Abstract
Estrogens are well known for their capacity to promote bone maturation and at high doses to induce growth plate closure and thereby stop further growth. High-dose estrogen treatment has therefore been used to limit growth in extremely tall girls. However, recent data suggest that this treatment may have severe side effects, including increased risk of cancer and reduced fertility. We hypothesized that estrogenic effects in bone are mediated via ERα signaling. Twelve-week-old ovariectomized female C57BL/6 mice were subcutaneously injected for 4 weeks with E2 or selective ERα (PPT) or ERβ (DPN) agonists. After killing, tibia and femur lengths were measured, and growth plate morphology was analyzed. E2- and PPT-treated mice had shorter tibiae and femur bones when compared to vehicle-treated controls, whereas animals treated with DPN had similar bone lengths compared to controls. Growth plate height and hypertrophic zone height were reduced in animals treated with E2 or PPT but not in those treated with DPN, supporting that the effect was mediated via ERα. Moreover, PCNA staining revealed suppressed proliferation of chondrocytes in the tibia growth plate in PPT- or E2-treated mice compared to controls. Our data show that estrogenic effects on bone growth and growth plate maturation are mainly mediated via ERα. Our findings may have direct implications for the development of new and more selective treatment modalities of extreme tall stature using selective estrogen receptor modulators that may have low side effects than high-dose E2 treatment.
Introduction
Longitudinal bone growth takes place in the growth plate, consisting of three layers: resting zone, proliferative zone and the hypertrophic zone. Bone growth is regulated by estrogens, acting either indirectly via the GH/IGF-I axis or directly via estrogen receptors (ER) expressed in the growth plate chondrocytes (Borjesson et al. 2013). Multiple studies have shown the importance of estrogens in regulating bone maturation and mineralization and in preventing bone loss. The most effective estrogen is 17β-estradiol (E2). The importance of E2 in the regulation of the bone growth is well known. However, the effects of E2 are dose dependent, where low levels stimulate bone growth in early puberty and high levels induce growth plate fusion in late puberty (Juul 2001). In addition to estrogens, phytoestrogens may also affect bone growth and growth plate fusion. Studies performed by our group have shown that resveratrol, a phytoestrogen structurally similar to diethylstilbestrol, delayed growth plate fusion and improved longitudinal bone growth in ovariectomized rabbits (Karimian et al. 2013). Moreover, selective estrogen receptor modulators (SERMs) have also been shown to regulate growth plate fusion. Our studies have demonstrated that raloxifene induced growth plate fusion and decreased chondrocyte proliferation in rabbits (Nilsson et al. 2003). In addition, tamoxifen, another SERM, was shown to suppress longitudinal and cortical bone growth in male rats (Karimian et al. 2008).
The expression of ERα and ERβ in bone and growth plate cartilage has previously been demonstrated (Borjesson et al. 2013). Moreover, gender- and skeletal region-specific variations in ER expression was recently investigated in the rat demonstrating expression of ERα as well as ERβ in spinal and tibial growth plate chondrocytes in both males and females (Li et al. 2012).
The important role of estrogen signaling in growth plate closure was demonstrated in the studies of patients with aromatase deficiency or an inactivation mutation of the ERα gene (Smith et al. 1994, Morishima et al. 1995, Bilezikian et al. 1998, Quaynor et al. 2013). In addition, a clinical study of an 18-year-old female patient with a homozygous ERα mutation interfering with estrogen signaling has recently suggested that ERα mutations cause estrogen resistance. The symptoms of the patient with a homozygous ERα mutation were similar to the phenotypical changes in the mouse orthologue knockout (Quaynor et al. 2013). In contrast, excessive ER activation, as seen in tall adolescent girls with high doses of estradiol (100–1000 µg/day), leads to premature growth plate closure (Goldzieher 1956, Normann et al. 1991). Unfortunately, high-dose estradiol treatment not only limits further bone growth but also has severe side effects including reduced fertility (Venn et al. 2004, Hendriks et al. 2011) and increased risks of breast and endometrial cancers, as well as melanoma (Collaborative Group 1997, Weiderpass et al. 1999, Benyi et al. 2014). It is important to point out that for contraception much lower doses of estradiol (20–30 µg/day) are nowadays being used (Bachmann & Kopacz 2009).
Several studies using different mouse models have suggested that ERα is the main ER mediating estrogenic effects in bone (Lindberg et al. 2002, Moverare et al. 2003). In particular, mice with chondrocyte-specific deletion of Esr1-encoding ERα were shown to have longer bones than wild-type mice, suggesting ERα-mediated growth plate fusion (Borjesson et al. 2010). Therefore, estrogen signaling via ERα has a regulatory effect on longitudinal bone growth and bone development. In contrast to ERα, ERβ was shown only to act in the presence of ERα by moderating its effects in female mouse bone (Sims et al. 2002, Lindberg et al. 2003). Thus, previous studies suggest that ERβ may affect bone growth mainly in the presence of ERα. Based on the current data, we hypothesized that specific ER signaling may have selective effects in growth plate cartilage. To test this, we treated 12-week-old ovariectomized female C57BL/6 mice with E2 and selective ERα and ERβ agonists while tibia and femur bone growth was monitored. We aimed to study the effects of selective ERα and ERβ agonists on longitudinal bone growth.
Materials and methods
Reagents
17β-estradiol-3-benzoate (E2) was purchased from Sigma-Aldrich and the ERβ-selective agonist DPN (2,3-bis (4-hydroxyphenyl)propionitrile) and the ERα-selective agonist PPT (1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole) were obtained from Tocris Bioscience (Bristol, UK).
Primary rabbit anti-PCNA antibody (RbPAb + PCNAab 18197) was provided by Abcam. Normal donkey serum and secondary CY3-conjugated AffiniPureF (ab) Fragment Donkey Anti-Rabbit IgG antibody were purchased from Jackson ImmunoResearch Laboratories. Fluorescence mounting medium was obtained from Dako. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Sigma-Aldrich.
Animals
Twelve-week-old female C57BL/6 mice (n = 9–10/group) were purchased from Charles River Laboratories. Animals were housed in the animal facility at the University of Gothenburg and kept in 12-h day and night condition with standard chow and tap water ad libitum. The study was approved by the ethical committee for animal experiments at the University of Gothenburg (ethical permit no. 205-2007).
Ovariectomy and treatment
Mice were randomized into 5 different groups. Four groups were ovariectomized and one group was sham-operated. Both ovariectomy and sham operations were performed at twelve weeks of age under intraperitoneal anesthesia with ketamine (Ketalar; Pfizer) and medetomidine (Domitor; Orion Pharma, Espoo, Finland). Carprofen (Orion Pharma) was used postoperatively for pain relief. A midline incision was followed by flank incisions of the peritoneum, and the ovaries were removed with sterile scissors. The skin incision was closed with metallic clips. The sham-operated mice were treated in the same way, except that the ovaries were not removed. Sham-operated mice were subcutaneously (s.c.) injected with 100 μL of vehicle (10% ethanol and 90% Miglyol 812, Omya Peralta, Hamburg, Germany) (n = 10). The ovariectomized mice were treated s.c. with 100 μL of E2 (1 µg/mouse; 0.04 mg/kg/day) (n = 9), PPT (175 µg/mouse; 7 mg/kg/day) (n = 10), DPN (105 µg/mouse; 4.2 mg/kg/day) (n = 10) or vehicle (n = 10). All the groups received injections 5 days per week for 4 weeks.
Tissue samples collection
The femur and tibia were dissected out and fixed in 4% phosphate-buffered formalin for 24 h. Decalcification was done in 10% EDTA for 2 weeks and bones were stored in 70% ethanol. For histological analysis, tibias were embedded in paraffin. Five micrometer-thick sections were prepared from the paraffin blocks. Uteruses were collected and weighed prior to analysis.
Body weight, uterus, femur and tibia measurements
Body weight was measured at the beginning and the end of the experiment. Also, at the end of the experiment, mice were killed and uteruses were weighted. The length of the femur and tibia was measured ex vivo using a pocket vernier caliper (Helios-Preisser, Gammertingen, Germany).
Quantitative histology of mouse growth plates
Tibia growth plates were analyzed ex vivo. For quantitative histology of tibia growth plates, 5-µm-thick paraffin-embedded tibia sections were prepared and stained with Alcian blue/van Gieson. The quantitative analysis was performed by measuring two-third of the growth plate sections at 10× magnification. Images were captured by a Nikon Eclipse E800 light microscope (Nikon) connected to the digital camera (Hamamatsu C4742-95, Hamamatsu City, Japan) with a digital color camera system (Olympus DP70). Olympus MicroImage software (version 4.0; Olympus Optical) was applied for imaging. The height of the whole tibia growth plates was calculated as an average of 20 measurements per growth plate. The height of the proliferative and the hypertrophic zones was measured in 20 columns per tibia growth plates and was presented as an average. Hypertrophic chondrocytes were defined as cells with height bigger than 7 µm. All the measurements were obtained in a blind manner by Image Pro Plus, version 6.3 software (NIH).
Immunofluorescence/immunocytochemistry for the detection of PCNA expression
For immunohistochemistry, the tissue sections were deparaffinized for 40 min at 60°C, rehydrated in xylene, 100% ethanol, 95% ethanol and 75% ethanol, for 5 min in each solution and finally washed with water. Antigen retrieval was performed in sodium citrate buffer (10 mM, pH 6.0) for 15 min at 95°C. After retrieval, the slides were incubated with 1.5% donkey serum in PBS for 1 h at room temperature (RT), with primary rabbit anti-PCNA antibody (1:250 dilution) overnight at 4°C and with secondary donkey anti-rabbit antibody (1:250 dilution) for 1 h at RT. Nuclear staining with DAPI was performed for 15 min at RT. After the staining, the slides were mounted with Dako Fluorescence Mounting Medium.
TUNEL assay (apoptosis detection)
The TUNEL assay was performed according to the manufacturer’s instructions. Briefly, apoptotic chondrocytes were identified by terminal deoxynucleotidyltransferase (TdT)-mediated deoxy-UTP nick end labeling (TUNEL) immunohistochemistry applying the TdT-FragEL DNA fragmentation kit (Oncogene Research, Boston, MA, USA) as described previously by our group (Chagin et al. 2004). Alexa-546 (Invitrogen) (red florescence) positive cells represented apoptotic chondrocytes.
Statistical analysis
All the data are shown as mean ± s.e.m., and the differences between control and treated groups were calculated by one-way ANOVA by multiple comparisons vs control group (Holm–Sidak method). ***P < 0.001, **P < 0.01 and *P < 0.05 were considered as statistically significant.
Results
Effects of E2 and selective ERα and ERβ agonists on tibia and femur growth
To analyze the effects of E2 and selective ERα and ERβ agonists, mice were ovariectomized and treated with E2, PPT or DPN. Tibia and femur longitudinal growth was decreased by either E2 or PPT treatment compared to vehicle (Fig. 1A and B; P < 0.01). However, DPN did not have any effect (Fig. 1A and B). Longitudinal growth of tibia and femur was decreased in sham-operated mice with intact ovaries compared to OVX vehicle (Fig. 1A and B; P < 0.01).
Effects of E2 and ERα and ERβ agonists on tibia and femur length. The study groups included ovariectomized mice treated with E2, ERα agonist PPT, ERβ agonist DPN or vehicle alone and sham-operated mice. The group treated with E2 consisted of 9 mice, the other treatment groups contained 10 mice each. Values are means ± s.e.m. *P < 0.05 and **P < 0.01. Tibia length (A) and femur length (B).
Citation: Journal of Endocrinology 232, 3; 10.1530/JOE-16-0263
Effects of E2 and selective ERα and ERβ agonists on tibia growth plate cartilage
Histological evaluation of tibia sections was performed to investigate the effects of E2 and selective ERα and ERβ agonists on the growth plate. In OVX mice, the growth plate height was decreased by E2 or PPT treatment compared to vehicle (Fig. 2A and B; P < 0.001), whereas DPN had no effect on growth plate height. In addition, the growth plate height was also decreased in sham-operated mice compared to vehicle-treated OVX mice (Fig. 2A and B; P < 0.001).
Additional histological analysis of tibia growth plate zones in OVX mice showed that the height of both proliferative and hypertrophic zones was decreased by E2 or PPT treatment compared to vehicle (Fig. 2C and D; P < 0.01). However, no effect was detected on DPN treatment. In addition, the height of both proliferative and hypertrophic zones was also decreased in sham-operated mice compared to OVX vehicle (Fig. 2C and D; P < 0.001).
Interestingly, the hypertrophic zone was more affected than the proliferative zone by either E2 or PPT treatment. As a consequence, the proliferative/hypertrophic ratio observed was higher in mice treated with either E2 or PPT than in other treatment groups (Fig. 2E; P < 0.001).
Effects of E2 and ERα and ERβ agonists on tibia growth plate length and proliferative and hypertrophic chondrocytes. Alcian blue/van Gieson staining of mouse proximal tibia growth plate. R = resting zone; P = proliferative zone and H = hypertrophic zone (A), quantitative morphological studies showing the total growth plate height. (B), the height of the proliferative zone (C), the height of the hypertrophic zone (D) and ratios of proliferative/hypertrophic zones (E). Magnifications 10×. The group treated with E2 consisted of 9 mice, and the other treatment groups contained 10 mice each. Values are means ± s.e.m., ***P < 0.001.
Citation: Journal of Endocrinology 232, 3; 10.1530/JOE-16-0263
Effects of E2 and selective ERα and ERβ agonists on chondrocyte proliferation and apoptosis
The effects of E2 and selective ERα and ERβ agonists on the proliferation capacity of growth plate chondrocytes were analyzed by PCNA staining. The histological evaluation of PCNA staining of tibia growth plate cartilage showed lower proliferation in E2- or PPT-treated OVX mice compared to OVX vehicle (Fig. 3A; P < 0.001; Supplementary Fig. 1, see section on supplementary data given at the end of this article). However, proliferation was not affected by DPN treatment. Besides, proliferation in tibia growth plate cartilage was also decreased in sham-operated mice compared to vehicle-treated OVX mice (Fig. 3A; P < 0.001). In addition, the effects of E2 and selective ERα and ERβ agonists on apoptosis were studied by TUNEL assay. No significant differences in apoptosis level were detected between the different treatment groups (Fig. 3B).
Proliferation and apoptotic detection in tibia growth plate chondrocytes. The effects of E2 and selective ERα and ERβ agonists on chondrocyte proliferation and apoptosis were analyzed by the PCNA (A) and TUNEL (B) techniques, and percentages of positive cells were calculated. The group treated with E2 consisted of 9 mice, and the other treatment groups contained 10 mice each. Values are means ± s.e.m. ***P < 0.001.
Citation: Journal of Endocrinology 232, 3; 10.1530/JOE-16-0263
Effects of E2 and selective ERα and ERβ agonists on body and uterus weights
Treatments with E2 or selective ERα and ERβ agonists did not have any effect on body weight. However, body weight was decreased in sham-operated mice compared to OVX vehicle (Table 1; P < 0.001). With regard to the effects on uterus weight, treatments with either E2 or PPT increased uterus weight in OVX mice (Table 1; P < 0.001). However, uterus weight was not affected by DPN treatment in OVX mice (Table 1). As expected, uterus weight was increased in sham-operated mice when compared to vehicle-treated OVX mice (Table 1; P < 0.001).
Body and uterus weights of female mice treated with E2 or selective ERα and ERβ agonists.
| Body weight (g) | Uterus weight (mg) | |
|---|---|---|
| Sham (n = 10) | 19.5 ± 0.4*** | 33.1 ± 4.2*** |
| Vehicle (n = 10) | 21.3 ± 0.2 | 15.1 ± 1.4 |
| E2 (n = 9) | 21.1 ± 0.3 | 106.3 ± 7.8*** |
| PPT (n = 10) | 20.9 ± 0.3 | 26.4 ± 1.7*** |
| DPN (n = 10) | 21.7 ± 0.3 | 12.2 ± 1.1 |
P < 0.001 vs vehicle.
Discussion
We here show that selective ERα agonist PPT or E2 treatment decreased tibia and femur longitudinal growth in female OVX mice. In addition, the height of the tibia growth plate cartilage was decreased by PPT or E2 treatments. These changes were observed in both the proliferative and the hypertrophic zones of the tibia growth plate. Similar findings in the group of animals treated with E2 and ERα agonist, but not with ERβ agonist, suggest that the observed effects of E2 are mediated via ERα.
With regard to ERα-mediated regulation of bone growth, ERα-expressing growth plate chondrocytes are potential targets for ERα-selective agonists. Moreover, ERα was shown to be a regulator of chondrocyte proliferation mediating longitudinal bone growth using a transgenic mouse model (Ikeda et al. 2012). Our findings are in line with previous studies in global and chondrocyte-specific ERα knockout models demonstrating inhibitory effect of ERα on bone growth (Borjesson et al. 2010, 2012). In addition, the expression of constitutively active mutant ERα in chondrocytes was shown to reduce chondrocyte proliferation and impair longitudinal bone growth in female mice (Ikeda et al. 2012). Our study is the first to demonstrate that ligand-dependent activation of ERα causes suppression of bone growth. We have shown that E2 and PPT treatments negatively affect the height of both proliferative and hypertrophic zones. Both hypertrophic and prehypertrophic chondrocytes were shown to be decreased in a mouse model expressing constitutively active mutant ERα in chondrocytes (Ikeda et al. 2012).
In contrast to the selective ERα agonist (PPT), the selective ERβ agonist (DPN) did not show any effect on longitudinal bone growth or growth plate chondrocytes proliferation. Previous studies showed regulation of skeletal growth by ERβ in female (Lindberg et al. 2001) but not in male mice (Vidal et al. 2000). Furthermore, a recent in vitro study suggested an inhibitory effect of ERβ on chondrocyte proliferation (Zeng et al. 2016). Taking all this into account, it is still possible that ERβ activation may have an effect on bone growth.
With regard to the effects on proliferation, mechanistic studies of growth plate cartilage revealed suppressed proliferation of chondrocytes by PPT or E2 in the tibia growth plate. In contrast, the histological analysis showed no effects on apoptosis in all treatment groups, although genes involved in apoptosis were previously found to be regulated by ERα in bone (Chokalingam et al. 2012). In line with our data, a recent study found no differences in apoptosis between ERα−/− and WT mice (Borjesson et al. 2012). However, molecular mechanisms of ERα-mediated bone growth inhibition remain to be identified. Gene expression analysis, such as RNA sequencing, could be applied to identify the spectrum of target genes regulated via ERα in the growth plate.
Previous studies have found different effects of estradiol on growth plate closure in humans and mice. High doses of estradiol induce growth plate closure in humans but not in mice at the end of puberty (Weise et al. 2001). Therefore, care should be taken in extrapolating these findings in mice to humans. With regard to clinical applications of our findings, toxicological studies must be performed to evaluate the potential adverse effects of selective ERα agonists. In particular, ligand-dependent ERα activation may induce tumorigenic side effects, especially in women, as ERα has been implicated in the progression of breast cancer (Cortez et al. 2010). Furthermore, a study of male ERα knock-in mice treated with PPT showed that ERα signaling is important for male mouse reproductive tract development (Sinkevicius et al. 2009).
Studies of PPT activity in estrogen target tissue in vivo showed that PPT was as effective in stimulating uterine weight as E2; however, it required higher concentration than E2 to achieve the stimulating effect (Harris et al. 2002). In contrast with these observations, in our study, PPT inhibited bone growth, but had very weak uterotrophic effect compared to E2. These results suggest that it may be possible to optimize doses of PPT to suppress growth plate proliferation, without substantially affecting uterus and mammary glands. Currently, a limited number of studies used selective ERα agonists in vivo (Arias-Loza et al. 2008), and further experiments are still needed to determine the potential side effects of PPT and other ERα agonists.
To conclude, we here demonstrate that a selective ERα agonist has the capacity to suppress longitudinal bone growth in treated mice. Our findings may have direct implications for the development of a specific treatment of extreme tall stature.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/JOE-16-0263.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by Stockholm City Council, Swedish Research Council, Pfizer Inc and a grant from Karolinska Institutet.
Authors’ contribution statement
M I, L S, C O and M L designed the experiments. M I and M L performed the experiments. Data were analyzed by M I and M L. M I drafted the manuscript. Critical review of the manuscript was done by L S, C O and M L. Final version was approved by L S, M I, C O and M L.
Acknowledgments
The authors thank Dr Emma Ericsson for helping with the software’s introduction at the beginning of the project.
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