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European Journal of Trauma

Review Article

Think Bone
The Novel Paradigm of Central Bone Mass Control
Michael Haberland, Arndt F. Schilling, Johannes M. Rueger, Michael Amling1

Abstract Our understanding of the molecular physiology of the skeleton has been transformed by recent advances in human and mouse genetics and molecular endocrinology. Through the successful convergence of genetics and clinical observation, novel insights marking a breakthrough in the understanding of the molecular physiology of the skeletal system were gained. The paradigm that osteoblast and osteoclast function are mechanistically linked, being dogma in the bone field for decades, is overcome by experimental data showing that the maintenance of the skeletal system is, at least partially, controlled by the hypothalamus. Leptin was identified as one key molecule in the central regulation of bone mass, and its importance was demonstrated across several species. Indeed, in vivo leptin signaling is able to overcome the deleterious effect of hypercortisolism and hypogonadism on the skeleton. This review presents a perspective on the data supporting the hypothesis of central bone mass control. Key Words Bone Molecular genetics Leptin Transgenic mice Skeletal maintenance Central regulation
Eur J Trauma 2001;27:21825 DOI 10.1007/s00068-001-1153-0

Introduction Being daily confronted with pathologies of the skeletal system, our need to understand its physiology and regulating mechanisms is great indeed. Take osteoporosis for example, a debilitating disease, still causing a daily 8 24 mio. disease-associated costs in Europe alone [14], and creating multiple clinical problems for the orthopedic surgeon, it is clear that our progress in understanding and treating skeletal disease is still pretty slow. For the last years, the process of gaining knowledge about the skeleton consisted mainly of descriptive morphology, histomorphometry, endocrinology, and cellular studies of bone turnover [2, 3]. There has been a dramatic increase in the pace of discovery when genetics entered the field. Taken together with the molecular and biochemical studies of bone cells in vitro and in vivo, made possible through recent advances in molecular biology, a number of genes have been identified that are involved in skeletal abnormalities of mice and humans [13]. In the beginning there was a lot of skepticism about using small rodents like mice as models for understanding the genetics of human disease, but the successful convergence of mouse and human genetics in skeletal biology in many instances slowly has overcome this skepticism [40]. Patients with cleidocranial dysplasia (CCD) showed the same phenotype as mice which lack an allele of the transcription factor cbfa1, a molecule essential for osteoblast differentation and function [10, 32, 33]. And indeed it was demonstrated that patients suffering from CCD lack exactly this allele in their genome. Jansens metaphyseal dysplasia suddenly became explainable on a molecular level when a number of mouse experiments showed the importance of the PTHrP receptor for chondrocyte apoptosis. The conse-

Department of Trauma Surgery, Hamburg University School of Medicine, Hamburg, Germany.

Received: June 20, 2001: accepted: August 3, 2001

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quent analysis of patients suffering from Jansens disease demonstrated an activating mutation of the PTHrP receptor, thus making apoptosis of the chondrocyte and subsequent calcification of the hyaline matrix impossible [20, 21, 25, 41]. Mice with a mutation in the vitamin D receptor developed a phenotype mimicking human rickets [4, 26, 27], and Stickler syndrome could be duplicated in mice with mutations in collagen type XI [19, 36]. In mouse models with an accelerated senescence (klotho, SAMP6), the skeleton showed a phenotype very similar to the one found in patients with senile osteoporosis [22, 23, 45]. These and other experiments gained a lot of insight in the molecular mechanisms of skeleton disease, but to date no therapeutic benefit could be obtained from these advances. Gene therapy is still far from being used routinely in medical practice, and so far the only gain is academic. Most skeletal diseases confronting the clinician in his daily work are not due to a genetic defect but rather to a defective regulation in the skeletal repair mechanisms. In contrast to our increasing knowledge about the genetics of developmental skeletal disease, progress in understanding its physiology on a molecular level has been very slow. This relative ignorance of what should be called molecular physiology of the bone explains why we know so little about the pathophysiology of the most frequent skeletal diseases, the so-called acquired diseases, and why effective treatment of these diseases, especially osteoporosis, is still not possible. But as it is true for the genetically determined hereditary diseases mentioned above, in recent years mouse genetics revolutionized the understanding of skeletal molecular physiology and pathophysiology. These new results may lead to a shift in our physiological understanding of the skeleton and the emergence of novel paradigms. These in turn should help us to devise new (approaches to) treatments for such degenerative skeletal diseases as osteoporosis. Interestingly, the most exciting insights resulted from the dialog of clinical observation and mouse genetics. It is ancient knowledge for the clinician that gonadal failure favors osteoporosis while obesity protects against it [15, 35, 37, 38, 46]. But these connections could never be sufficiently explained. Our working hypothesis has been that these two observations suggest that body mass, bone remodeling, and reproduction are all controlled by similar mechanisms. Since body weight and reproduction are controlled centrally, we hypothesized

that bone remodeling might also be under central control. Aside from the the large body of evidence indicating the existence of local regulatory mechanisms of bone remodeling, which will not be reviewed here, there is ample evidence for a central regulation of bone remodeling in multiple species, which is consistent with the idea that the two clinical observations mentioned above are functionally linked. The same evidence indicates that this central regulation is not accessory or secondary but dominant, since its modulation can overcome the deleterious effect of gonadal failure on bone remodeling. The Paradigm of Local Bone Mass Control Bone remodeling is the physiological process by which bone mass is maintained constant in vertebrates between the end of puberty and gonadal failure [17]. In comparison with other homeostatic systems, bone is unusual as it harbors a cell, namely the osteoclast, whose only function is to destroy or resorb the organ hosting it [5, 43]. After resorption of old bone by the osteoclast, new bone tissue is laid down by the boneforming cells, the osteoblasts. The fact that bone remodeling occurs simultaneously in multiple skeletal locations has generally been viewed as indirect evidence that it is controlled locally. Additionally, the well-documented role of cells of the osteoblast lineage favoring osteoclast differentiation in vitro, has long been viewed as proof that bone remodeling is primarily an autocrine/paracrine process [39]. Multiple experimental evidence have demonstrated that this regulation does exist. Recently, the cloning of two cytokines acting in a paracrine fashion, namely osteoprotegerin (OPG), which is an inhibitor of osteoclast differentiation [42, 47], and of osteoprotegerin ligand (OPGL), also called RANKL, which is an osteoclast differentiation factor [24], have again shown that there is indeed a local control of bone formation. So for the last 25 years it has been believed in the bone field that the balance between bone formation and bone resorption is kept by the osteoblast and the osteoclast communicating with each other on a paracrine level, and that one could not function without the help of the other, saying that bone resorption and bone formation are mechanistically linked. Challenging the Paradigm of Local Control The acceptance of the fact that local control is involved in the regulation of bone remodeling should not lead to

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exclusion of other modes of regulation. The old paradigm of local control has been challenged from two different fronts, namely mouse genetics and recent advances in endocrinology. In mouse genetic experiments, where either the osteoclast or the osteoblast was deleted, the dissociation between osteoclastic bone resorption and osteoblastic bone formation could be demonstrated. In a mouse model where a gene (the src gene) that is essential for osteoclast function has been deleted, the number of osteoclasts is higher than normal, but they lack the typical ruffled border, and they are not functional, i.e., they cannot resorb bone [2830]. If the paradigm of a

functional coupling of osteoclast and osteoblast would hold true, the osteoblast function should also be diminished. When these mice were analyzed, they showed an osteopetrosis, a clear indicator for continuing osteoblast bone formation in the absence of osteoclast bone resorption, and thus providing an example for an uncoupled function of osteoblast and osteoclast (Figure 1). In another mouse model, we were able to show that this principle also works the other way round. A mouse model was generated that harbored an inducible suicide gene in its differentiated osteoblasts. When the osteoblasts are ablated by induction of the suicide gene, these mice develop a short stature and an osteopenic phenotype due to continued osteoclast resorption in face of abolished bone formation [8]. In humans, these findings can be confirmed in osteopetrotic patients in whom the osteoclasts are either absent or nonfunctional, yet bone formation is not halted. These findings show that the function of either osteoblast or osteoclast is not dependent on the others function. The observation that bone remodeling is a tightly controlled process, taking place at multiple locations in the skeleton, is consistent with the possibility that bone remodeling is under endocrine control. This would not be very surprising as most other homeostatic functions of the body are known to be under endocrine, if not neuroendocrine, control. Accordingly, it is well known that hormones, such as sex steroid and parathyroid hormones among others, can affect bone resorption [34, 38], and the cytokines OPG and OPGL themselves can, in addition to their paracrine mode of action mentioned above, act in a systemic manner on osteoclast differentiation [24, 42]. This raises the following question: Is bone formation also under endocrine control? In contrast to the Figures 1A to 1D. Osteopetrosis in the src/ mouse, an example for continuing osteoblast emerging knowledge demonstrating function despite a lack of functional osteoclasts. In comparison to wild-type controls (A, C), the the existence of an endocrine regulalumbar vertebrae of src/ mice (B, D) show a massive increase of trabecular bone volume (A, B low magnification, C, D high magnification; von Kossa staining of undecalcified sections). tion of bone resorption, we know

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much less about the molecular mechanisms regulating the rate of bone formation by the osteoblasts. Indirect evidence for an endocrine control of the osteoblast has been gained from the osteoblast ablation model mentioned above. If in these mice the expression of the suicide gene was stopped, osteoblasts repopulated the bone and bone mass was quickly restored with an astonishing precision to virtually the same amount as in wild-type age-matched littermates. This extreme precision indicates that the osteoblast has two speeds to make bone. First, it quickly deposits a large amount of bone matrix to restore bone mass, but once this is achieved it slows down so the bone mass does not increase beyond that in wildtype mice. These findings could be regarded in favor of the hypothesis that osteoblast function, as it has been shown for osteoclast function, is under endocrine control. However, the main input toppling the old paradigm of local control came from two clinical observations. The first is that obesity protects against bone loss, and the second that menopause favors bone loss. Translated into a molecular vocabulary, these observations led us to hypothesize that bone mass, body weight, and gonadal function might somehow be connected, and may be regulated by the same secreted molecules without presaging the mode of action on their target organs. As the endocrine regulation of body mass and gonadal function has been well established, the testing of this hypothesis could help on the quest for identifying a possible endocrine factor in osteoblast control. At this phase, recent advances in molecular endocrinology came to help, i.e., the discovery of leptin and its role in influencing body mass and fertility. Leptin the Most Powerful Endocrine Inhibitor of Bone Formation In the candidate gene approach taken to identify a potential endocrine regulator of bone formation, leptin seemed from the beginning the most promising one. Leptin, a polypeptide hormone, is synthesized by adipocytes and functions as a starvation and adiposity signal after binding to its receptor localized primarily in the hypothalamus. If there are few adipocytes in the body (i.e., a szenario of starvation), leptin levels are low and food intake is stimulated. If fat cells are abundant as in adiposity, high leptin levels reduce the food intake [16, 48]. Leptin is the product of the ob gene, whose activity is missing in ob/ob mice, a mouse model of obesity. Rodents and humans genetically deficient in leptin signaling are

obese [1, 6]. The absence of leptin signaling also causes sterility (i.e., hypogonadism) and hypercortisolism, conditions usually associated with bone loss. Normally, the influence of the absence of gonadal function together with the hypercortisolism should antagonize the mechanical influence of obesity on bone integrity in ob/ob mice and result in a low bone mass phenotype. Surprisingly, the leptin-deficient ob/ob mice showed a two- to threefold higher bone mass than wild-type mice (Figure 2) [9]. Another mutant mouse strain, the db/db mouse has a mutation in the leptin receptor (db) and the same series of phenotypes as the ob/ob mice [7, 9]. Likewise, fa/fa rats, harboring an inactivating mutation in the gene encoding the leptin receptor, are obese, hypogonadic, and have a high bone mass (HBM). The ob/ob and db/db mice as well as the fa/fa rats are the only animal models known so far where a state of hypogonadism coexists with an HBM phenotype. Taking additionally into account their hypercortisolism, a state which usually leads to a decrease in osteoblast function and osteoporosis, these animal models are an invaluable resource to study bone remodeling and its diseases. The HBM phenotype of the ob/ob and db/db mice, which is caused by an increase in bone formation, is not secondary to their obesity as it is observed in young ob/ob mice before they become obese [9]. More importantly, this phenotype is dominant, being observed in heterozygous mice (ob/+ and db/+), and is specific to the absence of leptin signaling since it is not observed in non-ob mouse models of obesity. The fact that the HBM phenotype is dominant whereas the obesity phenotype is recessive, demonstrates that the control of bone mass by leptin is not an accidental function of a body weight-regulating hormone. It rather indicates that the control of bone formation is a function of leptin, which is as important as the control of body weight. It also demonstrates that the HBM phenotype is not secondary to any endocrine abnormalities observed in ob/ob and db/db mice, since ob/+ and db/+ mice have none of them. The HBM phenotype of the ob/ob and db/db mice develops despite two osteoporosisfavoring conditions such as hypercortisolism and hypogonadism, demonstrating the great importance of leptin regulation in bone formation. Indeed, no other animal model has been identified so far harboring an HBM phenotype despite the coexistence of these two conditions. Bone histomorphometry analysis performed before and after correction of the hypogonadism of ob/ob mice showed that leptin regulates bone mass through inhibition of bone formation; it has no overt effect on osteo-

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Figure 2. Leptin deficiency leads to a high bone mass phenotype. Ob/ob mice (right) in which leptin signaling is absent have a marked increase in trabecular bone volume, despite their hypogonadic and hypercortisolic state, compared to wild-type controls (left). Lumbar spine, micro-CT image, Scanco Medical, Switzerland.

clast differentation and function. Importantly, leptin action on bone formation did not involve osteoblast differentation as ob/ob and db/db mice have a normal number of osteoblasts. This latter observation indicates that any possible local mode of leptin on bone must affect already differentiated osteoblasts and not their progenitors. Towards the Paradigm of Central Bone Mass Control How does leptin act to control bone formation? Does it influence the osteoblast through an autocrine,

paracrine, or endocrine mechanism? Or does it simply control bone formation as it controls body weight following its binding to its hypothalamic receptor? Before addressing these crucial questions, one has to summarize what is the phenotype of the ob/ob and db/db mice and the critical implications this phenotype conveys. These mutant mouse strains do make more bone with a normal number of osteoblasts. In other words, this is a functional phenotype, not a differentiation phenotype. This implies that if leptin acts locally it has to do so through the presence of functional receptors in differentiated primary osteoblasts, not on osteoblast progenitors. A multiplicity of experiments, biochemical, molecular, and genetic, failed to detect any expression of leptin or of a signal-transducing receptor in osteoblasts, thus virtually ruling out an autocrine, paracrine, or endocrine mechanism of regulation. Additionally, osteoblasts isolated from receptor-deficient db/db mice behave like wild-type osteoblasts in vitro [9]. These data indicate that leptin does not directly target osteoblasts. It was conceivable that in the absence of leptin, adipocytes release a molecule favoring bone formation. Studies using a transgenic mouse strain deprived of white fat, however, proved the contrary. These mice, which are called fat-free mice, have a very low level of leptin since they have virtually no adipocytes [31]. Nevertheless, they had an HBM phenotype, thus ruling out the formal possibility that in the absence of leptin, adipocytes release an activator of bone formation. The remaining possibility to be tested was the most simple and yet the most novel, namely that leptin controls bone formation following its binding to hypothalamic nuclei where the leptin receptor is particularly abundant. It is also the most simple explanation because this mode of action is also the one used by leptin to control body weight. It is the most unusual because a central regulator of bone remodeling has never been demonstrated in vivo. The Paradigm of Central Bone Mass Control To test the hypothesis that leptin acts through a central relay, we had to devise a strategy to administer it directly to its presumed place of action, namely the hypothalamus, without any leptin reaching systemic circulation. As the hypothalamus lies directly beneath the third ventricle, we performed intracerebroventricular (ICV) infusion of leptin in nanomolar concentrations, where even with the most sensitive tests no leptin could be

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detected systemically. If leptin deficiency at the hypothalamic receptors causes the HBM phenotype in ob/ob mice, it should be possible to correct it by application of leptin to the hypothalamus. Indeed, ICV infusion of leptin in ob/ob mice led to a rapid and massive decrease in their bone mass. Similarly, ICV infusion of leptin in wild-type mice led to the development of a severe osteopenic phenotype, demonstrating that bone remodeling or at least its bone formation aspect is under the control of the hypothalamus. No leptin could be detected in the serum of these ICVtreated animals, this latter control demonstrating unambiguously that leptin can regulate bone formation without contacting the osteoblast directly [9]. These findings, in line with the mode of regulation of body weight and gonadal function, do not close the door to any other possible mode of action of leptin yet to be demonstrated in vivo. Rather they should be viewed as providing investigators in the bone field with a new conceptual framework to better understand bone physiology (Figure 3). To date we still do not know whether there is a single linear genetic or biochemical pathway explaining leptins role in body weight control following binding to its hypothalamic receptor [11, 12, 18]. Likewise, we do not yet know what the mechanisms are that convey to the osteoblasts the information leptin delivers into the hypothalamus. Nevertheless, leptins action on body weight and on bone mass seems to use different pathways, as indicated by its dominant action on bone formation rate and its recessive action on body mass. Clearly, one of the challenges ahead of the field will be to identify mechanisms downstream of leptin but maybe the more important aspect will be to define the endpoint molecules regulating leptins action on bone. Is leptin the only systemic regulator of bone formation? Most likely not, as the existence of a negative regulation of bone mass suggests that positive regulators of bone formation may exist and await to be identified. Conclusions Going back to our initial hypothesis that reproduction, body weight, and bone mass may share common regulation loops, how do these findings explain the observation that gonadal failure favors osteoporosis and adiposity protects from it? It seems that adipose patients, having many fat cells and therefore high leptin levels, should show a low bone mass rather than a high bone mass phenotype. Howev-

Figure 3. Central regulation of bone mass. Leptin, after its synthesis in adipocytes, binds to its receptor (Ob-Rb) in the hypothalamus and influences bone formation, body weight, and fertility.

er, it has been demonstrated that obese people often harbor a leptin resistance. The molecular basis of this leptin resistance is not well understood but it results in a partial functional deficiency of leptin, a situation similar to the one of ob/+ and db/+ mice, and leading therefore to HBM. These findings are in line with another clinical observation, that patients with generalized lipodystrophy, a condition marked by a nearly complete absence of adipocytes and white fat, also exhibit an HBM phenotype. The in vivo analysis of the role of leptin during bone remodeling has taught us several important lessons. The first, most general and, we feel, most important one is that there is a lot of essential information or suggestive evidence to be found in terms of molecular hypotheses for many physiological processes in the classical clinical literature. This wealth of knowledge, when paired with a molecular approach, should open many new avenues for investigators to follow, and demonstrates the importance of an intensive dialog between clinician and researcher. The second and, in fact, critical lesson is that bone remodeling is as much a centrally controlled

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process as it is a local one. This central regulation is of paramount importance since its disruption is the only known biological setting in which the deleterious consequences of hypogonadism on bone metabolism are overcome. An implication of this finding is that the most typical and frequent bone remodeling disease, namely osteoporosis, is, at least partly, a central or hypothalamic disease. As such, these studies may be viewed as establishing a novel paradigm in our understanding of bone remodeling. This does not mean, however, that we now understand everything about bone remodeling. On the contrary, this shift of concepts raises far more questions than it answers. In a parabiosis experiment [44], in which the circulatory system of two mice was connected, only the one mouse receiving an ICV application of leptin had a decrease in bone mass, while the other mouse sharing the same bloodstream but not obtaining any leptin had a normal bone phenotype, raising the possibility that leptin regulation of bone mass is not mediated through a soluble factor. If these findings hold true, the remaining mechanism of action would be that the information from the hypothalamus is transmitted to the bone via use of the nervous system, and thereby implying that it is indeed the brain that controls bone. In terms of potential therapeutics, the identification of leptin as such a powerful inhibitor of bone formation also has important implications. Conceivably, since the HBM phenotype is dominant whereas the obesity phenotype is recessive, it should be possible to design drugs acting on this pathway, that would have a protective effect on skeleton integrity without leading to obesity. References
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Correspondence Address Michael Amling, MD Department of Trauma Surgery Hamburg University School of Medicine Martinistrae 52 20246 Hamburg Germany Phone (+49/40) 42803-6083, Fax -8010 e-mail: amling@uke.uni-hamburg.de

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