Anna Börjesson
Centre for Bone and Arthritis Research Institute of Medicine at Sahlgrenska Academy University of Gothenburg, Gothenburg, Sweden
Gothenburg 2013
Cover illustration:
Enhancement of a µCT image of vertebral trabecular bone. Anna Börjesson designed the cover and Anette Hansevi performed the µCT.
The role of estrogen receptor α in the regulation of bone and growth plate cartilage
© Anna Börjesson 2013 anna.borjesson@gu.se ISBN 978-91-628-8578-6 http://hdl.handle.net/2077/30567 Printed in Gothenburg, Sweden 2013 Kompendiet, Gothenburg
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Academy, University of Gothenburg, Gothenburg, Sweden
Estrogens are important endocrine regulators of skeletal growth and maintenance in both females and males. Studies have demonstrated that the estrogen receptor (ER)-α is the main mediator of these estrogenic effects in bone. Therefore, estrogen signaling via ERα is a target both for affecting longitudinal bone growth and for bone remodeling. However, treatment with estradiol (E2) would lead to an increased risk of side effects such as venous thromboembolism and breast cancer. An improved understanding of the signaling pathways of ERα will therefore be essential in order to find better bone specific treatments, with minimal adverse effects, for different estrogen-related bone disorders. The aim of this thesis was to characterize the intracellular ERα signaling pathways in bone versus other tissues by studying different domains of ERα, and also to find which target cells that are important for the ERα mediated regulation of bone.
The intracellular signaling via ERα activation function (AF)-2 in mice was shown to be crucial for the estrogenic effects on all parameters evaluated, whereas the ERα AF-1 signaling was tissue dependent: with a crucial role in uterus but not in cortical bone. Thus, SERMs activating ERα AF-1 minimally could retain beneficial effects in cortical bone while minimizing effects on reproductive organs.
Further studies of ERα signaling in mice showed that ERα was indispensible for the reduction of longitudinal bone growth and reduced growth plate height in old mice (resembling growth plate closure in humans). In addition, it was shown that specific inactivation of ERα AF-1 results in a hyperactive ERα, since old mice lacking ERα AF-1 displayed fused growth plates. Studies using mice with cartilage specific inactivation of ERα revealed that local ERα in the growth plate chondrocytes is not involved in the regulation of the early pubertal longitudinal bone growth, while it is crucial for the effects of E2 to reduce longitudinal growth in sexually mature mice.
By examining mice lacking ERα in neuronal cells it was found that central ERα has an effect on bone. It was shown that, although peripheral ERα signaling is positive for the bone, centrally expressed ERα in nervous tissue has a negative impact on bone. Thereby, neuronal cells are important targets for estrogen, mediating ERα signaling pathways that affect bone remodeling.
The studies presented in this thesis have characterized signaling pathways of estrogen in bone versus other tissues. A better knowledge about the estrogenic signaling pathways may in turn facilitate the design of new, bone specific treatment strategies with minimal adverse effects.
Keywords: estrogen receptor α, bone, growth plate, estrogen ISBN: 978-91-628-8578-6
dessa processer är det möjligt att stärka benmassan med östrogenbehandling samt att manipulera den slutliga längden hos en person genom att antingen tillföra östrogen eller hämma östrogenets verkan. Tyvärr har östrogenbehandling visats ge ökad risk för allvarliga biverkningar som blodproppar och bröstcancer. Studier har visat att östrogen utövar effekter på skelettet framförallt genom att binda till östrogenreceptor α (ERα), som då ger signaler som påverkar olika processer i skelettet. Signaler från ERα är inte bara viktiga för östrogenets effekter i skelettet utan påverkar också flera andra organ i kroppen. En bättre förståelse för hur ERα signalerar är därför viktig för att hitta nya, bättre, behandlingar som bara ger positiva effekter på skelettet utan att ge biverkningar i andra organ. Syftet med denna avhandling var att utreda signaleringsvägarna för östrogen via ERα i skelettet kontra övriga vävnader, genom att studera olika delar av ERα, samt att hitta målceller som är viktiga för att förmedla signaler via ERα som leder till beneffekter.
Vi har visat att östrogensignalering via den del av ERα som kallas aktiverings funktion 2 (AF-2) är nödvändig för att östrogen ska ge effekter på alla de vävnader vi har utvärderat. Däremot visades att betydelsen av östrogensignalering via AF-1 delen av ERα skilde sig mellan olika vävnader, med en viktig roll i livmodern men inte i kortikalt ben. Därmed skulle östrogenliknande preparat som har minimal påverkan på AF-1 delen av ERα kunna ge positiva effekter på ben utan att ge negativa effekter på reproduktionssystemet.
Tillväxtbrosket finns i skelettets tillväxtzoner och våra studier har visat att ERα är absolut nödvändig för att östrogen ska kunna reducera tillväxtzonens höjd samt minska längdtillväxten i äldre möss. Dessutom har vi visat att äldre möss som saknar AF-1 delen av ERα, till skillnad från normala möss, har helt förbenade tillväxtzoner och att detta resulterar i att de är kortare. Vi har också studerat möss som saknar ERα i broskcellerna som utgör tillväxtbrosket och visat att ERα i tillväxtbrosket inte behövs för att reglera den pubertala längdtillväxten, men är viktig för att minska längdtillväxten och tillväxtzonens höjd i äldre möss. Broskcellerna i tillväxtbrosket är därmed viktiga målceller för att östrogen ska kunna reducera tillväxtzonens höjd och därmed minska längdtillväxten.
Vi har också visat att signalering via ERα i nervceller har en påverkan på ben. Möss som saknar ERα enbart i nervceller har en ökad benmassa, vilket visar att ERα i nervvävnad normalt har en negativ inverkan på ben. Vi har härmed visat att även nervceller är viktiga målceller för östrogen och att de utövar en effekt på ben via ERα.
Resultaten som presenteras i denna avhandling har kartlagt östrogens signaleringsvägar via ERα i ben och andra vävnader. En ökad förståelse för hur östrogen signalerar i kroppen kan leda till utveckling av bättre benspecifika behandlingar, med så få biverkningar som möjligt.
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This thesis is based on the following studies, referred to in the text by their Roman numerals.
I. Börjesson AE, Windahl SH, Lagerquist MK, Engdahl C, Frenkel B, Movérare-Skrtic S, Sjögren K, Kindblom JM, Stubelius A, Islander U, Antal MC, Krust A, Chambon P, Ohlsson C Roles of transactivating functions 1 and 2 of estrogen receptor-α in bone. Proc Natl Acad Sci U S A.
2011;108:6288-93.
II. Börjesson AE, Windahl SH, Karimian E, Eriksson EE, Lagerquist MK, Engdahl C, Antal MC, Krust A, Chambon P, Sävendahl L, Ohlsson C The role of estrogen receptor-α and its activation function-1 for growth plate closure in female mice. Am J Physiol Endocrinol Metab.
2012;302:E1381-9.
III. Börjesson AE, Lagerquist MK, Liu C, Shao R, Windahl SH, Karlsson C, Sjögren K, Movérare-Skrtic S, Antal MC, Krust A, Mohan S, Chambon P, Sävendahl L, Ohlsson C The role of estrogen receptor α in growth plate cartilage for
longitudinal bone growth. J Bone Miner Res. 2010;25:2414- 24.
IV. Ohlsson C, Engdahl C, Börjesson AE, Windahl SH, Studer E, Westberg L, Eriksson E, Koskela A, Tuukkanen J, Krust A, Chambon P, Carlsten H, Lagerquist MK Estrogen receptor-α expression in neuronal cells affects bone mass.
Proc Natl Acad Sci U S A. 2012;109:983-8.
Reprints were made with permission from the publishers.
ABSTRACT ... 3
LIST OF PUBLICATIONS ... 5
CONTENTS ... 6
ABBREVIATIONS ... 8
INTRODUCTION ... 11
General introduction ... 11
Growth plate physiology ... 11
Bone remodeling ... 13
Structure of estrogen receptors ... 14
ERα is the main ER in bone ... 16
Signaling pathways of estrogen ... 16
Primary target cells involved in bone regulation ... 16
Intracellular signaling... 17
Estrogen and postmenopausal osteoporosis ... 19
Estrogen and modulation of longitudinal bone growth... 19
Selective ER modulators (SERMs)... 20
AIM ... 21
METHODOLOGICAL CONSIDERATIONS ... 22
Animal models ... 22
Gonadectomy and E2 treatment ... 22
Measurements of bone parameters ... 23
Dual X-ray absorptiometry ... 23
Peripheral quantitative computerized tomography ... 23
Micro computed tomography ... 23
Histomorphometry of the bone ... 24
Quantitative histology of the growth plate ... 24
Immunohistochemistry ... 25
Three-point bending ... 25
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Analysis of gene expression with real time PCR ... 25
Western blot ... 26
Flow cytometry ... 27
Serum measurements... 27
RESULTS AND DISCUSSION ... 28
Investigating intracellular E2-signaling for bone metabolism and bone growth ... 28
Roles of transactivating functions 1 and 2 of ERα in bone (Paper I) ... 29
The role of ERα and its AF-1 for growth plate closure in female mice (PaperII) ... 30
The role of ERα in growth plate cartilage for longitudinal bone growth (Paper III) ... 32
ERα expression in neuronal cells affects bone mass (Paper IV) ... 34
SUMMARY ... 36
CONCLUSION ... 37
ACKNOWLEDGEMENT ... 38
REFERENCES ... 39
AF-1 AF-2 aa AP-1 aBMD BMD BMU bp BV/TV cAMP cDNA CIITA CNS Col2α1 Col2α1-ERα-/-
DBD DXA E2 EGF ELISA ERα ERα-/- ERαAF-10 ERαAF-20 ERαflox/flox ERβ ERE ERK Estrogen- resistant man FACS FITC FSH GH
Activation function 1 Activation function 2 Amino acid
Activator protein 1
Areal bone mineral density Bone mineral density Basic multicellular unit Base pairs
Trabecular bone volume/total bone volume Cyclic adenosine monophosphate
Complimentary DNA
Class II, major histocompatibility complex, transactivator Central nervous system
Collagen type II α 1
ERαflox/flox mice expressing Cre recombinase under the
control of the Col2α1 promoter, generating mice with deletion of ERα in chondrocytes
DNA binding domain Dual X-ray absorptiometry 17β-estradiol
Epidermal growth factor
Enzyme-linked immunosorbent assay Estrogen receptor α
Total estrogen receptor α knockout Deletion of ERα AF-1
Deletion of ERα AF-2
Exon 3 of the ERα gene is flanked by the loxP sequence Estrogen receptor β
Estrogen response elements
Extracellular-signal-regulated kinases
Man with a non-functional ERα due to a point mutation in exon 2 of ERα
Fluorescence-activated cell sorting Fluorescein isothiocyanate
Follicle-stimulating hormone Growth hormone
9 Gnx
GPCR GPER-1 IGF-I IL K-ERα-/-
KO LBD LH mRNA MUP
Nestin- ERα-/-
OPG Orx Ovx PCNA PCR PE PKA pQCT RANKL RIA RT-PCR SRC-1 SRC-1 KO SERM SP-1 TNFα µCT vBMD WHI WT
Gonadectomy
G protein-coupled receptor
G protein-coupled estrogen receptor-1, also called GPR30 Insulin-like growth factor 1
Interleukin
A mouse model generated in the Korach and Smithies laboratories with low expression of some truncated ERα isoforms. First believed to be a total ERα knockout.
Knockout
Ligand binding domain Luteinizing hormone Messenger RNA Major urinary protein
ERαflox/flox mice expressing Cre recombinase under the
control of the Nestin promoter, generating mice with deletion of ERα in neuronal cells
Osteoprotegerin Orchidectomy Ovariectomy
Proliferating cell nuclear antigen Polymerase Chain Reaction Phycoerythrin
Protein kinase A
Peripheral quantitative computerized tomography Receptor activator of nuclear κB ligand
Radioimmunoassay Real time PCR
Steroid receptor coactivator 1
Mice with a specific deletion of SRC-1 Selective estrogen receptor modulator Specificity protein 1
Tumor necrosis factor α Micro computed tomography Volumetric bone mineral density Women’s health initiative Wild type
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Estrogens are important endocrine regulators of skeletal growth and maintenance in both females and males. Experimental and human studies have demonstrated that the estrogen receptor (ER)-α is the main mediator of these estrogenic effects in bone (1-8). Therefore, estrogen signaling via ERα is a target both for affecting longitudinal bone growth and for bone remodeling. 17β-estradiol (E2) is the most potent estrogen and it is important for preventing bone loss. E2 also affects longitudinal bone growth and low levels of E2 during sexual maturation is essential for the longitudinal growth spurt, whereas high E2 levels in late puberty result in growth plate closure and thereby cessation of longitudinal bone growth in humans (9). Estrogen deficiency after menopause reduces bone mineral density (BMD) which leads to an increased risk of fractures (10). Since E2 is important for the different stages of longitudinal bone growth it is possible to affect the longitudinal bone growth by manipulating the estrogen signaling in young patients with idiopathic short stature or constitutionally tall stature. It is also possible to prevent bone loss in postmenopausal women by E2 administration (3,10).
However, there are several estrogen responsive tissues expressing ERs e.g.
the reproductive system, central nervous system (CNS), and blood vessels (11-13). Therefore, estrogen treatment would not only have positive effects on the bone but could also lead to an increased risk of adverse effects, such as venous thromboembolism and breast cancer (14,15). Thus, it would be beneficial to develop bone-specific estrogen treatments. To achieve this, it is important to further characterize the estrogenic signaling pathways in bone versus other tissues in vivo.
The growth plates are a cartilaginous tissue found at the distal ends of the long bones between the metaphyseal and epiphyseal bone (Fig 1). They consist of three principal zones of chondrocytes: the resting zone, the proliferative zone and the hypertrophic zone, where the hypertrophic chondrocytes are the most differentiated (Fig 1) (16,17). Longitudinal bone growth of the long bones occurs at the growth plates through a process called endochondral ossification. In this process the chondrocytes proliferate, become more differentiated and finally become hypertrophic. The hypertrophic chondrocytes enlarge and secrete matrix proteins which form a
Figure 1. Schematic drawing of a femur and a magnified growth plate. (Growth plate: Image courtesy of Prof Tim Arnett, UCL).
scaffold for the formation of bone tissue (18). Terminal hypertrophic chondrocytes undergo apoptosis at the metaphyseal border of the growth plate. There is a vascular invasion in the cartilage at this site and the cartilage is then remodeled into bone by bone cells migrating to this area, resulting in bone elongation (18-20).
Low E2 levels enhance skeletal growth during early sexual maturation (i.e.
the pubertal growth spurt), whereas high E2 levels during late puberty result in growth plate fusion and thereby cessation of longitudinal bone growth in humans (9). The mechanisms of action for these two seemingly opposite effects of estrogens on longitudinal bone growth are not fully understood but clearly depend on maturational stage and serum levels of E2 (9,21).
Estrogens are crucial regulators of the growth hormone/insulin-like growth factor 1 (GH/IGF-I) axis, and therefore, some of the effects of estrogens on skeletal growth might be indirect via modulation of the GH/IGF-I axis, while other effects of estrogens on skeletal growth may be direct (9,22-24). Low- dose E2 treatment has been shown to increase serum GH and IGF-I, which may contribute to the pubertal growth spurt. An effect via the GH/IGF-I axis is supported by the fact that ER blockade down-regulates the GH/IGF-I axis (3,9). The key role for E2 in the regulation of longitudinal bone growth and growth plate closure in humans was demonstrated by the findings that both males and females with estrogen deficiency, caused by a mutation in the aromatase gene, do not show a pubertal growth spurt and continue to grow after sexual maturation due to unfused growth plates (3,25-28). ERα was demonstrated to be the main mediator of this effect when a man with a similar phenotype was shown to have a non-functional ERα due to a point mutation in exon 2 (estrogen-resistant man). This man had elevated serum E2 levels, to which he was non-responsive (6,29). In contrast, the growth phenotype of the aromatase deficient patients could be rescued by E2
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treatment (3,25-27). The mechanism for the estrogenic effects on growth plate fusion is not well understood. However, it has been demonstrated that estrogen accelerates the fusion of the growth plate in rabbits by advancing the senescence of the growth plate via proliferative exhaustion of the chondrocytes (20).
The process in which osteoclasts resorb bone and osteoblasts form new bone is called bone remodeling. This process is important for enabling the bone to respond and adapt to load induced strain, replace old or damaged bone tissue, and to maintain calcium homeostasis (30). In a healthy skeleton the number and activity of osteoclasts and osteoblasts are balanced so that the bone resorption and formation is equivalent. Bone remodeling is a constant process in both the dense cortical bone and in the spongy trabecular bone. The remodeling occurs in basic multicellular units (BMUs), in which osteoclasts and osteoblasts collaborate to remodel the bone in a bone remodeling cycle (Fig 2) (1,31,32). This cycle starts with recruitment of preosteoclasts to the bone surface. At the surface the preosteoclasts fuse and become mature multinucleated osteoclasts. The osteoclasts start resorbing bone by digesting the mineral matrix. At the end of the resorption-phase preosteoblasts migrate to the resorption site, mature and start forming new bone by producing matrix, which is subsequently mineralized (31,32). Some of the osteoblasts become surrounded by bone matrix and differentiate into osteocytes. A network is formed in the bone by the osteocytes via canaliculi. Osteoblasts and bone lining cells are also connected to this network, enabling communication between cells. This network senses the loading of the bone and signals to the BMUs to start the bone remodeling cycle, thus it is important for adapting the bone to mechanical load (33).
Estrogen is important for maintaining the balance between bone resorption and bone formation. At menopause, when the E2 levels drop, there is an imbalance in bone resorption and bone formation leading to an accelerated bone loss (1). Estrogen has fundamental effects on bone metabolism by several different mechanisms, acting both direct and indirect on the skeleton.
Estrogen affects the expression of certain factors in osteoblastic cells e.g.; it increases osteoprotegerin (OPG) and decreases receptor activator of nuclear κB ligand (RANKL) and tumor necrosis factor (TNF)-α, the result of the regulation of these genes is that bone resorption is suppressed (34-36). It has been suggested, by Khosla et al (1), that the most consistent effect of estrogen on bone is to i) induce commitment of precursor cells to the osteoblast lineage at the expense of the adipocyte lineage and ii) prevent apoptosis of osteoblastic cells (1,37-41). In addition, estrogen also reduces bone
resorption by inhibiting differentiation and promoting apoptosis of osteoclasts (40,42-46).
Figure 2. Bone remodeling cycle. I) Multinucleated osteoclasts resorb bone by digesting the mineral matrix and resorption pits are formed. II) Osteoblasts are recruited to the resorption pit and start to form bone. III) The osteoblasts produce bone matrix which is subsequently mineralized. Some of the osteoblasts become surrounded by bone matrix and differentiate into osteocytes. After a complete bone remodeling cycle the resorbed bone has been replaced by new bone.
Different bones contain different ratios of cortical and trabecular bone, where the vertebra contains mostly trabecular bone while the diaphysis of the long bones contains mainly cortical bone. The cortical bone constitutes 80% of the skeleton and is likely the major contributor to overall fracture risk (1,47). The trabecular bone constitutes 20% of the skeleton, still the trabecular bone has a larger surface area than the cortical bone due to its spongy structure and it also has a higher metabolic activity. Although the trabecular bone loss is accelerated at menopause, it has been suggested that the trabecular bone is not as sensitive as the cortical bone to the estrogen deprivation at menopause, since the trabecular bone loss starts already in young-adult life when the levels of estrogens are normal (1).
The first estrogen receptor discovered; ERα, was cloned in 1986, although its existence was suggested already in 1962 (48-50). Ten years later a second estrogen receptor; ERβ, was cloned (51). In 2005 there were reports suggesting that a seven-transmembrane G protein-coupled receptor (GPCR) named G protein-coupled ER-1 (GPER-1 or GPR30) was a membrane- associated ER (52-54). However, there are also contradictory studies suggesting that GPER-1 is not an estrogen receptor (54-58).
Both ERα and ERβ belong to the nuclear receptor superfamily of ligand- activated transcription factors and they display, overall, high sequence homology. The primary structure of these receptors is divided into six
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functional domains, A-F (Fig 3) (51,59-61). The best conserved region, in these ERs, is the C domain (>95% (51,60)), where the DNA binding domain (DBD) and the area important for dimerization is found. This means that ERα and ERβ can dimerize to form both homo- and heterodimers and it also implies that they bind to the same DNA sequences (62). The A/B domains in the N-terminus is the least conserved region (<20%) and contains the ligand independent activation function-1 (AF-1) (61). The ligand binding domain (LBD) is found in the E/F domains in the C-terminus together with the ligand dependent AF-2, this region is not very well conserved (E ~55%, F <20%
(51,59,60)). The differences in this region suggest that, although both receptors have the same affinity to 17β-estradiol they can also bind other ligands, which may be receptor-specific. The D domain is not well conserved (~30%), it comprises the nuclear localization signal and is also a hinge, which increases the flexibility between the C- and E/F domains (51,61). The most abundant ERα isoform is the 66 kDa protein, but there is also a truncated less expressed 46 kDa ERα isoform that lacks most of the A/B domains (63).
Both AFs are important for recruiting cofactors essential for gene regulation.
The cofactors that bind to the AFs are suggested to be dependent on the ER, the cell type and promoter context (64,65). When a ligand binds to an ER the ER undergoes a conformational change so that helix 12 in the LBD folds in the agonistic orientation (66). Helix 12 has a key role in forming the ERα AF-2, together with helices 3, 4 and 5, which can attract cofactors important for gene regulation, hence the AF-2 is ligand dependent (66-68). Although the ERβ AF-1 is suggested to be weaker than the ERα AF-1 (69), they both interact with cofactors independently of ligand being bound to the receptor or not, therefore the AF-1 is ligand independent (70,71). In vitro studies of ERα have shown that the E2-induced transactivation is dependent on either one of the AFs but that most promoter contexts require synergism between them for full transcriptional activity (64,70,72-74).
Figure 3. Schematic picture of the different domains of ERα and ERβ, A-F. The sequence homology between the receptors is given in percent. A/B, important for transcriptional activation/repression; C, responsible for DNA binding and dimerization; D, comprises a hinge and the nuclear localization signal; E/F, important for transcriptional activation/repression and contains the ligand binding domain.
The bone-sparing effects of estrogen are primarily mediated via ERα. This has been demonstrated using different mouse models lacking ERα, ERβ, both ERα and ERβ or GPER-1. ERα has been shown to be crucial for mediating the estrogenic effects in both trabecular and cortical bone, while ERβ plays a less important role and GPER-1 has been demonstrated to be dispensable for the estrogenic preservation of bone mass (2,4,5,54,75,76). Female and male mice that are estrogen deficient due to ovariectomy (ovx) or orchidectomy (orx) lose bone. However, both trabecular and cortical bone mass can be restored by E2 treatment in both genders. In contrast, the bone mass cannot be restored in E2 treated ovx or orx mice lacking ERα (ERα-/- mice), demonstrating a crucial role for ERα in both the female and male skeleton (2,4,5,75). ERβ has only been shown to slightly modulate the effects of ERα in female mice but not in male mice (2,8,77-79). This modulating effect of ERβ was shown by studying female adolescent ERβ-/- mice. These mice displayed an increased cortical bone mineral content but no trabecular phenotype (79). Also 1-year-old female ERβ-/- mice were studied and were shown to maintain the cortical phenotype, seen in the adolescent mice, but also had a higher trabecular bone mass compared to controls (78). This suggests that ERβ normally has a slightly inhibiting effect on ERα in the female bone (78,79). In addition, estrogen-regulated transcriptional activity in bone was evaluated in female ERα-/-, ERβ-/- and ERαβ-/- mice, demonstrating that ERβ reduces ERα-regulated gene transcription in bone in the presence of ERα but partially replace ERα in the absence of ERα (80). Although both ERα and ERβ have an AF-1, this region is not well conserved. The ERβ AF-1 is weaker than the ERα AF-1 and it has been shown that the estrogenic effect mediated by ERα is slightly repressed when an ERα/ERβ heterodimer is formed (69).
Both ERs have been shown to be expressed throughout the skeleton in osteoblasts, osteoclasts, osteocytes and chondrocytes (81-84). The expression of ERs in these different bone cells demonstrates that the estrogenic effects can be exerted locally in the skeleton. The relative importance of the different estrogenic target cells for bone regulation is not yet completely understood. It would be valuable to fully characterize the primary target cells for estrogen, and how these cells exert effects on bone regulation when stimulated with E2, in order to have cell specific treatments. To investigate the importance of the different bone cells for the estrogenic skeletal effects, mouse models have
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been developed that specifically lack ERα in one cell type. Specific deletion of ERα in osteoclasts in female, but not male, mice led to trabecular bone loss and this effect was due to a decreased apoptosis of the osteoclasts (42,43).
Further experiments are required in order to find the target cells for the estrogenic effects in trabecular bone in male mice and in cortical bone in both male and female mice.
Bone is traditionally considered to be regulated by peripheral signaling;
controlled by hormones (e.g. estrogen), autocrine/paracrine signals, and mechanical loading. However, bone is an innervated tissue and lately central signaling, via the CNS, has also been recognized to have a major role in bone regulation (85). The first clear evidence showing this was when mice deficient of leptin were studied. These mice were hypogonadal but despite this they had a high bone mass which was shown to be reduced by intracerebroventricular injections of leptin (86). In addition, mice lacking the β2-adrenergic receptor, which binds the main sympathetic neurotransmitter noradrenaline, are resistant to the central bone-reducing effects of leptin, demonstrating that central leptin signaling may exert its negative effects on bone via the sympathetic nervous system (87,88).
At the cellular level the ERs are generally considered to have four signaling pathways, three of them are classified as ligand dependent and one is classified as ligand independent (Fig 4) (89). The ERs are transcription factors that either directly or indirectly bind to the DNA. The AF-1 and AF-2 in the ERs are important for recruiting certain coactivators, which in turn can interact with the transcriptional preinitiation complex and thereby initiate transcription of specific estrogen regulated genes (90,91). There are two genomic ligand dependent signaling pathways involving dimerization and translocating the ERs from the cytosol to the nucleus when ligand binds; 1) in the classical (direct) genomic pathway the ER-ligand complex binds directly to estrogen response elements (ERE) on the DNA and regulates gene transcription, 2) in the non-classical (indirect) genomic pathway other transcription factors are bound to the DNA and the ER-ligand complex binds to these transcription factors and thereby regulates gene transcription (Fig 4) (62,89,92). Fos/Jun and SP-1 are examples of transcription factors involved in the non-classical genomic pathway and these transcription factors can interact with both the ERs and specific sites on the DNA, not harboring EREs. Fos/Jun proteins form the transcriptional complex AP-1 (activator protein 1), which interacts with AP-1 responsive elements in the DNA (93,94). The SP-1 (specificity protein 1) complex interacts with GC-rich SP-1 motifs in the DNA (95). When these transcription factors interact with the
ER-ligand complex gene transcription is activated. The ligand dependent non-genomic pathway is not very well understood. The rapid responses of this pathway imply that no gene regulation occurs. Instead, it involves signaling cascades, which are activated when ligand binds to an ER that could be located either in the membrane or in the cytoplasm (Fig 4) (89,96).
Some of these rapid responses to E2 are mediated by second messenger systems, e.g. cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA), while other responses are mediated by membrane-based ion fluxes, involving e.g. Ca2+ and Ca2+-dependent K+ channels, which are capable of responding to estrogens (96,97). GPER-1 has been suggested to be involved in this non-genomic pathway and there is also some evidence for a membrane localized ERα (52,54,98). The ligand independent pathway does not involve any ligand binding to the ER, instead other factors are involved (e.g. growth factors (GFs) like epidermal growth factor (EGF) and IGF) that can activate extracellular-signal-regulated kinases (ERKs) which in turn can activate the ER by phosphorylation (Fig 4) (89,99-102). The phosphorylated ER translocates to the nucleus where it will recruit coactivators and mediate gene transcription (89,103). ERα has several sites for phosphorylation and most of them are found in the AF-1 (104). Phosphorylation of the ER does not only occur independent of ligand and has been shown to increase after E2 binds to the ER (103).
Figure 4. Intracellular signaling pathways of estrogen receptors.
I) In the classical genomic pathway E2 binds to ERs, which dimerize and translocates into the nucleus where they bind to EREs in promoters of certain estrogen regulated genes. II) In the non-classical genomic pathway the ERs interact with other transcription factors that are bound to specific promoter regions instead of binding directly to the DNA. III) The non-genomic pathway involve an ER in the membrane, mediating rapid signaling cascades without involving gene regulation, e.g. via cAMP that activates phosphorylation cascades. IV) In the ligand independent pathway no E2 binds to the ERs. Instead the ERs can be phosphorylated via growth factor signaling, and the phosphorylated ERs bind to specific promoter regions.
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The estrogen levels decrease drastically at menopause which can lead to symptoms affecting everyday life; e.g. hot flushes and mood swings. Usually these symptoms are transient but the decreased levels of estrogen will also lead to bone loss which may result in postmenopausal osteoporosis. Menopausal symptoms can be reduced by treatment with estrogen plus progestin, which also leads to prevention of bone loss. However, when the long term effects of estrogen plus progestin therapy was evaluated in the Women’s Health Initiative (WHI) study it was shown that healthy postmenopausal women had a number of adverse effects e.g. increased risk of venous thromboembolism and breast cancer, although the risk of fractures was reduced. The part of the WHI study including the estrogen plus progestin therapy was stopped early because the risks were concluded to outweigh the benefits (105,106). The results from the WHI study clearly showed that estrogen plus progestin therapy leads to a decreased risk of fractures but that long-term treatment is not an option due to the several adverse effects reported. Therefore, there is an interest in developing bone- specific estrogen treatments that would not lead to the adverse effects shown in the WHI study (107).
Estrogen is important both for the longitudinal growth spurt and for cessation of growth by inducing growth plate closure (9). It is possible to manipulate the longitudinal bone growth by affecting the estrogenic signaling pathways. High- dose E2 treatment will result in growth plate closure and thereby cessation of growth, leading to a shorter adult stature than expected. In contrast, treatment with aromatase inhibitors (attenuating the conversion of androgens into estrogens) will delay the closure of the growth plates, thereby increasing the final height (108-110). The long-term effects of high-dose estrogen treatment or treatment with aromatase inhibitors are only recently being evaluated. It has been suggested that high-dose estrogen treatment in adolescent girls may lead to reduced fertility later in life (111-113). There are also concerns for increased risks of breast or gynecological cancers (109,114). In addition, boys treated with aromatase inhibitors were shown to have a high rate of vertebral body deformities after treatment (115). Further characterization of the estrogenic signaling pathways in bone and growth plate will therefore be important in order to find better treatments for manipulating longitudinal bone growth, without increasing the risk of adverse effects later in life.
SERMs have the ability to bind to an ER and act as an ER agonist or antagonist in a tissue-specific manner. Tamoxifene was the first developed SERM and it was used for breast cancer treatment since it had an antagonizing effect in breast tissue. In addition, Tamoxifene was shown to be an agonist in bone but unfortunately also an agonist in uterus increasing the risk of endometrial cancer (116,117). Another SERM; Raloxifene, was shown to be an ER agonist in bone and an ER antagonist in breast and it was the first SERM approved for the prevention and treatment of postmenopausal osteoporosis (118). More SERMs have been developed but all available SERM treatments for osteoporosis today, including Raloxifene, will not only lead to a reduced fracture risk but also adverse effects such as thromboembolism (118-120). Therefore, further studies of the signaling pathways of estrogen are required in order to find more specific treatment strategies.
In vitro studies have shown that the SERMs have a bulky side chain, which upon binding to ERα protrudes from the ligand binding pocket. This hinders the optimal conformational change of the LBD of the ERα by preventing the folding of helix 12 in the agonistic orientation. Instead, helix 12 is able to bind to the static region of ERα AF-2; formed by residues from helices 3, 4 and 5. Helix 12 then prevents/or limits the interaction between the static region of ERα AF-2 and certain coactivators and corepressors. Because of the lack of cofactors binding to the ERα AF-2 after SERM binding, ERα AF-1 is suggested to be the main mediator of the SERM effects (64,66-68,121-123).
Variation in the expression of cofactors and the recruitment of cofactors to the ER in different cell types appear to have an important role for the tissue specific effects of the SERMs (66,122). Regarding treatment strategies for osteoporosis, development of new SERMs that would have the positive effects of estrogen in bone and not any of the negative effects in other tissues would be optimal. In addition, a SERM treatment only directed towards the growth plate in very tall girls could reduce their final height without leading to systemic effects. To achieve this, the characterization of the ER signaling pathways in bone versus other tissues is of importance in order to develop better SERMs.
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The aim of this thesis was to characterize the ER signaling pathways in bone versus other tissues in order to have a better understanding of how to develop SERMs that would improve treatment of osteoporosis and estrogen related growth disorders, with minimal adverse effects.
The specific aims were:
To evaluate the importance of the ERα AF-1 and ERα AF-2 for the estrogenic effects in bone versus other tissues.
To study the role of ERα and its AF-1 for the regulation of longitudinal bone growth.
To determine whether the estrogenic effects on longitudinal bone growth is mediated locally, by ERα in the chondrocytes.
To investigate if centrally expressed ERα is important for the regulation of bone mass.
Mice are commonly used as models for studying different human diseases and treatments. The mouse genome is quite similar to ours and it can be manipulated quite easily. In addition, the life cycle of a mouse is short and they are relatively inexpensive to breed. Therefore, mice lacking or overexpressing certain genes can quite easily be developed and studied. Thus, mice have become the most common animal model used in medical research.
Mice and humans have similar bone physiology, but there is a discrepancy regarding growth plate closure. In humans the growth plates close after puberty, due to high E2 levels, leading to a cessation of longitudinal bone growth while in mice the growth plates are never fully closed. However, old mice have a reduced longitudinal bone growth and high-dose E2 treatment can fuse the growth plates in adult mice. Rabbits are an example of another animal model that has been used for studying the growth plate, since they close their growth plate at sexual maturation. However, it is much more difficult to generate genetically modified rabbits than mice and rabbits are more expensive to keep as they require larger space. Therefore, we have chosen mice as models for studying both bone remodeling and growth plate physiology.
The main production of sex steroids in rodents takes place in the ovaries or the testicles and the sex steroid levels are drastically decreased after gonadectomy (ovariectomy (ovx) of female mice and orchidectomy (orx) of male mice). In humans, there is a significant production of androgens from the adrenals that can be converted into estrogens, but in mice the androgen production from the adrenals is considered insignificant. The lack of sex steroids due to ovx and orx of mice leads to a decrease in bone mass which corresponds to the decrease in bone mass seen in postmenopausal women or castrated men. In addition there is a striking decrease of the uterus weight and an increase in the thymus weight in the ovx mice compared to ovary-intact mice. This thesis investigates the estrogenic responses, in both the skeleton and other estrogenic target tissues, in mice with deletion of the whole ERα, the ERα AF-1, the ERα AF-2 or deletion of ERα specifically in cartilage or neuronal cells compared to WT mice. Some of our mouse models have disturbed negative feedback regulation of serum sex steroids, which leads to elevated serum levels of sex steroids. To avoid confounding effects of their endogenous sex steroids these mice were gonadectomized and treated with either E2 or placebo to examine their estrogenic responses. The E2 doses
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used in papers I, II and IV are slightly supraphysiological while the E2 doses in paper III are supraphysiological.
The dual X-ray absorptiometry (DXA) is commonly used for measuring BMD both in the clinical setting and in animal research. It is a non-invasive method, which is an advantage when longitudinal studies are performed. Due to the two emitted X-ray beams with different energy levels, the DXA can distinguish between soft tissue and bone, since these tissues absorb the energy differently. The disadvantage with the DXA technique is that it produces a two-dimensional (2D) image and does not take the third dimension (3D) into account. This could become a problem when studying growing animals where the skeletal size could change drastically. The BMD received is therefore called the areal BMD (aBMD, g/cm2) and should not be mistaken for the true volumetric BMD (vBMD, g/cm3). The DXA analyses in papers I, II, III and IV were performed using the Lunar PIXImus mouse densitometer (Wipro GE Healthcare, Madison, WI, USA), which was calibrated before use.
In contrast to the DXA, the peripheral quantitative computerized tomography (pQCT) can measure the true vBMD and also separate the trabecular bone from the cortical bone. The disadvantage of this method, compared to DXA, is the slightly higher radiation dose and that the analyses are more time consuming. The pQCT technique is based on a rotating X-ray source which moves around the bone being analyzed, creating a 3D image. The trabecular bone in this thesis was measured, ex vivo, in the metaphysis of the long bone, with the growth plate as a reference point, and is defined as the inner 45% of the total cross-sectional area. The cortical bone was measured, ex vivo, in the middiaphyseal region of the long bone. In this thesis the pQCT analyses in papers I, III and IV were performed using the pQCT XCT RESEARCH M (version 4.5B; Norland, Fort Atkinson, WI, USA), operating at a resolution of 70 µm.
The micro computed tomography (µCT) gives a 3D image of the bone with a higher resolution than the pQCT, but the analyses are more time consuming than the pQCT analyses. The µCT creates an image where the
microarchitecture of the analyzed bone can be seen. The image separates trabecular and cortical bone and can also provide information about the trabecular network by calculating the trabecular number and thickness, operating at a resolution of 5 µm. The bone is placed on a rotating stage, which is located between an X-ray source and a charged-coupled detector (CCD) array. The spatial resolution of the image is primarily determined by the focal spot size of the X-ray source, the detector’s array resolution, and the bone’s position with respect to the source and the detector. The imaging system provides a series of X-ray projections from a range of angles around the bone. Each projection represents the value of the X-ray attenuation line integral through the bone along the line from the X-ray source to the X-ray detector element. Imaging the bone at equiangular-spaced views over 180 degrees provides a complete set of projection data. Image reconstruction creates a 2D image from the measured projection data and a 3D image is calculated by reconstructing and stacking individual 2D slices. Different algorithms are then used to calculate several bone parameters. In papers I and IV a model 1072 scanner (Skyscan N.V., Aartselaar, Belgium) was used for the µCT analyses.
Histomorphometry is a classical method for evaluating bone parameters. The dissected and fixed bone of interest is embedded in plastic (e.g. L R White Resin; Agar Scientific) and thereafter sectioned. The structure and organization of the bone sections are analyzed under a light microscope.
Different kinds of staining are also used in order to better visualize specific cells or bone compartments. The quantity of e.g. trabecular bone volume or osteoclasts is measured by static histomorphometry (papers I and IV), while dynamic histomorphometry measures the changes in the bone over time by using fluorescent double labeling. In papers I and IV dynamic histomorphometry was studied by injecting mice with calcein at day 1 and 8 before termination, inducing double labeling. The calcein is incorporated on the bone surfaces and by double labeling the amount of new bone formed in one week can be determined.
Quantitative histology is used to evaluate growth plate parameters. The dissected and fixed bone of interest is embedded in paraffin and then sectioned and stained with e.g. Alcian blue/van Gieson, to better visualize the growth plate tissue. The sections are studied under a light microscope and all measurements of the growth plate, in this thesis, are made on the central three-fourths of the growth plate. In papers II and III, quantitative histology was performed to measure the growth plate height, the heights of the different
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zones of the growth plate and also the height of the terminal hypertrophic chondrocytes.
This is a commonly used method for identifying proteins of interest in bone sections and can aid in histomorphometric analyses. Sections are prepared from paraffin embedded fixed bones. This method takes advantage of the fact that antibodies recognize antigens (proteins). Therefore, specific antibodies have been developed that recognize certain antigens. Prior to sectioning, the bones are fixed by formaldehyde which leads to cross-linking of proteins.
This could make the antigens inaccessible for the antibodies, which in turn could lead to false negatives. Trying to avoid this, antigen retrieval is performed on all sections so that the antibodies will have the possibility to find their antigens in the section. Unspecific binding of the antibodies may lead to false positives, and one has to be cautious when evaluating the results and always use appropriate controls. Immunohistochemistry was used in paper II to detect ERα and type X collagen (marker for hypertrophic chondrocytes) and in paper III to detect proliferating cell nuclear antigen (PCNA, detecting proliferating chondrocytes).
Three-point bending is used for testing the mechanical properties of a bone.
In our studies we have used the Instron 3366, Instron Corp. (Norwood, MA, USA) testing machine, the bone is placed in the machine and load is applied to the bone until it breaks. Since the bone breaks, other analyses of the bone should have been performed prior to the three-point bending test. The test is performed on the mid diaphysis of a long bone and is therefore mainly corresponding to the mechanical strength of the cortical bone. The load deformation curves from the three-point bending are registered and from these the biomechanical parameters are calculated by raw-files produced by Bluehill 2 software version 2.6 (Instron Corp., Norwood). Maximal load at failure and stiffness was calculated from three-point bending tests in paper IV.
Gene expression is usually evaluated with the real time PCR (RT-PCR) method, which is very sensitive for measuring mRNA levels. The results will show the relative expression levels of a specific gene in different tissue samples. The mRNA is prepared from the tissue of interest and is then reverse transcribed into cDNA. The amount of cDNA is measured by amplifying the specific cDNA sequence. There are two common methods for
this quantification of cDNA and both are using fluorescence. The first method uses a dye, usually SYBR Green, which fluoresces only when it is bound to double stranded DNA. Primers for a specific gene are added and after each amplification step the fluorescence from SYBR Green will increase. The SYBR Green dye binds to all double stranded DNA and it can therefore also bind to unspecific PCR products and primer dimers. In our studies, we have used the TaqMan method which is more specific than the SYBR Green method. In the TaqMan method, fluorescence is emitted only when the complimentary sequence of a specific TaqMan probe is amplified.
This method is more specific since the probe is designed to anneal to a specific cDNA sequence between the two PCR primers. The probe has both a fluorescent dye and a quencher of fluorescence attached to it and when the probe is intact no fluorescence is emitted, due to the quencher. When the Taq-polymerase starts replicating the DNA from the primers the probe is eventually cleaved and the quencher is no longer close to the fluorescent dye.
Fluorescence is then emitted and the emitted light is proportional to the amount of amplified cDNA. The amplification can be followed over time and the amount of cDNA is doubled in each cycle making the amplification exponential. It is possible to analyze two different mRNAs at the same time with TaqMan, making it possible to correlate the amount of the specific gene to an internal standard, usually the 18S. RT-PCR analysis in paper I and IV was performed using the ABI Prism 7000 Sequence Detection System (PE Applied Biosystems).
Western blot is a commonly used method for identifying specific proteins in a homogenized tissue sample. This technique separates denatured proteins by size, using gel electrophoresis. The proteins on the gel are then transferred to a membrane where the protein of interest is visualized by antibody staining.
In order to know that similar amounts of the tissue samples are loaded onto the gel the membrane is also stained for β-actin, which should be expressed at similar amounts in all samples. To be able to quantify the amount of the protein of interest in different samples a densitometry method is used and the protein of interest is correlated to the amount of β-actin in the sample.
Quantification of proteins from a Western blot by densitometry is not a perfect method, but it is a much cheaper method than mass spectrometry. In paper III, the amount of ERα in cartilage is quantified by using densitometry and Image Gauge software (Fujifilm). In paper I, the aim was to show that the ERαAF-20 protein was expressed and therefore no quantification was made.
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Flow cytometry is a powerful tool for sorting and phenotyping cells from a heterogeneous mix of cells in a relatively short time. The cells are labeled with different fluorophore-conjugated antibodies, which emit fluorescence after they pass an excitation source. The cells can then be sorted, one cell at a time, based upon the specific emitted wavelength of the fluorophore.
However, cells contain endogenous fluorophores and this autofluorescence could interfere with the fluorescence from the fluorophore-conjugated antibodies used to sort the cells. This has been taken into account when we have analyzed our samples. In paper I bone marrow cells were stained with phycoerythrin (PE)-conjugated antibodies to CD19 for detection of B lymphocytes and in paper IV fluorescein isothiocyanate (FITC)-conjugated antibodies to CD3 for detecting T lymphocytes. The cells were then subjected to fluorescence-activated cell sorting (FACS) on a FACSCalibur (BD Pharmingen) and analyzed by using FlowJo software.
Commercially available radioimmunoassay (RIA) kits were used to assess serum concentrations of E2, luteinizing hormone (LH), testosterone, and IGF-I. Serum leptin and serotonin levels were measured using enzyme-linked immunosorbent assays (ELISA). Serum follicle-stimulating hormone (FSH) levels were measured using an immunofluorometric assay. There are several available immunoassays for measurements of mouse serum E2 levels.
However, none of the available assays can detect small changes in mouse serum and are therefore not capable of distinguishing E2 levels between ovary-intact (sham) mice and ovx mice. In papers I, II, III and IV, the aim was to test if the different knockout (KO) mouse models had disturbed negative feedback regulation of serum E2. A RIA kit from Siemens Medical Solutions Diagnostics is able to detect such changes in serum E2 levels and was used for evaluating the serum E2 levels in papers I, II, III and IV (124).
The most accurate measurements of mouse serum E2 is gained from gas chromatography/tandem mass spectrometry, but unfortunately this method is very expensive and also requires large sample volumes (200 µl) in order to get accurate measurements and was therefore not feasible to use (124).
The two transactivation functions; ERα AF-1 and AF-2, have been shown to interact with cofactors (coactivators and corepressors) and are therefore important for mediating the gene-regulating effects of ERα. In vitro studies have shown that most promoter contexts requires a synergism between ERα AF-1 and AF-2 for full transcriptional activity but that ERα AF-1 or AF-2 alone also can mediate transactivaton. The importance of the respective AF is dependent on the promoter context and the cell type, which is probably due to the cofactors found in the cell type and their expression levels (64,70,73,74).
Further characterization of these AFs in vivo is of importance to understand if they can act independently to mediate gene regulation and if this is dependent on the tissue. The previous experiments evaluating the ERα AF-1 and AF-2 have been performed in vitro and only recently the importance of each AF for the estrogenic effects in vivo could be evaluated. This was possible after the development of two new mouse models lacking either the ERα AF-1 (ERαAF-10 (125)) or the ERα AF-2 (ERαAF-20 (75)). The ERαAF-10 mice have a deletion of 441 bp of exon 1, corresponding to amino acid (aa) 2 to 148, with a preserved translational initiation codon in exon 1 (ATG1). The ERαAF-10 mice do not express any full-length 66 kDa protein (75,125).
Instead they express a truncated 49 kDa ERα protein that lacks AF-1 and also the physiologically occurring, but less abundantly expressed, 46 kDa ERα isoform, initiated by a second translational initiation codon in exon 2 (ATG2). ERαAF-20 mice have a deletion of the AF-2 core, which resides within exon 9 and corresponds to aa 543 to 549 (Fig 3) (75). The sizes of the ERαAF-20 proteins are slightly smaller than the WT ERα proteins of 66 kDa and 46 kDa respectively (corresponding to the 7 aa truncation located in the AF-2 region; (75)). In order to evaluate the involvement of the AFs for the bone protective effects of estrogen (Paper I, (75)) and also for the effects on growth plate closure and longitudinal bone growth (Paper II, (126)) the ERαAF-10 and ERαAF-20 mice were studied together with mice completely devoid of ERα protein (ERα-/- (127)) and wild type (WT) control mice.
