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Estrogen receptor and Wnt signaling interact to regulate early gene expression in response to mechanical strain in osteoblastic cells
Biochemical and Biophysical Research Communications 394 (2010) 755–759
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Estrogen receptor and Wnt signaling interact to regulate early gene expression in response to mechanical strain in osteoblastic cells Astrid Liedert a,*, Liane Wagner a, Lothar Seefried b, Regina Ebert b, Franz Jakob b, Anita Ignatius a a b
Institute of Orthopedic Research and Biomechanics, University of Ulm, Ulm, Germany Orthopedic Department, Orthopedic Center for Musculoskeletal Research, University of Würzburg, Würzburg, Germany
a r t i c l e
i n f o
Article history: Received 4 March 2010 Available online 17 March 2010 Keywords: Bone remodeling Mechanotransduction Estrogen receptor signaling Wnt signaling Cyclooxygenase-2
a b s t r a c t Bone mass homeostasis is regulated by an interaction of various factors, including growth factors, systemic hormones and mechanical loading. Two signal transduction pathways, the estrogen receptor (ER) and the Wnt/b-catenin signal transduction pathway, have been shown to have an important role in regulating osteoblast and osteoclast function and to be involved in mechanotransduction. Therefore, dysfunction of these pathways can lead to osteoporotic bone loss. However, less is known about the modulation of gene expression by the interaction of these pathways in response to mechanical strain. We performed in vitro stretch experiments using osteoblastic MC3T3-E1 cells to study the effect of both pathways and mechanical strain on the expression of cyclooxygenase-2 (Cox-2), which is involved in the synthesis of prostaglandins, modulators of bone formation and resorption. Using specific agonists and antagonists, we demonstrated a regulation by an interaction of these pathways in mechantransduction. Estradiol (E2) had a sensitizing effect on mechanically induced Cox-2 expression, which seemed to be ligand-specific as it could be abolished using the antiestrogen ICI182,780. However, mechanical strain in the presence of Wnt signaling activators diminished both the E2 sensitizing effect and the stimulatory effect of Wnt signaling in the absence of strain. This interaction might be one regulatory mechanism by which mechanical loading exerts its role in bone mass homeostasis. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction In the adult skeleton bone mass is maintained by the balance of bone resorption and bone formation, which is ensured by the coordinated activities of osteoclasts and osteoblasts. This process, termed bone remodeling, is influenced by numerous factors, including growth factors, systemic hormones and the mechanical environment [1]. All these factors modulate signal transduction pathways, which act together to regulate gene expression resulting in adjustment of bone mass. Two of these signal transduction pathways are the estrogen receptor (ER) and the Wnt/b-catenin signaling pathway, which have been shown to be activated and to interact with other pathways in response to mechanical stress in osteoblastic cells [2]. Activation of the ER signaling pathway can occur in a liganddependent way by binding of estradiol (E2) to intracellular ERs as well as independently of E2-binding by cross-talk with peptide growth factors [3]. Nongenomic effects of estrogen are mediated by membrane-associated ERs, which may involve, e.g., the G-protein-
* Corresponding author. Address: Institute of Orthopedic Research and Biomechanics, University of Ulm, Helmholtzstr. 14, 89081 Ulm, Germany. Fax: +49 731 500 55302. E-mail address: [email protected] (A. Liedert). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.065
coupled receptor GPR30, now termed GPER, and are able to initiate various cell responses, including cell proliferation [4]. The Wnt/bcatenin signaling pathway is activated by specific Wnt glycoproteins binding simultaneously to members of the frizzled (Fzd)-family of cell surface receptors and their coreceptors low-density lipoprotein receptor-related protein (Lrp) 5 or 6. By inducing a cascade of signaling events the Wnt proteins stabilize b-catenin. The latter migrates into the nucleus, where it binds to both the transcription factors lymphoid enhancer binding factor-1 (Lef-1) and T-cell factor (Tcf), thereby modulating b-catenin target gene expression [5]. Dysfunction of both pathways can be associated with osteoporotic bone loss. In postmenopausal osteoporosis, withdrawal of estrogen together with mechanical loading may result in an impaired bone mass adaption leading to insufficient bone strength and fragility fractures [6]. The osteoporosis-pseudoglioma syndrome (OPPG), a rare autosomal recessive disorder of severe juvenile osteoporosis and congenital blindness, results from loss-offunction mutations in the Lrp-5 gene [7]. Thus, both pathways play an important role in bone remodeling. They regulate expression of genes, which modulate osteoclast and osteoblast function [5,8]. One of these genes is cyclooxygenase-2 (Cox-2) that is upregulated immediately in response to mechanical stimulation in osteoblasts [9]. It has also been shown
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to be activated by E2 and Wnt-3a in human uterine microvascular endothelial cells and mouse mammary epithelial cells, respectively [10,11]. Cox-2 can be transiently induced by local and systemic factors, and regulates prostaglandin (PG) synthesis in bone together with cyclooxygenase-1 (Cox-1), which is constitutively expressed. Cox-2-deficient mice exhibit significantly lower bone mineral density than wild-type mice and show hyperparathyroidism [12]. Prostaglandins play a critical role in bone remodeling as local regulators of bone resorption and formation. PGs can increase receptor activator of nuclear factor kappa B ligand (Rankl) expression in osteoblasts, thereby enhancing the Rankl-induced differentiation of osteoclast precursors into osteoclasts [13]. Additionally, PGs also have stimulatory effects on proliferation and differentiation of osteoblasts. They have a biphasic effect on bone formation and this effect appears to be modulated in the presence of growth factors and systemic hormones. Because PG concentration has a crucial impact on the biphasic effect a strict regulation of PG synthesis by Cox-2 can be assumed. Thus, the aim of our study was to investigate the regulation of Cox2 expression by the ER and Wnt/b-catenin signaling pathway in response to mechanical strain in osteoblastic cells. Using specific agonists and antagonists we examined the influence of activation and interaction of these pathways on mechanically induced Cox2 expression.
2. Material and methods 2.1. Cell culture Cell culture experiments were performed with the murine osteogenic cell line MC3T3-E1 (DSMZ, Braunschweig, Germany). The cells were cultured in alpha-minimum essential medium (aMEM, Biochrom, Berlin, Germany) supplemented with 10% FCS (PAA Laboratories, Cölbe, Germany), 1% L-glutamine (Biochrom), and 1% penicillin/streptomycin (Gibco, Karlsruhe, Germany) in 5% CO2 at 37 °C and saturation humidity. Culture medium was replaced twice a week. Confluent cultures were trypsinized with 1 trypsin/EDTA solution (Biochrom) and passaged at a density of 200,000 cells/dish on pre-coated (10% FCS for 1 week) flexible silicone dishes (60 30 mm). 2.2. Mechanical cell stimulation Mechanical cell stimulation was performed at day 5 after cell seeding by homogenous cyclic stretching as described previously [14]. We used strain (sinusoidal) at 1% and a frequency of 1 Hz for 1800 cycles (equivalent to 30 min) or 5400 cycles (equivalent to 1.5 h). Serum reduction was performed with 2% serum at day 4, when cell confluence was reached. Control dishes were prepared in parallel using the identical procedure but no load was applied. 2.3. Treatment of the cell cultures with agonists and antagonists Agonists and antagonists were added to the controls and to cultures to be stimulated, 3 h and 3.5 h, respectively, prior to loading. 17b-Estradiol (E2, 10 nM, Sigma–Aldrich, Taufkirchen, Germany) was used to activate the estradiol dependent signal transduction pathways, LiCl (a glycogen synthase kinase3-b (GSK3-b) inhibitor, 10 mM, Sigma–Aldrich) or mouse Wnt-3a (3 nM, R&D Systems, Wiesbaden, Germany) was added to activate the Wnt/b-catenin signal transduction pathway. ICI182,780 (100 nM, Biozol, Eching, Germany) was used to inhibit the ER pathway. Mouse Dickkopf-1 (Dkk-1, 18 nM, R&D Systems) was added to inhibit the Wnt/b-catenin pathway, respectively. Control cell culture dishes were prepared in parallel with an equal volume of the respective vehicle.
2.4. RT-PCR and quantitative real-time PCR Mechanically stimulated and unstimulated cells were harvested and lysed immediately after the loading period. Total RNA was isolated and RT-PCR was performed as described previously [15]. Quantitative real-time RT-PCR was performed with the Platinum Sybr Green qPCR SuperMix-UDG Kit (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions in a total volume of 25 ll, using specific primer pairs for amplifying products of mouse Cox-2 (GenBank Accession No. NM_011198) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh, control, GenBank Accession No. NM_008084). A 66-bp Cox-2 product was amplified using the sense primer 50 -AAG GGT GTC CCT TCA CTT CT-30 and the antisense primer 50 -CAT TGA TGG TGG CTG TTT TG-30 . An 81-bp Gapdh product was amplified using the sense primer 50 -ACC CAG AAG ACT GTG GAT GG-30 and the antisense primer 50 -GGA TGC AGG GAT GAT GTT CT-30 . For standardization, amplification products were cloned into pCR4-TOPO vector (Invitrogen). Plasmid DNA was isolated with Plasmid Midi Kit (Qiagen), sequenced by MWG Biotech AG (Germany), linearized, and used as a standard. 2.5. Western blotting Cells were washed once with ice-cold PBS immediately after loading and lysed in 500 ll SDS sample buffer (125 mM Tris–HCl (pH 6.8), 1% w/v SDS, 8.5% glycerol) per dish. Protein concentration was measured with the BCA protein assay (Perbio Science, Pierce, Bonn, Germany). Samples were heated to 95 °C for 5 min. Aliquots of 10 lg protein were resolved by SDS–PAGE (10% resolving gel, according to Laemmli, 1970), transferred to nitrocellulose membranes (Bio-Rad Laboratories, Münschen, Germany) and blocked with blocking buffer (10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 3% w/v BSA, 0.1% Tween 20) for 1 h at room temperature. After washing three times for 5 min with Tris-buffered saline/Tween (10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20) membranes were incubated with Cox-2 specific antibodies (Zytomed, Berlin, Germany, 1:10000) overnight at 4 °C with gentle shaking. The membranes were washed three times for 5 min with Tris-buffered saline/Tween and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Perbio Science, 1:1000) for 1 h at room temperature. Again the membranes were washed three times for 5 min with Tris-buffered saline/Tween. For the ECL reaction, immunoblots were developed for 5 min in SuperSignal West Pico Chemiluminescent Substrate (Perbio Science) and the respective proteins were visualized using the Fusion Molecular Imaging System (Vilber Lourmat, Eberhardzell, Germany). To demonstrate equivalency of protein loading specific bactin antibodies (Cell Signaling, New England Biolabs, Frankfurt, Germany, 1:1000) were used. 2.6. Statistical analysis Independent experiments were performed a minimum of three or four times in duplicate (n = 6–8). Data obtained were analyzed for significance (value p 6 0.05) using Student’s t-test or analysis of variance (ANOVA). The results were presented as mean value ± standard error of the mean (SEM).
3. Results 3.1. Sensitizing effect of ER signaling on mechanically induced Cox-2 expression As expected, mechanical strain resulted in an increase of Cox-2 mRNA expression (1.8-fold, p 6 0.05) immediately after mechani-
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3.2. Abolishment of Wnt/b-catenin induced Cox-2 expression by mechanical strain
Fig. 1. Sensitizing effect of ER signaling on mechanically induced Cox-2 expression. Relative gene expression of Cox-2 without and after mechanical strain in the presence of the ER signaling agonist E2 and antagonist ICI182,780 in MC3T3-E1 cells. Mechanical loading was performed over a time period of 0.5 h at a frequency of 1 Hz with an amplitude of 1%. E2 (10 nM) and ICI182,780 (100 nM) were added to the controls and cell cultures to be stimulated, 3 h and 3.5 h, respectively, prior to loading. Expression was determined by real-time RT-PCR, and the results were normalised to Gapdh and expressed as means ± SEM (*p 6 0.05) compared to control cells.
cal stimulation in MC3T3-E1 cells (Fig. 1). Pretreatment of the cells with E2 without subsequent mechanical loading did not change Cox-2 expression. In contrast, E2 and mechanical loading increased Cox-2 mRNA levels up to 7.2-fold (p 6 0.05). The addition of ICI182,780, a selective high affinity estrogen receptor antagonist had neither a significant effect on Cox-2 expression without subsequent mechanical loading nor did it change significantly the mechanically induced expression of Cox-2. However, it abolished the sensitizing effect of E2 on the mechanically induced Cox-2 expression, suggesting that the mechanosensitizing effect of E2 was mediated by ligand (E2)-dependent receptor signaling.
Addition of LiCl, a Wnt/b-catenin signaling activator (GSK3-b inhibitor), resulted in an increase of Cox-2 expression up to 14.0fold (p 6 0.05, Fig. 2). Mechanical strain in the presence of LiCl led to the abolishment of the Wnt/b-catenin signaling induced Cox-2 expression. We verified these effects with the natural Wnt/ b-catenin signaling activator Wnt-3a. As shown in Fig. 3, Wnt-3a led to significant up-regulation of Cox-2 expression (2.6-fold, p 6 0.05), whereas mechanical stimulation in addition to Wnt-3a treatment diminished the Wnt-3a stimulated Cox-2 expression. These results showed that mechanically induced pathways interact with the activated Wnt/b-catenin signaling pathway reducing the expression of the early response gene Cox-2. Protein data as judged by western blotting were in agreement with the mRNA data (Fig. 2B). The physiological Wnt/Lrp-5/b-catenin antagonist Dkk-1 inhibited the Wnt-3a mediated activation of Cox-2 expression, as expected, though this inhibition was not complete under the conditions used (Fig. 3). Treatment with Dkk-1 alone did not result in a significant change of Cox-2 expression, whereas Dkk-1 in the presence of mechanical strain led to a slight, but significant increase of expression compared to the up-regulation by mechanical strain alone (Fig. 3). This stimulation in the presence of Dkk-1 on the effect of mechanical loading could also be observed when Wnt-3a was present, additionally. This argues that Dkk-1 might have an effect on strain-mediated Cox-2 expression independent of Wnt-3a signaling. 3.3. Abolishment of the ER dependent mechanosensitizing effect on Cox-2 expression by activation of the Wnt/b-catenin signaling pathway The Wnt/b-catenin signaling agonist LiCl decreased the mechanosensitizing effect of E2 on Cox-2 expression (Fig. 2,
Fig. 2. Abolishment of Wnt/b-Catenin induced Cox-2 expression and ER dependent mechanosensitizing effect on Cox-2 expression by mechanical strain. (A) Relative gene expression of Cox-2 without and after mechanical strain in the presence of the Wnt/b-catenin signaling agonist LiCl (10 mM) in MC3T3-E1 cells. Mechanical loading was performed over a time period of 0.5 h at a frequency of 1 Hz with an amplitude of 1%. The agonist was added to the controls and cell cultures to be stimulated 3 h prior to loading. Expression was determined by real-time RT-PCR, and the results were normalised to Gapdh and expressed as means ± SEM (*p 6 0.05) compared to control cells. (B) Representative immunoblot showing protein levels of Cox-2 without and after mechanical load in the presence of E2 (10 nM) and Wnt-3a (3 nM), b-actin was measured as loading control. Mechanical loading was performed over a time period of 1.5 h.
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Fig. 3. Abolishment of Wnt-3a induced Cox-2 expression by mechanical strain. Relative gene expression of Cox-2 after mechanical and without mechanical strain in the presence of the physiological agonist Wnt-3a and the physiological antagonist Dkk-1 of the Wnt/b-catenin signal transduction pathway in MC3T3-E1 cells. Wnt-3a (3 nM) and Dkk-1 (18 nM) were added to the controls and cell cultures to be stimulated, 3 h and 3,5 h, respectively, prior loading. Mechanical loading was performed over a time period of 0.5 h at a frequency of 1 Hz with an amplitude of 1%. Expression was determined by real-time RT-PCR, and the results were normalised to Gapdh and expressed as means ± SEM (*p 6 0.05) compared to control cells.
p 6 0.05). This revealed that activation of the Wnt/b-catenin signaling pathway led to the abolishment of the ER dependent mechanosenitizing effect on Cox-2 expression. Interestingly, E2 in the mechanically unstimulated cell almost abolished the stimulatory effect of LiCl on Cox-2 expression (p 6 0.05), which indicated an inhibitory effect of ER signaling on Wnt/b-catenin induced Cox2 expression. 4. Discussion Using inducible Cox-2 expression we demonstrated in the present study that the Wnt/b-catenin and the ER signaling pathway interact to modulate mechanically induced early response gene expression in osteoblastic cells. We showed that E2 is able to sensitize osteoblastic cells for the immediate response to mechanical loading and potentiates strain-induced Cox-2 mRNA expression. Moreover, we demonstrated that the increase of Cox-2 expression on mechanical loading with or without E2 was diminished by the activation of the Wnt/b-catenin signaling pathway. Additionally, mechanical loading itself down-regulated Cox-2 expression induced by the activation of the Wnt/b-catenin signaling pathway both with and without E2 present. Expression of Cox-2, which catalyzes the rate limiting step in prostaglandin synthesis, namely the conversion of arachidonic acid to prostaglandin H2, is known to be rapidly induced by mechanical loading in vitro and in vivo [16]. Cox-2 dependent PG biosynthesis occurs over several hours and is active in the early and delayed PG response to mechanical loading of bone. Prostaglandins are important mediators of bone remodeling, as they regulate both bone formation and bone resorption. The bone forming-activity of PGE2 depends on its stimulatory effect on osteoblast proliferation and differentiation. The bone-resorptive activity of PGE2 is mediated by receptor activator of NF-jB ligand (Rankl, Tnfsf11) as well as by its involvement in IL-1 and IL-6 stimulated bone resorption. A strict localization-, concentration-, and time-dependent regulation of PG synthesis can be expected. We showed that E2 was able to sensitize osteoblastic cells to mechanical loading, as Cox-2 expression was strongly elevated in the presence of E2 in mechanically loaded compared to loaded MC3T3-E1 cells without E2 (Fig. 1). A mechanosensitizing effect of E2 on prostaglandin production has been shown in bone cells
from elderly women and from osteoporotic donors [17,18]. Because E2 did not significantly affect Cox-1 and Cox-2 mRNA expression, the authors suggested that estrogen modulates bone cell mechanosensitivity via the prostaglandin synthetic pathway independently of Cox expression. The discrepancy with our study might be due to the different cell type, as it can be presumed that primary osteoblasts from elderly women were less responsive to mechanical stimulation owing to decreased ERa activity [19]. ERa has been shown to be required for mechanically induced responses of bone cells in vitro and in vivo [20]. A dose-dependent effect of estrogen and parathyroid hormone on the mechanical response of osteoblasts and osteocytes, respectively, has already been documented, respectively [21,22]. In addition to their role as ligand-dependent mediators of the effects of estrogen, ERs have also been shown to participate in mechanotransduction of bone cells in a ligand-independent manner [23]. Using the high affinity ER antagonist ICI182,780, we demonstrated the reversal of the sensitizing effect of E2 on the mechanically induced Cox-2 expression, thereby confirming the involvement of the ER-dependent signaling pathway in the mechanosensitizing mechanism. This result is consistent with the finding of Armstrong et al. [24] that Wnt/b-catenin signaling contributes to early responses of bone cells to mechanical strain and that its effectiveness is ER dependent. Cox-2 has been shown to be a Wnt/b-catenin inducible gene [11]. The regulation of Cox-2 expression by Lef-1 and b-catenin has been demonstrated in murine chondrocytes [25]. As expected, treatment with LiCl, which inhibits GSK3-b, thereby activating the Wnt/b-catenin signaling pathway, led to a significant up-regulation of Cox-2 expression in osteoblastic cells (Fig. 2A). Both the increase of Cox-2 by mechanical stimulation alone and by LiCl alone was abolished by mechanical load in the presence of LiCl, respectively. It has been shown that mechanical strain in the presence of a Wnt/b-catenin signaling activator can result in a synergistic transcriptional response on Wnt/b-catenin target genes in MC3T3-E1 cells [26]. Interestingly, the authors demonstrated less or even a lack of up-regulation of most of these genes in cells after treatment with the Wnt/b-catenin pathway activator alone compared to the expression of cells after application of mechanical strain without activator. In contrast, we found a higher increase of Cox-2 expression after the addition of the activator alone than after application of mechanical load without activator. We did not detect any mechanosensitizing effect on the response, but an abolishment of the increase of Cox-2 expression after mechanical loading on additionally treating with LiCl. This difference might be due to the longer loading period (strain for 5 h) used in the study of Robinson et al. [26]. We verified our results by using the physiological activator Wnt-3a (Fig. 3). In comparison with mechanically loaded MC3T3-E1 cells, those treated with Wnt-3a alone showed a higher increase of Cox-2 expression. Treatment with Wnt-3a and mechanical load led to the diminishing of the elevated Cox-2 expression. These changes correlated with decreased Cox-2 protein levels after Wnt-3a treatment and mechanical loading (Fig. 2B). Thus it can be proposed that in the present study activated Wnt/b-catenin signaling counteracts with mechanically induced signaling. Studies have provided evidence that mechanical loading is able to counteract cytokine-sensitive pathways in chondrocytes [27,28]. Treatment with the physiological Wnt/Lrp-5/b-catenin pathway antagonist Dkk-1 alone did not lead to a significant effect on Cox-2 expression, thus demonstrating that the Wnt/b-catenin pathway was not involved in basal expression of Cox-2 (Fig. 3). However, application of mechanical strain in the presence of Dkk-1 resulted in a significantly higher up-regulation of expression compared to the expression after mechanical loading without Dkk-1. This stimulatory effect was not affected by Wnt-3a, suggesting that Dkk-1 exerts a Wnt-3a independent effect on mechanical regulation of
A. Liedert et al. / Biochemical and Biophysical Research Communications 394 (2010) 755–759
Cox-2 expression. This deserves further investigation. Using CIMC4 and UMR106 cells, respectively, as well as a different strain period, Case et al. [29] and Sunters et al. [30], too, have shown that Dkk-1 did not block strain-induced transcription from the Wnt/ Lrp-5 target gene promoter. Activation of the ER signaling pathway led to down-regulation of Wnt/b-catenin signaling dependent Cox-2 expression induced by LiCl (Fig. 2A). On the other hand, LiCl abolished the activating effect of E2 in the presence of mechanical strain. Thus, LiCl and E2 are potent activators of Cox-2 expression in the mechanically unstimulated and stimulated cell, respectively, and both of them have a strong inhibitory effect on the activation caused by the other. 5. Conclusion In conclusion, we have shown that ER and Wnt/b-catenin and mechanically induced signaling interact to modulate early response gene expression. We demonstrated a sensitizing effect of E2 on mechanically induced Cox-2 expression, which seemed to be due to ligand-dependent ER signaling and was diminished by simultaneous activation of Wnt/b-catenin signaling. Moreover, we showed an activation of early gene expression by Wnt/b-catenin signaling in the unloaded cell that was diminished by simultaneous activation of ER signaling. Our results suggest that the interaction of both pathways might be one regulatory mechanism by which mechanical loading exerts its role in maintaining bone mass in the healthy skeleton. This interacting regulatory system appears to be well suited to fine-tune early gene expression in response to different loading situations, and its disturbance during postmenopause, when E2 levels are declined, might be involved in the disturbed equilibrium of bone formation and bone resorption, ultimately leading to postmenopausal osteoporosis.
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This work was financially supported by the Deutsche Forschungsgemeinschaft (D.F.G., Grant No. IG18/5-1; Grant No. JA 504/10-1).
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Estrogen receptor and Wnt signaling interact to regulate early gene expression in response to mechanical strain in osteoblastic cells Astrid Liedert a,*, Liane Wagner a, Lothar Seefried b, Regina Ebert b, Franz Jakob b, Anita Ignatius a a b
Institute of Orthopedic Research and Biomechanics, University of Ulm, Ulm, Germany Orthopedic Department, Orthopedic Center for Musculoskeletal Research, University of Würzburg, Würzburg, Germany
a r t i c l e
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Article history: Received 4 March 2010 Available online 17 March 2010 Keywords: Bone remodeling Mechanotransduction Estrogen receptor signaling Wnt signaling Cyclooxygenase-2
a b s t r a c t Bone mass homeostasis is regulated by an interaction of various factors, including growth factors, systemic hormones and mechanical loading. Two signal transduction pathways, the estrogen receptor (ER) and the Wnt/b-catenin signal transduction pathway, have been shown to have an important role in regulating osteoblast and osteoclast function and to be involved in mechanotransduction. Therefore, dysfunction of these pathways can lead to osteoporotic bone loss. However, less is known about the modulation of gene expression by the interaction of these pathways in response to mechanical strain. We performed in vitro stretch experiments using osteoblastic MC3T3-E1 cells to study the effect of both pathways and mechanical strain on the expression of cyclooxygenase-2 (Cox-2), which is involved in the synthesis of prostaglandins, modulators of bone formation and resorption. Using specific agonists and antagonists, we demonstrated a regulation by an interaction of these pathways in mechantransduction. Estradiol (E2) had a sensitizing effect on mechanically induced Cox-2 expression, which seemed to be ligand-specific as it could be abolished using the antiestrogen ICI182,780. However, mechanical strain in the presence of Wnt signaling activators diminished both the E2 sensitizing effect and the stimulatory effect of Wnt signaling in the absence of strain. This interaction might be one regulatory mechanism by which mechanical loading exerts its role in bone mass homeostasis. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction In the adult skeleton bone mass is maintained by the balance of bone resorption and bone formation, which is ensured by the coordinated activities of osteoclasts and osteoblasts. This process, termed bone remodeling, is influenced by numerous factors, including growth factors, systemic hormones and the mechanical environment [1]. All these factors modulate signal transduction pathways, which act together to regulate gene expression resulting in adjustment of bone mass. Two of these signal transduction pathways are the estrogen receptor (ER) and the Wnt/b-catenin signaling pathway, which have been shown to be activated and to interact with other pathways in response to mechanical stress in osteoblastic cells [2]. Activation of the ER signaling pathway can occur in a liganddependent way by binding of estradiol (E2) to intracellular ERs as well as independently of E2-binding by cross-talk with peptide growth factors [3]. Nongenomic effects of estrogen are mediated by membrane-associated ERs, which may involve, e.g., the G-protein-
* Corresponding author. Address: Institute of Orthopedic Research and Biomechanics, University of Ulm, Helmholtzstr. 14, 89081 Ulm, Germany. Fax: +49 731 500 55302. E-mail address: [email protected] (A. Liedert). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.065
coupled receptor GPR30, now termed GPER, and are able to initiate various cell responses, including cell proliferation [4]. The Wnt/bcatenin signaling pathway is activated by specific Wnt glycoproteins binding simultaneously to members of the frizzled (Fzd)-family of cell surface receptors and their coreceptors low-density lipoprotein receptor-related protein (Lrp) 5 or 6. By inducing a cascade of signaling events the Wnt proteins stabilize b-catenin. The latter migrates into the nucleus, where it binds to both the transcription factors lymphoid enhancer binding factor-1 (Lef-1) and T-cell factor (Tcf), thereby modulating b-catenin target gene expression [5]. Dysfunction of both pathways can be associated with osteoporotic bone loss. In postmenopausal osteoporosis, withdrawal of estrogen together with mechanical loading may result in an impaired bone mass adaption leading to insufficient bone strength and fragility fractures [6]. The osteoporosis-pseudoglioma syndrome (OPPG), a rare autosomal recessive disorder of severe juvenile osteoporosis and congenital blindness, results from loss-offunction mutations in the Lrp-5 gene [7]. Thus, both pathways play an important role in bone remodeling. They regulate expression of genes, which modulate osteoclast and osteoblast function [5,8]. One of these genes is cyclooxygenase-2 (Cox-2) that is upregulated immediately in response to mechanical stimulation in osteoblasts [9]. It has also been shown
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to be activated by E2 and Wnt-3a in human uterine microvascular endothelial cells and mouse mammary epithelial cells, respectively [10,11]. Cox-2 can be transiently induced by local and systemic factors, and regulates prostaglandin (PG) synthesis in bone together with cyclooxygenase-1 (Cox-1), which is constitutively expressed. Cox-2-deficient mice exhibit significantly lower bone mineral density than wild-type mice and show hyperparathyroidism [12]. Prostaglandins play a critical role in bone remodeling as local regulators of bone resorption and formation. PGs can increase receptor activator of nuclear factor kappa B ligand (Rankl) expression in osteoblasts, thereby enhancing the Rankl-induced differentiation of osteoclast precursors into osteoclasts [13]. Additionally, PGs also have stimulatory effects on proliferation and differentiation of osteoblasts. They have a biphasic effect on bone formation and this effect appears to be modulated in the presence of growth factors and systemic hormones. Because PG concentration has a crucial impact on the biphasic effect a strict regulation of PG synthesis by Cox-2 can be assumed. Thus, the aim of our study was to investigate the regulation of Cox2 expression by the ER and Wnt/b-catenin signaling pathway in response to mechanical strain in osteoblastic cells. Using specific agonists and antagonists we examined the influence of activation and interaction of these pathways on mechanically induced Cox2 expression.
2. Material and methods 2.1. Cell culture Cell culture experiments were performed with the murine osteogenic cell line MC3T3-E1 (DSMZ, Braunschweig, Germany). The cells were cultured in alpha-minimum essential medium (aMEM, Biochrom, Berlin, Germany) supplemented with 10% FCS (PAA Laboratories, Cölbe, Germany), 1% L-glutamine (Biochrom), and 1% penicillin/streptomycin (Gibco, Karlsruhe, Germany) in 5% CO2 at 37 °C and saturation humidity. Culture medium was replaced twice a week. Confluent cultures were trypsinized with 1 trypsin/EDTA solution (Biochrom) and passaged at a density of 200,000 cells/dish on pre-coated (10% FCS for 1 week) flexible silicone dishes (60 30 mm). 2.2. Mechanical cell stimulation Mechanical cell stimulation was performed at day 5 after cell seeding by homogenous cyclic stretching as described previously [14]. We used strain (sinusoidal) at 1% and a frequency of 1 Hz for 1800 cycles (equivalent to 30 min) or 5400 cycles (equivalent to 1.5 h). Serum reduction was performed with 2% serum at day 4, when cell confluence was reached. Control dishes were prepared in parallel using the identical procedure but no load was applied. 2.3. Treatment of the cell cultures with agonists and antagonists Agonists and antagonists were added to the controls and to cultures to be stimulated, 3 h and 3.5 h, respectively, prior to loading. 17b-Estradiol (E2, 10 nM, Sigma–Aldrich, Taufkirchen, Germany) was used to activate the estradiol dependent signal transduction pathways, LiCl (a glycogen synthase kinase3-b (GSK3-b) inhibitor, 10 mM, Sigma–Aldrich) or mouse Wnt-3a (3 nM, R&D Systems, Wiesbaden, Germany) was added to activate the Wnt/b-catenin signal transduction pathway. ICI182,780 (100 nM, Biozol, Eching, Germany) was used to inhibit the ER pathway. Mouse Dickkopf-1 (Dkk-1, 18 nM, R&D Systems) was added to inhibit the Wnt/b-catenin pathway, respectively. Control cell culture dishes were prepared in parallel with an equal volume of the respective vehicle.
2.4. RT-PCR and quantitative real-time PCR Mechanically stimulated and unstimulated cells were harvested and lysed immediately after the loading period. Total RNA was isolated and RT-PCR was performed as described previously [15]. Quantitative real-time RT-PCR was performed with the Platinum Sybr Green qPCR SuperMix-UDG Kit (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions in a total volume of 25 ll, using specific primer pairs for amplifying products of mouse Cox-2 (GenBank Accession No. NM_011198) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh, control, GenBank Accession No. NM_008084). A 66-bp Cox-2 product was amplified using the sense primer 50 -AAG GGT GTC CCT TCA CTT CT-30 and the antisense primer 50 -CAT TGA TGG TGG CTG TTT TG-30 . An 81-bp Gapdh product was amplified using the sense primer 50 -ACC CAG AAG ACT GTG GAT GG-30 and the antisense primer 50 -GGA TGC AGG GAT GAT GTT CT-30 . For standardization, amplification products were cloned into pCR4-TOPO vector (Invitrogen). Plasmid DNA was isolated with Plasmid Midi Kit (Qiagen), sequenced by MWG Biotech AG (Germany), linearized, and used as a standard. 2.5. Western blotting Cells were washed once with ice-cold PBS immediately after loading and lysed in 500 ll SDS sample buffer (125 mM Tris–HCl (pH 6.8), 1% w/v SDS, 8.5% glycerol) per dish. Protein concentration was measured with the BCA protein assay (Perbio Science, Pierce, Bonn, Germany). Samples were heated to 95 °C for 5 min. Aliquots of 10 lg protein were resolved by SDS–PAGE (10% resolving gel, according to Laemmli, 1970), transferred to nitrocellulose membranes (Bio-Rad Laboratories, Münschen, Germany) and blocked with blocking buffer (10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 3% w/v BSA, 0.1% Tween 20) for 1 h at room temperature. After washing three times for 5 min with Tris-buffered saline/Tween (10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20) membranes were incubated with Cox-2 specific antibodies (Zytomed, Berlin, Germany, 1:10000) overnight at 4 °C with gentle shaking. The membranes were washed three times for 5 min with Tris-buffered saline/Tween and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Perbio Science, 1:1000) for 1 h at room temperature. Again the membranes were washed three times for 5 min with Tris-buffered saline/Tween. For the ECL reaction, immunoblots were developed for 5 min in SuperSignal West Pico Chemiluminescent Substrate (Perbio Science) and the respective proteins were visualized using the Fusion Molecular Imaging System (Vilber Lourmat, Eberhardzell, Germany). To demonstrate equivalency of protein loading specific bactin antibodies (Cell Signaling, New England Biolabs, Frankfurt, Germany, 1:1000) were used. 2.6. Statistical analysis Independent experiments were performed a minimum of three or four times in duplicate (n = 6–8). Data obtained were analyzed for significance (value p 6 0.05) using Student’s t-test or analysis of variance (ANOVA). The results were presented as mean value ± standard error of the mean (SEM).
3. Results 3.1. Sensitizing effect of ER signaling on mechanically induced Cox-2 expression As expected, mechanical strain resulted in an increase of Cox-2 mRNA expression (1.8-fold, p 6 0.05) immediately after mechani-
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3.2. Abolishment of Wnt/b-catenin induced Cox-2 expression by mechanical strain
Fig. 1. Sensitizing effect of ER signaling on mechanically induced Cox-2 expression. Relative gene expression of Cox-2 without and after mechanical strain in the presence of the ER signaling agonist E2 and antagonist ICI182,780 in MC3T3-E1 cells. Mechanical loading was performed over a time period of 0.5 h at a frequency of 1 Hz with an amplitude of 1%. E2 (10 nM) and ICI182,780 (100 nM) were added to the controls and cell cultures to be stimulated, 3 h and 3.5 h, respectively, prior to loading. Expression was determined by real-time RT-PCR, and the results were normalised to Gapdh and expressed as means ± SEM (*p 6 0.05) compared to control cells.
cal stimulation in MC3T3-E1 cells (Fig. 1). Pretreatment of the cells with E2 without subsequent mechanical loading did not change Cox-2 expression. In contrast, E2 and mechanical loading increased Cox-2 mRNA levels up to 7.2-fold (p 6 0.05). The addition of ICI182,780, a selective high affinity estrogen receptor antagonist had neither a significant effect on Cox-2 expression without subsequent mechanical loading nor did it change significantly the mechanically induced expression of Cox-2. However, it abolished the sensitizing effect of E2 on the mechanically induced Cox-2 expression, suggesting that the mechanosensitizing effect of E2 was mediated by ligand (E2)-dependent receptor signaling.
Addition of LiCl, a Wnt/b-catenin signaling activator (GSK3-b inhibitor), resulted in an increase of Cox-2 expression up to 14.0fold (p 6 0.05, Fig. 2). Mechanical strain in the presence of LiCl led to the abolishment of the Wnt/b-catenin signaling induced Cox-2 expression. We verified these effects with the natural Wnt/ b-catenin signaling activator Wnt-3a. As shown in Fig. 3, Wnt-3a led to significant up-regulation of Cox-2 expression (2.6-fold, p 6 0.05), whereas mechanical stimulation in addition to Wnt-3a treatment diminished the Wnt-3a stimulated Cox-2 expression. These results showed that mechanically induced pathways interact with the activated Wnt/b-catenin signaling pathway reducing the expression of the early response gene Cox-2. Protein data as judged by western blotting were in agreement with the mRNA data (Fig. 2B). The physiological Wnt/Lrp-5/b-catenin antagonist Dkk-1 inhibited the Wnt-3a mediated activation of Cox-2 expression, as expected, though this inhibition was not complete under the conditions used (Fig. 3). Treatment with Dkk-1 alone did not result in a significant change of Cox-2 expression, whereas Dkk-1 in the presence of mechanical strain led to a slight, but significant increase of expression compared to the up-regulation by mechanical strain alone (Fig. 3). This stimulation in the presence of Dkk-1 on the effect of mechanical loading could also be observed when Wnt-3a was present, additionally. This argues that Dkk-1 might have an effect on strain-mediated Cox-2 expression independent of Wnt-3a signaling. 3.3. Abolishment of the ER dependent mechanosensitizing effect on Cox-2 expression by activation of the Wnt/b-catenin signaling pathway The Wnt/b-catenin signaling agonist LiCl decreased the mechanosensitizing effect of E2 on Cox-2 expression (Fig. 2,
Fig. 2. Abolishment of Wnt/b-Catenin induced Cox-2 expression and ER dependent mechanosensitizing effect on Cox-2 expression by mechanical strain. (A) Relative gene expression of Cox-2 without and after mechanical strain in the presence of the Wnt/b-catenin signaling agonist LiCl (10 mM) in MC3T3-E1 cells. Mechanical loading was performed over a time period of 0.5 h at a frequency of 1 Hz with an amplitude of 1%. The agonist was added to the controls and cell cultures to be stimulated 3 h prior to loading. Expression was determined by real-time RT-PCR, and the results were normalised to Gapdh and expressed as means ± SEM (*p 6 0.05) compared to control cells. (B) Representative immunoblot showing protein levels of Cox-2 without and after mechanical load in the presence of E2 (10 nM) and Wnt-3a (3 nM), b-actin was measured as loading control. Mechanical loading was performed over a time period of 1.5 h.
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Fig. 3. Abolishment of Wnt-3a induced Cox-2 expression by mechanical strain. Relative gene expression of Cox-2 after mechanical and without mechanical strain in the presence of the physiological agonist Wnt-3a and the physiological antagonist Dkk-1 of the Wnt/b-catenin signal transduction pathway in MC3T3-E1 cells. Wnt-3a (3 nM) and Dkk-1 (18 nM) were added to the controls and cell cultures to be stimulated, 3 h and 3,5 h, respectively, prior loading. Mechanical loading was performed over a time period of 0.5 h at a frequency of 1 Hz with an amplitude of 1%. Expression was determined by real-time RT-PCR, and the results were normalised to Gapdh and expressed as means ± SEM (*p 6 0.05) compared to control cells.
p 6 0.05). This revealed that activation of the Wnt/b-catenin signaling pathway led to the abolishment of the ER dependent mechanosenitizing effect on Cox-2 expression. Interestingly, E2 in the mechanically unstimulated cell almost abolished the stimulatory effect of LiCl on Cox-2 expression (p 6 0.05), which indicated an inhibitory effect of ER signaling on Wnt/b-catenin induced Cox2 expression. 4. Discussion Using inducible Cox-2 expression we demonstrated in the present study that the Wnt/b-catenin and the ER signaling pathway interact to modulate mechanically induced early response gene expression in osteoblastic cells. We showed that E2 is able to sensitize osteoblastic cells for the immediate response to mechanical loading and potentiates strain-induced Cox-2 mRNA expression. Moreover, we demonstrated that the increase of Cox-2 expression on mechanical loading with or without E2 was diminished by the activation of the Wnt/b-catenin signaling pathway. Additionally, mechanical loading itself down-regulated Cox-2 expression induced by the activation of the Wnt/b-catenin signaling pathway both with and without E2 present. Expression of Cox-2, which catalyzes the rate limiting step in prostaglandin synthesis, namely the conversion of arachidonic acid to prostaglandin H2, is known to be rapidly induced by mechanical loading in vitro and in vivo [16]. Cox-2 dependent PG biosynthesis occurs over several hours and is active in the early and delayed PG response to mechanical loading of bone. Prostaglandins are important mediators of bone remodeling, as they regulate both bone formation and bone resorption. The bone forming-activity of PGE2 depends on its stimulatory effect on osteoblast proliferation and differentiation. The bone-resorptive activity of PGE2 is mediated by receptor activator of NF-jB ligand (Rankl, Tnfsf11) as well as by its involvement in IL-1 and IL-6 stimulated bone resorption. A strict localization-, concentration-, and time-dependent regulation of PG synthesis can be expected. We showed that E2 was able to sensitize osteoblastic cells to mechanical loading, as Cox-2 expression was strongly elevated in the presence of E2 in mechanically loaded compared to loaded MC3T3-E1 cells without E2 (Fig. 1). A mechanosensitizing effect of E2 on prostaglandin production has been shown in bone cells
from elderly women and from osteoporotic donors [17,18]. Because E2 did not significantly affect Cox-1 and Cox-2 mRNA expression, the authors suggested that estrogen modulates bone cell mechanosensitivity via the prostaglandin synthetic pathway independently of Cox expression. The discrepancy with our study might be due to the different cell type, as it can be presumed that primary osteoblasts from elderly women were less responsive to mechanical stimulation owing to decreased ERa activity [19]. ERa has been shown to be required for mechanically induced responses of bone cells in vitro and in vivo [20]. A dose-dependent effect of estrogen and parathyroid hormone on the mechanical response of osteoblasts and osteocytes, respectively, has already been documented, respectively [21,22]. In addition to their role as ligand-dependent mediators of the effects of estrogen, ERs have also been shown to participate in mechanotransduction of bone cells in a ligand-independent manner [23]. Using the high affinity ER antagonist ICI182,780, we demonstrated the reversal of the sensitizing effect of E2 on the mechanically induced Cox-2 expression, thereby confirming the involvement of the ER-dependent signaling pathway in the mechanosensitizing mechanism. This result is consistent with the finding of Armstrong et al. [24] that Wnt/b-catenin signaling contributes to early responses of bone cells to mechanical strain and that its effectiveness is ER dependent. Cox-2 has been shown to be a Wnt/b-catenin inducible gene [11]. The regulation of Cox-2 expression by Lef-1 and b-catenin has been demonstrated in murine chondrocytes [25]. As expected, treatment with LiCl, which inhibits GSK3-b, thereby activating the Wnt/b-catenin signaling pathway, led to a significant up-regulation of Cox-2 expression in osteoblastic cells (Fig. 2A). Both the increase of Cox-2 by mechanical stimulation alone and by LiCl alone was abolished by mechanical load in the presence of LiCl, respectively. It has been shown that mechanical strain in the presence of a Wnt/b-catenin signaling activator can result in a synergistic transcriptional response on Wnt/b-catenin target genes in MC3T3-E1 cells [26]. Interestingly, the authors demonstrated less or even a lack of up-regulation of most of these genes in cells after treatment with the Wnt/b-catenin pathway activator alone compared to the expression of cells after application of mechanical strain without activator. In contrast, we found a higher increase of Cox-2 expression after the addition of the activator alone than after application of mechanical load without activator. We did not detect any mechanosensitizing effect on the response, but an abolishment of the increase of Cox-2 expression after mechanical loading on additionally treating with LiCl. This difference might be due to the longer loading period (strain for 5 h) used in the study of Robinson et al. [26]. We verified our results by using the physiological activator Wnt-3a (Fig. 3). In comparison with mechanically loaded MC3T3-E1 cells, those treated with Wnt-3a alone showed a higher increase of Cox-2 expression. Treatment with Wnt-3a and mechanical load led to the diminishing of the elevated Cox-2 expression. These changes correlated with decreased Cox-2 protein levels after Wnt-3a treatment and mechanical loading (Fig. 2B). Thus it can be proposed that in the present study activated Wnt/b-catenin signaling counteracts with mechanically induced signaling. Studies have provided evidence that mechanical loading is able to counteract cytokine-sensitive pathways in chondrocytes [27,28]. Treatment with the physiological Wnt/Lrp-5/b-catenin pathway antagonist Dkk-1 alone did not lead to a significant effect on Cox-2 expression, thus demonstrating that the Wnt/b-catenin pathway was not involved in basal expression of Cox-2 (Fig. 3). However, application of mechanical strain in the presence of Dkk-1 resulted in a significantly higher up-regulation of expression compared to the expression after mechanical loading without Dkk-1. This stimulatory effect was not affected by Wnt-3a, suggesting that Dkk-1 exerts a Wnt-3a independent effect on mechanical regulation of
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Cox-2 expression. This deserves further investigation. Using CIMC4 and UMR106 cells, respectively, as well as a different strain period, Case et al. [29] and Sunters et al. [30], too, have shown that Dkk-1 did not block strain-induced transcription from the Wnt/ Lrp-5 target gene promoter. Activation of the ER signaling pathway led to down-regulation of Wnt/b-catenin signaling dependent Cox-2 expression induced by LiCl (Fig. 2A). On the other hand, LiCl abolished the activating effect of E2 in the presence of mechanical strain. Thus, LiCl and E2 are potent activators of Cox-2 expression in the mechanically unstimulated and stimulated cell, respectively, and both of them have a strong inhibitory effect on the activation caused by the other. 5. Conclusion In conclusion, we have shown that ER and Wnt/b-catenin and mechanically induced signaling interact to modulate early response gene expression. We demonstrated a sensitizing effect of E2 on mechanically induced Cox-2 expression, which seemed to be due to ligand-dependent ER signaling and was diminished by simultaneous activation of Wnt/b-catenin signaling. Moreover, we showed an activation of early gene expression by Wnt/b-catenin signaling in the unloaded cell that was diminished by simultaneous activation of ER signaling. Our results suggest that the interaction of both pathways might be one regulatory mechanism by which mechanical loading exerts its role in maintaining bone mass in the healthy skeleton. This interacting regulatory system appears to be well suited to fine-tune early gene expression in response to different loading situations, and its disturbance during postmenopause, when E2 levels are declined, might be involved in the disturbed equilibrium of bone formation and bone resorption, ultimately leading to postmenopausal osteoporosis.
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Acknowledgments [23]
This work was financially supported by the Deutsche Forschungsgemeinschaft (D.F.G., Grant No. IG18/5-1; Grant No. JA 504/10-1).
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