Raloxifene therapy interacts with serum osteoprotegerin in postmenopausal women

Maturitas 56 (2007) 38–44

Raloxifene therapy interacts with serum osteoprotegerin in postmenopausal women Enrico M. Messalli ∗ , Giampaolo Mainini, Cono Scaffa, Angela Cafiero, Pier Luigi Salzillo, Angelo Ragucci, Luigi Cobellis Department of Gynaecology, Obstetrics and Reproductive Medicine, Second University of Naples, Largo Madonna delle Grazie 1, 80138 Naples, Italy Received 29 November 2005; received in revised form 7 May 2006; accepted 14 May 2006

Abstract Objectives: Osteoprotegerin (OPG) is a protein expressed by osteoblasts that, linking the receptor activator of nuclear factor ␬B (RANK) ligand (RANKL), produced by osteoblasts, blocks the process of osteoclastic differentiation and modulates osteoclastic apoptosis. Raloxifene (RAL) stimulates the production of OPG from osteoblasts, as demonstrated in vitro, carring out their antiresorption activity, at least in part, as means of the OPG/RANK/RANKL system. The aim of this study was to evaluate in vivo if the RAL treatment of postmenopausal women was associated to changes in serum OPG; moreover, to evaluate the serum changes of bone turnover modulators interleukin-6 (IL-6) and C-telopeptides of type-1 collagen (CrossLaps). Methods: A prospective, randomized, placebo-controlled study was designed. A group of consecutive healthy postmenopausal women (n = 40) referred to II Menopause Centre of the Department of Gynaecology of Second University of Naples for climacteric syndrome was enrolled and divided in two groups: (n = 20) postmenopausal women received for 6 months oral raloxifene (60 mg/day) versus (n = 20) postmenopausal women received placebo tablets. Results: Serum OPG levels in postmenopausal women after RAL treatment are statistically significant increased (P < 0.001) versus baseline (P = 0.007) versus placebo. Conclusions: These in vivo data demonstrate that RAL could improve osteoporosis, also through an increase of OPG production by osteoblasts. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Osteoprotegerin; Raloxifene; Menopause; Osteoporosis

1. Introduction ∗

Corresponding author. Tel.: +39 081 8581149; fax: +39 081 5665610. E-mail address: [email protected] (E.M. Messalli).

The decrease in estrogen circulating levels during the menopausal transition represents the main causes

0378-5122/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2006.05.007

E.M. Messalli et al. / Maturitas 56 (2007) 38–44

of bone loss [1]. A rapid decrease is, however, evident into the first 5–10 years following the menopause [2]. The physiological bone remodelling in this period is characterized mainly by a relevant prevalence of the resorption due to the osteoclastic activity. Estrogens prevent bone loss also by regulating the production of several cytokines that modulate osteoclastic bone resorption, including interleukin6 (IL-6), interleukin-1␤ (IL-1␤), receptor activator of nuclear factor ␬B (RANK) ligand (RANKL), Ctelopeptides of type-1 collagen (CrossLaps) and osteoprotegerin (OPG) by cells of the osteoblastic lineage [3]. OPG is a soluble protein expressed by the osteoblasts. It acts as a decoy receptor for RANKL blocking the process of osteoclast differentiation and modulating apoptosis process in these cells [4,5]. OPG is, therefore, a negative regulator of osteoclastmediated bone resorption. Data obtained in in vitro studies have demonstrated that estrogens stimulate the OPG production by osteoblasts [4,6,7]. However, the role of OPG in physiological bone remodelling and its regulation in vivo is under investigation [8–10]. The interaction of pro-inflammatory cytokines, including IL-6, mainly produced by the stromal cells/osteoblasts in bone tissue, with menopausal decrease of ovarian hormones are not entirely understood, but experimental and clinical studies support a link between the increased activity of these cytokines and postmenopausal bone loss [11,12]. Raloxifene (RAL) is a selective estrogen receptor modulators (SERMs) used to increase bone density, with a molecular mechanism, not well known [13]. An osteoblast release of OPG is stimulated by raloxifene, as well as demonstrated in vitro for estrogens [6,7]; this antiresorptive activity, at least in part, is carried out throughout the OPG/RANK/RANKL system and other cytokines, in particular IL-6. An increase of OPG levels, and on the contrary, an IL-6 fall in concentrations have been demonstrated in in vitro data [4,6,7]. The aim of our study was to evaluate if the modulator effect on the bone turnover of raloxifene treatment in postmenopausal women is associated to changes in the serum levels of OPG, IL-6 and CrossLaps.

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2. Materials and methods 2.1. Study group The study was approved by the Institutional Review Board and informed written consent was obtained from the participants. A group of consecutive healthy postmenopausal women (n = 49) referred to II Menopause Centre of the Department of Gynaecology, Obstetrics and Reproductive Medicine of Second University of Naples for climacteric syndrome was enrolled. The final number of randomized subjects was 40, while 9 patients refused to entry in the study protocol, requiring a specific therapy for low bone density. Women were randomly divided into two groups (using a computer-generated randomization the subjects from numbers 1 to 40 were randomly divided into two groups on a one-to-one basis, either active or placebo group). Active group (n = 20) received for 6 months oral raloxifene (one tablet daily of raloxifene hydrochloride 60 mg: raloxifene 56 mg); placebo group (n = 20) received placebo tablet. The patients were selected according to the following inclusion criteria: spontaneous menopause for at least 1 year, low bone density (T-score < −1). The exclusion criteria were: BMI > 30 (high BMI is not a risk factor for fractures); bone disorders except osteoporosis; use of bone-active agents less than 6 months before the study; use of HRT less than 6 months before the study; uterine-adnexal diseases; contraindications to raloxifene (history of a prior thromboembolic disease, liver disease); allergy to raloxifene. 2.2. Biochemical measurements Serum osteoprotegerin, interleukin-6 and Ctelopeptides of type-1 collagen (CrossLaps) levels were determined; T-score and Z-score at baseline (T0 step) and after 6 months of treatment (T1 step) were measured. A blood sample was obtained by venipuncture between 08:00 and 10:00 a.m., and serum was immediately separated and stored at −20 ◦ C. Serum OPG was measured by enzyme-linked immunosorbent assay—ELISA (Biomedica Gruppe,

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Wien, Austria): OPG as pmol/l. The detection limit of the assay was 0.14 pM, whereas the intra- and interassay CVs were 6.7% and 8.9%, respectively. CrossLaps were assayed by ELISA method (Nordic Bioscience Diagnostic A/S, Herlev, Denmark): CrossLaps as ng/ml. The detection limit of the assays and the intra- and interassay assay variations expressed as CVs were 0.01 ng/ml, 5.3%, and 6.3%, respectively. Serum levels of IL-6 were measured by sandwich ELISA (Bender MedSystems, Vien, Austria): IL-6 as pg/ml. The sensitivity of the assay was 0.7 pg/ml, while the intra- and interassay assay variations expressed as CVs were 6.2% and 7.0%, respectively. Bone mineral density (BMD) evaluation was performed with a quantitative ultrasonometry (QUS) (DBM Sonic Bone Profiler-Igea S.r.l. Carpi, Mo, Italy). Ultrasound frequency of 1.25 MHz was used to measure amplitude-dependent speed of sound (Ad-SoS) through the metaphysis of the proximal phalanx. Measurements were performed on the nondominant hand of the subject at the distal metaphysis of the first phalanges of the last four fingers. The results, reported as m/s, T-score and Z-score, were calculated with the software provided by manufacturer, and correlating the results with a database of the measurements obtained on an Italian population sample. The coefficient of variation (CV) was 1.92%, determined by repeated measurements in a subgroup of 12 subjects (three measurement per person on two different days). The ranges indicative of low bone density for the QUS method were calculated on the basis of the results of a large epidemiological study on European women, indicating as osteopenia, a T-score value of <−l S.D. and as osteoporosis <−3.2 S.D. [14]. 2.3. Statistical analysis All values are depicted as the mean values ± standard deviation (S.D.) and range. The statistical analysis was performed by paired Student t-test and Wilcoxon matched pairs test with nonparametric distribution of values. Statistical analyses were performed using the Statistical Package for Social Sciences version 10.0 (SPSS, Inc., Chicago, IL). All tests were two-sided, and P < 0.05 was regarded as significant.

3. Results The study period of 6 month was ended by 37 patients out of 40 (19 in the active group and 18 in the placebo group). The patient’s characteristics of the two groups (active and placebo group) are described in Table 1. In the active group at baseline (T0 step), serum OPG levels were respectively, 3.37 ± 0.55 pmo/l (2.61–4.70), IL-6 5.88 ± 4.76 pg/ml (0.70–15.93) and CrossLaps 0.53 ± 0.17 ng/ml (0.26–0.83). After 6 months of raloxifene treatment (T1 step), serum OPG levels significantly rise in concentration: 4.57 ± 1.08 pmol/l (2.04–5.79) versus baseline values or placebo group (P < 0.001 versus baseline and P = 0.007 versus placebo). At the same time, after raloxifene treatment, IL-6 and CrossLaps serum levels registered a not statistical significant change (P > 0.05): 4.81 ± 3.43 pg/ml (0.23–12.36) and 0.46 ± 0.27 ng/ml (0.10–0.86), respectively (Table 2). The mean percentage changes in serum OPG, IL-6 and CrossLaps after 6 months treatment with raloxifene versus placebo were depicted in Fig. 1. T-score and Z-score at T1 step registered a not statistical significant change (P > 0.05) from

Table 1 Baseline characteristics of the participants by intervention groups (mean ± S.D.)

Ages (year) Body mass index (kg/m2 ) Age of menopause (year) Duration of menopause (year) Serum OPG (pmol/l) Serum IL-6 (pg/ml) Serum CrossLaps (ng/ml) T-score (phalangeal ultrasonography) Z-score (phalangeal ultrasonography)

Active group n = 19

Placebo group n = 18

55.7 ± 6.1 26.1 ± 1.8

54.9 ± 5.8 26.7 ± 2.0

48.5 ± 4.5

48.1 ± 4.3

7.0 ± 6.0

6.7 ± 5.8

3.37 ± 0.55 5.88 ± 4.76 0.53 ± 0.17

3.27 ± 0.61 5.49 ± 4.71 0.52 ± 0.19

−3.61 ± 1.00

−3.54 ± 0.95

−2.15 ± 0.70

−2.13 ± 0.69

OPG, Osteoprotegerin; IL-6, interleukin-6; telopeptides of type-1 collagen. P > 0.05.

CrossLaps,

C-

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Table 2 Serum osteoprotegerin (OPG), interleukin-6 (IL-6) and C-telopeptides of type-1 collagen (CrossLaps) at baseline and after 6 months treatment with raloxifene 60 mg/day vs. placebo (mean ± S.D.) Active group

(pmol/l)*

OPG IL-6 (pg/ml)** CrossLaps (ng/ml)** * **

Placebo group

T0(baseline)

T1(6 months)

T0(baseline)

T1(6 months)

3.37 ± 0.55 5.88 ± 4.76 0.53 ± 0.17

4.57 ± 1.08 4.81 ± 3.43 0.46 ± 0.27

3.27 ± 0.61 5.49 ± 4.71 0.52 ± 0.19

3.63 ± 0.92 5.09 ± 3.75 0.50 ± 0.24

P < 0.001 vs. baseline, P = 0.007 vs. placebo. P > 0.05.

Fig. 1. Percental changes in serum osteoprotegerin (OPG), interleukin-6 (IL-6) and C-telopeptides of type-1 collagen (CrossLaps) after 6 months treatment with raloxifene 60 mg/day vs. placebo (mean).* P < 0.001 vs. baseline, P = 0.007 vs. placebo, ** P > 0.05.

−3.61 ± 1.00 to −3.67 ± 0.92 and from −2.15 ± 0.70 to −2.37 ± 0.98, respectively (Table 3).

4. Discussion Our results in postmenopausal women confirm the hypothesis that Raloxifene treatment interacts with OPG levels. Estrogens act on bone with a twice activity [15]: one mediated by the estrogen receptors present on the bone cell [6,7,16], the other, indirect and delayed,

that acts through a change of calcium metabolism both at level of intestine, kidneys and parathyroids [17]. Estrogen deficiency determines an inhibition of mature osteoblasts and osteocytes, while it promotes faster osteoblastic apoptosis [18]. At the same time, estrogen deficit interferes with the osteoclastic lineage increasing its recruitment and activation due to the stimulation of osteoblast cytokines release: IL-1, IL6, TNF-␣ [19,20]. The interference on the osteoclastic activity acts also through a reduction of TGF-␤ and osteoprotegerin production.

Table 3 T-score and Z-score (phalangeal ultrasonography) at baseline and after 6 months treatment with raloxifene 60 mg/day vs. placebo (mean ± S.D.) Active group

T-score Z-score P > 0.05.

Placebo group

T0(baseline)

T1(6 months)

T0(baseline)

T1(6 months)

−3.61 ± 1.00 −2.15 ± 0.70

−3.67 ± 0.92 −2.37 ± 0.98

−3.54 ± 0.95 −2.13 ± 0.69

−3.71 ± 0.95 −2.37 ± 0.94

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The importance of OPG in bone metabolism is suggested by the positive correlation between the gene production of OPG [5] and the bone mass increase in animal models [1,21]. In humans, OPG is implicated in the pathogenesis of postmenopausal osteoporosis [22] and of other metabolic diseases, characterized by bone loss [23]. In fact, in a group of women monitored for thyroid carcinoma, postmenopausal patients showed higher levels of serum OPG and lower levels of BMD compared to premenopausal patients [24]. Other authors [8] state that circulating OPG levels reflect an antiresorptive activity in bone or are significantly related to BMD [25], while a negative relationship between OPG and bone turnover status have been reported [26–28]. In the last few years, many studies were published focusing the attention of the media on possible side effects of hormone replacement therapy on breast [29] and cardiovascular system [30,31]. Consequently, more data have been provided in patients with osteoporosis by the use of a class of drugs called SERMs, of which RAL was the first validated in the reduction of vertebral fractures in osteoporotic menopausal women [32,33]. RAL acts on bone in a similar manner to estrogens, but its osteoblastic actions remain to be fully clarified. Studies in vitro show that RAL modulates the bone homeostasis inhibiting osteoclastogenesis and the bone resorption with dose-dependent activity [7]. In fact, the inhibition of osteoclastic differentiation happens only at low concentration. The mechanism, however, seems to be more complex so that it could imply direct and indirect activities on bone cells [6,17], and a down-regulation of the osteoblastic production of IL-6 and other cytokines has been suggested [4,7]. RAL stimulates the production of OPG and inhibits the production of IL-6 from cultured human osteoblasts, confirming the hypothesis that RAL stimulant action on the osteoblastic differentiation is linked to an increased production of OPG [6]. On the contrary, RAL and estradiol seem to downregulate the OPG production by human osteoblasts in culture [4,34]. This effect is probably due to the lower expression of ER in cell cultured by these authors. In in vitro model, OPG protein release is higher in cells overexpressing ER-␣, because RAL acts mainly on ER-␣, and therefore, these effects might be the consequence

of different expression of ER-␣ by human osteoblasts [4,7,9,16,35,36]. For the first time, our in vivo data confirm in vitro results (Table 2) [6,7,9]; in fact, a statistically significant serum OPG level increase (P < 0.001 versus baseline and P = 0.007 versus placebo) is showed in postmenopausal women with low bone density taking RAL. The postmenopausal osteoporosis improving throughout RAL administration is probably modulated by OPG production. The link between OPG levels and bone markers is also under debate. Rogers et al. [37] hypothesize a significant positive relationship between OPG and bone mineral density at total body, and a significant weak positive relationship between circulating OPG and serum estradiol has been showed. They also suggest that circulating levels of OPG may reflect OPG activity in bone and are related to circulating endogenous levels of estradiol [37]. This statement is questioned by other authors [8,38], because circulating OPG levels are depended on several factors and paracrine mechanisms involved in osteoporosis genesis [8] and on non-skeletal sources [26]. This last hypothesis might also explain the different results reported by other authors [10,26] about serum OPG after hormonal or RAL therapy. In particular, one of the main differences between our group and Bashir’s subjects is the mean age. It is possible that in the elderly women, the different expression of ER is responsible of a different biological answer to RAL therapy. The decreases of IL-6 and CrossLaps are in line with in vitro results [4,6,39], but the variations are not significant (P > 0.05). This study is extended to a limited number of patients, and the results about these two markers should be confirmed in a more size sample [40]. In fact, the S.D.s of IL-6 and CrossLaps parameters are relatively higher compared to OPG, with data not powered in order to define the possible effect of therapy. Although 6 months are not sufficient for clinically evident BMD changes, it is easier to detect a wide change of biochemical bone marker than a narrow change of BMD with a bone densitometer [41]. Low bone density assessments have been stated with an ultrasound method, not considered as the gold standard in the diagnosis of the low bone density women, but able [42] to determine bone density decrease related

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to sexual hormone deficit in respect to the conventional DEXA technique. [5]

5. Conclusion [6]

Raloxifene treatment in postmenopausal women shows a significant increase in OPG levels after 6 months of therapy, providing for the first time in vivo data. This report could be directly related to the hypothesis about the RAL therapeutic effect on bone loss in postmenopause mediated by OPG antiresorptive actions. The bone turnover is reduced by RAL, with a consequent increase in the bone mass through a direct positive action on the differentiation and maturation of the osteoblasts, as demonstated in vitro [6,7]. The indirect action of RAL mediated by OPG is evident through a slower differentiation and a faster apoptosis of the osteoclasts. In conclusion, these hypothesis might contribute to the better understanding of RAL action mechanism, opening further therapeutic use of OPG or antiRANKL drugs, as already tested by other authors [9,43].

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