Effects of 17β-estradiol on adiponectin regulation of the expression of osteoprotegerin and receptor activator of nuclear factor-κB ligand

Bone 51 (2012) 515–523

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Original Full Length Article

Effects of 17β-estradiol on adiponectin regulation of the expression of osteoprotegerin and receptor activator of nuclear factor-κB ligand Qing-Ping Wang b, Li Yang a, Xian-Ping Li c, Hui Xie a, Er-Yuan Liao a, Min Wang c,⁎, Xiang-Hang Luo a,⁎⁎ a b c

Institute of Endocrinology & Metabolism, The Second Xiangya Hospital of Central South University, 139# Middle Renmin Road, Changsha, Hunan 410011, PR China Department of Clinical Laboratory, The Shaoxing Hospital of China Medical University, 1# Huayu Road, Shaoxing County, Zhejiang 312030, PR China Department of Clinical Laboratory, The Second Xiangya Hospital of Central South University, 139# Middle Renmin Road, Changsha, Hunan 410011, PR China

a r t i c l e

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Article history: Received 16 December 2011 Revised 23 March 2012 Accepted 16 May 2012 Available online 23 May 2012 Edited by: M. Noda Keywords: Estrogen Adiponectin OPG RANKL

a b s t r a c t Adiponectin may exert a negative effect on bone metabolism by regulating osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) expression. However, the action of adiponectin on bone may be influenced by estrogen in women. The present study was undertaken to investigate the effects of 17β-estradiol (E2) on adiponectin-regulated OPG and RANKL expression in human osteoblast. Human osteoblasts were treated with α-MEM containing 10 μg/ml adiponectin alone or together with 10− 10 to 10− 8 M E2 for 12–48 h. Cells were also treated with α-MEM containing 10 μg/ml adiponectin together with 10− 8 M E2 plus p38 agonistanisomycin or estrogen receptor (ER) antagonist ICI182780 for 48 h. The effects of E2 were also investigated by knockdown of ERs or overexpression of p38 MAPK in osteoblasts. Further, we examined the effects of E2 on adiponectin-dependent osteoclastogenesis by the co-culture systems of osteoblast and CD14+ peripheral blood monocytes (PBMCs). Real-time quantitative PCR (RT-PCR) and ELISA were used to detect OPG/RANKL mRNA and their corresponding protein expression, Western Blot was used to analyze the phosphorylated p38 (p-p38) levels. The results showed that E2 blocked adiponectin-induced p38 phosphorylation, decreased adiponectin-regulated OPG/RANKL mRNA and protein expression in a dose- and time-dependent manner. ICI182780 or knockdown of ERs abolished the effects of E2 on adiponectin-dependent p38 phosphorylation and OPG/RANKL expression. Furthermore, anisomycin or overexpression of p38 also reserved the effects of E2 on adiponectin-dependent p38 phosphorylation and OPG/RANKL expression. E2 inhibited adiponectindependent osteoclastogenesis in the co-culture systems of osteoblast and CD14+ PBMCs, whereas anisomycin, ICI182780, knockdown of ERs and overexpression of p38 significantly reversed this response. In conclusions, our findings demonstrated, through blocking the activation of adiponectin-induced p38 MAPK, E2 suppressed the adiponectin-regulated OPG/RANKL expression and then inhibited osteoclastogenesis, which suggested that estrogen would suppress the effect of adiponectin on bone metabolism. © 2012 Elsevier Inc. All rights reserved.

Introduction Adiponectin, a recently described adipocyte-produced hormone, is highly and specifically expressed in differentiated adipocytes and is abundantly present in plasma [1–3]. Mediated via two adiponectin receptors (AdipoRs), AdipoRs 1 and 2, adiponectin increases the activities of AMP kinase, mitogen-activated protein kinase (MAPK), and peroxisome proliferator-activated receptors-α ligand, playing significant roles in physiology and pathophysiology [4–7].

⁎ Correspondence to: M. Wang, Department of Clinical Laboratory, The Second Xiangya Hospital of Central South University, 139# Middle Renmin Road, Changsha, Hunan 410011, PR China. Fax: + 86 731 85292142. ⁎⁎ Correspondence to: X.H. Luo, Institute of Endocrinology & Metabolism, The Second Xiangya Hospital of Central South University, 139# Middle Renmin Road, Changsha, Hunan 410011, PR China. Fax: +86 731 85295999. E-mail addresses: [email protected] (M. Wang), [email protected] (X-H. Luo). 8756-3282/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2012.05.011

Adiponectin and its receptors have been found to be produced by human bone-forming cells [8], suggesting that adiponectin is a hormone linking bone and fat metabolism. The association between serum adiponectin and bone metabolism had been studied by several groups. Ealey et al. [9] and Peng et al. [10] reported that serum adiponectin was negatively associated with BMD at a number of skeletal sites even after adjusting for fat mass. Jürimäe et al. [11] similarly reported a significant negative association between adiponectin and whole body BMC and BMD as well as lumbar spine BMD. In elderly men, adiponectin was associated with low BMD [12,13]. These clinical findings suggested that adiponectin had a negative effect on bone metabolism. Furthermore, our recent study in vitro demonstrated that adiponectin could stimulate receptor activator of nuclear factor-κB ligand (RANKL) and inhibit osteoprotegerin (OPG) expression in human osteoblasts through the AdipoR1/p38 MAPK pathway, and these responses contributed to the adiponectin-induced osteoclast formation [14]. Thus, adiponectin could be exerting its effect on bone metabolism through promotion of the bone-resorbing RANKL pathway.

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Nevertheless, adiponectin has been demonstrated to have divergent effects on bone metabolism in pre- and postmenopausal women. Some studies showed that serum adiponectin was found to be a predictor of BMD in postmenopausal women rather than premenopausal women [15–17]. In a study on adolescent women, although a negative correlation was found between the adiponectin levels and BMD, it was not apparent after adjustment for fat mass [18]. Consistent with the study by Richards et al. [15] and others [16,17], our most recent study found that the relationship between serum adiponectin and BMD was negatively significant only in postmenopausal Chinese women but not in premenopausal women; furthermore, in that study, the significant positive correlations between adiponectin and bone-specific alkaline phosphatase (BAP), bone cross-linked N-telopeptides of type I collagen (NTX) were found only in postmenopausal women [19]. Taken together, these data indicated that menopausal status may influence the relationship between adiponectin and bone metabolism. Encouraged by our previous findings that adiponectin is able to exert its effect on bone metabolism through promotion of the bone-resorbing RANKL pathway [14] and the facts that menopausal status may influence the relationship between adiponectin and bone metabolism, we further undertook in vitro to assess the effects of 17β-estradiol (E2) on adiponectin regulation of human osteoblast OPG/RANKL expression. Materials and methods Reagents Recombinant human adiponectin was purchased from R&D systems, Inc. (Minneapolis, MN, USA). Recombinant human OPG and RANKL were purchased from R&D systems (Minneapolis, MN, USA). OPG and RANKL protein ELISA kits were purchased from Biomedica Group (Windham, NH, USA). E2, anisomycin, ICI 182780, TRACP staining assay, and 1α,25-dihydroxyvitamin D3 (1,25 vitD) were purchased from Sigma (St Louis, MO, USA). Adiponectin-free fetal bovine serum (FBS) was prepared by the passage of FBS through anti-adiponectin antibody Seharose 4B affinity columns (Amersham Pharmacia Biotech) to remove adiponectin as described previously [20]. Primary human osteoblast cultures Bone samples were obtained with informed consent from donors and after approval by the Local Research Ethics Committee. Primary cultures of normal human osteoblast were prepared from trabecular bone obtained during surgery following road traffic accident victims as previously described [21–23]. Briefly, samples were rinsed extensively with serum-free α-MEM (Sigma Chemical Corp., St. Louis, MO, USA) and digested with type IV collagenase (Sigma). The digested chips were cultured in phenol red-free α-MEM containing 10% fetal bovine serum (FBS, Gibco-BRL Corp., Grand Island, NY, USA), 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml ascorbic acid at 37 °C. After 15 days, the cells migrated from within the bone particles and reached confluence after 25 days. They were then passaged and subcultured in α-MEM containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ ml ascorbic acid (Sigma). These cultured human osteoblasts were characterized and display an osteoblastic phenotype, as previously described [21–23]. Real-time quantitative PCR assay for OPG and RANKL mRNA expression In experiments, human osteoblasts were cultured in 25 cm2 flasks in α-MEM containing 10% FBS and 50 μg/ml ascorbic acid. After day 4 of culture, cells were treated with vehicle (serum-free medium) or 10 μg/ml adiponectin or 10 μg/ml adiponectin plus 10 − 10 to 10 − 8 M E2 for 48 h. Cultures were also exposed to E2 at 10 − 8 M with or without 10 μg/ml adiponectin for 12–48 h.

Real-time quantitative PCR analysis was done using Roche Molecular LightCycler (Roche Applied Science, Indianapolis, IN, USA) as described previously [21–23], which is a combined thermal cycler and fluorescence detector with the ability to monitor the progress of individual PCR reactions optically during amplification. Total RNA from cultured cells was isolated using Trizol reagent (Gibco), and reverse transcription was performed using 1.0 μg total RNA and the reverse transcription system (Promega, Madison, WI, USA). Amplification reactions were set up in 25 μl reaction volumes containing amplification primers and SYBR Green PCR Master Mix (PE Applied Biosystems). A 1 μl volume of cDNA was used in each amplification reaction. Preliminary experiments were carried out for primer concentration optimization. Primer sequences are detailed as previously described. For OPG, the PCR primers were 5-CGTCAAGCAGGAGTGCAATC-3 and 5-CCAGCTTGCACCACTCCAA-3, yielding a 126 bp fragment. For RANKL, the PCR primers were 5-TCGTTGGATCACAGCACA TCA-3 and 5-TATGGGAACCAGATGGGATGTC-3, yielding a 141 bp fragment. For β-actin, the PCR primers were 5-CCCAGCCATGTACGTTGCTA-3 and 5-AGGGCATA CCCCTCGTAGATG-3, yielding a 126 bp fragment. PCR was performed as follows: 94 °C for 15 s, 60 °C for 10 s, and 72 °C for 10 s for 40 cycles followed by a 10 min incubation at 72 °C. The identities of PCR products were confirmed by direct sequencing using an automatic DNA sequence (PE Applied Biosystems). Amplifications were performed, and calibration curves were run in parallel in triplicates for each analysis. Each sample was analyzed six times during each experiment. The experiments were carried out at least twice. Amplification data were analyzed using the Sequence Detector System Software (PE Applied Biosystems). Relative quantification was calculated by normalizing the test crossing thresholds (Ct) with the β-actin amplified control Ct. The results were normalized to β-actin and expressed as percentage of controls. OPG and RANKL protein expression assay The human osteoblasts were cultured in 24-well plates (5×104 cells/ well) in α-MEM containing 10% FBS and 50 μg/ml ascorbic acid. After day 4 of culture, cells were treated with vehicle (serum-free medium) or 10 μg/ml adiponectin or 10 μg/ml adiponectin plus 10− 10 to 10− 8 M E2 for 48 h. Cultures were also exposed to E2 at 10− 8 M with or without 10 μg/ml adiponectin for 12–48 h. For investigating the RANKL protein expression in cultured osteoblasts, the cell layers were homogenated with Triton lysis buffer (50 mM Tris–HCl, pH 8.0 containing 150 mM NaCl, 1% Triton X-100, 0.02% sodium azide, 10 mM EDTA, 10 μg/ml aprotinin, and 1 μg/ml aminoethylbenzenesulfonyl fluoride). Then the conditioned media were collected for RANKL protein expression assay using RANKL ELISA (Biomedica Group, Windham, NH, USA). OPG protein expression was determined through measuring the levels of OPG in the culture media using OPG ELISA (Biomedica Group, Windham, NH, USA). Cells were also harvested and counted. RANKL and OPG protein levels were normalized to cell numbers in each well. Detection of p38 MAPK by Western blot analysis Our previous study has shown that adiponectin induces activation of p38 MAPK in osteoblasts [14]. To study the effects of E2 on p38 MAPK signaling pathway in human osteoblasts, Western blot analysis was performed for detecting the activity of p38 kinase. The human osteoblasts were treated respectively with vehicle (serum-free medium), 10− 8 M E2, 10 μg/ml adiponectin, 10 μg/ml adiponectin plus 10− 10 to 10− 8 M E2, 10 μg/ml adiponectin plus 10− 8 M E2 plus 10 ng/ml p38 agonist-anisomycin or 10− 8 M estrogen receptor (ER) antagonist ICI 182 780 for 48 h. Then, cell monolayers were washed quickly with cold PBS containing 5 mM of EDTA and 0.1 mM of Na3VO4, and lysed with a lysis buffer. Protein concentrations

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were determined by Bradford assay. 40 μg of protein from each cell layer was loaded onto a 7.5% polyacrylamide gel. After electrophoresis, the SDS-PAGE separated proteins were transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech.). The membrane was blocked with 5.0% nonfat milk in PBS and incubated with anti-p-p38 and antiβ-actin antibodies at 1:500 in PBS for 2 h. Then, the membrane was then incubated with goat anti-mouse or rabbit IgG conjugated with horseradish peroxidase at 1:1000 in PBS for 1 h. Blots were processed using an ECL Kit (Santa Cruz) and exposed to X-ray film. Densitometries of the bands were scanned and quantified using the Bio-Rad Gel Doc 2000 System (Bio-Rad Laboratories, Hemel Hempstead, UK). Data were normalized against those of the corresponding β-actin bands. Results were expressed as fold increase over control.

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overexpression of p38 MAPK or knockdown of ERs with siRNA osteoblasts was also co-cultured with CD14+ PBMCs to further investigate the effects of E2 on adiponectin-dependent osteoclastogenesis. The cultures were stained for TRACP (a marker enzyme of osteoclasts) and osteoclasts were counted as our previously described [14]. Statistical analyses All experiments were repeated at least six times. SPSS 13.0 was used for the statistical analyses. Data are presented as the mean± SD. Comparisons were made using a one-way ANOVA or paired t test. p b 0.05 was considered statistically significant. Results

Osteoblasts infection with lentivirus One day before infection, osteoblasts were plated in 60-mm-diameter dish and cultured for 24 h in medium without antibiotics. After 24 h of culture, cells were infected and then cultured. siRNAs for ERs (ERα and ERβ) were used to knockdown the expression of ERs in human osteoblasts. The coding sequences of siRNAs for ERs (ERα, 5′-AAAGATTGGC- CAGTACCAATG-3′; ERβ, 5′-AATTCTCCT TCCTCCTACAA-C-3′) were cloned into pGCL-GFP (Genechem, Shanghai, China) to generate pGCL-GFP-siRNA-ERα and pGCL-GFP-siRNA-ERβ. Then, the lentivectors containing the siRNA-ERα and siRNA-ERβ expression construct were co-transfected together with the packaging plasmids of pHelper 1.0 and pHelper 2.0 into 293T cell using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions, to produce lentivirus-siRNA-ERα and lentivirus siRNA-ERβ. The culture supernatants containing the lentivirus were harvested and centrifuged. Osteoblasts grown to 70–80% confluence were infected with the lentiviruses at MOI 30 in the presence of 5 μg/ml Polybrene (Sigma, St. Louis, MO). 48 h after infection, the GFP-positive osteoblasts were sorted and collected by FACS. Cells were also infected with lentiviral control vector (Genechem, China). The infection efficiency was assessed by RT-PCR analysis. To overexpress p38 MAPK in human osteoblasts, the coding sequence of p38 MAPK (the longest version) was cloned into pCDHCMV- MCS-EF1-copGFP (System Bioscience, Mountain View, CA). Then the pCDH-CMV- MCS-EF1-copGFP-p38 MAPK expression construct was co-transfected with the packaging plasmids into 293T cell for 48 h. After transfection, the lentivirus-containing supernatants were harvested and osteoblasts were infected with the lentiviruses for 48 h, and then the GFP-positive osteoblasts were sorted and collected by FACS. RT-PCR analysis was performed to assess the infection efficiency. After infection, osteoblasts were cultured with 10− 8 M E2, 10 μg/ml adiponectin, 10 μg/ml adiponectin plus 10− 8 M E2 for 48 h, respectively. Uninfected osteoblast was cultured with media alone as a control. OPG and RANKL expression were analyzed by RT-PCR and ELISA assay, and the p38 kinase levels were detected by western blotting analysis and normalized to β-actin. Co-culture of osteoblasts and CD14+ PBMCs and osteoclasts formation CD14+ peripheral blood mononuclear cells (PBMCs) were isolated and purified as osteoclast precursors as previously described [14]. Osteoblasts were seeded onto 24-well plates and cultured to subconfluence. CD14 + PBMCs (1 × 10 6 cells/well) were added to osteoblasts as previously described [14] and co-cultured for 14 days in DMEM containing 10% adiponectin-free FBS in the presence of 10 − 7 M 1α, 25-dihydroxyvitamin D3 (1,25 vitD). The cultures were treated with media alone, 10 μg/ml adiponectin, 10 μg/ml adiponectin+ 10− 8 M E2, 10 μg/ml adiponectin +10− 8 M E2+ 10 ng/ml anisomycin, 10 μg/ml adiponectin +10− 8 M E2+10− 8 M ICI 182780 respectively, and treated with 80 ng/ml OPG or/and 20 ng/ml RANKL as controls. In addition,

Effects of E2 dose on adiponectin regulation of OPG and RANKL mRNA expression Fig. 1A shows the effects of E2 on adiponectin-inhibited OPG mRNA expression in a dose-dependent manner in osteoblasts. After 48 h of culture, the OPG mRNA expressions in the cells treated with 10 μg/ml adiponectin alone were significantly smaller than those in α-MEM controls (44.03 ± 4.49% of controls, p b 0.01). When the cells were treated with 10 μg/ml adiponectin together with 10 − 10 to 10 − 8 M E2, the adiponectin inhibition of OPG mRNA expressions was gradually attenuated with the increasing concentrations of E2. Compared to 10 μg/ml adiponectin alone, 10 μg/ml adiponectin together with 10− 10 M E2 caused the OPG mRNA expressions an insignificant increase (p>0.05), while 10− 9 M to 10− 8 M E2 caused significant increases in the OPG mRNA expressions (all p b 0.01). Fig. 1B shows the dose response of the effects of E2 on adiponectinpromoted RANKL mRNA expression in cultured osteoblasts. In cells treated with 10 μg/ml adiponectin alone, after 48 h of culture, the RANKL mRNA expressions were significantly greater than those in α-MEM controls (189.23 ± 13.02% of controls, p b 0.01). However, in cells treated with 10 μg/ml adiponectin together with 10− 10 M E2, the increases of RANKL mRNA expression were attenuated slightly but significantly (p b 0.05) compared to those in cells treated with 10 μg/ml adiponectin alone. While in cells treated with 10 μg/ml adiponectin together with 10− 9 M to 10− 8 M E2, the RANKL mRNA expressions attenuated more greatly (p b 0.01). Effects of E2 dose on adiponectin regulation of OPG and RANKL protein expression Fig. 2 shows the effects of E2 on the adiponectin-inhibited OPG and adiponectin-stimulated RANKL protein expressions in osteoblasts. In cells treated with 10 μg/ml adiponectin alone, the concentrations of OPG were more than three times lower than those in α-MEM controls (2.93± 0.20 ng vs. 9.43 ±1.61 ng, pb 0.01), whereas the RANKL levels were nearly twice higher than those in α-MEM controls (2.29 ± 0.21 ng vs. 1.34 ±0.15 ng, p b 0.01). However, the cells treated with adiponectin together with E2 (including the cells treated with 10 μg/ml adiponectin alone, 10 μg/ml adiponectin together with 10− 10 to 10− 8 M E2) had a significant increase in OPG but decrease in RANKL protein productions in an E2 dose-dependent manner (both p b 0.01). Effects of the time course of E2 on adiponectin regulation of OPG and RANKL expression Figs. 3A, B shows the effects of the time course of E2 on OPG mRNA and protein expressions in osteoblasts treated with 10 μg/ml adiponectin plus 10− 8 M E2 (the cells treated with 10 μg/ml adiponectin alone as controls). Compared to the controls, after 12 h of culture, the OPG mRNA and protein increased slightly (116.5±6.50% and 121.17±6.93% of controls, pb 0.05); after 24 h or 48 h in culture, the OPG mRNA expression

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A

B

Fig. 1. Effects of E2 dose on adiponectin regulation of OPG and RANKL mRNA expression in human osteoblast. Cells were exposed to α-MEM control, 10 μg/ml adiponectin, 10 μg/ml adiponectin 10− 8 M E2 plus 10− 10 to 10− 8 M E2 for 48 h. RANKL and OPG mRNA expression were determined by RT-PCR. Results are expressed as % of controls. A: The dose response of E2 on adiponectin-regulated OPG mRNA expression in osteoblast. The bar represents the mean±SD (n=6) a, b pb 0.01, vs. α-MEM control; *pb 0.01, vs. 10− 8 M E2 treatment; #pb 0.01, vs. 10 μg/ml adiponectin treatment. B: The dose response of E2 on adiponectin-regulated RANKL mRNA expression in osteoblast. The bar represents the mean ± SD (n = 6) c, d p b 0.01, vs. α-MEM control; *p b 0.01, vs. 10− 8 E2 treatment; #p b 0.05 or p b 0.01, vs. 10 μg/ml adiponectin treatment. Note: AD, adiponectin.

(147.35±8.88% and 185.47±9.65% of controls respectively, pb 0.01), and the protein expression (155.08±9.97% and 240.10±11.42% of controls, pb 0.01) increased more obviously. Figs. 3C, D shows the effects of the time course of E2 on RANKL mRNA expressions and protein productions in cells treated with 10 μg/ml adiponectin plus 10 − 8 M E2. ANOVA analysis showed, after the cells being cultured for 12 h to 48 h, E2 caused significant decreases in RANKL mRNA expressions and protein productions in a time-dependent manner (both p b 0.01). Compared to the controls, after the cells being cultured for 12 h to 48 h, the RANKL mRNA expressions decreased by 9.13 ± 3.19%, 20.28 ± 4.13% and 51.09 ± 4.91%

A

respectively (all p b 0.01), and the RANKL protein productions decreased by 7.93 ± 2.07%, 21.83 ± 4.07% and 44.10 ± 4.84% respectively (p b 0.05 or p b 0.01). These results showed, with time, E2 gradually attenuated the decrease of OPG and increase of RANKL expression regulated by adiponectin.

The effects of E2 on p38 MAPK signaling pathway in human osteoblasts Because our previous study has shown that the adiponectin regulation of OPG and RANKL expression is mediated via p38 MAPK pathway,

B

Fig. 2. Effects of E2 dose on the adiponectin regulation of OPG and RANKL protein expressions. Cells were exposed to α-MEM control, 10 μg/ml adiponectin, 10 μg/ml adiponectin plus 10− 10–10− 8 M E2 for 48 h. RANKL and OPG protein were determined by ELISA. Results are expressed as ng/5 × 104 cells. A: The effects of E2 on adiponectin-inhibited OPG protein expressions in human osteoblast. The bar represents the mean ± SD (n= 6) a, b p b 0.01, vs. α-MEM control; *p b 0.01, vs. 10− 8 M E2 treatment; #p b 0.01, vs. 10 μg/ml adiponectin treatment. B: The effects of E2 on adiponectin-stimulated RANKL protein productions in human osteoblast. The bar represents the mean± SD (n= 6) c, d p b 0.01, vs. α-MEM control; *p b 0.05 or 0.01, vs. 10− 8 M E2 treatment; #p b 0.01, vs. 10 μg/ml adiponectin treatment. Note: AD, adiponectin.

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Fig. 3. Effects of the time course of E2 on the adiponectin regulation of OPG and RANKL expression. Cells were exposed to 10 μg/ml adiponectin plus 10− 8 M E2 for 12–48 h. RANKL and OPG mRNA expression were determined by RT-PCR and the proteins were detected by ELISA. Results are expressed as % of controls (the cells treated with 10 μg/ml adiponectin alone). A: The time course of E2 on adiponectin-regulated OPG mRNA expression in osteoblast. B: The time course of E2 on adiponectin-regulated OPG protein expressions in osteoblast. C: The time course of E2 on adiponectin-regulated RANKL mRNA expression in osteoblast. D: The time course of E2 on adiponectin-regulated RANKL protein expressions in osteoblast. The dots represent the percent of controls at every time point (n = 6; *p b 0.05, **p b 0.01, vs. 10 μg/ml adiponectin control).

we examine whether E2 affects the adiponectin-induced p38 MAPK activation. Western blot analysis (Fig. 4A) showed that, treatment of cells with adiponectin + E2, the p38 activities, as demonstrated by phosphorylated p38 (p-p38) levels, were inhibited when compared to that of only treatment with 10 μg/ml adiponectin. Moreover, with the concentrations of E2 increase, the p-p38 levels decreased. However, when 10 ng/ml p38 agonist-anisomycin or 10 − 8 M ER antagonist ICI 182780 was added into the media (10 μg/ml adiponectin plus 10 − 8 M E2), both the levels of p-p38 were similar to that of only treatment with 10 μg/ml adiponectin. These outcomes were further confirmed by lentivirus-infected osteoblasts (Fig. 4B). Compared to osteoblasts treated with 10 μg/ml adiponectin plus 10 − 8 M E2 (Fig. 4A), knockdown of ERs with siRNAs abrogated the effects of E2 on the adiponectin-induced phosphorylation of p38 kinase, while overexpression of p38 MAPK markedly strengthened the actions of adiponectin and then attenuated the effects of E2 on phosphorylation of p38 kinase (Fig. 4B). These data indicate that E2 inhibit adiponectin-induced p38 MAPK activation via ERs in osteoblasts. E2 suppressed adiponectin-regulated OPG and RANKL expression through blocking the adiponectin-induced p38 MAPK activation The results above demonstrated that E2 inhibit adiponectin-induced p38 MAPK activation via ER in osteoblasts, however, E2 may exert its effects through other pathways by binding to the ER. Therefore, we test whether E2 suppresses adiponectin-regulated OPG and RANKL expression through blocking the adiponectin-induced p38 MAPK activation. When p38 agonist-anisomycin was added into the culture with 10 μg/ml adiponectin plus 10 − 8 M E2, the OPG mRNA and protein expressions in human osteoblasts were obviously lower than those of without anisomycin (p b 0.01), while the RANKL mRNA expressions and protein productions were increased markedly (p b 0.01). In p38 MAPK overexpression osteoblasts treated with 10 μg/ml adiponectin

plus 10 − 8 M E2, more significant decrease of OPG mRNA/protein and increase of RANKL mRNA/protein productions were observed. The ER antagonist-ICI 182780 and knockdown of ERs with siRNAs had the similar effects with anisomycin. Moreover, the effects of E2 on adiponectin-regulated OPG/RANKL expression were completely blocked when ICI 182780 was added into the media containing 10 μg/ml adiponectin plus 10− 8 M E2, or when ERs were knockdowned. Compared to those treated with only 10 μg/ml adiponectin, the levels of OPG mRNA, RANKL mRNA, OPG protein and RANKL protein in the cultures treated with above media had no difference (all p>0.05). These results suggested that blocking the adiponectin-induced p38 MAPK activation was a key mechanism by which E2 suppressed adiponectin-regulated OPG and RANKL expression. The effects of anisomycin, ICI 182780, overexpression of p38 MAPK and knockdown of ERs with siRNAs on the OPG/RANKL expressions in osteoblasts cultured with adiponectin and E2 are shown as Fig. 5. Effects of E2 on adiponectin-dependent osteoclastogenesis in co-culture of osteoblasts and CD14+ PBMCs Fig. 6A shows representative microscopic views of the inhibition effect of E2 on adiponectin-dependent osteoclastogenesis. Adiponectin (10 μg/ml) increased TRACP+multinucleated osteoclast formation in co-culture of osteoblasts and CD14+PBMCs compared with media alone (pb 0.01). However, the adiponectin-induced osteoclast formations were suppressed by the presence of E2 (p b 0.01). As controls, 20 ng/ml RANKL induced osteoclast formation obviously, whereas 80 ng/ml OPG blocked the increasing osteoclast formation by adiponectin or RANKL. Fig. 6B shows adiponectin-dependent osteoclastogenesis after impairing the action of E2. When p38 agonist-anisomycin or ER antagonist-ICI 182780 was added into the co-cultures in the presence of adiponectin and E2, the suppression of TRACP + multinucleated osteoclast formation by E2 was reversed. Similarly, knockdown of ERs with siRNAs abrogated the effects of E2 on the adiponectin-induced osteoclast formations. In overexpression of p38 MAPK osteoblasts,

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Fig. 4. Effects of E2 on adiponectin-induced p38 MAPK activation in osteoblast. A. Cells were treated with α-MEM control, 10− 8 M E2, 10 μg/ml adiponectin, 10 μg/ml adiponectin plus 10− 10 M to 10− 8 M E2, 10 μg/ml adiponectin plus 10− 8 M E2 plus p38 agonist-anisomycin or ER antagonist ICI 182780 for 48 h. p-p38 expression was analyzed by western blotting. Results are expressed as folds increase over control. The bar represents the mean ± SD (n = 6) *p b 0.01, vs. α-MEM control. B. After knockdown of ERs or overexpression of p38 MAPK, cells were cultured with 10− 8 M E2, 10 μg/ml adiponectin, 10 μg/ml adiponectin plus 10− 8 M E2 for 48 h. Uninfected osteoblast was cultured with media alone as a control. p-p38 expression was analyzed by western blotting. Results are expressed as folds increase over control. The bar represents the mean ± SD (n = 6) *p b 0.01, vs. media control. Note: AD, adiponectin.

the increase of osteoclast formation was more obvious, suggesting that E2 was not able to suppress the increase of osteoclast formation by adiponectin in these settings. These data showed that E2 suppressed adiponectin-dependent osteoclastogenesis through p38 MAPK- and ERs-regulated OPG/RANKL production. Discussions This in vitro study demonstrated that, through blocking the activation of adiponectin-induced p38 MAPK, E2 inhibited the adiponectin-regulated OPG/RANKL expression. The E2-elicited changes, up-regulation of OPG and down-regulation of RANKL, in adiponectinregulated OPG/RANKL expression suggested that estrogen may be involved in the mediation of the regulations of adiponectin on bone metabolism. Adiponectin has emerged as an element in the regulation of bone metabolism. Our previous in vitro study demonstrated that adiponectin was able to inhibit OPG and stimulate RANKL expression in human osteoblasts, and these responses contributed to the adiponectin-induced osteoclast formation in the co-culture of osteoblast and peripheral blood mononuclear cell systems [14]. The OPG/RANKL system is thought to play a major role in the regulation of osteoclastogenesis and to be the chief mechanism by which osteoblast and osteoclast activities are coupled to coordinate bone formation and bone resorption [24–26]. RANKL, a membrane-bound molecule, is a member of the tumor necrosis factor (TNF) ligand family and has been shown to initiate intracellular signaling transduction directly in the osteoclast precursor or osteoclast, which induces osteoclast differentiation and promotes osteoclast activity [27,28]. OPG, a soluble receptor secreted by osteoblasts and other cell types, acts as a decoy receptor by blocking the interaction of

RANKL with its functional receptor RANK [29], thereby inhibiting osteoclastogenesis. Thus, our previous in vitro study illustrated that adiponectin might promote more bone resorption and then exerted its negative effect on bone metabolism. Nevertheless, clinically relevant studies have indicated that estrogen seems to influence the negative relationship between adiponectin and bone metabolism, since this negative relationship disappears in premenopausal women [15–17]. There are many studies about adiponectin or estrogen in the regulation of bone metabolism, but no study has explored the actual association of estrogen with adiponectin in bone metabolism. Therefore, the effect of estrogen on adiponectin-dependent bone metabolism is unclear. The present study is the first report to investigate the effects of E2 on adiponectin-regulated OPG and RANKL expression measured in the human osteoblast cultures. In the present study, as in our previous report [14], we confirmed again that adiponectin could induce RANKL but inhibit OPG expression in human osteoblast. However, when human osteoblasts were cultured with both E2 and adiponectin, the adiponectin-induced RANKL and adiponectin-inhibited OPG expression were attenuated by E2 in a time- and dose-dependent manner. While, in our coculture systems of osteoblasts and CD14 + PBMCs, E2 inhabited adiponectin-dependent osteoclastogenesis. These results suggested, through up-regulation of OPG expression inhibited by adiponectin and down-regulation of RANKL expression promoted by adiponectin, the high circulating estrogen levels in premenopausal women would attenuate even completely suppress the negative effect of adiponectin on bone metabolism. This is consistent with the clinical findings that adiponectin have divergent effects on bone metabolism in pre- and postmenopausal women [15–17]. Indeed, it is well known that estrogen is an important factor in the maintenance of bone homeostasis, its deficiency at the time of

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Fig. 5. Effects of anisomycin, ICI 182780, overexpression of p38 MAPK or knockdown of ERs on the inhibition of E2 on OPG/RANKL expressions in osteoblast. Osteoblasts were treated with α-MEM, 10− 8 M E2, 10 μg/ml adiponectin, 10 μg/ml adiponectin plus 10− 8 M E2 respectively. Cells were also treated with 10 μg/ml adiponectin plus 10− 8 M E2 plus anisomycin or ICI 182780, or treated with 10 μg/ml adiponectin plus 10− 8 M E2 after overexpression of p38 MAPK or knockdown of ERs. The OPG/RANKL mRNA and protein were detected by RT-PCR and ELISA. A, B: The effects of anisomycin, ICI 182780, overexpression of p38 MAPK or knockdown of ERs on the OPG/RANKL mRNA expressions in osteoblast. Results are expressed as % of α-MEM control; C, D: The effects of anisomycin, ICI 182780, overexpression of p38 MAPK or knockdown of ERs on the OPG/RANKL protein productions in osteoblast. Results are expressed as ng/5 × 104 cells. The bar represents the mean± SD (n= 6). $p b 0.01, vs. the other groups; *p b 0.01, vs. the other groups except 10 μg/ml adiponectin + 10− 8 M E2 + ICI 182780, siRNA + 10 μg/ml adiponectin + 10− 8 M E2 treatment; #p b 0.01, vs. the other groups except 10 μg/ml adiponectin, and siRNA + 10 μg/ml adiponectin + 10− 8 M E2 treatment; &p b 0.01, vs. the other groups except 10 μg/ml adiponectin, 10 μg/ml adiponectin + 10− 8 M E2+ ICI 182780 treatment. Note: AD, adiponectin.

menopause is associated with bone loss whereas this can be prevented by estrogen replacement therapy. Although the mechanisms by which estrogen has this impact have remained somewhat elusive, many studies have so far addressed that estrogens can stimulate the production of OPG and/or decrease that of RANKL [30–35]. However, the OPG and RANKL are regulated by various hormones, cytokines, and mesenchymal

transcription factors, variations in the balance of OPG and RANKL are thought to critically contribute to the pathology of osteoporosis and other bone diseases [36,37]. While the effects of alterations in the OPG/ RANKL pathway are various, including the so-called “genomic” or “classical” pathways and the “nongenomic” pathways. Our previous study demonstrated the adiponectin-regulated OPG and RANKL expression in

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A

B

Fig. 6. Effects of E2 on adiponectin-dependent osteoclastogenesis in co-culture of osteoblasts and CD14+ PBMCs. Osteoblasts were seeded onto 24-well plates and cultured to subconfluence. CD14+ PBMCs were added to osteoblasts and were co-cultured for 14 days in DMEM containing 10% adiponectin-free FBS in the presence of 10−7 M 1α, 25-dihydroxyvitamin D3 (1,25 vitD). After overexpression of p38 MAPK or knockdown of ERs, osteoblasts were also co-cultured with CD14+ PBMCs in similar media. The cultures were stained for TRACP. TRACP+red multinucleated cells were counted as osteoclasts.A. Representative microscopic view of the inhibition effect of E2 on adiponectin-dependent osteoclastogenesis at a magnification of ×200. The co-cultures were treated respectively with (a) media alone; (b) 10 μg/ml adiponectin; (c) 10 μg/ml adiponectin+10− 8 M E2; (d) 20 ng/ml RANKL; (e) 80 ng/ml OPG+20 ng/ml RANKL; (f) 80 ng/ml OPG + 10 μg/ml adiponectin. B. Adiponectin-dependent osteoclastogenesis after impairing the action of E2. The co-cultures were treated with 10 μg/ml adiponectin+ 10− 8 M E2 + 10 ng/ml anisomycin, 10 μg/ml adiponectin+ 10− 8 M E2 + 10− 8 M ICI 182780; or treated with 10 μg/ml adiponectin+ 10− 8 M E2 after overexpression of p38 MAPK or knockdown of ERs. Results are expressed as osteoclast numbers/cell. The bar represents mean ± SD (n= 6). *p b 0.01 vs. media control; #p b 0.01 vs. 10 μg/ml adiponectin+ 10− 8 M E2 treatment). Note: AD, adiponectin.

human osteoblasts was mediated via a “nongenomic” pathway, the activation of p38 MAPK [14]. MAPKs are well known to play an essential role in controlling cell proliferation, differentiation, gene expression, survival, and programmed cell death by phosphorylating the specific serines and threonines of target protein substrates [38]. Studies have shown that MAPKs are involved in gene expression in bone cells [39,40]. Kusumi et al. [41] reported that cyclic tensile strain inhibited RANKL and stimulated OPG synthesis in osteoblast through p38 MAPK pathway. Li et al. [42] showed that annexin II stimulates RANKL expression through MAPK pathway. E2 exerts most of its effects through the so-called “genomic” or “classical” pathway by binding to the ERs (ERα and ERβ) and interacting with the estrogen response element on the promoter region of a target gene [43]. However, in addition to this so-called “genomic” or “classical” pathway, it has also been shown that E2 can exert early and rapidly physiological effects through “nongenomic” pathways such as protein kinase A, phosphotidylinositol-3 kinase, and MAPK signaling pathways [44,45]. To gain further insight into the mechanisms by which estrogen effects on adiponectin regulating OPG and RANKL expression, we tested whether E2 would affect the adiponectin-induced p38 phosphorylation or not. Interestingly, similar to the studies reported by Hsieh et al. [46] and Wang et al. [47], our results showed that E2 blocked the adiponectin-induced p38 phosphorylation. Whereas, when the action of E2 was suppressed by ICI182780 or knockdown of ERs, the adiponectin-induced p38 phosphorylation was not blocked. Furthermore, anisomycin or overexpression of p38 also reserved the p38 phosphorylation. These results indicated that the effect of E2 on adiponectin-regulated OPG/RANKL expression was mediated through p38 and ERs. More importantly, all the effects of E2 on adiponectin-

dependent OPG/RANKL expression and osteoclastogenesis were blunted after impairing the action of E2 by using ICI182780, anisomycin, knockdown of ERs or overexpression of p38, further confirming that E2 suppressed adiponectin-regulated OPG/RANKL expression in human osteoblasts and then inhibited osteoclastogenesis through binding to its ER and inhibiting the activation of adiponectin-induced p38 MAPK. In conclusion, the present study has provided evidence that estrogen inhibit the adiponectin-dependent OPG/RANKL expression and then inhibit osteoclastogenesis, at least in part, though blocking the activation of adiponectin-induced p38 MAPK, suggesting that estrogen would suppress the negative effect of adiponectin on bone metabolism. Thus, the effects of estrogen on the adiponectin-regulated RANKL and OPG expression may contribute to the mechanisms by which a negative correlation was found between serum adiponectin level and BMD in the postmenopausal women but not in the premenopausal women. Acknowledgments This work was supported by Grant-30700891 and Grant-30672197 from the China National Natural Science Foundation and Grant20060533031 from the Specialized Research Fund for the Doctoral Program of Higher Education. References [1] Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF. A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 1995;270:26746–9. [2] Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1

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