals, which mimic the dimensions of natural bone mineral, were electrosprayed on glass substrates, as a suitable synthetic biomedical material for osteoblast outgrowth was explored. A variety of topographic patterns were deposited and the influence of these designs on osteoblast alignment and cell differentiation was investigated. Patterned cell growth and enhanced cell differentiation were seen. Osteoblasts were also cultured on apatite nanocrystals chemically modified with either carbonate or silicon ions. Enhanced cell proliferation and early formation of mineral nodules were observed on apatite nanocrystals with silicon addition. This work highlights the importance of the combined effects of surface topography and surface chemistry in the guidance of cell behaviour.

Synthesis of osteoprotegerin and RANKL by megakaryocytes is modulated by oestrogen

To investigate the mechanisms by which megakaryocytes (MKs) may influence bone remodelling, CD34+ cells were cultured for 6, 9 and 12 d with or without 17β‐oestradiol (E) and immunolocalized for osteoprotegerin (OPG), receptor activator of nuclear factor (NF)‐κB ligand (RANKL) and CD61. Specific protein expression was measured quantitatively by image analysis. Fluorescence‐based immunocytochemistry was used to co‐localize OPG and RANKL with CD61. OPG and RANKL mRNA was assessed in CD61+ cells with or without E at 24 and 48 h. At 6 d, OPG and RANKL expression was unchanged by E treatment. At 9 d, the E‐treated cultures with maturing MKs showed a 1·72‐fold (P < 0·01) increase in OPG expression and a 1·8‐fold (P < 0·01) reduction in RANKL. Maximal OPG expression was seen at 12 d with a threefold induction of expression (P < 0·001), whilst RANKL levels were further suppressed by 2·3‐fold compared with controls (P < 0·001). CD61 co‐localized with OPG and RANKL. mRNA data were consistent with that of protein, with a 90‐fold induction in OPG expression and a 34‐fold suppression of RANKL expression by E (P < 0·001). Thus, E stimulates megakaryocytopoiesis and modulates OPG and RANKL expression, providing evidence that MKs may play a role in bone remodelling and, in particular, in E‐induced changes in osteoclastogenesis and bone resorption.

Osteoclast formation and bone resorption are inhibited by megakaryocytes

It has been previously reported that addition of megakaryocytes (MKs) to osteoblasts in vitro results in increased osteoblastic collagen and osteoprotegerin (OPG) production, suggesting a role for MKs in bone formation. To further investigate this role, we have studied the effects of MKs on osteoclast formation and activity. Human osteoclasts were generated from CD14 monocytes isolated from peripheral blood and cultured in the presence of M-CSF and sRANKL on dentine and calcium phosphate substrates. MKs were generated from CD34+ cells isolated from either human peripheral blood or cord blood and cultured in liquid medium for 6 days, after which time maturing MKs (CD61-positive cells) were isolated and added to monocyte cultures. After 6 and 9 days of culture, the number of osteoclasts identified morphologically and by TRAP staining was counted. Cells were removed and the area of resorption was identified by von Kossa staining and quantitatively assessed by image analysis. The addition of MKs to osteoclast cultures at day 0 inhibited the number of osteoclasts formed 1.9-fold (p>0.003), whereas addition at 3 days had no effect on osteoclast number. The presence of MKs inhibited resorption 8.7-fold when co-cultured with osteoclasts from day 0 (p>0.004), but only by 3.1-fold when co-cultured from day 3 (p>0.01). In dose-response experiments, it was found that 1-10% of MKs added to monocyte cultures elicited the greatest inhibition of resorption. Similar osteoclast cultures were treated with CD61-negative cells (non-MKs) to confirm that the inhibition of osteoclast formation and activity was specifically due to MKs. Experiments with a cell-impermeable membrane indicated that both cell to cell contact and release of soluble factor(s) were involved in mediating these effects. These results show that MKs inhibit osteoclast formation and activity. The most pronounced effects were seen when MKs and osteoclasts were co-cultured from day 0, suggesting that MKs act primarily on osteoclast precursors.

Effects of estrogen on collagen synthesis by cultured human osteoblasts depend on the rate of cellular differentiation

Estrogen is known to act on osteoblasts according to their stage of differentiation and estrogen receptor (ER) isoform expression. The aim of this study was to determine when type I collagen (COL1) synthesis by cultured low‐passage, human bone‐derived osteoblasts (hOBs) is upregulated in response to estrogen. Cell lines from female donors aged 1 and 66 years were cultured for 11 days on collagen in growth medium supplemented with human serum, hydrocortisone, and β‐glycerophosphate. Young‐donor hOBs grew more quickly than old‐donor hOBs and did not mineralize. Old‐donor hOBs formed mineralized nodules 5 days after reaching confluence. Changes in mRNA levels with time for ERs, type I collagen, and alkaline phosphatase reflected the faster differentiation of the old‐donor cells. The ERβ/ERα ratio fell threefold in young‐donor hOBs but rose 300‐fold in old‐donor hOBs. Increased ERβ/ERα ratios prevented ligand‐dependent downregulation of ERα transcription, resulting in reduced proliferation in old‐donor hOBs. Upregulation of COL1 mRNA expression in response to estrogen was confined to intermediate stages of differentiation, resulting in significant increases in COL1 mRNA by estradiol only in young‐donor cells. Since the young and old‐donor hOBs were cultured under identical conditions, our results indicate that the response of hOBs to estrogen is largely dependent on intracellular mechanisms that control the timing of cellular differentiation.

Estrogen stimulates differentiation of megakaryocytes and modulates their expression of estrogen receptors α and β

Estrogen has multifunctional effects influencing growth, differentiation, and function in many tissues. High‐dose estrogen has been shown to produce anabolic skeletal effects in the skeleton of postmenopausal women with increased megakaryocyte (MK) population in the bone marrow, suggesting a possible role for these cells in bone remodelling. To investigate if estrogen stimulates megakaryocytopoiesis and affects on estrogen receptor (ER) expression, CD34(+) cells were cultured for 6, 9, and 14 days plus or minus low‐dose or high‐dose 17β estradiol (E). Cells were immunolocalised for CD61, CD41, ERα and β. ER mRNA expression was assessed by RT‐PCR. Cells formed more CD61 positive MK colonies with low‐ and high‐dose E treatment (P < 0.001) at 6 and 9 days. CD41 expression was increased dose‐dependently in MK (3‐ and 5‐fold P < 0.001) at 9 days. E‐stimulated ERα expression at 6 days (P < 0.001) whilst ERβ was dose‐dependently increased only at 9 days (P < 0.01). ERα mRNA was increased at 6 days but not at 14 days whilst ERβ mRNA expression was only increased at 14 days with E treatment. These results demonstrate that E stimulates the colony forming potential of CD34(+) cells to a more megakaryocytic phenotype in vitro. This finding together with the stimulation of ER protein and mRNA expression adds to the increasing evidence for a role for MKs in estrogen‐induced bone formation.