α CofactorRIZGene TheVitaminDReceptorGeneandtheEstrogenReceptor- GeneticVariabilityinHumanBonePhenotypes

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(1)Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 135. Genetic Variability in Human Bone Phenotypes The Vitamin D Receptor Gene and the Estrogen Receptor-α Cofactor RIZ Gene ELIN GRUNDBERG. ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2006. ISSN 1651-6206 ISBN 91-554-6528-5 urn:nbn:se:uu:diva-6784.

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(202) List of papers included in the thesis. This thesis is based on the following papers1, which will be referred by their roman numerals: A poly adenosine repeat in the human vitamin D receptor gene is assoI ciated with bone mineral density in young Swedish women. Grundberg E, Brandstrom H, Ribom EL, Ljunggren O, Kindmark A, Mallmin H. Calcif Tissue Int. 2003 Nov;73(5):455-62.. II. Genetic variation in the human vitamin D receptor is associated with muscle strength, fat mass and body weight in Swedish women. Grundberg E, Brandstrom H, Ribom EL, Ljunggren O, Mallmin H, Kindmark A. Eur J Endocrinol. 2004 Mar;150(3):323-8. III. Imbalanced Expressed VDR Haplotypes in Primary Human Trabecular Bone Cells are Associated with BMD and Fractures in Men: the MrOS Study in Sweden and Hong Kong E Grundberg, E Lau, T Pastinen, A Kindmark, O Nilsson, Ö Ljunggren, O Johnell, D Mellström, I R Johnell, A Holmberg, P C Leung, T Kwok, C Ohlsson, E Orwoll, H Mallmin, T J Hudson and H Brändström. In manuscript. IV. A deletion polymorphism in the RIZ gene, a female sex steroid hormone receptor coactivator, exhibits decreased response to estrogen in vitro and associates with low bone mineral density in young Swedish women. Grundberg E, Carling T, Brandstrom H, Huang S, Ribom EL, Ljunggren O, Mallmin H, Kindmark A. J Clin Endocrinol Metab. 2004 Dec;89(12):6173-8.. V. Site- and gender-specific association between the deletion/insertion polymorphism in the ERĮ-cofactor RIZ gene and bone mineral density in elderly men and women. E Grundberg, K Åkesson, A Kindmark, P Gerdhem, A Holmberg, O Johnell, D Mellström, Ö Ljunggren, E Orwoll, C Ohlsson, H Mallmin, and H Brändström. Submitted. 1. Reprints were made with the kind permission of the Springer Science and Business Media, the European Journal of Endocrine Society and the Endocrinology Society..

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(204) Contents. PREFACE....................................................................................................11 HUMAN GENETICS .................................................................................12 THE HUMAN GENOME ........................................................................13 GENETIC VARIABILITY ......................................................................13 Haplotype structures in the human genome.........................................14 BONE METABOLISM ..............................................................................16 BONE STRUCTURE AND CELL TYPES.............................................16 OSTEOBLAST ISOLATION AND CULTIVATION ............................17 BONE REMODELING............................................................................18 Bone formation by osteoblasts.............................................................19 Bone resorption by osteoclasts ............................................................19 Regulation of bone remodeling ...........................................................20 CALCIUM HOMEOSTASIS...................................................................21 PEAK BONE MASS AND BONE LOSS ...............................................21 OSTEOPOROSIS.....................................................................................22 Primary osteoporosis ...........................................................................23 Secondary osteoporosis .......................................................................23 Bone densitometry ...............................................................................24 Diagnosis .............................................................................................24 GENETICS OF OSTEOPOROSIS ..........................................................26 Linkage analysis ..................................................................................28 The candidate gene approach: an overview.........................................29 CANDIDATE GENE: THE VITAMIN D RECEPTOR..........................29 Vitamin D and its receptor...................................................................29 Biological actions of vitamin D ...........................................................31 VDR and genetic variability ................................................................32 CANDIDATE GENE: THE ERĮ COFACTOR RIZ ...............................33 Estrogen and its receptor .....................................................................33 Biological actions of estrogen .............................................................34 The ERĮ cofactor RIZ1 .......................................................................35.

(205) PRESENT STUDY......................................................................................37 AIMS........................................................................................................37 MATERIAL AND METHODS: CANDIDATE GENE APPROACHES..38 Subjects................................................................................................38 Bone Densitometry ..............................................................................40 Muscle strength measurements (The Uppsala T-score Cohort)...........40 Body Composition (The Uppsala T-score Cohort)..............................41 Assessment of fractures .......................................................................41 Genotyping ..........................................................................................42 Statistical Analyses..............................................................................43 MATERIAL AND METHODS: FUNCTIONAL APPROACHES...........44 CAT Assay ..........................................................................................44 Allelic Imbalance Assay ......................................................................44 Haplotype Construction .......................................................................46 RESULTS AND DISCUSSION ..............................................................47 Genetic variations in the VDR gene are associated to human bone phenotypes: association and functional studies...................................47 A deletion/insertion polymorphism in the ERĮ cofactor RIZ gene is associated with human bone phenotypes: association and functional studies ..................................................................................................50 GENERAL DISCUSSION AND FUTURE PERSPECTIVES ...............53 The Vitamin D receptor gene ..............................................................53 The ERĮ cofactor RIZ gene.................................................................55 CONCLUSIONS......................................................................................58 ACKNOWLEDGEMENTS .......................................................................59 REFERENCES............................................................................................61.

(206) Abbreviations. ANOVA BMD CAT DIP DNA DXA ER ERE FP GLM HGP HMT IL-6 LD NIH OPG OR PCR PTH QCT QTL RFLP RIZ RNA SD SNP TGF-ȕ UTR VDR VNTR WHO. Analysis of variance Bone mineral density Chloramphenicol acetyl transferase Deletion/insertion polymorphism Deoxy ribonucleic acid Dual X-ray absorptiometry Estrogen receptor Estrogen response element Fluorescence polarization General linear model Human Genome Project Histone methyl transferase Interleukin-6 Linkage disequilibrium National Institute of Health Osteoprotegerin Odds ratio Polymerase chain reaction Parathyroid hormone Quantitative computed tomography Quantitative trait loci Restriction fragment length polymorphism Retinoblastoma interacting zinc finger Ribonucleic acid Standard deviation Single nucleotide polymorphim Transforming growth factor-ȕ Untranslated region Vitamin D receptor Variable length of tandem repeat World Health Organization.

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(208) PREFACE. Genetics of complex diseases, such as osteoporosis, is a rapidly developing field with many technologies and strategies reported for detection of the genes influencing the diseases. However, the name “complex disease” indicates that the results from relevant studies are not simple to interpret. Therefore, the present subject needs consideration from two different perspectives; genetics and bone biology. The introduction to the present study is divided in two sections named Human Genetics and Bone Metabolism, respectively, which hopefully will provide sufficient information for the general understanding of the purpose of this thesis. I gratefully acknowledge Professor Alan Boyde, Biophysics OGD, QMUL, Dental Institute, London, UK, for his kind permission of reprinting the cover picture named ‘Porotic Bone’. The picture is a scanning electron micrograph, of an 89 year human female osteoporotic bone, showing typical architectural features of osteoporotic changes in ageing human bone. This work was carried out at the Department of Medical Sciences, Uppsala University and was financially supported by grants from the Swedish Society for Medical Research, the Swedish Research Council, Seda and Signe Hermanssons Foundation, Börjessons Foundation, The Swedish Society of Medicine and Wallenberg Consortium North. Research travel grant was received from Rönnows travel foundation and my participations in conferences were supported by grants from The Swedish Society of Medicine and Rektors Wallenbergmedel.. Elin Grundberg, Uppsala 26th of March, 2006. 11.

(209) HUMAN GENETICS. The pioneering genetic studies were carried out in the late 19th century by Gregor Mendel, a Movarian monk who performed breeding experiments with pea plants. Mendel hypothesized that inheritance was controlled by unit factors and that plants inherited two unit factors for a specific trait, one from (1) . each parent. These so-called unit factors are what we today call genes However, the cellular molecule that provides the basis for Mendelian genetics were unknown until the middle of the 20th century when Oswald Avery and co-workers published 1944 the first results that demonstrated that DNA or deoxyribonucleic acid is the genetic material (2). However, since proteins and enzymes were known to be the molecular phenotype of cells the question remained: how could the genetic information residing in DNA be transformed into proteins and how could it be inherited? This extensive issue would partially be explained in the mid 1950s by studies of the structure of the DNA by James Watson and Francis Crick. They combined previously known data about DNA and proposed that DNA consists of two molecules that are wound around each other to form a righthanded helix that is stabilized by hydrogen bonding interactions with complementary base pairs between the two molecules. Watson and Crick pointed out that this proposed structure, the double helix, provided a hint as to how DNA could be self-replicating (3-5). Shortly afterward the structure of DNA was presented, the involvement of ribonucleic acid (RNA) molecules in protein synthesis was established and the central dogma of molecular biology was a fact. Today we know that each chromosome contains many genes, which are the basic physical and functional units of heredity. Genes are specific sequences of bases (e.g. A, C, T, G) that encode instructions on how to make proteins.. Box 1: Genetics vs. Genomics The WHO definition of Genetics is the study of heredity but the term is sometimes confused with Genomics, which in contrast is defined as the study of genes and their functions, and related techniques.. 12.

(210) THE HUMAN GENOME As the clinical feature of the DNA is a nucleotide sequence with three-letter codes of bases encoding amino acids, knowledge of the exact sequence will provide information of all the specific genes in the human genome. This was achieved approximately 50 years after the presentation of the double helix by Watson and Crick. In 1990 the US Department of Energy and the National Institute of Health presented the Human Genome Project (HGP) with the primary aim of determining the nucleotide sequence of the entire human nuclear genome. The main goals included identification of all the genes in human DNA, determination of the sequences of the 3000 Mb base pairs that make up human DNA as well as improvement of tools for data analysis and establishment of databases to store all information. In order to achieve these goals, the genetic makeup of five model organisms were studied as well; the bacterium Escherichia coli, the yeast Saccaromyces cerevisiae, the roundworm Caenorhabditis elegans, the fruit fly Drosophila Melanogaster and the mouse Mus Musculus*. The project originally was planned to last 15 years, but rapid technological advances enabled its completion in only 13 years, by 2003 (6). Currently, 349 genomes have been completed of which 41 are eukaryotic, and many are ongoing projects. The information is publicly available on the website of the National Center for Biotechnology Information (http://www.ncbi.nih.gov/) and on GOLD™ (Genomes OnLine Database v 2.0 http://www.genomesonline.org/).. GENETIC VARIABILITY During the HGP and even earlier, when the human genome was sequenced, many small regions of DNA that vary among individuals were identified (6, 7) even though any two genomes are 99.9% identical. Sequence variations are either mutations or polymorphisms and often arise in the genome due to copying errors during DNA replication with a frequency of 1 per 1300 bases (8-12) . At present, about 10.4 million different sequence variations in the human genome are reported in the public SNP database (dbSNP, http://www.ncbi. nlm.nih.gov/SNP/; build 125), of which 4.9 million are validated. A mutation is defined as any change in a DNA sequence away from normal that directly cause human diseases. Such disease alleles are generally rare in the population since they reduce fitness.. * Information from http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml. 13.

(211) In contrast, a polymorphism is a DNA sequence variation that is common in the population and usually does not alone cause a disease but may influence characteristics such as height and hair color. However, these common sequence variations can also predispose alone or in combination with other gene variants to common disease and influence the health. To be classed as a polymorphism, the least common allele must have a frequency of 1% or more in the population. If the frequency is lower, the allele is regarded as a mutation. Approximately 90% of sequence variation among individuals is due to polymorphisms where the most common type is single-nucleotide polymorphisms (SNP) (Fig 1) (13). The remaining 10% of the genetic variants are deletion/insertion polymorphisms (DIP) and various length of tandem repeats (VNTR), which are also known as micro- or minisatellites (8). Since coding DNA accounts for about 1.5% of the human genome, most SNPs are found in non-coding regions, such as within introns and do not directly affect the function of the specific protein (14). However, SNPs in the coding regions of a gene can either change the amino-acid sequence or be synonymous or silent, which means that the codon change caused by the SNP does not result in an altered amino acid (11, 12, 15).. Haplotype structures in the human genome Although the HGP has been completed, many polymorphisms reported in public SNP databases are not validated and are also known to differ between populations. Moreover, the polymorphisms in the genome are often not independent of one other, i.e. a particular variant at one site on a chromosome can predict the presence of a particular variant at another site. These phenomenon when polymorphisms are associated to each other are known as linkage disequilibrium (LD) (16-18) and the combination of variants along a chromosome is termed a haplotype (Fig 1) (19). However, these issues described above were the rationale for the initiative of the SNP Consortium Allele Frequency Projects which aimed to determine the frequency of 60,000 SNPs in three major world populations (Africa, Europe, Asia) and also to characterize haplotype pattern across 51 autosomal regions in the human genome (20). The results showed that the human genome can be grouped into haplotype blocks, which are highly correlated across populations. These results were the basis for the International HapMap Project, established in October 2002. The HapMap Project aimed to construct a haplotype map of the human genome from populations with ancestry from parts of Africa, Europe and Asia and to identify so-called "tag" SNPs that uniquely identify the haplotypes (21, 22).. 14.

(212) SNP. SNP. SNP. SNP. SNP. Chr 1 AACAGG..CCACGCGGT..CGACTTC..GGTTC..ATGCTTGCA Chr 2 AACAAG..CCACGCGGT..CTACTTC..GGCTC.. ATGATTGCA Chr 3 AACAGG..CCACGCCGT..CTACTTC..GGTTC.. ATGATTGCA. Haplotype 1. GGGTC. Haplotype 2. AGTCA. Haplotype 3. GCTTA. Tag SNPs. G T / / C C. Figure 1. SNP vs Haplotype. Adjacent SNPs identified in DNA from three individuals. The SNPs are inherited together and thus constitute haplotypes. "Tag" SNPs within haplotypes are identified that uniquely identify those haplotypes. By genotyping two tag SNPs shown in this figure, one can identify which of the three haplotypes shown here are present in each individual. Box 2: The origin of haplotypes The chromosome pairs are inherited from the person's father and mother but chromosomes do not pass from each generation to the next as identical copies but undergo a process known as recombination. The members of each chromosome pair come together and exchange pieces, which is passed on to the next generation. Over the course of many generations, segments of the ancestral chromosomes in an interbreeding population are shuffled through repeated recombination events. Some of the segments of the ancestral chromosomes have not been broken up by recombination, and these are the haplotypes. As modern humans spread throughout the world, the frequency of haplotypes came to vary from region to region through random chance, natural selection, and other genetic mechanisms. As a result, a given haplotype can occur at different frequencies in different populations, especially when those populations are widely separated and unlikely to exchange much DNA through mating. New changes in DNA sequences, mutations or polymorphisms, have created new haplotypes.. 15.

(213) BONE METABOLISM. The human skeleton consists of more than 200 bones of varying size, shape and composition. Bone is a specialized form of connective tissue that serves as both a tissue and an organ system within higher vertebrates. Bones support body structure, protect internal organs, and together with muscles facilitate movement, and actively participate in maintaining the mineral homeostasis in the body (23).. BONE STRUCTURE AND CELL TYPES Up to 90% of bone is composed of an extracellular matrix, comprising an organic and an inorganic component (24). The organic makeup, accounting for approximately one third of the total weight of the extracellular matrix, consists primarily of collagen where type I collagen predominates, but noncollagenous proteins can be found as well (25). Collagens are responsible for the strength of the bone and the molecules have a characteristic triple helical conformation which extracellularly forms fibrils (26). However, the inorganic component, accounting for the remaining part of the extracellular matrix, is an important and major reservoir of minerals in the body. The mineral salts are primarily calcium and phosphate in the form of hydroxyapatite (23). Morphologically, bones are classified as cortical or as trabecular (cancellous) (Fig 2). Cortical bone represents nearly 80% of the skeletal mass, but not of the skeletal surface, and forms a protective outer shell around every bone in the body. Cortical bone is compact and has a high resistance to bending and torsion, which is important in the middle of long bones, the diaphysis. In contrast, trabecular bone is less dense, more elastic and has a higher turnover rate than cortical bone and is made up of a network of tiny strands of bone called trabeculae. Trabecular bone predominates in the vertebrae as well as at the ends of long bones, the epiphysis (27). The remaining part of the skeleton, apart from the extracellular matrix, consists of cells and blood vessels. Four different cell types can be found in human bones: osteoblasts, osteoclasts and bone lining cells located along the surface of the bones, and osteocytes located in the interior of bone (23). Osteoblasts and osteoclasts play a major role in the bone remodeling cycle (see section below) whereas the function of the bone lining cells and osteocytes is not fully elucidated. It has been speculated that bone lining cells can be pre16.

(214) cursors for osteoblasts or function as a barrier between extracellular fluid and bone (23, 27). However, the osteocytes are the most abundant cell type of the bone (28) and since their location is within the bone matrix osteocytes are responsible for the maintenance of the bone matrix and also known to both synthesize and resorb matrix to a limited extent (29).. Figure 2. Organization of cortical and trabecular bones. Cortical bones surround the trabecular, spongy part of bones where the centre contains the bone marrow. In the cortical bones, units of structure called osteons can be found. A Harvesian canal forms the centre of each osteon and carries capillaries, arterioles, venules and nerves. Canaliculi are small canals that connect the Harvesian canal with the lacunae, containing the osteocytes. (Figure from Wikipedia, the free encyclopedia, released into the public domain by its author).. Besides the difference in location of the cells in the bone, bone cells are also different based on their origin. Osteoblasts, osteocytes and bone lining cells originate from mesemchymal stem cells, from which muscle cells, fibroblasts and adipocytes are also derived (29-31). In contrast, osteoclasts originate from hemopoietic stem cells where mononuclear progenitors of the monocyte/macrophage family are fused and a multinucleated giant cell are formed, the osteoclast (32, 33).. OSTEOBLAST ISOLATION AND CULTIVATION Cell culture has been practiced for nearly a century (34) but its application to bone cells became routine in the 1960s when Peck and co-workers initiated the use of primary bone cells (35). When conducting cell cultivation of bone cells, a choice has to be made between using primary cells or cell lines. 17.

(215) The methods used to release primary cells from bone are based on two classical techniques of culture initiation: proteolytic digestion and explantoutgrowth. Digestion of tissues with protease-like collagenase or trypsin is the most common method for releasing cells from calvariae (skull bone) from fetal or neonatal mice, which are an excellent source of bone cells for culture since they proliferate readily in vitro (35-37). The isolated cells are viable, proliferate during culture, and exhibit high activity of the osteoblast marker alkaline phosphatase (ALP). However, explant-outgrowth is the most commonly used technique for obtaining osteoblastic cells from more mature bone, particularly from adult human biopsies or surgical specimens. This approach takes substantially longer than digestion methods to generate cells for experiments but involves less damage to the cells from proteases. Normally, small pieces of bone are freed of loosely adherent cells by rinsing (38) and osteoblast-like cells (most likely bone lining cells, osteocytes, preosteoblasts and mature osteoblasts) migrate off the bone onto a substrate such as the culture dish surface. Although the cell source cannot be defined unambiguously (39, 40), the cells isolated from bone explants have been shown to exhibit a full range of osteoblastic features (41). Several cell lines derived from human osteosarcomas have also been used as models of osteoblastic cells, e.g. SaOs and MG-63. The advantage of using cell lines over freshly isolated cells lies in the ready availability of large numbers of cells, the homogeneity of the cell cultures, and the expected invariability of the phenotype. In the long run, however, cell lines appear unstable to some extent. In addition, their clonal selection has favored rapidly growing cells, but has not necessarily selected for the whole range of bonespecific gene expression characteristic of primary bone cells. Short-term bone cell cultures are thought to more closely manifest the properties cells express in the tissue (42).. BONE REMODELING Bone mass is maintained constant in vertebrates through bone remodeling, where the key players are the osteoblasts and the osteoclasts (Fig 3). These cells are also known as the bone-forming cells and the bone-resorption cells (30) . The remodeling cycle begins with the activation of resting osteoblasts on the surface of bone and marrow stromal cells that recruit and differentiate multinucleated giant cells to osteoclasts (43). Osteoclast precursors express two receptors on the cell surface, c-fms and RANK which bind to the macrophage colony stimulating factor (M-CSF) and RANKL expressed by osteblasts progenitors (44, 45). These interactions are necessary to promote the osteoclastogenesis. However, M-CSF is a secreted product whereas RANKL is a surface-residing molecule, indicating the importance of contact between 18.

(216) osteoclast precursors and osteoblasts for the osteoclastogenesis (46). In 1997, protein osteoprotegerin (OPG) was also found to be involved in the osteoclastogenesis (47, 48) by competing with RANK for RANKL (46) and inhibits the osteoclastogenesis. The quantity of bone resorbed is dependent on the balance between the expression of OPG and RANKL (49).. Bone formation by osteoblasts Osteoblasts, in addition to signal osteoclasts, are responsible for the production of the bone matrix by secretion of type I collagen and non-collagenous proteins followed by the mineralization of the skeleton. Calcium phosphate granules are found within osteoblastic vesicles (50) and are deposited within and between adjacent collagen fibrils. The mineralization process increases the density and strength of bone but the organization and amount of collagen remains the same resulting in a both elastic and strong bone tissue. During mineralization the osteoblasts are surrounded by bone matrix, stop dividing and become osteocytes (23, 27).. Bone resorption by osteoclasts The initial event in bone resorption is the attachment of osteoclasts to the target bone matrix. On attachment, the osteoclasts form a “ruffled membrane” which is a unique feature that permits it to recognize and degrade bone (32). Thereafter, the osteoclasts secrete HCl into the resorptive microenvironment (51) and the inorganic matrix of hydroxyapatite is dissolved. The remaining organic component of the bone matrix is degraded in the acidic milieu by a lysosomal protease, cathepsin K (52, 53). By-products of the resorption are endocytosed by the osteoclasts and transported to and released at the cell’s antiresorptive surface (54). Box 3: Metabolic Bone Disorders Osteomalacia is a disorder characterized by disturbed mineralization of the newly formed osteoid in the bone matrix due to lack of available calcium and/or phosphorus. Osteoporosis is described as a systematic skeletal disease characterized by low bone mass strength predisposing a person to an increased risk of fracture. Osteopetrosis is a rare genetic disease characterized by abnormally dense bone, due to defective resorption of immature bone. Osteogeneis imperfecta (OI) is a large and miscellaneous group of genetic conditions caused by an abnormality in the collagen protein resulting in easily fractured bones. Paget’s disease is a disorder characterized by a greatly accelerated remodeling process; osteoclastic resorption is massive and new bone formation by osteoblasts is extensive, resulting in irregular thickening and softening of the bones.. 19.

(217) Figure 3. The bone remodeling cycle. Figure from a slide series entitled “Die Knochenbiopsie inder klinischen Osteologie”, from Prof. R. Bartle, Medical Clinic II, University of Munich, Germany.. Regulation of bone remodeling In order to maintain the balance between bone formation and resorption, the remodeling cycle is under the control of a number of factors, which coordinate the remodeling sequence. Prostaglandin E2 (PGE), interleukin-6 (IL-6), fibroblast growth factor and transforming growth factor (TGF)-ȕ are all local factors shown to regulate the bone remodeling (55-57). However, these locally produced modulators are often the result of hormonal action where key players are sex steroids and vitamin D (see sections below). However, parathyroid hormone (PTH) has been proposed to be the main hormone regulating bone remodeling. Interestingly, PTH has a dual role in the remodeling: while continuous infusion of PTH causes bone loss, intermittent administration induces bone formation (58) . In addition, studies of the hormone leptin, which acts on its receptor in the hypothalamus (59), have indicated presence of a central regulation of bone remodeling both in humans and in animals (60, 61). Recently, the Wnt signaling pathway, including LRP5/6 and Wnt proteins, was discovered as another pathway involved in the regulation of bone mass (62-65). This homeostatic system of bone remodeling is, like all others, subject to genetic influences, which has been demonstrated in several twin studies (see section Genetics of osteoporosis) and prompted studies of candidate genes in association to bone phenotypes.. 20.

(218) CALCIUM HOMEOSTASIS. Bone mass. The concentration of calcium in the human body is highly regulated in order to reach the optimal range suitable for the many cellular functions affected by calcium. Calcium serves as a second messenger in many signal transductions pathways intracellular whereas it also plays an important role extracellularly in e.g., neural transmission, muscle function and blood coagulation. However, 99% of the body’s calcium content is found in the bone tissue, providing the strength of the bones. The skeleton turns over approximately 250 mg/day mediated by the osteoblasts and the osteoclasts. The normal range of calcium in the blood is 8.5 - 10.5 mg/dL, mainly either ionized or bound to proteins (e.g. albumin or globulin). The homeostatic system of calcium is regulated by the hormones PTH and vitamin D acting in bone, kidney and the gastro intestinal tract. When calcium levels in serum are low, PTH is secreted as a result of activation of the calcium-sensing receptor on the parathyroid glands (66). PTH indirectly increases the calcium absorption from the intestine through activation of vitamin D production in the kidneys. PTH increases reabsorption of calcium from the urine and mobilizes calcium via direct effect on bone (67).. Women Peak bone mass. Men. Bone loss 0. 10. 20. 30. 40. 50. 60. 70. 80. Age (years). Figure 4. Peak bone mass and bone loss in men and women.. PEAK BONE MASS AND BONE LOSS The amount of bone in all individuals increases during childhood and adolescence, and much more bone is deposited than withdrawn, so the skeleton grows in both size and density. Up to 90 percent of peak bone mass is acquired by the age of 18 in girls and age 20 in boys (Fig 4) (68, 69). The amount of bone tissue in the skeleton can keep growing until around age 30 in men and women; then, bones have reached their maximum strength and density, known as peak bone mass. In women, total bone mass tends to decrease be21.

(219) tween age 30 and menopause. Later, during the first few years after menopause, most women experience rapid bone loss, which then slows but continues throughout the postmenopausal years (70). Peak bone mass is mainly genetically determined, but is also affected by contribution of calcium, diet and exercise during childhood and puberty (71, 72) . Moreover, twin studies have suggested that the rate of bone loss is less strongly influenced by genetics than peak bone mass (73). Taken together, in the ageing person, the balance between bone formation and resorption are disturbed and the amount of resorption exceeds the formation. Once the imbalance has become clinically significant, a person is diagnosed with osteoporosis.. OSTEOPOROSIS Osteoporosis is a major health problem and affects millions of people worldwide (74). Generally, 1 of 3 women between 70-79 years of age suffer from osteoporosis and 1 of 5 men (75-78). An internationally accepted definition of osteoporosis was presented at a consensus development conference in 1991 and 1993 (79, 80), but was modified by the The National Institute of Health (NIH) in 2000(81). In fact, osteoporosis was not specifically defined as a disease until 1994 by The World Health Organization (WHO) (82) but currently it is considered as a priority health issue along with other major noncommunicable diseases. Box 4. Definitions of osteoporosis Osteoporosis is a systemic skeletal disease characterised by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Osteoporosis is a skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture.. The definition of osteoporosis described as compromised bone strength, reflects both a person’s bone mineral density (BMD) and bone quality (83). BMD, expressed as gram per area or volume, is determined by peak bone mass and subsequent bone loss whereas bone quality refers to bone turnover, microarchitecture and mineralization (84). Osteoporosis is regarded as a significant risk factor for fracture both in men and in women (75 233, 85, 86) and is commonly classified as either primary or secondary osteoporosis. Besides sex and age, other life style factors can also influence BMD and the risk of fractures (87, 88). Smoking (89, 90), alcohol (91-93), physical inactivity (94-96) and low body weight (97-99) are some examples of risk factors whereas physical 22.

(220) activity and fitness reduce the risk of osteoporosis and fractures (100, 101). However, the genetic component to osteoporosis is large (73, 102) and a parental history of fractures confers an increased risk of fractures that is independent of BMD (103). (See Genetics of Osteoporosis).. Primary osteoporosis Sex and age (81, 104) are the major underlying predictors of primary osteoporosis which is sometimes also classified as postmenopausal osteoporosis (type I) and senile osteoporosis (type II). Type I generally develops in women due to the drastic decrease in estrogen at menopause, which results in a rapid depletion of calcium from the skeleton due to imbalanced bone remodeling (105). In total, 5 - 20% of women between the age of 50 and 75 suffer from postmenopausal osteoporosis (106) and, on average, a 50-year-old white woman has a 16% lifetime risk of experiencing a vertebral fracture during her lifetime (76). However, since men in their 50s do not experience the rapid loss of bone mass that women do, due to decrease in hormone levels, type I osteoporosis is not as common in men as in women (106). In contrast, type II osteoporosis typically occurs after the age of 70 in both men and women, although women are affected twice as frequently as men. With age, the bone remodeling process is no longer coordinated and imbalanced resorption and formation result in a decline in bone strength.. Secondary osteoporosis The loss of bone mass in secondary osteoporosis is caused by lifestyle factors including medications, diseases and nutritional deficiencies (81). Among men, 30 - 60% of all osteoporosis cases are associated with secondary causes, and the most common include hypogonadism (107), glucocorticoid medications (108), and alcoholism (91-93). In perimenopausal women suffering from osteoporosis, 50% of the cases are due to hypoestrogenemia (109), use of glucocorticoids (110) and thyroid hormone excess (111). Indications for BMD assessment according to ISCD* -Women aged 65 or older -Postmenopausal women under age 65 with any risk factor -Men aged 70 or older -Adults with a fragility fracture -Adults with a disease or condition associated with low bone mass -Adults taking medication associated with low bone mass -Anyone being considered for pharmacological therapy * International Society of Clinical Densitometry. 23.

(221) Bone densitometry The only reliable way to determine bone strength and loss of bone mass in order to make a diagnosis is to measure BMD, which accounts for approximately 60-70% of the variation in bone strength (112-116). A variety of methods are available to assess BMD but the method of first choice is Dual X-ray Absorptiometry (DXA), which provides a two-dimensional, areal image of the bone density. One important advantage with this technique, besides it being painless and non-invasive, is that the radiation exposure during scanning is low. (117-120). The basic principle of DXA data acquisition is based on the differences between bone and soft tissue attenuation at high and low Xray levels. DXA uses two X-ray beams at high and low energy where the first scan at high energy identifies bone and non-bone areas. Thereafter, pure fat and non-bone lean tissue are identified by comparing the attenuation at low energy X-rays. The end result is determination of lean mass, fat mass, and bone mass and when bone mass is divided by the area measured, BMD is acquired (121). However, one should emphasize that BMD measured by DXA is not a volumetric density but an areal density. In order to achieve a more accurate bone density, quantitative computed tomography (QCT) has been applied (122, 123) . The advantage of QCT is its ability to isolate the area of interest from surrounding tissues. It can, therefore, localize an area in a vertebral body of only trabecular bone leaving out elements most affected by e.g. degenerative change and sclerosis. However, the QCT radiation dose is about 10 times that of DXA, and QCT tests may be much more expensive than DXA.. Diagnosis Osteoporosis is defined as BMD 2.5 standard deviations (SD) or more below the average value for premenopausal women. Premenopausal women are used as reference because the distribution or density of bone mineral content in young healthy adults (peak bone mass) is approximately Gaussian normal, irrespective of the technique used. Therefore, BMD values in individuals can be expressed in relation to the reference population in SD units. This ability reduces the difficulties associated with differences in calibration between instruments. When SDs are used in relation to the young population, this measurement is referred to as the T score. The current recommendation of the International Osteoporosis Foundation and WHO is to use the National Health and Nutrition Examination Survey (NHANES) reference database in women aged 20-29 years as the reference range (124). It is generally agreed that measurement at the hip is the gold standard in terms of site since it has the highest predictive value for hip fracture (125) which is the most severe complication of osteoporosis. However, lumbar 24.

(222) spine measurements are also commonly used as fracture prediction (Fig 5) (126) . Recently, it was suggested that multiple site may improve the prognostic value of the BMD test to predict fractures (127, 128). This was examined in 19,071 individuals (68% women) from six population-based cohorts but results indicated that taking the minimum value of two measurements does not improve predictive ability (129). World Health Organization Diagnostic Criteria For Osteoporosis Normal BMD value within 1 SD of young-adult mean (T-score at or above -1) Osteopenia BMD value between -1 SD and -2.5 S.D. below young-adult mean (T-score between -1 and -2.5) Osteoporosis BMD value at least -2.5 SD below young adult mean (T-score at or below -2.5) Severe osteoporosis BMD value at least -2.5 SD below young adult mean and presence of fracture. Figure 5. DXA measurements Spine measurements are made in the lumbar region. Testing in the thoracic spine is not possible because of the overlying sternum. The accuracy of measurements in individual vertebral bodies is less than that in a larger region; thus, the composite of the L1-4 or L2-4 is recommended for clinical use. In the proximal femur, measurements are made in the femoral neck and the trochanter regions. The total hip measurement is a combination of the femoral neck, trochanter, and intertrochanteric regions. Reprinted with the kind permission from Current Medicine, Inc. Taken from McClung M, Atlas of Clinical Endocrinology: Osteoporosis.. 25.

(223) GENETICS OF OSTEOPOROSIS Medical genetics was revolutionized during the 1980s by the application of genetic mapping to locate the genes responsible for simple Mendelian diseases (130). However, most diseases and traits do not follow simple inheritance patterns and are called complex or polygenic, which means that multiple genes contribute to the phenotype either individually or through interactions with each other or the environment (131). This is the case for the pathogenesis of osteoporosis where the different skeletal determinants of osteoporotic fracture risk are known to be under the control of a number of genetic and environmental factors. This has widely been examined in studies of twins, which is a common approach for investigation of heritability of complex traits and diseases (132). A study of 500 normal female twins, aged 50 to 70, showed a strong genetic component of BMD at different sites measured (Fig 6). The estimated heritability ranged from 0.46 to 0.84 (73), which has been confirmed by others (133, 134). Heritability of bone mineral density (%) 100 80 60 40 20 0 Lumbar spine. Femoral neck. Trochanter. Figure 6. Heritability of BMD. Twin study of 38 monozygotic and 27 dizygotic twin pairs suggesting that environmental factors are more important in the etiology of osteopenia of the hip than that of the lumbar spine. (73). However, there is evidence to suggest gender and site specificity of inheritance of BMD. Although the proportion of bone strength variance explained by genetic factors is similar for men and women, a gender-specific genetic component is shown in some skeletal regions (135). Duncan et al found that lumbar spine BMD correlated more strongly in male-male comparisons; and hip BMD, in female–female comparisons (136). Studies in laboratory mice have also highlighted the important role of gender in the genetic basis of peak bone mass (137). Regarding site specificity, Judex et al showed that the genetic control of bone mass and morphology in mice, even within a given 26.

(224) bone, is highly site-specific, which encouraged the search for BMD sitespecific genes (138). Moreover, both muscle strength and body weight are considered as important predictors of BMD (Fig 7) (139) but studies of twins have demonstrated that the genetic component to muscle mass and strength accounts for little of the genetic component to BMD (140). This confirms the rationale for research into bone- and muscle-specific genes. However, muscle variables themselves have a moderate genetic component with heritability estimates of approximately 0.5. Heritability of muscle strength and mass (%) 100 80 60 40 20 0 Grip. Leg extensor. Lean mass. Figure 7. Heritability of muscle strength and muscle mass. 227 pairs of monozygous (MZ) twins and 126 pairs of dizygous (DZ) twins were measured for muscle parameters. All three muscle variables had a moderate genetic component with heritability estimates of 0.52 for lean body mass, 0.46 for leg extensor strength, and 0.30 for grip strength (all p < 0.05)(140).. Since the importance of BMD in osteoporotic fractures is well established and the heritability of BMD is substantial, one could suggest that the genetic liability to osteoporotic fractures might reflect that for BMD. Michaëlsson et al showed that the genetic variation in liability to fracture differed considerably by type of fracture and age (Fig 8). The heritability of hip fractures before the age of 69 years was 0.68, but decreased significantly with older age (141). A twin study of risk of wrist fracture in healthy female volunteer between 18 and 80 years of age showed that the heritability of wrist fractures is 0.54 (142). Furthermore, the genetic influence on the risk was not significantly reduced when BMD was included as a co-variate, indicating that fracture and low BMD have their own specific genetic risk factors that are unlikely to be shared between the two traits.. 27.

(225) Heritability of osteoporotic fractures (%) 100 80 60 40 20 0 <69 y. 69-79 y. >79 y. Figure 8. Heritability of fractures. 6021 twins with any fracture were studied. The results indicated that the importance of genetic factors in propensity to fractures depends on fracture site and age. (141). Even though twin studies have indicated the importance of overall genetic influence on bone phenotypes, the challenge is, however, to identify the specific genes or regions that contribute to the pathogenesis of osteoporosis. Currently, there are two basic strategies for searching genes that influence complex traits such as BMD: linkage analysis and candidate gene approaches (143, 144).. Linkage analysis Linkage analysis in either humans or animals determines whether a phenotypic locus of interest is transmitted with genetic markers of known chromosomal position. Results of linkage analyses are expressed in LOD scores which estimate the ratio of the odds that the candidate locus is linked to the trait (145). Linkage studies in humans were developed for identifying genes responsible for monogenic diseases (130). Previously linkage analysis has been applied to investigate genes regulating quantitative traits (e.g. BMD) and these regions are therefore called quantitative trait loci (QTL) (146). However, since complex traits in humans are genetically heterogeneous, the statistical power to detect genes with modest effect is low (147) and as a consequence, only a few of the identified QTL for BMD have been replicated. Notably, one QTL for femoral neck BMD identified on chromosome 1p36 has been replicated in several studies. Originally, Devoto et al reported linkage of the 1p36 locus to hip BMD (148, 149). This was confirmed by Wilson et al, who studied the linkage of 1p36 to BMD in 1097 unselected female twin pairs (150). In addition, Karasik et al was able to confirm the result of a bone QTL on 1p36 (151), which further strengthened the initial theory. These findings have prompted investigation of candidate genes located within the 1p36 for association to BMD and osteoporosis (152-157). 28.

(226) The candidate gene approach: an overview In candidate gene studies one seeks to test the association between a particular genetic variant (i.e. polymorphism) and a phenotype (131). Such an association study is dependent on knowledge about the gene and the frequency of the polymorphism (158). The candidate gene is often chosen on the basis that it has biological effects on bone metabolism or that the region is within a QTL. However, the phenotype could either be assessed in a populationbased study, with a normal distributed phenotype, or in a case-control study with patients diagnosed for osteoporosis compared with controls without the disease (159). If the variant is more frequent in persons with the disease or associated with low BMD in the normal population, then one could infer that the candidate gene polymorphism is responsible for the effect observed. However, it could also be because the genetic polymorphism studied is in LD with a disease gene at a locus near the locus in question (16-18). Thus, in order to interpret association studies of genotype and phenotype and to identify disease-causing loci, it is necessary to analyze haplotypes instead of single non-coding SNPs (160). Currently, it is suggested that positive findings should be demonstrated to be robust by either achieving sufficiently stringent statistical thresholds or by confirmation in independent datasets. Moreover, studies should have power enough to identify small effects (161). In the field of genetics of osteoporosis, many association studies are published but most results have been variable. It has been proposed that this is most likely due to the absence of a major gene effect on BMD, failure to consider gene-environment and gene-gene interactions, small sample sizes and lack of functional effect of the polymorphism studied (161, 162). Moreover, most studies have examined single polymorphisms rather than haplotypes. The classical candidate genes studied in association to osteoporosis include the vitamin D receptor, the estrogen receptor and collagen type I whereas LRP5 and SOST, involved in the Wnt signaling pathway, are novel genes recently also shown to be associated to bone phenotypes (163).. CANDIDATE GENE: THE VITAMIN D RECEPTOR Vitamin D and its receptor In 1919, Sir Edward Mellanby found that rickets was caused by a nutritional deficiency in a fat-soluble antirickitic molecule, identified as vitamin D2 (164). Later studies led to structural identification of vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) which are both produced by the action of sunlight in the skin (reviewed in (165)). Currently, it is well known that the metabolites of the vitamin D3 are the major circulating derivates of vitamin D in the human body (166, 167).. 29.

(227) The hormone vitamin D3 in the human body is obtained from the diet or by the action of sunlight on 7-dehydrocholestrol in the skin (168, 169). Vitamin D3 is then 25-hydroxylated in the liver by the enzyme 25-hydroxylase to 25hydroxy-vitamin D3 (25(OH)D3) (170). The levels of 25(OH)D3 increase in proportion to vitamin D intake, which is the reason for the use of 25(OH)D3 as an indicator of vitamin D status (171). Moreover, the 25(OH)D3 is converted to the active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by the enzyme 1Į-hydroxylase, which occurs mainly in the kidney (172, 173). In contrast, the active form of vitamin D3 is catabolized by the enzyme 24hydroxylase by oxidation, leading to loss of biological activity (174). However, since the 1,25(OH)2D3 is the key regulator in the maintenance of calcium homeostasis (see section above), the regulation of the metabolite is strictly coordinated. For hypocalcemia, with low calcium levels, PTH activates the 1Į-hydroxylase gene transcription and decreases 24hydroxylase activity. This results in inhibited degradation of 1,25(OH)2D3 and increased hydroxylation of 25(OH)D3 to active 1,25(OH)2D3, which can increase the serum calcium. The inverse effect is seen in hypercalcemia, with high calcium levels, where the 1Į-hydroxylase is depressed and the activity of the 24-hydroxylase is increased (67, 175). Moreover, it has also been shown that calcium, independent of PTH, can alter the levels of 1,25(OH)2D3 by direct suppression of the 1Į-hydroxylase activity (165). Furthermore, 1,25(OH)2D3 controls its own homeostasis through simultaneous suppression of 1Į-hydroxylase and stimulation of 24-hydroxylase (176). The vitamin D metabolites, described above, are all lipophilic molecules with low solubility in the aqueous media and are therefore transported in the circulation bound to plasma proteins, e.g. the vitamin D-binding protein (DBP) (177). The 1,25(OH)2D3-DBP complex is taken up by target cells and then either targeted by intracellular vitamin D-binding proteins (IDBP) to 24-hydroxylase degradation or receptor binding. The biological actions of 1,25(OH)2D3 are mediated by binding to its receptor, the vitamin D receptor (VDR), in a heterodimer with the retinoid X receptor (RXR). VDR is a member of the superfamily of nuclear receptors for steroid hormones, which all act as ligand-activated transcription factors. The VDR protein has a DNA and a ligand-binding domain, which mediates nuclear localization, heterodimerization and transactivation (178). On binding of 1,25(OH)2D3 to VDR in the cytoplasm of the target cell, a conformational change of the receptor occurs in order to facilitate the recruitment of motor proteins (179). These proteins are responsible for the translocation of the cytoplasmic VDR to the nucleus. The second step is the association between the VDR with its protein partner RXR, followed by binding of the heterodimer to 1,25(OH)2D3 target genes (180). The binding site is restricted to specific DNA sequences in the promoter region of the target genes, called vitamin D responsive elements (VDREs). VDREs normally consist of two repeats of 30.

(228) the half-site sequence AGGTCA separated by several non-specified bases (180, 181) . It has been speculated that repression or activation of target gene transcription by VDR is dependent on type of response element (182). However, response element has been isolated on several genes, either with a direct role in well-known vitamin D pathways or in genes not traditionally considered as vitamin D targets. Examples of such genes include osteocalcin (183) , osteopontin (184), PTH (185), fibronectin (186) and integrin ȕ3 (187). However, in order to activate or repress target gene transcription, interactions with coregulators are necessary, so-called coactivators or corepressors. SRC-1 (188) and CBP/p300 (189) are VDR coactivators whereas NcoR and SMRT (165) function as corepressors, and all remodel the chromatin and thus facilitate the transcription regulation by the vitamin D complex. Non-genomic rapid effects of 1,25(OH)2D3 have been described although these processes are not fully elucidated (190, 191).. Biological actions of vitamin D The classical vitamin D-responsive tissues include the intestine, skeleton, kidney and the parathyroid glands, all involved in the maintenance of the calcium homeostasis (Fig 9). The effect of vitamin D has been recently studied in mouse models lacking either the 1Į-hydroxylase (1Į(OH)ase-/-) or the VDR (VDR-/-; 1Į(OH)ase-/- VDR-/-) or both (192). In the small intestine, 1,25(OH)2D3 bound to VDR stimulates the absorption of dietary calcium and phosphate. This is performed by the VDR receptor-complex by activating the epithelial calcium uptake channels (TRPV5 and TRPV 6) and the basolateral plasma membrane calcium channels (PMCA1) which pumps calcium from the cell to the bloodstream (193). Notably, as proved in the mouse models, the calcium absorption requires both 1,25(OH)2D3 and VDR. Studies of the bone remodeling in the presence of a defective 1,25(OH)2D3-VDR system with normal PTH levels indicated that 1,25(OH)2D3 may exert effects on both bone resorption and formation analogous to those of PTH. However, it is well known that 1,25(OH)2D3 regulates the osteoclastogenesis by upregulation of RANKL on the surface of the osteoblast and represses the expression of OPG. In the kidney, 1,25(OH)2D3 enhances renal calcium reabsorption and accelerates PTHdependent calcium transport in the distal tubule (194). Several putative VDR binding sites have been located in the human promoter of the renal TRPV5 (165) . 1,25(OH)2D3 by binding to VDR has been shown to inhibit PTH synthesis through transrepression of the gene (195), indicating its role as a potent modulator of the parathyroid function (Fig 9).. 31.

(229) Figure 9. The role of vitamin D in the bone and mineral metabolism. Reprinted with the kind permission from Current Medicine, Inc. Taken from Blumberg J, McKayD. Atlas of Clinical Endocrinology:Human Nutrition and Obesity.. There is strong evidence indicating that abnormalities in the vitamin D endocrine system are associated to disorders not only related to calcium homeostasis as osteoporosis but muscle functions and susceptibility to prostate, breast and colon cancer (196, 197). It has been shown that the 1,25(OH)2D3VDR system arrests the cancerous cell cycle, mainly by inducing gene transcription (198). Moreover, 1,25(OH)2D3 –induced apoptosis is an important contributor to the growth-suppressing properties of hyperproliferative disorders (199). It has been demonstrated that skeletal muscle is a target organ for 1,25(OH)2D3 that directly affects muscle cell metabolism through various pathways (200). Vitamin D deficiency is associated with muscle weakness (200, 201) . The 1,25(OH)2D3 is also a regulator of the adipogenesis and adipocyte function (202, 203) indicating a possible role in the susceptibility to obesity as well (204).. VDR and genetic variability The human VDR was cloned in 1988 (205) and shown to be over 100 kb in size and to consist of at least 14 exons. The gene maps to chromosome 12q13.1 (206), just downstream from the collagen type II alpha I gene (207, 208). 32.

(230) However, it has been demonstrated that the human VDR gene comprise an extensive promoter region capable of generating multiple tissue-specific transcripts. In total, 14 different transcripts were identified, all differed at their 5’ end but identical at the 3’end (209). VDR is one of the genes that have been most extensively studied in relation to BMD and also the first polymorphic candidate gene found significantly associated with BMD (210). Studies have supported the original findings reported by Morrison et al concerning the VDR polymorphisms and BMD whereas others have found an inverse association (211-215). Further complicating the picture, other investigators have found no significant correlations between VDR alleles and BMD (216 , 217 , 218). Most of those studies had small sample sizes and were carried out in samples collected for other reason and with heterogeneous populations, which may partly explain these conflicting results. Moreover, most of the VDR association studies on various traits have been focused on the different adjacent SNPs usually analyzed as the ApaI, TaqI and BsmI RFLPs, but their effects are poorly understood. However, it has been demonstrated that these SNPs are in strong LD with a poly adenosine (A) microsatellite at the VDR 3´UTR (210, 219). Recently, highresolution SNP, haplotype and LD maps of the VDR gene were developed for four European populations. The comparative analyses revealed that the LD pattern in the VDR gene are identical in all four populations, indicating that the tag SNPs selected in one European population effectively predict the non-tag SNPs in the other Europeans, for this region (220). Following this study, a comprehensive investigation of the genomic organization of the VDR gene region in different ethnic groups (European, Asian and African Americans) was reported, with haplotype structures and tag SNPs (221).. CANDIDATE GENE: THE ERĮ COFACTOR RIZ Estrogen and its receptor The most potent form of estrogen in the body is estradiol, synthesized from testosterone or estrone via the aromatase or 17ȕ-hydroxysteroid dehydrogenase enzymes. Of the circulating estradiols, most hormones are bound to albumin or sex hormone binding globulin (SHBG) (222). It is currently well established that estradiol is not only a female hormone but also a male hormone (223). In the 1970s it was demonstrated that estrogens are directly produced in the testes and not only converted from testosterone (224, 225). A large study on peripheral levels of estradiol in men aged 38 - 70 showed a mean total serum estradiol concentration of 110±54 pmol/l (mean±SD) (226). These concentrations are comparable to those in women in the early follicular phase of the menstrual cycle, whereas concentrations in postmenopausal. 33.

(231) women decrease to concentrations significantly lower than in men of the same age (227). More than 40 years ago it was established that estradiol mediates the biological effects by binding to a receptor protein (228), which was cloned several years later and named the estrogen receptor (ER) (229, 230). Until 1995, it was assumed that there was only one ER, mediating all effects of estrogens but in 1995, a second receptor, ERȕ, was discovered (231) and the former ER is now called ERĮ. ERs are, as VDR described above, members of the superfamily of nuclear receptors for steroid hormones and thus contain the characteristic DNA and ligand binding domains (232). The classical mechanism of estrogen action includes diffusion of estrogens through the capillary wall and the cell membrane bilayer of the target cell. Once estrogen is in the cytoplasm, it is not yet clear whether a transport protein is required for movement to the nucleus or whether estrogen occurs through passive or active transport of the nuclear pore (233). On binding of estrogen to the receptor in the nucleus, ER is “activated” by alterations in the conformation, which triggers dimerization (234) and ER forms homodimers, or heterodimers of ERĮ and ERȕ (235, 236). Thereafter, ligand bound-ER dimers bind to estrogen responsive elements (ERE) in the regulatory region of target genes and thus induce a DNA bend (237-239) which promotes recruitment of cofactors (240). These proteins exist in multiple complexes, possess multiple enzymatic activities and bridge ERs, either to chromatin components such as histones, or components of the basal transcription machinery, or both (241). Today it is well known that ERĮ and ERȕ can also modulate gene expression without directly binding to the DNA but by binding to other DNA-bound transcription factors and thus inhibit or activate target gene expression (242, 243).. Biological actions of estrogen Since osteoporosis is associated with estrogen deficiency in both men and women, estrogen must play an important role in regulating the skeletal homeostasis. The suggested mechanism is that estrogens inhibit bone turnover by reducing bone resorption. A role for the ER in development of osteopenia and in skeletal growth and maintenance is exemplified by the case of an estrogen-resistant male patient carrying a loss-of-function mutation in the gene encoding ERĮ (244). This patient demonstrated incomplete epiphyseal closure and reduced BMD. Similar skeletal abnormalities are seen in patients with a mutation in the gene encoding aromatase (245). Moreover, a role for the ERs in bone has been studied in both cell culture and rodent model system. In 1988, Komm et al demonstrated ER expression and estradiol-mediated induction of type I collagen and TGF-ȕ, a local regulator of bone remodeling, in human clonal osteoblast-like osteosarcoma cells (246). This was confirmed in primary human osteoblast cells (247). There are indications that es34.

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