Selective Estrogen Receptor Modulator Inhibits Osteocyte Apoptosis ...

Calcif Tissue Int (2007) 81:139–144 DOI 10.1007/s00223-007-9049-6

Selective Estrogen Receptor Modulator Inhibits Osteocyte Apoptosis during Abrupt Estrogen Withdrawal: Implications for Bone Quality Maintenance C. Huber Æ S. Collishaw Æ J. R. Mosley Æ J. Reeve Æ B. S. Noble

Received: 27 February 2007 / Accepted: 6 June 2007 / Published online: 20 July 2007 Ó Springer Science+Business Media, LLC 2007

Abstract Estrogens exert positive effects on the quantity and quality of bone, including the maintenance of osteocytes through the inhibition of their apoptosis. Ideally, selective estrogen receptor modulators (SERMs) confer all of the positive bone-associated effects of estrogens without any adverse effects. In a similar way to estrogen, the raloxifene analog LY 117018 has been shown to prevent bone loss in ovariectomized (OVX) rats. In this study, we investigated whether the osteocyte-sparing effect of 17bestradiol can be mimicked by the SERM LY 117018 in a rat model of OVX. Twenty-four juvenile female rats were divided into four treatment groups: sham-operated (SHAM), OVX, OVX + 17b-estradiol (OVX+E2), and OVX + LY 117018 (OVX+SERM). At 7 or 14 days following the start of treatment, the radius and ulna were removed. The percentage of apoptotic osteocytes, determined using an in situ nick-translation method, was increased (2.5–fold at 7 days and sixfold at 14 days) in the OVX group compared with SHAM in both the radius and ulna. Treatment of OVX animals with either 17b-estradiol at a dose rate of 0.125 mg/kg/day or LY 117018 at a dose rate of 3 mg/kg/day prevented these increases in osteocyte apoptosis similarly. These observations demonstrate that

C. Huber S. Collishaw J. R. Mosley B. S. Noble (&) Musculoskeletal Tissue Engineering Collaboration, Department of Medicine, University of Edinburgh, 49 Little France Crescent, Chancellor’s Building, University of Edinburgh, Edinburgh EH16 4SB, UK e-mail: [email protected] J. Reeve Bone Research Division, University Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Level 5, Box 157, Cambridge CB2 2QQ, UK

LY 117018 exerts a powerful inhibitory effect upon osteocyte apoptosis directly after estrogen loss, in a similar way to the known effect of 17b-estradiol replacement. These results point to the potential benefits of SERMs on both the quantity and quality of bone in E2-depleted rats. Keywords Osteocyte Apoptosis 17b-Estradiol Selective estrogen receptor modulator

The presence of osteocytes within bone has been associated with its ability to respond to mechanical stimuli [1], remodel efficiently [2–6], and repair accumulated microdamage [7–9]. Estrogen withdrawal due to either menopause or ovariectomy (OVX) is associated with bone loss [10–12] and a high prevalence of osteocyte death [13, 14]. Previous studies have identified an increase in the proportion of apoptotic osteocytes in OVX rats and in women undergoing estrogen suppression when treated for endometriosis with gonadotropin-releasing hormone analogs. Selective estrogen receptor modulators (SERMs) have been shown to mimic the positive effects of estrogen on bone and cholesterol metabolism without being associated with its negative effects on reproductive tissues such as the uterus and breast [15]. During the last decade, raloxifene has effectively replaced estrogen as a treatment against postmenopausal osteoporosis [16]. The SERM LY 117018, a structural analog of raloxifene, has been shown to maintain bone mineral density in oophorectomized rats [17–19] and reduce the increased bone turnover associated with estrogen deficiency [20], as evidenced by the reduced production of biochemical markers of bone turnover [17, 18]. We present evidence that the SERM LY 117018 can prevent osteocyte apoptotic death induced by suppressing estrogen in a rat model of OVX.

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Materials and Methods Animals and Treatments Twenty-four weight-matched (range 250–270 g) female Sprague-Dawley rats were purchased (Charles River, Margate, UK) a minimum of 7 days before the start of the experiment. A standard rodent diet (RM1; Special Diet Services, South Witham, UK) and tap water were available ad libitum. They were randomized into four treatment groups (n = 6 per group), and each group was subdivided to provide two time points for analysis (7 and 14 days) such that each subgroup contained three animals. At day 0, each animal was anesthetized. Three of the treatment groups underwent OVX and an osmotic pump was implanted subcutaneously (Alzet, Cupertino, CA). The pump delivered one of three treatments: 17b-estradiol (Calbiochem, Nottingham, UK) at a dose rate of 0.125 mg/kg/day (OVX+E2), SERM 117018 (Eli Lilly, Indianapolis, IN) at a dose rate of 3 mg/kg/day (OVX+SERM), or vehicle alone (b-hydroxycyclodextrin). The fourth (control) group was subjected to sham OVX and an osmotic pump was implanted to deliver vehicle alone (SHAM+VEH). After either 7 or 14 days, as dictated by the experimental subgroup, the animals were killed. The radius and ulna were harvested, briefly immersed in 5% polyvinyl alcohol (Sigma, Poole, UK) and chilled to –70°C in supercooled hexane (BDH, Poole, UK) prior to storage and analysis. All animal experimentation was conducted in compliance with national ethical guidelines.

C. Huber et al.: SERM Inhibits Osteocyte Apoptosis

available at http://www.rsb.info.nih.gov/nih-image/). In all cases, a total of at least 1,000 osteocytes were assessed per bone. Nick-Translation Assay for In Situ Analysis of Osteocyte Apoptosis The percentage of osteocytes demonstrating significant levels of DNA fragmentation, a characteristic of apoptosis, was determined using the previously described DNA nicktranslation technique [21]. Briefly, bone sections were transferred to Tespa-coated slides and fixed in 4% paraformaldehyde (Sigma, Poole, UK). Sections were demineralized in 0.25 mol/L ethylenediaminetetraacetic acid (EDTA, pH 7.4), (Sigma, Poole, UK) for 10 minutes, washed three times in phosphate-buffered saline, and subsequently incubated in the nick-translation mix (3 mmol/L digoxigenin [DIG]-labeled deoxyuridine triphosphate; 3 mmol/L each of deoxyguanosine triphosphate, deoxyadenosine triphosphate, and deoxycytosine triphosphate; 50 mmol/L Tris HCl, pH 7.5; 5 mmol/L MgCl2; and 0.1 mmol/L dithiothreitol and 0.5 mL/100 mL DNA polymerase I) for 45 minutes at 37°C. Sections were incubated with fluorescein isothiocyanate (FITC)-labeled anti-DIG antibody (all reagents obtained from Roche, Lewes, UK) for 1 hour at room temperature and counterstained with PI at 2.5 ng/mL. Negative control sections were provided by treatment with the nick-translation mixture in the absence of DNA polymerase I. In Situ Cell Viability Assessment

Quantification of Osteocyte Viability and Apoptosis Transverse undecalcified cryostat sections of 7 lm thickness were obtained from the region of the secondary spongiosa with cortical medullary distinction moving in the direction of the diaphysis for 2.5 mm. The sampling site used was approximately 0.6 mm below the base of the growth plate in order to exclude primary spongiosa. Three nonconsecutive sections from each bone were used to calculate the percentage of apoptotic osteocytes by estimating the number of apoptotic osteocytes (nicktranslation assay, see below) relative to the total number of these cells which were stained positive with propidium iodide (PI). Adjacent sections were used to determine the density of viable osteocytes, i.e., the number of lactate dehydrogenase (LDH)-positive osteocytes per millimeter squared, using the in situ LDH viability assay (see below). A minimum of nine nonoverlapping fields of view were randomly selected from each section so that generally >70% of the total bone area in each section was analyzed using the software package NIH image (developed at the National Institutes of Health and

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Viable cells were identified in cryostat sections by virtue of their LDH activity. Histochemical staining was undertaken using the method of Noble and Stevens [22]. Briefly, sections were incubated in 1.75 mg/mL disodium salt (a-nicotinamide adenine dinucleotide), (Roche, Lewes, UK), 60 mmol/L lactic acid, 3 mg/mL nitroblue tetrazolium (Sigma, Poole, UK) and 40% Polypep (Sigma, Poole, UK) to stabilize (pH 8.0) for 3 hours at 37°C. Sections were rinsed and fixed in 4% paraformaldehyde. Sections were examined under transmitted light, and the number of LDH-positive osteocytes per millimeter squared was quantified using NIH Image. Statistical Analysis All statistical analyses were conducted using quantitative data analysis with SPSS release 11.5 for Windows (SPSS, Chicago, IL). One-way analysis of variance was performed followed by Dunnett’s post hoc test in order to allow comparison of all treatment groups against the control group (SHAM). A Tukey-Kramer post hoc test was


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LY 117018 SERM for 7 days protected osteocytes from OVX-induced apoptosis in both the radius (0.63% vs. 1.27%, respectively) and the ulna (0.53% vs. 1.05%, respectively; P < 0.05) (Fig. 1A). After 14 days, E2 reduced the increase in apoptotic osteocytes due to OVX treatment in the radius (0.38% vs. 1.78%, P = 0.02) and the ulna (0.68% vs. 1.4%, P = 0.013) (Fig. 2B). The percentage of osteocytes displaying apoptotic morphology was, however, higher following treatment with 17b-estradiol compared to SHAM (P = 0.012). Similarly, the proportion of apoptotic osteocytes due to OVX was also decreased following treatment with the LY 117018 SERM for 14 days in both the radius (0.23% vs. 1.78%, P = 0.009) and the ulna (0.16% vs.1.4%, P < 0.001) (Fig. 2B). Osteocyte Viability Fig. 1 In situ demonstration of osteocytes containing fragmented DNA in response to OVX. Section of cortical bone was reacted with the nick-translation mixture and counterstained with PI (red) to identify nuclear DNA. White arrows illustrate osteocytes staining positive for DNA fragmentation, identified as an orange signal, whereas FITC-green signal was colocalized with PI nuclear staining (x20). Bar = 100 lm

employed to test for comparisons between the treatment groups. Results are expressed as means ± standard error (SE), and P < 0.05 (two-way) was considered to be statistically significant.

Osteocyte viability was demonstrated using the presence of LDH demonstrated histochemically as the number of viable osteocytes per millimeter squared (Fig. 3). As previously noted [14], OVX did not affect the proportion of viable osteocytes in either the radius or the ulna after 7 or 14 days relative to SHAM (P > 0.05 in all cases) (Fig. 4A, B). Further, treatment with 17b-estradiol or LY 117018 had no effect on the number of viable osteocytes at 7 or 14 days postoperation in either of the two bones compared to SHAM animals (Fig. 4A, B). Percent Change in Body Weight

Results OVX Induces Osteocyte Apoptosis in a TimeDependent Manner Osteocyte apoptosis was demonstrated in situ using the nick-translation technique (Fig. 1). After 7 days of surgery, OVX led to a 2.5-fold increase in the percentage of apoptotic osteocytes compared to SHAM in both the radius (P = 0.02) and the ulna (P = 0.015) as estimated using nick translation (Fig. 2A). Fourteen days after OVX, osteocyte apoptosis was further increased to sixfold compared to SHAM in both the radius (P = 0.01) and the ulna (P < 0.001) (Fig. 2B). 17b-Estradiol and LY 117018 SERM Prevent OVXInduced Osteocyte Apoptosis Slow release of 17b-estradiol (OVX+E2) at a dose of 0.125 mg/kg/day for 7 days following OVX reduced the percentage of OVX-induced apoptotic osteocytes in both the radius (0.41% vs. 1.27%, respectively; P = 0.014) and the ulna (0.36% vs. 1.05%, respectively; P = 0.01) to levels similar to SHAM (P > 0.05) (Fig. 2A). Treatment with the

Animals were weighed before treatment and regularly thereafter. As shown in Table 1, no statistically significant difference was observed in the percentage change in body weight at 7 days following OVX. However, by day 14 postsurgery, comparison of the body weights prior to and following treatment revealed significant differences between the four experimental groups (P < 0.05). Therefore, we went on to identify the percentage change in body weight following different treatments. After 14 days, OVX+VEH rats displayed an increase in the percentage rate of body weight gain compared to SHAM (17.21 ± 1.89% vs. 5.74 ± 0.63%, P = 0.003). Treatment with either 17b-estradiol or LY 117018 for 14 days significantly reduced the percentage increase in body weight in OVX rats to SHAM levels or below (–0.34 ± 1.45% and 1.75 ± 0.74% vs. 5.74% ± 0.63, P = 0.035).

Discussion These data show that in a rat model of abrupt estrogen withdrawal administration of the SERM LY 117018 prevents the osteocyte apoptosis associated with OVX.

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Fig. 2 17b-Estradiol and LY 117018 SERM prevent OVXinduced osteocyte apoptosis in situ after (A) 7 and (B) 14 days of treatment in both the radius (left panel) and the ulna (right panel). Sections taken from both the radius and the ulna of all treatment groups (OVX+VEH, SHAM+VEH, OVX+E2, and OVX+SERM) were examined for the presence of fragmented DNA material using an in situ nick-translation technique. Results are expressed as the mean percentage of total osteocytes displaying positive staining for fragmented DNA ± SE. *P < 0.05, ** P < 0.001 relative to SHAM, P < 0.05 relative to OVX+E2 treatment

Fig. 3 Cell viability determined in situ using LDH activity in rat sections. Blue arrow shows a viable LDH-positive osteocyte in proximity to a lacuna with no staining (white arrow) (x20). Bar = 100 lm

LY 117018, which is similar in structure to raloxifene with the exception of having a five- instead of a sixmember amide ring, has been shown to exert positive effects on estrogen-deficient bone, as evidenced by improvement of bone mineral density [17–19] and a

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reduction in the increase in bone turnover due to OVX [18, 20]. However, little is known about the effects of SERMs on osteocytes. These cells have previously been shown to be vulnerable to estrogen loss in two species [13, 14]. Sudden estrogen withdrawal resulted in a substantial increase in the number of osteocytes undergoing apoptosis as evidenced at 24 and 3 weeks [13, 14]. In this study, apoptotic osteocytes in both the radius and the ulna were identified as early as 7 days following OVX [21]. In this study, administration of LY 117018 at the known bone-sparing dose of 3 mg/kg/day [19, 20] resulted in the protection of osteocytes from OVX-induced apoptosis in both the radius and ulna 7 and 14 days following surgery. However, as shown previously [14], the number of cells affected was apparently insufficient to significantly reduce the number of viable osteocytes per millimeter squared compared to control animals. This lack of effect on complete cell death is most likely due to efficient removal of dead or dying cells as a result of the rapid rate of remodeling in rodent bone [13]. In contrast, in more slowly remodeling human bone, the increased osteocyte apoptosis associated with estrogen loss was shown to result in a measurable cumulative loss of osteocytes [14]. The molecular antiapoptotic mechanism of action by which estrogenic compounds prevent osteocyte apoptosis was not addressed in this study. In vitro studies by


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Fig. 4 The number of viable osteocytes (LDH-positive cells) per bone area (mm2) (A) 7 and (B) 14 days postoperation in both the radius (left panel) and ulna (right panel) is not altered by the treatments. Sections taken from both the radius and the ulna of all treatments (OVX+VEH, SHAM+VEH, OVX+E2, and OVX+SERM) were studied for the presence of cellular LDH activity. Results are expressed as the mean ratio of the number of LDH-positive osteocytes per area (mm2) for every treatment

Table 1 Effects of 17b-estradiol and LY 117018 on body weight in OVX rats after 7 and 14 days of treatment Treatments (n = 3)

Percentage change in body weight Day 7

Day 14

SHAM+VEH

3.40 ± 0.63

5.74 ± 0.63

OVX+VEH

7.61 ± 0.59

17.21 ± 1.89*

OVX+E2 (0.125 mg/kg/day)

–4.48 ± 1.13

–0.34 ± 1.45**

OVX+SERM (3 mg/kg/day)

2.83 ± 2.08

1.75 ± 0.74***

For each group, body weights before and after treatments are expressed as mean ± SE *P = 0.003, SHAM+VEH vs. OVX+VEH at day 14; **P = 0.035, SHAM+VEH vs. OVX+E2 at day 14; ***P = 0.035, SHAM+VEH vs. OVX+SERM at day 14

Kousteni et al. [23, 24] have suggested that sex steroids exert nongenotrophic antiapoptotic effects on osteoblasts and osteocytes involving the activation of a mitogen-activated protein signaling pathway. Alternatively, in vitro studies in our group have shown that both 17b-estradiol and LY 117018 are capable of preventing osteocyte apoptosis in a nongenomic, nuclear estrogen receptorindependent manner by exerting antioxidant activities on osteocytes [25]. In conclusion, treatment with the SERM LY 117018 inhibited the increase in osteocyte apoptosis associated with estrogen loss, mimicking the bone-sparing effects of

17b-estradiol in a rodent model of OVX. There is growing interest in the current hypothesis that osteocytes are important in maintaining bone quality and modulating bone formation, possibly through sclerostin secretion [26] and resorption [9]. These results highlight an important bioactivity that may explain part of the action of SERMs in vivo. It is possible that the effects of SERMs in preventing osteocyte apoptosis will in future be harnessed to increase their effectiveness in the treatment of a number of clinical conditions including those of old age. Acknowledgments This project was supported by funding from Eli Lilly (Indianapolis, IN), the Medical Research Council, and Scottish Higher Education Funding Council.

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