Endocrine Research

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Authors

M. Á. Brennan1, 2, 3, M. G. Haugh1, 2, F. J. O’Brien4, 5, L. M. McNamara1, 2

Affiliations

Affiliation addresses are listed at the end of the article

Key words ▶ estrogen deficiency ● ▶ osteoblasts ● ▶ osteocytes ● ▶ mineralization ● ▶ apoptosis ●

Abstract

received 28.08.2013 accepted 28.11.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1363265 Published online: January 20, 2014 Horm Metab Res 2014; 46: 537–545 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence L. M. McNamara, PhD Department of Mechanical and Biomedical Engineering National University of Ireland Galway Galway Ireland Tel.: + 353/91/492251 Fax: + 353/91/563991 [email protected]

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Recent studies have demonstrated increased bone mineral heterogeneity following estrogen withdrawal in vivo. Such changes likely contribute to fracture risk during post-menopausal osteoporosis since tissue mineralization is correlated with bone strength and stiffness. However, the cellular mechanisms responsible for increased mineral variability have not yet been distinguished. The objective of this study is to elucidate how alterations in mineral distribution are initiated during estrogen depletion. Specifically, we tested 2 separate hypotheses; (1) estrogen deficiency directly alters osteoblast mineralization and (2) estrogen deficiency increases bone cell apoptosis. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLOY4) were pretreated with or without estrogen (17β-estradiol) for 14 days. Estrogen deficiency was subsequently induced by either withdrawing estrogen from cells or blocking estrogen

Introduction

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During normal physiology, bone tissue is continually being renewed by osteoclast and osteoblast cells acting in concert; however, during postmenopausal osteoporosis excessive resorption occurs without adequate new bone formation [1]. Such cellular changes are believed to occur as a results of estrogen deficiency following the menopause and lead to low bone mass, a deteriorated micro-architecture and consequently increased fracture risk [2]. It is believed that estrogen plays a role in maintaining bone mass by different means, specifically through the ERα in osteocytes of trabecular bone in male mice [3], but via osteoclasts in trabecular bone of female mice [4] and through osteoblast progenitors in cortical bone [4]. It has also been shown that

receptors using an estrogen antagonist, fulvestrant (ICI 182,780). Cell number (Hoechst DNA), alkaline phosphatase activity (p-NPP), mineralization (alizarin red) and apoptosis (Caspase 3/7) were evaluated. Whether estrogen withdrawal altered apoptosis rates in the presence of an apoptosis promoting agent (etoposide) was also determined. Interestingly, estrogen withdrawal from cells accustomed to estrogen exposure caused significantly increased osteoblast mineralization and osteocyte apoptosis compared with continued estrogen treatment. In contrast, blocking estrogen receptors with fulvestrant abrogated the mineralization induced by estrogen treatment. When apoptosis was induced using etoposide, cells undergoing estrogen withdrawal increased apoptosis compared to cells with continued estrogen treatment. Recognizing the underlying mechanisms regulating bone cell mineralization and apoptosis during estrogen deficiency and their consequences is necessary to further our knowledge of osteoporosis.

estrogen deficiency exacerbates bone loss due to aging by diminishing the cells resistance to oxidative stress [5]. The quantity of mineral, together with its distribution within the bone tissue matrix are among factors governing the mechanical strength of bone [6, 7]. It has recently been demonstrated that the distribution of tissue-level mineral is more heterogeneous during estrogen deficiency, and particularly that these changes occur along the inter-trochanteric fracture line of sheep femora [8]. Such alterations in tissue mineral distribution may be a contributing factor for reduced mechanical strength [9, 10]. However, the cellular mechanisms responsible for such changes have not yet been distinguished. The degree of bone mineralization is influenced by the frequency of bone remodeling as well as

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Estrogen Withdrawal from Osteoblasts and Osteocytes Causes Increased Mineralization and Apoptosis

538 Endocrine Research Materials and Methods

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Cell culture Two bone cell lines, MC3T3-E1 osteoblast-like cells and MLO-Y4 osteocyte-like cells, were used in this study. MC3T3-E1 cells are an accepted, commercially available, model of primary osteoblast function as they express high amounts of alkaline phosphatase, produce mineral and they have demonstrated the ability to differentiate into osteoblasts and osteocyte-like cells in vitro [31]. MLO-Y4 cells (a kind gift from Prof. Lynda Bonewald, University of Missouri) possess many similar characteristics to primary osteocytes such as low expression of alkaline phosphatase and production of numerous dendritic processes [32]. MC3T3-E1 cells, purchased from LGC Standards, Middlesex, UK, were maintained in α-MEM supplemented with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/ml penicillin and 100 μg/ml streptomycin. MLO-Y4 cell cultures were maintained in α-MEM supplemented with 2.5 % fetal bovine serum (FBS) and 2.5 % calf serum (CS), 2 mM L-glutamine, 100 U/ml penicillin/100 μg/ml streptomycin in culture flasks coated with 0.15 mg/ml collagen (type I from rat tail, C3867 Sigma Aldrich).

Estrogen pretreatment In the present study, in vitro administration of 17β-estradiol, a naturally occurring estrogen derived from cholesterol, was used to simulate normal conditions when circulating estrogen is available to bone cells. MC3T3-E1 and MLO-Y4 cells were cultured at 37 °C in a humidified 5 % CO2 environment in their aforementioned standard media with or without addition of 1 · 10 − 8 M 17β-estradiol (Sigma Aldrich). This estrogen dosage is within the normal physiological circulating serum levels in mice [33–35]. Cell culture media were replenished every 3–4 days. The pretreatment duration (14 days) was determined following preliminary experiments and was deemed to be an appropriate duration to allow cells to become accustomed to estrogen before subsequent withdrawal.

Estrogen deficiency mineralization experiments The effects of estrogen deficiency on MC3T3-E1 and MLO-Y4 cells were evaluated by either (1) withholding estrogen from estrogen pretreated cells (estrogen withdrawal) or (2) by blocking estrogen receptors with 1 · 10 − 7 M fulvestrant (ICI 182,780). Fulvestrant competes with estrogen for binding to the estrogen receptor with a binding affinity that is 89 % that of 17β-estradiol [36], and has been used previously to mimic estrogen deficiency in human [13] and animal osteoblasts in vitro [37]. MC3T3-E1 cells were plated in triplicate at a density of 3 · 104 cells/ml, in 24 well plates and cultured with osteogenic media supplemented with 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate and 10 nM dexamethasone. MLO-Y4 cells were plated in triplicate at a density of 1.5 · 104 cells/ml in collagen coated tissue culture plates and cultured with aforementioned standard media without osteogenic promoting agents. Both MC3T3-E1 and MLO-Y4 cells were subsequently cultured under separate experimental conditions; (E2) Sustained estrogen exposure: cells were cultured in their appropriate media (osteoblast osteogenic media or osteocyte standard media respectively) with continued supplementation of 17β-estradiol (1 · 10 − 8 M), (E1) Reduced estrogen exposure: cells were cultured in their appropriate media with the addition of reduced concentration of 17β-estradiol (1 · 10 − 10 M), (E–) Estrogen withdrawal:

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mineral deposition rates. The changes in osteoclast activity occurring at the onset of estrogen deficiency have been well established [1, 2]. However, recent evidence suggests that alterations in osteoblast activity may be the first event that occurs following estrogen withdrawal and that altered osteoclast activity and consequential increased bone resorption may be later events in the osteoporotic bone loss cascade [11, 12]. Many studies have investigated the response of osteoblasts to estrogen and have yielded conflicting results: studies show that estrogen decreases [13] and conversely increases cell proliferation [14, 15]. Expression of alkaline phosphatase (ALP), and osteocalcin (OC), have been shown to be stimulated, inhibited, or unresponsive to estrogen [13, 14, 16]. Furthermore, estrogen treatment has been shown to enhance [17, 18], or have no effect [15] on mineral production by osteoblastic cells, whereas blocking estrogen receptors with the estrogen antagonist fulvestrant has been shown to have negative effects on the mineralization of osteoprecursor cells [19]. Importantly, it has been shown that only osteoblasts that were first pretreated with estrogen in vitro and subsequently underwent estrogen withdrawal, paralleled the activities of cells from ovariectomized (OVX) mice [20]. Cells without estrogen pretreatment prior to culture without estrogen behaved significantly differently to OVX cells [20]. However, in spite of this finding [20], to date no in vitro study has investigated whether altered mineralization by bone cells occurs as a consequence of estrogen withdrawal. Osteocytes residing in lacunae within the mineralized bone matrix are believed to have the ability to modify the mineral content of their surrounding matrix, by either dissolving mineralized matrix from their perilacunar regions [21, 22] or by producing new mineralized tissue [23, 24]. Since osteocytes possess receptors for estrogen, their activities may be affected when estrogen production is deficient during postmenopausal osteoporosis. It has been demonstrated that an increase in osteocyte apoptosis occurs in hip fracture patients [25], following drug induced estrogen withdrawal in women [26] and also in an ovariectomized mouse model [5, 27]. Mineral infilling of empty osteocyte lacunae following osteocyte apoptosis, a phenomenon referred to as micropetrosis [28, 29], might explain alterations in bone mineralization during estrogen deficiency. Although it has been shown that estrogen treatment prevents drug induced osteoblast and osteocyte apoptosis in vitro [28, 30], whether apoptosis occurs as a direct consequence of estrogen withdrawal in vitro (and thereby alters mineralization by micropetrosis) has not yet been investigated. The objective of this study is to explore how alterations in bone tissue mineral distribution are initiated during estrogen withdrawal. Specifically, 2 separate hypotheses by which estrogen deficiency might alter bone tissue mineralization were tested; (1) estrogen withdrawal directly alters mineralization by bone cells and (2) estrogen deficiency increases bone cell apoptosis and thereby leads to a micropetrosis type response wherein osteocytes hyper-mineralize their surrounding matrix. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLO-Y4) were pretreated in vitro with estrogen. Estrogen withdrawal was evaluated by depleting cells of estrogen or by treatment with an estrogen antagonist, fulvestrant, which blocks estrogen receptors. Specifically, the aim was to explore whether cell number, alkaline phosphatase activity, mineralization, and apoptosis were altered as a direct consequence of estrogen withdrawal.

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Table 1 Experimental design.

Study 2: Osteocyte Apoptosis

Cell lines

Pretreatments

Experimental conditions

Cellular Assays

MC3T3-E1 Osteoblast

14 days Estrogen

E2 (1 · 10 − 8 M 17β-Estradiol) E1 (1 · 10 − 10 M 17β-Estradiol) E– (Osteogenic media) E– & F (1 · 10 − 7 M Fulvestrant) E1 & F E2 & F

All cells assayed at 1, 4, 7, and 14 days for: Proliferation (DNA Hoechst) ALP activity (ρNPP) Mineralization (Alizarin Red)

MLO-Y4 Osteocyte MLO-Y4 Osteocyte

Untreated 14 days Estrogen

E2 (1 · 10 − 8 M 17β-Estradiol) E1 (1 · 10 − 10 M 17β-Estradiol) E– (Osteogenic media) E– & F (1 · 10 − 7 M Fulvestrant) E2 & F

With/without Etoposide

Cells assayed after 4, 12, 24, 30, 48, and 72 h for: Apoptosis (Caspase 3/7)

Both the MC3T3-E1 osteoblast line and MLO-Y4 osteocyte cell lines were used to investigate the effects of varying concentrations of estrogen and estrogen withdrawal on cell proliferation and osteogenic differentiation. The effects of estrogen withdrawal on osteocyte apoptosis was also assessed

cells were cultured in their appropriate media without the addition of estrogen, (E– & F) Estrogen receptor inhibition: cells were cultured in their appropriate media with the addition of 1 · 10 − 7 M fulvestrant, (E1 & F) Estrogen receptor inhibition with reduced estrogen exposure: cells were cultured in their appropriate media with the addition of 1 · 10 − 10 M 17β-estradiol and 1 · 10 − 7 M fulvestrant, (E2 & F) Estrogen receptor inhibition with sustained estrogen exposure: cells were cultured in their appropriate media with the addition of 1 · 10 − 8 M 17β-estradiol and 1 · 10 − 7 M fulvestrant. The detailed experimental design is sum▶ Table 1. All cells were maintained at 37 °C in a marized in ● humidified 5 % CO2 environment and were cultured for 1, 4, 7, and 14 days. Media were replenished every 3–4 days. All compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.

Cells were washed in sterile PBS and lysed in their plates in 500 μl molecular grade water by means of 3 freeze/thaw cycles ( − 80 °C, 25 °C). The lysate was assayed for DNA content using a fluorescent dye Hoechst 33258. A standard curve was generated using 0–500 ng DNA of calf thymus DNA with 0.1 μg/ml of Hoechst dye (Sigma-Aldrich). Fluorescent assay buffer (100 mM TrisHCl, pH 7.4, with 10 mM EDTA and 2 M NaCl) was added to each well of a 96-well plate (50 μl/well). Standards (50 μl) and samples were added to each well in triplicate. Finally 100 μl Hoechst 33258 dye solution (0.1 μg/ml) was added to each well. The plate was incubated for 10 min in the dark at room temperature, and then fluorescence (Ex360 nm/Em 460 nm) was read on a Microplate Reader (Wallac 1420 VICTOR 2 TM, PerkinElmer, Boston, MA, USA).

Alkaline phosphatase expression Osteocyte apoptosis experiments To evaluate the effects of estrogen deficiency on osteocyte apoptosis, MLO-Y4 cells that were pretreated with 17β-estradiol for 14 days were plated in collagen coated 96 well plates at a density of 4 · 104 cells/mL. Cells were cultured in 100 μl media under the following conditions for 24 h; (E2), (E1), (E–), (E– & F) or (E2 & F). Etoposide, an apoptosis inducer, was also employed to investigate whether estrogen deficient cells are more susceptible to apoptosis compared with cells exposed to estrogen. After 24 h of culture, half of the wells were treated with etoposide, to promote apoptosis, whereas the other half did not receive etoposide but their media was replenished. Etoposide was prepared as a stock solution (1 mM) in dimethyl sulfoxide (DMSO). Final concentration when added to culture media was 50 μM. The detailed ▶ Table 1. experimental design is summarized in ●

Cellular Assays

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The glycoprotein alkaline phosphatase (ALP) was evaluated as it is an osteoblast differentiation marker, which plays an important role in the mineralization process [38]. It was measured at days 1, 4, 7, and 14, as temporal changes in ALP expression occur during osteoblastic differentiation from predifferentiated cells to a mature phenotype [39]. Specifically immature MC3T3-E1 cells initially produce low levels of ALP, but as these cells differentiate into mature osteoblasts they upregulate ALP production and finally as they differentiate into osteocytes they decrease ALP production [39, 40]. Cells were washed in sterile PBS and lysed in their plates in 500 μl molecular grade water by means of 3 freeze/thaw cycles ( − 80 °C, 25 °C). ALP activity was quantified in cell lysate using an ALP Colorimetric Assay Kit (Abcam). ALP enzyme converts the p-nitrophenyl phosphate (pNPP) substrate to an equal amount of colored p-nitrophenol (pNP). Colorimetric absorbance was measured at 405 nm using a micro-plate reader (Wallac Victor, PerkinElmer Life Science). Relative colorimetric readings were converted to ALP activity by creating a standard ALP curve.

Hoechst 33258 for DNA quantification In order to assess the effects of estrogen withdrawal on cell number, measurements of DNA content were performed after 1, 4, 7, and 14 days of culture. DNA content quantification was also assessed in order to normalize ALP production and mineralization to the quantity of DNA at each time point. This permits an understanding of whether changes in mineralization were related to the overall mineral capacity of individual cells or changes in the cell population available to produce that mineral.

Alizarin Red S staining for mineralization Mineralization was quantified after 7 and 14 days of culture. Cells were washed with sterile PBS solution and fixed with 10 % formalin for 10 min. Cells were thoroughly washed with distilled water and stained with 40 mM Alizarin Red-S in deionized water (pH 4.2) for 20 min at room temperature on an orbital rotator. Alizarin red stain binds to mineral nodules present. Cultures were rinsed 3 times with distilled water to remove any unbound

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Study 1: Mineralization

540 Endocrine Research

stain and wells were subsequently destained using 100 nM cetylpyridinium chloride. The absorbance of the extracted stain was measured at a wavelength of 550 nm using a plate reader (Wallac Victor, PerkinElmer Life Science). Relative photometry units were converted to μmol/well of alizarin red by creating a standard curve. Alizarin red measurements were expressed as μmol/well of calcium since one mole of alizarin red bind to 2 moles of calcium [41]. Mineral production was subsequently normalized to quantity of DNA (n mol Ca/μg DNA).

Apoptosis quantification Cultures were assessed for apoptosis after 4, 12, 24, 30, 48, and 72 h of culture, with/without etoposide administration using the ApoTox-Glo Triplex Assay (Promega). Apoptosis was quantified by measuring Caspase 3/7 activity. A reagent (containing tetrapeptide sequence DEVD) was added to each test well in a ratio of 1:1 and briefly mixed by orbital shaking for 30 s. Wells were incubated for 1 h at room temperature. The reagent causes cell lysis and following caspase cleavage, a substrate for luciferase (aminoleciferin) was released which causes the production of light. Luminescence is directly proportional to the amount of caspase activity present and was measured on a micro-plate reader (Wallac Victor, PerkinElmer Life Science).

Statistical analysis Data were expressed as a mean ± standard deviation. Statistical differences between groups (n = 6 per group) were determined using an ANOVA crossed factor model, defined using the General Linear Model (GLM) ANOVA function. Comparison between

treatments and pretreatments were made using the TukeysKramer multiple comparison test (Minitab 16). Statistical significance was defined as p ≤ 0.05.

Results

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Effects of estrogen pretreatment on osteoblast and osteocyte activity In osteoblast cultures, DNA content (expressed as ng DNA/ well) was lower in cultures that were pretreated with estrogen compared to untreated cells at day 4, in the E–, E– & F, and E2 & F groups, (590 ± 361 vs. 1 375 ± 242, p < 0.01), (841 ± 363 vs. 1 503 ± 574, p < 0.01), and (643 ± 389 vs. 1 405 ± 690, p < 0.01), respectively. Higher ALP production was found in cultures with estrogen pre▶ Fig. 1a, b). treatment compared to untreated cultures (● In osteocyte cultures, no difference in DNA content was found as a result of estrogen pretreatment. At every time point, with the exception of day 1, estrogen pretreatment resulted in higher osteocyte ALP production compared to untreated cultures ▶ Fig. 2a, b). When mineralization was investigated, in osteo(● blast cultures estrogen pretreatment resulted in higher mine▶ Fig. 3a). In osteocyte cultures, mineralization was ralization (● higher in estrogen pretreated cultures at day 7 in the E1 & F group (79.55 ± 41.64 vs. 37.05 ± 13.41, p < 0.03); however, no difference in mineralization was found as a consequence of estro▶ Fig. 3b). gen pretreatment at day 14 (●

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Fig. 1 Osteoblast ALP production during estrogen withdrawal. Comparison of ALP expression in cells with or without 14 days of estrogen pretreatment. Continued estrogen treatment controls (E2) are compared with lowering estrogen concentration (E1), estrogen withdrawal (E–), and blocking estrogen receptors (E– & F), (E1 & F) and (E2 & F) at a Day 1 and Day 4 and b Day 7 and Day 14. Groups sharing a letter are significantly different to each other, a (p < 0.01), b (p < 0.04), c (p < 0.03), d (p < 0.01). *Indicates significantly different to previously untreated cultures, within the same treatments and time points. Data is expressed as a mean ± standard deviation.

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Effects of estrogen deficiency on osteoblast activity Effect of estrogen deficiency on osteoblast number in vitro No significant difference in DNA content was found between estrogen deficiency groups (E–, E– & F, E2 & F) and controls (E2), in previously untreated cells, or those pretreated with estrogen for 14 days, at days 1, 4, 7, or 14.

Effect of estrogen deficiency on ALP production by osteoblasts in vitro At day 4, in estrogen pretreated cultures, significantly higher ALP production was observed when estrogen was subsequently withdrawn from cells (E–) compared to continued estrogen ▶ Fig. 1a). treatment (E2) (0.82 ± 0.26 vs. 0.39 ± 0.19, p < 0.01) (● Similarly, at day 14, in estrogen pretreated cells, estrogen withdrawal (E–) resulted in significantly higher ALP activity, compared to continued estrogen treatment (E2) (2.92 ± 0.70 vs. ▶ Fig. 1b). There was no statistical differ1.12 ± 0.38, p < 0.01) (● ence in ALP production by cells treated with fulvestrant compared to continued estrogen treatment (E2). In the previously untreated cell group, at day 7, significantly lower ALP activity was found in the estrogen blocker group (E2 & F) compared to estrogen controls (E2) (0.06 ± 0.01 vs. 0.6 ± 0.18, ▶ Fig. 1b). By Day 14, in previously untreated cultures, p < 0.04) (● significantly higher ALP production was found in the E– & F group compared to estrogen controls (E2) (1.21 ± 0.07 vs. 0.56 ± 0.12, p < 0.03).

Effect of estrogen deficiency on osteoblast mineralization in vitro Osteoblast mineralization results at Day 7 and Day 14 from cultures with or without 14 days estrogen pretreatment are pre▶ Fig. 3a. At day 14, in estrogen pretreated cultures, sented in ● significantly higher mineralization was observed in the estrogen withdrawal group (E–) compared to continued estrogen treatment (E2) (459.12 ± 170.56 vs. 247.69 ± 164.28, p < 0.01). Conversely, blocking estrogen receptors with (E– & F) and (E2 & F) treatments, resulted in significantly lower mineralization, compared with continued estrogen (E2) (52.40 ± 39.71 vs. 247.69 ± 164.28, p < 0.03) and (64.40 ± 32.63 vs. 247.69 ± 164.28, p < 0.05) respectively.

Effects of estrogen deficiency on osteocyte activity Effect of estrogen deficiency on osteocyte number in vitro In osteocytes that were pretreated with estrogen, lower DNA content was found in the estrogen blocker group (E2 & F) compared with the continued estrogen treatment controls (E2) (819.60 ± 380.71 vs. 1 315.25 ± 53.64, p < 0.01). No other difference in DNA content was found between treatment groups and controls (E2), in previously untreated cells, or those pretreated with estrogen for 14 days, at days 1, 4, 7, or 14.

Effect of estrogen deficiency on ALP production by osteocytes in vitro At Day 4, in estrogen pretreated cells, cultures treated with the estrogen blocker (E2 & F) showed significantly higher ALP production compared to continued estrogen treatment (E2)

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Fig. 2 Osteocyte ALP production following estrogen withdrawal. Comparison of ALP expression in cells with or without 14 days of estrogen pretreatment. Continued estrogen treatment controls (E2) are compared with lowering estrogen concentration (E1), estrogen withdrawal (E–), and blocking estrogen receptors (E– & F), (E1 & F) and (E2 & F). Groups sharing the same letters are significantly different to each other, a–e (p < 0.01). *Indicates significantly different to previously untreated cultures of the same subsequent treatment group and time point. Data is expressed as a mean ± standard deviation.

542 Endocrine Research

▶ Fig. 2a). By Day 7, in estro(0.06 ± 0.01 vs. 0.04 ± 0.01, p < 0.01) (● gen pretreated cultures, significantly higher ALP production was observed in estrogen blocker groups (E– & F) and (E2 & F), compared to continued estrogen treatment (E2), (0.14 ± 0.01 vs. 0.07 ± 0.02, p < 0.01) and (0.14 ± 0.01 vs. 0.07 ± 0.02, p < 0.01) ▶ Fig. 2b). There was no statistical difference in respectively (● ALP production following estrogen withdrawal (E–). At Day 1, in previously untreated cells, ALP expression was significantly lower in the E– group compared to cells with estrogen ▶ Fig. 2a). controls (E2) (0.03 ± 0.01 vs. 0.05 ± 0.01, p < 0.01) (● Conversely, cells exposed to the estrogen blocker (E– & F) had significantly higher ALP expression compared to the E2 group (0.06 ± 0.01 vs. 0.05 ± 0.01, p < 0.01).

Effect of estrogen deficiency on osteocyte mineralization in vitro Osteocyte mineralization results at Day 7 and Day 14 from cultures with or without estrogen pretreatment are presented in ▶ Fig. 3b. At Day 7, in the estrogen pretreatment group, detail in ● there was higher mineralization in the E1 & F group compared to the E2 control group (79.55 ± 41.64 vs. 31.49 ± 29.70, p < 0.03). No difference in mineralization was found in the E– group compared to the E2 group in pretreated or untreated cultures.

Effect of estrogen deficiency on osteocyte apoptosis in vitro Caspase 3/7 activity results, expressed as a percentage of con▶ Fig. 4. The effects of decreased trols (E2) are presented in ● estrogen concentration (E1), estrogen withdrawal (E–), or blocking estrogen receptors (E– & F), (E2 & F), on osteocyte apoptosis, as well as apoptosis induced using an apoptotic agent, etoposide are presented. Significantly higher apoptosis was observed by 24 h, when estrogen was withdrawn (E–) from osteocytes, compared to continued estrogen administration (E2) (110.5 ± 6.42 vs. 100, p < 0.05). At the 24 h time point cells were treated with the apoptosis inducing agent etoposide, and it was found that at 30 h (6 h after etoposide administration), there was significantly higher apoptosis in the etoposide group compared to cells not treated with etoposide, in the estrogen withdrawal group (E–) (122.91 ± 24.13 vs. 98.46 ± 12.24, p < 0.02). By 24 h of etoposide treatment (48 h time point), etoposide significantly elevated apoptosis in all treatment groups (p < 0.01). By 48 h of etoposide administration (72 h experimental time point), cell death signals diminished and significantly lower apoptosis was observed for all etoposide treated cultures, compared to those not treated with etoposide (p < 0.01), with the exception of the estrogen withdrawal (E–) group. Similarly, apoptosis in cultures without etopside treatment was signifi-

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Fig. 3 a Osteoblast and b osteocyte mineralization following estrogen withdrawal. Mineralization in cell groups with or without 14 days of estrogen pretreatment after 7 and 14 days. Continued estrogen treatment controls (E2) are compared with lowering estrogen concentration (E1), estrogen withdrawal (E–), and blocking estrogen receptors (E– & F), (E1 & F) and (E2 & F). *Indicates significantly different to previously untreated cultures of the same subsequent treatment group and time point. Groups sharing the same letters are significantly different to each other; a (p < 0.01), b (p < 0.03), c (p < 0.05), d (p < 0.03). Data is expressed as a mean ± standard deviation.

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cantly lower in the estrogen withdrawal group (E–) compared to continued estrogen treatment (E2) (52.94 ± 10.68 vs. 100, p < 0.01) as the cell death signals had diminished.

Discussion

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This study provides direct evidence that estrogen pretreatment and estrogen withdrawal alters bone cell biology in vitro. Estrogen pretreatment of both osteoblasts and osteocytes caused significantly increased ALP and mineralization compared to untreated cultures. When estrogen receptors were blocked using fulvestrant, by day 14 the mineralization effect of estrogen pretreatment on osteoblasts was abrogated. In contrast ALP production and mineralization by osteoblast cells increased even further when estrogen was withdrawn from cells that had become accustomed to estrogen exposure. Furthermore, estrogen withdrawal from osteocyte-like cells significantly increased apoptosis, both spontaneously and when apoptosis was artificially induced by administering etoposide. Together, these findings highlight the impact of estrogen deficiency on bone cell function, and suggest that osteoblast and osteocyte-like cells responded to estrogen withdrawal by increasing mineral production and apoptosis. It must be noted that there are a number of limitations associated with the current study. First, MC3T3-E1 and MLO-Y4 cells are both cell lines and may not represent the behavior of primary osteoblasts and osteocytes. However, it is difficult to isolate osteocytes from skeletally mature bone, although a technique has recently been developed [42], nevertheless even when primary osteocytes are isolated, they have limited proliferative capacity since they are terminally differentiated cells. Moreover, the use of cell lines eliminates the donor variability associated with primary cell culture and both MLO-Y4 and MC3T3-E1 have previously been used extensively to investigate osteocyte and osteoblast behavior in vitro [43–49]. Second, the withdrawal of estrogen in the current study is much more

abrupt than in vivo estrogen depletion during the menopause in females. However, ovariectomized animal models also undergo an immediate reduction in circulating estrogen levels and these have been shown to be a good representation of osteoporosis [50–52]. The higher ALP activity and mineralization observed in both osteoblasts and osteocytes when pretreated with estrogen is in agreement with previous studies [17, 18]. The current study reveals for the first time that higher ALP and mineral production occurs with osteoblastic cells following estrogen withdrawal in vitro. These findings are supported by certain in vivo studies. One study shows mineralized bone formation was increased in OVX rats compared to both sham operated rats and OVX rats treated with estrogen [53]. In addition, a study on osteoporotic females showed higher mineralization in osteoporotic compared to healthy bone [54] and drug induced estrogen suppression caused higher mineralization in young female patients [55]. Furthermore, osteoblasts from OVX rats produced more ALP and mineralized bone nodules compared to cells from sham operated rats [53]. The current study reveals a direct link between withdrawal of estrogen (from osteoblasts that were accustomed to its exposure) and increased mineralization, which provides a novel insight into these studies on bone tissue and cells from osteoporotic animals. Interestingly, it has been suggested that following estrogen withdrawal, alterations in osteoblast activity may occur prior to altered osteoclast activity [12]. There are several lines of evidence to support this concept, firstly, Krum et al. have showed that estrogens’ action on osteoclast apoptosis in vitro is mediated by Fas ligand production by osteoblasts [11]. In addition, it was shown in an osteopenia mouse model with altered osteoblastogenesis, that changes in osteoclast activity were secondary to impaired osteoblast function, as osteoclast activity in ex vivo was restored to normal following addition of osteoblast cells from normal mice [56]. Finally, it has been shown that the bone protective of estrogen on cortical bone are mediated through osteoblast precursors [3]. If the theory that a change in osteo-

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Fig. 4 Apoptosis, measured by caspase 3/7 activity, of estrogen pretreated osteocytes during decreased estrogen concentration (E1), estrogen withdrawal (E–), and blocking estrogen receptors (E– & F, and E2 & F), compared to % control (continued estrogen administration: E2) after a 24, b 30, c 48 and 72 h. Groups sharing letters are significantly different to one another, a (p < 0.05), b (p < 0.02), c (p < 0.01). *Indicates significantly different to cultures without etoposide. Data is expressed as a mean ± standard deviation.

544 Endocrine Research Conclusion

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In conclusion, altered osteoblast mineralization may be an early cell response to estrogen deficiency and could be an important event in the osteoporotic bone loss cascade. The apoptosis induction effect of estrogen withdrawal from osteocytes and the preventative effects of estrogen administration on osteocyte apoptosis, induced by apoptotic stimuli (etoposide) have been shown. Dysregulated apoptosis may have important implications for osteoporotic patients as increased apoptosis may compromise the osteocyte network, leading to bone resorption and increased fragility.

Acknowledgements

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The MLO-Y4 cell line was received as a kind gift from Professor Lynda Bonewald (School of Dentistry, University of Missouri, Kansas City, MO, USA). The authors would like to acknowledge funding from the Health Research Board (HRB), Ireland General Research Grant RP/2007/179, the European Research Council Grant ERC-2010-StG Grant 258992 BONEMECHBIO, and the Engineering and Physical Sciences Research Council (EPSRC), UK.

Conflict of Interest

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The authors declare that they have no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Affiliations Biomechanics Research Centre (BMEC), Department of Mechanical and Biomedical Engineering, National University of Ireland, Galway, Ireland 2 National Centre for Biomedical Engineering Sciences, NCBES, National University of Ireland, Galway, Ireland 3 Bioengineering Sciences Research Group, Faculty of Engineering and the Environment, University of Southampton, UK 4 Department of Anatomy, Royal College of Surgeons in Ireland, Dublin 2, Ireland 5 Trinity Centre for Bioengineering, Trinity College, Dublin 2, Ireland 1

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Brennan MÁ et al. Mineralization with Estrogen Withdrawal … Horm Metab Res 2014; 46: 537–545

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clast activity occurs downstream of altered osteoblast function is substantiated, the findings of the current study may indicate that the altered mineralization could play a role in the increased bone remodeling during osteoporosis. In the current study, it is shown that silencing estrogen receptors with fulvestrant in estrogen pretreated cells, causes significantly lower osteoblast mineralization compared to continued estrogen treated controls, in keeping with a previous study [19]. We also showed here that fulvestrant abrogated the increased mineralization by osteoblasts caused by estrogen pretreatment. In contrast, increased mineralization was found by withdrawing estrogen from cells using estrogen depleted media. These findings highlight the role of estrogen receptors in regulating osteoblast mineralization and reveals that blocking estrogen receptors affects mineralization differently than simply withdrawing estrogen from cultures. Furthermore, these findings were only present in estrogen pretreated cells, highlighting the significance of pretreating osteoblasts with estrogen followed by estrogen deficiency. The current study shows that estrogen withdrawal from osteocyte-like cells in vitro induces apoptosis. Previous in vivo studies have reported that estrogen deficiency is associated with an increase in apoptosis and depleted osteocyte number, in female patients [26] and also in ovariectomized animals [5, 27]. Estrogen treatment has previously been shown to reduce apoptosis induced by an apoptotic agent [27, 57]. The increased osteocyte apoptosis during estrogen withdrawal observed in the current study provides an insight into the results of in vivo studies [26, 27] by establishing a direct link between estrogen withdrawal and apoptosis of osteocytes. These results might also explain the increase in bone turnover characteristic of osteoporosis, since it has been suggested previously that osteocyte apoptosis increases bone remodeling [58, 59]. It is intriguing to speculate that the osteocyte apoptosis induced following estrogen withdrawal reported here may stimulate a micropetrosis type response and thereby leads to the altered tissue mineralization in vivo [28]. In the current study however, although significantly increased apoptosis was demonstrated, no difference in mineralization as a result of estrogen withdrawal from osteocytes was found. It is possible that calcification of debris left by apoptotic osteocytes occurs in vivo. Unlike osteoblasts, which lie on the bone surface, the debris from osteocyte apoptosis is not accessible to phagocytic cells and therefore mineral infilling may occur instead. Indeed, it has been shown recently that there is an increased proportion of hypermineralized lacunae in osteoporotic bone [60]. However, it must be noted that such debris would not persist in vitro due to media changes required to maintain cellular viability. Furthermore the micropetrosis response might not be captured during the duration of the experiments reported here. Therefore, this might explain the absence of altered osteocyte mineralization in our in vitro studies albeit that apoptosis is indeed occurring. Osteocyte apoptosis during estrogen deficiency could also directly negatively affect bone strength and mass since it has been demonstrated that the bone sparing effect of estrogen on trabecular bone in male mice is mediated through the estrogen receptor α in osteocytes [3]. Furthermore, estrogen deficiency has been shown to increase osteocyte apoptosis due to oxidative stress in a similar manner as occurs during aging [5]. Since osteocytes are mechanosensitive cells [61], increased apoptosis may impair bone’s ability to adaptively respond to mechanical loading and to repair micro-damage.

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