Oestrogen receptors and linear bone growth

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1 Acta Pædiatrica ISSN REVIEW ARTICLE Oestrogen receptors and linear bone growth Andrei S. Chagin Lars Sävendahl Pediatric Endocrinology Unit, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden Keywords Oestrogen receptors, Growth plate, Longitudinal bone growth, Selective oestrogen receptor modulators Correspondence Andrei S. Chagin, Pediatric Endocrinology Unit, Q2:08, Karolinska University Hospital, Stockholm, Sweden. Tel: Fax: Abstract In this review we summarize available data regarding linear growth in oestrogen receptor alpha (ER )- and oestrogen receptor beta (ER )-deficient mice. We discuss these findings in relation to known oestrogenic effects in humans and the possibility of applying this knowledge for the therapeutic modulation of longitudinal bone growth employing selective oestrogen receptor modulators (SERMs). We conclude that SERMs potentially could offer new possibilities to modulate bone growth by specifically targeting different oestrogen receptors within the growth plate. Received 22 March 2007; revised 15 May 2007; accepted 5 June DOI: /j x INTRODUCTION There are several potential clinical applications of pharmacologic modulating of growth plate fusion. Delaying growth plate fusion may improve final height in short children, whereas accelerating the fusion process would reduce further growth in patients predicted to become extremely tall. The significance of oestrogen as an initiator of the pubertal growth spurt in girls is well known (1). In boys, testosterone was believed to be the major trigger of pubertal growth until 1994 when a male patient carrying an inactivating mutation in oestrogen receptor alpha (ER ; herko man) was described. The patient presented with severe osteoporosis, a bone age of 14 years at his chronological age of 28, nonfused growth plates and, though being an adult, he was continuously growing (2). Additional evidence supporting a role for oestrogen in the process of growth plate fusion in both males and females comes from the characterization of patients with inactivating mutations of aromatase P450 (CYP19). Such inactivation eliminates the conversion of testosterone to oestrogen and, consequently, serum oestrogen levels are extremely low. The phenotype of such patients is similar to that of the herko man, including open growth plates, immature bones, lack of pubertal growth spurt and continuation of linear growth after becoming adults (3 6). Furthermore, inhibition of oestrogen synthesis by letrozole, an aromatase inhibitor, delays bone maturation and growth plate fusion in boys, thereby increasing the predicted adult height (7,8) and near-final height (9). In contrast to the herko man, treatment of aromatasedeficient patients with oestrogen results in rapid bone maturation, growth plate fusion and cessation of longitudinal bone growth (4). Sexual precocity in both girls and boys, which is characterized by a premature increase in sex steroid secretion, increased height velocity, accelerated epiphyseal maturation and reduced final adult height, provides another natural example of oestrogen action on longitudinal bone growth (10). These individuals demonstrate an early growth spurt, advanced bone maturation, premature growth plate fusion and, as a result, reduced final height. Furthermore, high-dose oestrogen treatment given to tall girls is well known to promote bone maturation, induce growth plate fusion and, thereby, reduce their final height (11 13). However, oestrogen therapy is associated with a multitude of undesired effects. These include a fear of increased risks for breast and uterus cancer (14), and negative effects on reproductive capacity (15). Consequently, there is a need for alternative ways to modulate growth plate fusion minimizing the side effects in other tissues. SELECTIVE OESTROGEN RECEPTOR MODULATORS (SERMs) SERMs represent a new category of drugs designed to prevent and treat diseases such as osteoporosis and breast C 2007 The Author(s)/Journal Compilation C 2007 Foundation Acta Pædiatrica/Acta Pædiatrica , pp

2 Table 1 Relative femur length in ER-deficient mice Sex Age Relative femur length Genotype Reference ( / +/+)( / / ) ( +/+ +/+) ( +/+ / ) Males A ERKO (12.1) = DERKO (12.1) < WT (12.5) = BERKO (13.1) 1 (31) WT (11.4) = BERKO (11.6) 1 (35) B ERKO (15.2) = DERKO (15.4) < WT (16.1) = BERKO (16.1) 1 (31) WT (15.0) = BERKO (15.2) 1 (35) ERKO (15.1) < WT (15.6) 2 (38) ERKO (15.7) > DERKO (15.0) < WT (15.8) = BERKO (16.1) 2 (33) C ERKO (15.2) < WT (15.8) 1 (39) WT (16.5) = BERKO (16.6) 1 (36) Females A ERKO (12.5) = WT (12.9) 1 (32) WT (11.2) = BERKO (11.3) 1 (35) B ERKO (11.9) < DERKO (13.2) < WT (13.7) < BERKO (14.5) 1 (37) WT (14.6) < BERKO (15.2) 1 (35) ERKO (12.0) < DERKO (12.8) < WT (13.4) < BERKO (14.0) 1 (34) ERKO (13.8) < WT (14.9) 1 (32) ERKO (15.4) = DERKO (15.9) = WT (15.3) = BERKO (15.9) 2 (33) ERKO (15.6) = WT (15.3) 2 (38) C WT (16.4) = BERKO (16.6) 1 (36) ERKO (12.0) < DERKO (13.2) < WT (14.0) = BERKO (14.0) 1 (34) ERKO (14.4) < WT (15.9) 1 (39) Relative femur lengths in wild-type (WT), ER knockout (ERKO), ER knockout (BERKO) and double ER plus ER knockout (DERKO) mice indicated as: < denotes significantly shorter femur; > denotes significantly longer femur; = denotes no significant difference in femur length. P-values < 0.05 were considered statistically significant. Absolute values for femur lengths (mm) are given within parentheses. Age A: days of age; age B: days of age; age C: 1 year and older. Genotype 1: mice generated in the laboratory of Dr. Smithies (remaining ER activity and no ER activity; 42). Genotype 2: mice generated in the laboratory of Dr. Chambon (no ER activity and remaining ER activity; 42). cancer. Unlike oestrogens, all of which are pure agonists, and antioestrogens, all of which are pure antagonists, SERMs display both pro- and antioestrogenic activities in tissuespecific manners (16). Potentially, SERMs may therefore offer a way to selectively target the bones and avoid the harmful effects of oestrogen in other tissues such as the breast and uterus. Indeed, promising data have been published confirming that raloxifene, a second-generation SERM, acts as an oestrogen agonist in bone tissue and oestrogen antagonist in the breast and uterus (17). The unique biological response of a tissue to SERMs involves three interacting factors: first, the types and levels of ERs expressed; second, the nature of the change in conformation with the ERs upon binding the SERM, which influences the recruitment of different coregulators; and, finally, the types and levels of different coregulator proteins present (18,19). Tamoxifen, a first-generation SERM, an ER antagonist in breast tissue and agonist in uterus, is widely used in the treatment of breast cancer and has also been reported to delay skeletal maturation in short pubertal boys (20) and in girls with precocious puberty attributable to McCune-Albright syndrome (21,22). In addition, tamoxifen administration in boys with pubertal gynaecomastia has been reported to decrease longitudinal bone growth (23). Similarly, tamoxifen inhibits bone growth in both male and female rodents and foetal rat bone explants (24 26). In contrast, another SERM, raloxifene, acts as an oestrogen receptor beta (ER ) agonist and induces growth plate fusion in rabbits without influencing uterus weight (27). These observations suggest that SERMs might allow the therapeutic modulation of longitudinal bone growth avoiding many side effects associated with oestrogen therapy. To allow the specific modulation of linear bone growth employing SERMs, it is essential to identify the specific roles of ER and ER in the regulation of bone growth. Most of the current knowledge about the role of ER and ER in the regulation of longitudinal bone growth has been gained from studies in ER-deficient mice. ER - AND ER KNOCKOUT MICE Mice in which ER (ERKO) or ER (BERKO) or both receptors (DERKO) have been knocked out have been created (28 30). Analyses of bone length in these mice have revealed that the effects of ER inactivation on bone growth are sexand age dependent (31 39). As demonstrated in Table 1 and discussed below, different effects are seen in young animals, that is, at days of age, the time of and immediately following sexual maturation (age A), in adult animals ( days old; age B) and in old animals (1 year and older; age C). Oestrogen receptor- (ER ) and bone growth in mice Knocking out ER does not affect femur length in male mice of any age (31,35,36) and in females at age A (35) and C (34,36; Table 1). However, bone growth is stimulated by inactivation of ER in female mice at age B (Table 1; 34,35,37). Interestingly, this inhibitory effect of ER (BERKO females have longer bones than wild type [WT], Table 1) is observed only at age B, an age when oestrogen levels are peaking (36). In another paper, ER was reported not to affect growth (33). However, it is important to point out that the strain of 1276 C 2007 The Author(s)/Journal Compilation C 2007 Foundation Acta Pædiatrica/Acta Pædiatrica , pp

3 BERKO mice with unaffected growth enrolled in the study by Sims et al. (33) has remaining ER activity (genotype 2 animals, Table 1; 30). It is essential to recognize that female ERKO and DERKO mice, secondary to a lack of negative feedback, do have elevated serum levels of oestrogen (33, 214, 36 and 115 pmol/l for female WT, ERKO, BERKO and DERKO mice, respectively; 37). Female ERKO mice (expressing ER ) have shorter bones than female DERKO animals (without ER ; 34,37). In male mice, knocking out ER does not lead to an elevation of serum oestrogen levels and furthermore, bone lengths in male ERKO mice are similar (31) or even greater (33) than in DERKO mice. Based on these data, it could be hypothesized that ER inhibits mouse bone growth only when activated by elevated serum levels of oestrogen (34). Additional support for this hypothesis is provided by the in vitro observations that 10-fold higher concentrations of 17 estradiol is required to activate the oestrogen response element (ERE) by ER than by ER (100 pmol/l vs. 10 pmol/l, respectively; 40,41). Finally, we have demonstrated that ER activation also has the capacity to induce growth plate fusion in old female mice (34). Our finding has recently been confirmed by another research group who demonstrated that old ERKO female but not male mice fuse their growth plates (39). Oestrogen receptor- (ER ) and bone growth in mice When analyzing the effects of ER gene ablation on bone length, clear differences appear depending on how the animals were created. The ERKO mice originating from Dr. Smithies s laboratory were generated by insertion of the Neo gene into exon 2 of the ER gene (hereafter referred to as ERKO-Neo2; 28), whereas the ERKO animals from Dr. Chambon s laboratory were generated by deletion of exon 3 from the ER gene (hereafter referred to as ERKO- 3; 30). Findings in ERKO- 3 mice, considered to be ER -null (42), has lead to the conclusion that ER does not influence bone length in female mice (33,38), and either stimulates (38) or has no effect (33) in male mice (genotype 2, Table 1). In contrast, in ERKO-Neo2 mice a clear stimulatory effect of ER on bone length has been described in both male (31,39) and female mice (34,37,39; genotype 1, Table 1). When trying to understand the mechanism behind the different bone phenotypes in ERKO- 3 and ERKO-Neo2 mice, it is important to recognize that ERKO-Neo2 mice do express a short (46-kDa) form of ER produced by alternative splicing (42). Although this form lacks the activation function-1 (AF-1) domain, it still contains DNA-binding (DBD) and ligand-binding (AF-2) domains (42,43). Consequently, this 46-kDa form activates ERE-dependent promoters in AF-2 dominant cells and can even display a dominant negative function (43). Interestingly, the patient lacking ER (herko) carries a mutation at residue 157 of the protein, which would allow the short 46-kDa spliced form of ER to be synthesized (43). In support of this, we have recently found that the mutated herko possesses relatively high oestrogenic activity (44) and indeed forms a short 46-kDa isoform (unpublished results). Clearly, the 46-kDa form of ER is involved in the regulation of bone growth in mice and presumably also in humans although the functional characteristics of this isoform of ER need to be further studied. SPECULATIONS Why humans but not most other species, including mice, have a pubertal growth spurt and also fuse their growth plates during puberty? This question is still unresolved but the data presented so far allow us to make some speculations. Patients with aromatase deficiency do not fuse their growth plates and continue to grow after sexual maturation, just like rodents, and oestrogen treatment induces growth plate fusion (3,4,45). This suggests that circulating oestrogen levels in rodents are not high enough to trigger growth plate fusion. Indeed, estradiol levels are roughly 10-fold higher in humans (Tanner stage B5 in girls) than in mice and rats (46 50). To induce growth plate fusion in mice, they need to be exposed to estradiol concentrations around 200 pmol/l for a year (conclusion based on Ref. 34, 37 and 39), a level that is comparable to what is normally observed in girls during mid-puberty (48,50). Circulating levels of estradiol in prepubertal boys and girls are below pmol/l and rise above pmol/l during middle (girls) or late (boys) puberty (48 50). As a higher estradiol concentration is needed to activate ER (100 pmol/l) than ER (10 pmol/l) (40,41), it can be speculated that activation of ER is essential for growth plate fusion to occur. This is supported by observations in ERKO mice (only ER present) that have high serum levels of estradiol and undergo growth plate fusion in contrast to DERKO mice (no ER,noER ) that do not fuse their growth plates despite similarly elevated serum estradiol levels (34). These observations in mice are in sharp contrast to the well-known male patient with inactivating mutation of ER (herko), who had high circulating oestrogen levels and open growth plates despite being an adult (2). Based on DNA sequence analysis in the herko man, it has been found that he is able to express a 46-kDa isoform of ER (44). This isoform of ER has been shown to repress ER activation and thus act in a dominant negative fashion (43). Based on these findings and taking in account that human ER lacks a functional AF-1 domain in contrast to murine ER (51), it could be speculated that the 46-kDa isoform of ER in the herko male has the capacity to suppress ER, at least in the growth plate, and if so, this patient could be considered as a functional ER /ER double knockout. If this is true, the results gained in the ER knockout mice and the herko man do not contradict each other and consequently, ER should be targeted when aiming to modulate growth plate fusion by SERMs. Our report of growth plate fusion in rabbits treated with the ER agonist raloxifene supports this strategy (27). Although the process of growth plate fusion is similar in rabbits and humans, data from clinical studies are needed before SERMs can be generally accepted for the manipulation of growth plate fusion in patients. SUMMARY We have summarized available data regarding bone growth in ER-deficient mice and discussed these findings in relation C 2007 The Author(s)/Journal Compilation C 2007 Foundation Acta Pædiatrica/Acta Pædiatrica , pp

4 to known oestrogenic effects in humans. We believe that this knowledge can be applied for the therapeutic manipulation of longitudinal bone growth in humans employing SERMs. The rapid development of more specific SERMs will offer new possibilities to more specifically target different ERs within the growth plate and thereby open new effective treatment options of growth disorders. ACKNOWLEDGEMENTS This study was supported by the Swedish Research Council, Sällskapet Barnavård, Stiftelsen Frimurare Barnhuset i Stockholm. We thank Dr. Lena Salin for critical review of the manuscript. References 1. Feuillan PP, Foster CM, Pescovitz OH, Hench KD, Shawker T, Dwyer A, et al. Treatment of precocious puberty in the McCune-Albright syndrome with the aromatase inhibitor testolactone. N Engl J Med 1986; 315: Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, et al. Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. 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