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0021-972x/96/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1996 by The Endocrine Society Vol. 81, No. 3 Printed m U.S.A. Invited Review of a Workshop: Anabolic Bone: Basic Research and Therapeutic RONALD N. MARGOLIS, ERNEST0 CANALIS, NICOLA AND Hormones Potential in C. PARTRIDGE Endocrinology Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (R.N.M.), Bethesda, Maryland 20892; the Department of Research, St. Francis Hospital and Medical Center (E.C.), Hartford, Connecticut 06105; the University of Connecticut School of Medicine (E.C.), Farmington, Connecticut 06030; and the Departments of Pharmacology and Physiological Sciences, St. Louis University School of Medicine (N.C.P.), St. Louis, Missouri 63104 ABSTRACT Age-, postmenopause-, and disease-related conditions that result in low bone mass represent important public health issues. Maintenance of bone mass is a balance between bone resorption and formation and is influenced by diet, body composition, activity level, and the interactions between and among a large number of hormones, growth factors, and cytokines. Recent research has emphasized establishing a more complete understanding of the hormonal regulation of bone and developing anabolic agents with therapeutic potential for the treatment of low bone mass. The NIDDK at the NIH recently spon- B sored a Workshop, entitled Anabolic Hormones in Bone: Basic Research and Therapeutic Potential, that attempted to define the current state of the art knowledge of hormones, growth factors, and cytokines that affect bone mass, with particular emphasis on those that could potentially have a role as anabolic agents in bone. This review presents a condensed proceedings of that workshop along with a summary of the optimal requisites for the development of anabolic agents with therapeutic potential in bone. (J Clin Endocrinol Metab 81:872-877, 1996) ONE PROVIDES a skeletal framework for the body and acts as a dynamic reservoir of mineral, particularly calcium and phosphorus. Bone turnover, the product of bone resorption and formation, is a tightly coupled process, with the net balance between the two determining the bone mass and the serum calcium level (1). Regulation of bone turnover requires the input of a large number of hormones, growth factors, and cytokines (1). In this context, an anabolic agent is one that can shift the net balance in the direction of increasedbone mass.Additionally, an antiresorptive agent can increase bone massif it shifts the regulatory balance toward net formation by suppressing resorption. Postmenopausal and aging-related osteoporosis, with attendant loss of bone mineral, results in widespread health problems (2, 3). Consequently, there is a great need to understand how to precisely regulate net homeostatic balancesin bone. Much of the current knowledge of bone development and growth has been built upon fundamental insights derived from animal and tissue culture studies aswell asfrom recent observations of the effects of mutations in bone-related proteins on bone structure and function. New knowledge derived from mutations in genes that cause skeletal abnormalities has provided needed information on both normal bone function and the effects of disease on these processes(see Ref. 4 for re- view). Still more understanding has been developed from resultant phenotypes derived from mouse models of hormone non- or overexpression, either from targeted disruption of a gene by homologous recombination (null allele) or from targeted overexpression in transgenic animals (4). Dissecting the role(s) of individual systemic hormones, growth factors, and cytokines in the regulation of bone turnover is a necessaryfirst step in identifying the appropriate stagesat which therapeutic interventions with anabolic agents can be developed to affect net bone formation. With this in mind, an NIDDK’ workshop entitled Anabolic Hormones in Bone: Basic Researchand Therapeutic Potential was held May l-2, 1995, at the NIH. The initial objective of the workshop was to focus on hormones, growth factors, and cytokines with demonstrated anabolic effects on bone. It gradually became clear that a large number of factors interact at the level of the osteoblast, osteoclast, and other cells to regulate the balance between net resorption and formation. Thus, the workshop was broadened to include agents that affect both sides of the regulatory equation and could be recognized as potential focal points for interventions directed at the sameoutcome: increased bone mass. PTHJPTH-related peptide (PTHrP) It haslong been known that chronic elevation of PTH results in bone resorption, asPTH is the most important hormone that regulates bone calcium levels (1). In addition to its resorptive actions, intermittent administration of PTH produces anabolic effects (3). The resorptive effects of PTH are well documented in patients with asymptomatic primary hyperparathyroidism (seeRef. 5 for review), in which chronic mild hypercalcemia reflects gradual erosion of bone mineral asa consequenceof the elevated levels of I’TH. Further indications of the resorptive Received August 8, 1995. Revision received September 27, 1994. Accepted November 3, 1995. Address all correspondence and requests for reprints to: Ronald N. Margolis, Ph.D., Endocrinology Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 45, Room 5AN.l2J, 45 Center Drive, MSC 6600, Bethesda, Maryland 20892-6600. E-mail: margolisrQep.niddk.nih.gov. ’ Cosponsored by the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the NIH Office of Research on Women’s Health. 872 INVITED effects of PTH were obtained when one of the factors linked to the hypercalcemia of malignancy was discovered to be PTHrP, a peptide with sequence and structural similarities in the Nterminal regions to PTH (5). Although PTH is restricted to production by the parathyroid gland, acting primarily as an endocrine factor, PTHrP is produced by a variety of tissues and cells, where it acts as a paracrine and/or autocrine factor (4-8). Gene knockout by homologous recombination has set the stage for major advances in our understanding of the precise role of PTH/PTHrP in skeletal and nonskeletal tissue development (see Ref. 4 for review). Despite lack of the PTHrP gene, mice survived to birth, although numerous developmental defects occurred, with major alterations in endochondral bone formation. These included premature ossification of the ribs and death during the perinatal period. Conversely, when overexpressed in transgenic mice, PTHrP was implicated in the development of numerous other tissues, including pancreatic islets, where it had an anabolic effect (9). These data confirm the importance of PTHrP as a locally active factor, mediating terminal differentiation and function of bone and other tissues. A major implication of these findings is the putative role of PTH and I’THrP as anabolic agents in bone. Although it appears that PTH and PTHrP have distinct roles in the regulation of calcium homeostasis, their actions converge on a common PTH/PTHrP receptor (10). This member of the seven-transmembrane G-protein-coupled receptor superfamily initiates complex cellular responses through two different signal transduction pathways coupled through increased CAMP production and/or stimulation of phospholipase C. The mechanism of the anabolic action of PTH and the signal transduction pathways involved are not known. Nevertheless, regulation of the PTH/PTHrP receptor becomes a point at which control of bone turnover can occur. The amount of receptor in the osteoblast is crucial to the ability of this cell to respond to PTH and/or PTHrP. In the osteosarcoma cell line ROS 17/2.8, a number of factors up-regulate PTH/PTHrP receptor levels, including glucocorticoids and transforming growth factor-p, (TGFP,) (11). These effects appear to be at the level of gene transcription. Human mutations of the PTH/PTHrP receptor result in Jansentype metaphyseal chondrodysplasia, a form of short limb dwarfism (12). In this rare disorder, patients have hypocalcemia and hypophosphatemia, with normal or near-normal serum levels of PTH and PTHrP. The mutation causes a chronic activation of the receptor, resulting in a gain of function, signaling the cell constantly through increased production of CAMP. Defects are seen in bone and kidney, target organs of PTH action. Among the several putative hormonal anabolic agents for bone only intermittent administration of PTH has demonstrated efficacy (1, 5, 13, 14). In a recent small scale clinical trial, intermittent PTH prevented bone loss in premenopausal women with endometriosis treated with gonadotropin release analogs known to cause osteopenia (14). The interplay between physiological levels of PTH/PTHrP and the common receptor in homeostatic control of serum calcium levels and bone mineral density is essential to long term maintenance of bone structure and function. The disruption of this normal balance, either during development or in the adult, can have profound effects on the skeleton (and other tissues). Clearly, further research is required to define the precise mechanism(s) of action of PTH/PTHrP in elicit- REVIEW 873 ing anabolic effects in bone. An understanding of when, where, and how to intervene must then follow to develop a better understanding of the potential therapeutic use(s) of PTH, PTHrP, and the appropriate analogs. Vitamin D Vitamin D (1,25-dihydroxyvitamin Ds), a member of the steroid/thyroid/retinoid supergene family of hormones, is the key hormone that regulates intestinal calcium absorption (1, 15). In addition to this role for vitamin D in calcium absorption, it has direct effects on bone cells. In small scale trials, vitamin D administration and dietary calcium supplementation increased bone mineral density at the femoral neck of elderly postmenopausal women (see Ref. 16 for review). In other studies, vitamin D alone decreased the vertebral fracture rate in patients with mild to moderate osteoporosis (16). Despite such promising clinical data, the risk of hypercalcemia and the lack of knowledge of the precise mechanism(s) of action of vitamin D have limited the putative anabolic uses of this hormone. Vitamin D is a primary resorbing factor in bone through its ability to stimulate the differentiation of undifferentiated bone cell precursors (17). Vitamin D has effects on the osteoblast, including regulation of the expression of extracellular matrix (ECM) proteins such as osteocalcin (18,19). The molecular mechanism of action of vitamin D appears to be through hormone binding to the receptor, leading to its heterodimerization with other accessory factors, specifically members of the retinoid subfamily (15). Binding of heterodimers of the VDR and a retinoid receptor (RXR) to specific consensus sequences in the promoter regions of vitamin D-dependent genes (e.g. osteocaltin) regulates gene expression in target cells. The study of human diseases such as vitamin D-dependent rickets has provided new insights by demonstrating that a mutation in the vitamin D receptor causes resistance to vitamin D (20). Work is underway to design and test vitamin D analogs that can provide therapeutic potential by increasing bone formation without concomitantly increasing serum calcium levels. Androgens Although the central role of estrogen (E2) deficiency in bone loss in postmenopausal women has received widespread attention, the role of anabolic steroids is not well established. Androgens have been used (and abused) for the enhancement of body composition, including muscle mass (21). During puberty in boys, administration of androgens accelerates the accumulation of calcium in bone, consistent with enhanced mineralization (22), and in elderly or hypogonadal men, administration of androgens has a positive effect on bone mass (23). The effects of androgens on body composition may involve indirect effects on the skeleton through increases in lean body mass (i.e. muscle) or direct effects on bone (24, 25). Indeed, human osteoblast-like cells express endogenous receptors and respond to adrenal androgens such as dehyroepiandrosterone sulfate with changes in gene expression consistent with a stimulation of bone formation. Anabolic steroids represent a potential area of future research, although their use has been hampered by the absence of definitive knowledge about their effects on bone 874 MARGOLIS, CANALIS, and concern over significant and undesired effects in other tissues. Development of analogs that can exploit the positive effects of androgen on bone without expressing less desirable secondary effects should be pursued. E, /progesterone (P) The role of sex steroids in the maintenance of bone mineral density is well established (see Ref. 16 for review), with the loss of cycling hormones at the menopause resulting in rapid and significant loss of bone mineral. Our understanding of the role of sex steroids in bone biology was enhanced after the discovery of functional E, and P receptors in osteoblasts and osteoclasts (16,26). Indeed, the presence of E, receptors (ER) in both osteoblasts and osteoclasts has fueled speculation on the potential effects of E,. Although E, appears to act on osteoclast-like cells to inhibit bone resorption (16), major attention has focused on the role of E2 in regulating the behavior of the osteoblast. E, has an antiresorptive effect on osteoclasts and alters the release of cytokines from osteoblasts and/or mononuclear cells involved in remodeling (reviewed in Refs. 4, 27, and 28). Cytokines, such as interleukin-l (IL-l) or IL-6, which recruit osteoclasts, are suppressed, whereas possible anabolic growth factors, such as insulinlike growth factor I (IGF-I) and TGFP, are stimulated by E, (see Refs. 27 for review). E, also has been implicated as a hormone responsible for increasing the levels of ECM-bound growth factors (29). Indeed, the role of the ECM in cellular adhesion and differentiation is an area of potential importance in the regulation of bone turnover. A specific example is the demonstration that PTH induces the production of fibronectin (FN) in osteoblasts. FN is a key element of the ECM potentially important in the recruitment, differentiation, and subsequent function of preosteoblasts. E, deficiency also interferes with FN production, with the absence of E, reducing the effects of PTH on FN production (30). Thus, interactions between E, and other hormones may play a major role in defining the net effect of hormonal action on bone turnover. Clearly the role(s) of steroid hormones acting directly and/or indirectly on bone requires further effort to achieve a mechanistic understanding. The recent use of gene knockout methodologies has resulted in a mouse with a null allele for the ER (31). Viable offspring provided a unique model to investigate the effects of the absence of ER on bone development. Perhaps equally important has been the discovery of a human mutation in the ER resulting in a man with E, resistance (see Ref. 4 for review). Initial study has revealed that the most striking skeletal effect was osteopenia and a failure of closure of the epiphyses, hinting at a role for E, in bone metabolism in both sexes. The administration of E2 was not effective in this individual, confirming the E, resistance. In addition, a man with a mutation causing a deficiency in aromatase, an enzyme that converts androgens to E,, also had marked osteopenia and failure of epiphyseal closure (32). Together, these data suggest a convergence in the role of E, regulation of bone turnover in women and men. In postmenopausal women, administration of E2 in a regimen of hormonal replacement therapy prevents the loss of bone mineral (16). However, in addition to effects on bone turnover, AND PARTRIDGE JCE & M . 1996 Vol81.No3 E2 has effects on a variety of other tissues, including breast and uterus. The use of E, alone in a regimen of hormonal replacement therapy to reduce osteopenia in postmenopausal women has been compromised by the small, but real, increase in the risk of developing breast and uterine tumors (16,33), with progestins now added to partly offset these effects. As P receptors (PR) are also present in bone cells, it is possible that P could have direct effects on bone (16). The effects of P at the molecular level are complicated by the recent demonstration that two isoforms of the PR exist, a full-length B form and a truncated A form (see Ref. 34 for review), each with cell- and promoter-specific actions. When both receptors are present, the A form acts as a dominant suppressor of the B form. Although this has immediate implications for antihormone therapy directed at PR-positive tumors of the breast, the corresponding effects on bone cells are entirely unknown. A promising approach to treating receptor-positive breast cancer has been the use of antihormone therapy. Tamoxifen (TAM) is an anti-E, in the breast, with partial agonist effects on bone, but it can also be a partial agonist in uterus (34). Preliminary data on E,-like analogs, such as raloxifene (LY 139481 HCI), a benzothiophene derivative that binds to the ER, have shown positive effects in bone without effects on the uterus (16). Clearly, more research is needed to develop analogs that express the agonist effects of E, in bone, but act as antagonists in other tissues (35). GHI IGF Although GH has general anabolic effects, including those in bone (36,37), questions regarding its efficacy and mode of action have limited its usefulness. GH stimulates the hepatic production of IGF-I, with IGFs also produced locally in various tissues, including bone, alone and under the control of other hormones (see Ref. 38 for review). IGF-I released by liver in response to GH circulates as part of a larger complex consisting of IGF-I and IGF-II, IGF-binding proteins (IGFBP), and an acid-labile subunit (see Ref. 13 for review). To date, six IGFBPs have been cloned, each of which binds IGFs at sites distinct from that on the cell surface receptor(s) (13,39). Tissue-specific proteases degrade binding proteins, thus affecting their total availability at local sites (13, 28, 40). The production of IGF-I by osteoblasts has led to speculation that IGF-I has both systemic and autocrine actions in bone (13). The effects of IGF-I on bone are anabolic, stimulating longitudinal growth and possibly bone mass (13, 41). The importance of IGF-I to normal development and growth has been demonstrated through gene knockout techniques (38). Mice homozygous for a null IGF-I allele are small and die at birth. The most noticeable deficiency in these mice is in muscle mass, although calcification of bones is diminished, suggesting a defect in the rate of ossification. IGF-I interacts with other bone-active hormones, including PTH and E,, with the former increasing the synthesis of IGF-I (13,28), perhaps accounting for some of the anabolic effects of PTH on bone (13). The ability of bone cells to respond to a variety of signaling molecules by increasing IGF production suggests that the convergence of a number of independent signals occurs at the level of this key effector. Thus, osteogenic protein-l [also known as bone morphogenetic protein-7 (BMP7)l and BMP-2, both members of the TGFf3 superfamily, also INVITED increasethe production of IGF-II and selectedIGFBPs (28).This effect may involve PTH aswell, asBMPs increasePTH binding to its bone cell receptor, resulting in increasedbone cell activity (42).Prostaglandins(e.g.PGE,) up-regulate transcription of the IGF-I genein osteoblast-likecells(43). Regulation of IGF-I gene expressionis complex, with several transcripts observed in cells reflecting a number of putative start sites for transcription, perhapsthe result of different signalsinteracting with different regions of the IGF-I promoter (43). IGFs affect primarily the differentiated function of the osteoblastwith modest mitogenic effects. In vitro coculturing experiments have suggested that IGFs also stimulate bone resorption by increasing osteoclastrecruitment (44). In addition to control at the level of gene expressionthe role of the IGFBPs and IGF receptors must be considered. There are two cell surface receptors: the type I, resembling the insulin receptor, a member of the receptor tyrosine kinase superfamily, and the type II or mannose-6-phosphatereceptor (13, 45). IGF-I and IGF-II bind to and appear to signal their anabolic activity through the type I receptor (13). IGFBPs govern IGF tissue availability, locally and systemically (13,40,42). IGFBP-3 is the predominant binding protein in serum and is a marker of GH action, as its synthesis by liver is sensitive to GH (46). Other IGFBPs, such asIGF’BP-4and IGFBP-5, may regulate the availability of IGF at the level of bone (13). At the tissue level, elements of the ECM, including glycosaminoglycans, play a role in the availability of IGFs (47). The releaseof IGFs from IGEBPs and the ECM is mediated by a family of proteases shown to degradeIGEBPs,releasingthe growth factor and thus increasing immediate tissue availability. Stability and selectivity may be governed by the ECM, which in some instances confers a degree of protection over proteolysis of IGFBPs (47). In addition, the synthesis of IGFBPs is regulated by IGFs and other growth factors (42). These revealing experimental data have served as the focal point for the design of studies to test the efficacy of IGFs for use in human patients. IGF-I administration to patients with GH resistancedue to a mutation in the GH receptor hasresulted in increasedlean body massand bone mineral (48). Short term treatment of postmenopausalwomen with IGF-I results in increased bone turnover (seeRef. 3 for review). Additional trials are required to establisha potential role for the IGFs astherapeutic agentsfor the treatment of low bone mass.The ability of IGFs to regulate bone turnover and cause a net anabolic effect on bone is governed by a host of tissue- and cell-specific interactions, including interactions among other growth factors, systemic hormones, ECM, and IGEBPs.If IGFs were to be used assystemic therapeutic agents in disordersof bone metabolism,questionsconcerning the optimal route of administration and potential side-effectsmust be answered. Other growth factors The multiplicity of responses of skeletal cells to systemic and local factors is nowhere more apparent than when considering the effect(s) of growth factors on bone. A prime example is the BMP group (4,28). This large family of growth factors, which acts through serine/threonine kinase receptors, regulates numerous cellular events, both during development and in the adult (28). Although some growth factors, such as tumor necrosis factor-a, appear to have resorptive effects on bone, others, including TGFP, REVIEW 875 have anabolic effects (28), often interacting with the signals generated by other systemic and local factors (4, 25, 28). Recent advances in our understanding of the structure and function of the receptors for the TGFP superfamily revealed a two-subunit model (49), with ligand binding to the type I receptor resulting in recruitment and serine/ threonine phosphorylation of the type II receptor. Among the key downstream events is stimulation of a2(1) collagen gene expression (see Ref. 4 for review), which is important to a variety of tissues and critical to bone function. TGFp also may regulate the differentiation of precursors to mature osteoblasts. Mechanical loading, which is important in defining skeletal growth, increases tissue levels of TGFP (50). TGFP is present in a latent form, with many factors, including binding to elements of the ECM, regulating this latency (28). Increased mechanical loading may activate the latent form of TGFP (50). Mutations in the mouse give rise to a condition known as short ear (seeRef. 4 for review). BMP-5 appearsto be the product of the short ear locus, which is altered in mice expressing short ear. The resulting phenotype includes a number of alterations, involving somethat develop both early and late in skeletal formation (4). BMPs and TGFP play key roles in the development of the skeleton and its maintenance and repair. Understanding their precisemolecular mechanismsof action is essential to consider their potential uses for the repair and treatment of bone disease,including osteoporosis. Fibroblast growth factors (FGFs) are members of a large family of heparin-binding growth factors with actions in a number of tissues (4, 28, 51). There are four FGF receptors (FGFRl-4), members of the protein tyrosine kinase superfamily of receptors (52). Originally studied for their role in angiogenesis,it is now clear that FGFs are involved in neural development, wound healing, and chondrogenesis (51). A seriesof human mutations in the receptors for FGF have been particularly revealing for defining the roles played by FGFs in skeletal development. Mutations in the FGFR3 cause achondroplasia, the most common form of inherited dwarfism (seeRef. 4 for review), and mutations in the FGFR2 gene cause Crouzon syndrome, characterized by premature fusion of the bonesof the skull (4). Administration of basic FGF by miniosmotic pumps near the tibia1 growth plate stimulated long bone growth and angiogenesis, suggesting the potential for therapeutic use (53). Further research defining the mechanisms of growth factor action may allow us to exploit those functions associated with anabolic effects in bone. Cytokines The precursors of osteoclastsand osteoblastsare present as relatively undifferentiated stem cells of the hematopoietic and mesenchymal lineage, respectively (4, 27). Consequently, agents that regulate the recruitment, replication, and/or differentiation of thesestem cellscan have a role in influencing the balance between formation and resorption (27,28). Cytokines represent a group of such factors (27). Included are the ILs, especially IL-l, IL-4, IL-6, and IL-11, which have been implicated in bone turnover (27,28,54). IL-11 and IL-6 are produced by osteoblasts(27,28). IL-6 is up-regulated in E, deficiency and may be responsiblefor the enhanced bone resorption in the E,-deficient state (27). Overproduction of IL-4 in transgenic 876 MARGOLIS, CANALIS, mice results in osteoporosis by decreasing the activity of osteoblasts (54). Administration of an antagonist to the IL-1 receptor in ovariectomized rats decreases bone loss by decreasing osteoclast-dependent bone resorption (27). One potential common point of convergence for these cytokines may be the signal transduction machinery, which operates through a common class of receptors (27, 55). The cytokine (or erythropoietin) receptor has an extracellular ligand-binding domain, but no apparent intracellular signal transduction machinery. Rather, the receptor is tightly bound to a glycoprotein of -130 kDa, known as gp130 (27,55), and it is this protein that appears to be essential for signal transduction through cytokine receptors in many cells, including osteoclasts. It is this receptor complex that is up-regulated in E, deficiency (27, 28). As communication between osteoblasts and osteoclasts is important in determining the coupling or uncoupling of formation and resorption, crosstalk fostered by levels and timing of IL release could be an important regulatory link. This may be the case with IL-11, which is released by osteoblasts and stimulates osteoclastogenesis (27). An important area for potential pharmacological intervention could be to alter the balance between osteoclastogenesis and osteoblastogenesis in favor of net bone formation. Colony-stimulating factor-l (CSF-1) has an important function in bone (4,28,56), and disruptions in the CSF-1 gene result in osteopetrosis (57), a disorder characterized by an absence of osteoclastic bone resorption. In some mouse models of osteopetrosis (e.g. the op/op mouse), administration of CSF-1 results in restoration of osteoclastogenesis, indicating a defect at the level of production of this cytokine and revealing a role in normal osteoclast function (57). In another model of osteopetrosis, mutations of a signaling protein tyrosine kinase called C-SK resulted in failure of development of mature osteoclasts, suggesting a defect in cytokine receptor signal transduction (4, 28). The similarity in phenotype that results from perturbation of either CSF-1 or C-SK suggests a convergence on the signal transduction pathway through C-SK in osteoclasts. CSF-1 is released by the osteoblast in response to PTH or PTHrP, and thus probably represents part of the overall bone remodeling sequence of regulatory events and another potential site for pharmacological intervention. Another factor involved in cytokine action is PGE,, which is produced and released by osteoblasts and participates in the regulation of bone resorption (4, 28, 58). PGE, production requires up-regulation of PGH synthase (4). This mitogen-inducible enzyme may be a potential point of intervention when considering how to generate anabolic effects in bone. Therapeutic potential When taken in the context of regulation of the dynamic counterbalanced process of bone turnover, the results of this workshop pointed toward anabolic agents primarily in the context of those that increase bone formation and secondarily those that decrease bone resorption and thus alter the balance of bone turnover toward net increase in bone mass. A number of inert agents that have positive effects on net accumulation of bone mineral, such as fluoride and bisphosphonates, have been used (3). However, novel agents acting by modulating the behavior of the cells responsible for regulating bone turnover may have a greater potential. Agents that increase the recruitment and subsequent differentiation/ proliferation of bone cell precursors or that act on the mature AND PARTRIDGE JCE & M . 1996 Vol81 . No 3 osteoblast would have the most direct effect on bone formation (45). Factors with antiresorptive effects or actions on the osteoclast or osteoblast should positively influence bone mass, but perhaps to a lesser extent than those that are primarily anabolic (59). Combinations of both types of agents could offer the most potential as therapeutic agents. Even more novel approaches may be required, perhaps using synthetic gene constructs that can modify specific signal transduction events in defined target cells (60). Other approaches that warrant consideration include a focus on agents that affect cell to cell and/or cell to matrix interactions, as the context of the cellular microenvironment can be as important as the presence/absence of a given hormone, growth factor, or cytokine in the overall regulation of bone turnover. Another consideration is whether a particular agent can be used systemically or must be present locally in the bone marrow or bone matrix to be effective. Regardless of the level of the regulatory process, several parameters must be met to obtain maximal therapeutic potential: 1) bone mass must be increased without sacrificing bone quality; the latter is essential to provide for maximal bone strength and resistance to fracture; 2) the magnitude of the effect must be clinically significant; only agents that improve bone strength and quality will improve quality of life; 3) the effects must be progressive and sustained; short term effects that are not sustainable will provide only transitory improvement; 4) the actions of such agents must be selective for bone, with no side-effects; agents that lack specificity or express unwanted secondary actions will not be effective or desired; and 5) ideally, such agents should be effective when taken orally once a day; ease of administration will encourage long term continued use. Although a number of promising agents are being tested in experimental animals, in tissue culture models, and in limited clinical trials, much basic research is yet required to fully understand the regulation of bone turnover. Major research questions focused on the roles of systemic and local factors in the regulation of bone cell development, differentiation, proliferation, and action need to be answered. Only then can the more practical questions about agents with therapeutic potential be resolved. Acknowledgments This NIDDK Workshop was cosponsored by the NIAMS and the NIH Office of Research on Women’s Health. The contributions to this workshop of all speakers and discussants is gratefully acknowledged: L. Attisano, L. Avioli, A. Baird, R. Baron, D. Baylink, L. PowellBraxton, D. Clemmons, C. Conover, J. Harrison, J. Hock, K. Horwitz, A. Kahn, A. Karaplis, S. Krane, D. LeRoith, J. Lian, R. Lindsay, S. Manolagas, R. Marcus, L. McCauley, D. McDonnell, G. Mundy, R. Nissenson, E. 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