Kong Wah Ng

Continued breast cancer risk reduction in postmenopausal women treated with raloxifene: 4-year results from the MORE trial

Raloxifene, a selective estrogen receptor modulator approved for the prevention and treatment of postmenopausal osteoporosis, has shown a significant reduction in breast cancer incidence after 3 years in this placebo-controlled, randomized clinical trial in postmenopausal women with osteoporosis. This article includes results from an additional annual mammogram at 4 years and represents 3,004 additional patient-years of follow-up in this trial. Breast cancers were ascertained through annual screening mammograms and adjudicated by an independent oncology review board. A total of 7,705 women were enrolled in the 4-year trial; 2,576 received placebo, 2,557 raloxifene 60mg/day, and 2,572 raloxifene 120mg/day. Women were a mean of 66.5-years old at trial entry, 19 years postmenopause, and osteoporotic (low bone mineral density and/or prevalent vertebral fractures). As of 1 November 1999, 61 invasive breast cancers had been reported and were confirmed by the adjudication board, resulting in a 72% risk reduction with raloxifene (relative risk (RR) 0.28, 95% confidence interval (CI) 0.17, 0.46). These data indicate that 93 osteoporotic women would need to be treated with raloxifene for 4 years to prevent one case of invasive breast cancer. Raloxifene reduced the risk of estrogen receptor-positive invasive breast cancer by 84% (RR 0.16, 95% CI 0.09, 0.30). Raloxifene was generally safe and well-tolerated, however, thromboembolic disease occurred more frequently with raloxifene compared with placebo (p=0.003). We conclude that raloxifene continues to reduce the risk of breast cancer in women with osteoporosis after 4 years of treatment, through prevention of new cancers or suppression of subclinical tumors, or both. Additional randomized clinical trials continue to evaluate this effect in postmenopausal women with osteoporosis, at risk for cardiovascular disease, and at high risk for breast cancer.

Localization of RANKL (receptor activator of NF kappa B ligand) mRNA and protein in skeletal and extraskeletal tissues.

RANKL (receptor activator of NFkappaB ligand) is a membrane-associated osteoblastic molecule, and along with macrophage-colony-stimulating factor, is crucial for osteoclast formation. RANKL is known to be strongly expressed in osteoblasts and lymphoid tissues. We have sought to determine the skeletal and extraskeletal sites of production of RANKL mRNA and protein using the techniques of in situ hybridization and immunohistochemistry. Expression of RANKL mRNA and protein were determined in the developmental progression of endochondral bone formation in mouse, intramembranous bone formation in a rabbit model (mRNA only), in human giant cell tumors of bone, and at extraskeletal sites in the mouse. RANKL mRNA was expressed in prehypertrophic and hypertrophic chondrocytes at day E15 embryonic mouse long bone, and its expression was maintained at these sites throughout development. In newborn and adult mice, high levels of RANKL mRNA were expressed in mesenchymal cells of the periosteum and in mature osteoblasts, while megakaryocytes within the marrow microenvironment expressed RANKL mRNA from 1 week of age. Immunohistochemical analysis revealed a similar localization pattern of RANKL protein at the sites described. In the intramembranous bone formation model, RANKL mRNA was expressed in mesenchymal cells and in actively synthesizing osteoblasts, but not in flattened lining osteoblasts or late osteocytes. Expression of RANKL mRNA and protein in osteoclasts was variable with those within resorption lacunae showing the strongest signal/staining. Likewise, expression varied in osteoclasts from giant cell tumor of bone with a minority of tartrate-resistant acid phosphatase-positive multinucleated cells having no detectable RANKL mRNA or protein. In extraskeletal tissues, RANKL mRNA and protein were detected in the brain, heart, kidney, skeletal muscle, and skin throughout mouse development, suggesting the possibility of several other functions of the molecule. RANKL was also developmentally regulated, as evidenced by its expression in the intestine, liver, and lung at E15 and newborn mouse but not in the adult.

Cell lines and primary cell cultures in the study of bone cell biology.

Bone is a metabolically active and highly organized tissue consisting of a mineral phase of hydroxyapatite and amorphous calcium phosphate crystals deposited in an organic matrix. Bone has two main functions. It forms a rigid skeleton and has a central role in calcium and phosphate homeostasis. The major cell types of bone are osteoblasts, osteoclasts and chondrocytes. In the laboratory, primary cultures or cell lines established from each of these different cell types provide valuable information about the processes of skeletal development, bone formation and bone resorption, leading ultimately, to the formulation of new forms of treatment for common bone diseases such as osteoporosis.

Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model

Expression of parathyroid hormone-related protein (PTHrP) messenger RNA (mRNA) and protein was investigated throughout the developmental progression of endochondral bone formation in mouse and intramembranous bone formation in an in vivo model in rabbit, using in situ hybridization and immunohistochemistry. Endochondral bone formation was investigated in a developing embryo, newborn, and adult mouse. In fetal long bones through to newborn (day 7), PTHrP mRNA and protein were consistently expressed in chondrocytes within the proliferative, transitional, and hypertrophic zones. In addition, high levels of PTHrP were also detected in osteoblasts on the surface of trabecular bone surfaces. Similarly, at the adult stage (week 7), PTHrP mRNA and protein were consistently expressed in chondrocytes at epiphyseal ends of the subarticular cartilage, within cortical periosteum, as well as in osteoblasts located at the metaphyseal trabecular bone surfaces. Using an in vivo intramembranous bone formation model in rabbits, expression of PTHrP mRNA and protein was demonstrated in preosteoblasts prior to trabecular bone formation (1-week bone harvest). As bone formed (2-, 3-, and 4-week bone tissue harvests), PTHrP mRNA and protein were highly expressed in actively synthesizing osteoblasts and in those osteocytes embedded within the superficial layers of the bone matrix. Lining osteoblasts and osteocytes buried deeply in the bone matrix displayed weak or no signal for PTHrP. The pattern of spatial and temporal expression of PTHrP demonstrated in cartilage cells and osteoblasts in the two systems suggests an important role of PTHrP in both endochondral and intramembranous bone formation.

Regulation of glucose metabolism and the skeleton

Complex interactions occur among adipose tissue, the central nervous system, bone and pancreas to integrate bone remodelling, glucose, lipid and energy metabolism. Data obtained largely from the judicious use of gain‐of‐function and loss‐of‐function genetic mouse models show that leptin, an adipocyte‐secreted product, indirectly inhibits bone accrual through a central pathway comprising the hypothalamus and central nervous system. Increased sympathetic output acting via β2‐adrenergic receptors present in osteoblasts decreases bone formation and causes increased bone resorption. Insulin is a key molecular link between bone remodelling and energy metabolism. Insulin signalling in the osteoblasts increases bone formation and resorption as well as the release of undercarboxylated osteocalcin. An increase in the release of bone‐derived undercarboxylated osteocalcin into the systemic circulation enables it to act as a circulating hormone to stimulate insulin production and secretion by pancreatic β‐cells and adiponectin by adipocytes. Insulin sensitivity increases, lipolysis and fat accumulation decreases while energy expenditure increases. Whether this model of integrative physiology involving the skeleton, pancreas and adipose tissue, so elegantly demonstrated in rodents, is applicable to humans is controversial. The mouse Esp gene, encoding an intracellular tyrosine phosphatase that negatively regulates insulin signalling in osteoblasts, is a pseudogene in humans, and a homolog for the Esp gene has so far not been identified in humans. A close homologue of Esp, PTP1B, is expressed in human osteoblasts and could take the role of Esp in humans. Data available from the limited number of clinical studies do not provide a sufficient body of evidence to determine whether osteocalcin or undercarboxylated osteocalcin affects glucose metabolism in humans.

Mechanisms Involved in Skeletal Anabolic Therapies

Abstract:  Since parathyroid hormone (PTH) is the only proven anabolic therapy for bone, it becomes the benchmark by which new treatments will be evaluated. The anabolic effect of PTH is dependent upon intermittent administration, but when an elevated PTH level is maintained even for a few hours it initiates processes leading to new osteoclast formation, and the consequent resorption overrides the effects of activating genes that direct bone formation. Identification of PTH‐related protein (PTHrP) production by cells early in the osteoblast lineage, and its action through the PTH1R upon more mature osteoblastic cells, together with the observation that PTHrP± mice are osteoporotic, all raise the possibility that PTHrP is a crucial paracrine regulator of bone formation. The finding that concurrent treatment with bisphosphonates impairs the anabolic response to PTH, adds to other clues that osteoclast activity is necessary to complement the direct effect that PTH has in promoting differentiation of committed osteoblast precursors. This might involve the generation of a coupling factor from osteoclasts that are transiently activated by receptor activator of nuclear factor‐κB ligand (RANKL) in response to PTH.

Osteoclast Inhibitory Lectin, a Family of New Osteoclast Inhibitors

We have identified two novel type II membrane-bound C-lectins, designated mOCILrP1 and mOCILrP2, of 218 and 217 amino acids, respectively, that share substantial identity with the murine osteoclast inhibitory lectin (OCIL). The extracellular domains of mOCILrP1 and mOCILrP2 share 83 and 75% identity, respectively, with the extracellular domain of mOCIL. When the extracellular domains were expressed as recombinant proteins, each inhibited osteoclast formation in murine bone marrow cultures treated with M-CSF and RANKL with similar potencies to mOCIL (IC50 of 0.2 ng/ml). Distinct but highly related genes encoded the three OCIL family members, with mOCIL and mOCILrP2 controlled by an inverted TATA promoter, and mOCILrP1 by a TTAAAA promoter. However only mOCIL was robustly regulated by calciotropic agents, while mOCILrP1 was not expressed, and mOCILrP2 was constitutively expressed in osteoblasts. Immunohistochemistry using antipeptide antibodies to the intracellular domain of mOCILrP1/mOCILrP2 and to mOCIL demonstrated that mOCIL and mOCILrP1/mOCILrP2 were concordantly expressed in osteoblasts, chondrocytes, and in extraskeletal tissues. Further, their cellular distribution was identical to that of RANKL. The identification of three distinct genes that were functionally related implies redundancy for OCIL, and their concordant expression with that of RANKL suggests that the RANKL:OPG axis may be further influenced by OCIL family members.

Isolation of a human homolog of osteoclast inhibitory lectin that inhibits the formation and function of osteoclasts.

Osteoclast inhibitory lectin (OCIL) is a newly recognized inhibitor of osteoclast formation. We identified a human homolog of OCIL and its gene, determined its regulation in human osteoblast cell lines, and established that it can inhibit murine and human osteoclast formation and resorption. OCIL shows promise as a new antiresorptive.

Stretch-induced PTH-related protein gene expression in osteoblasts.

Mechanical forces play a critical role in regulating skeletal mass and structure. We report that mechanical loading induces PTHrP in osteoblast‐like cells and that TREK‐2 stretch‐activated potassium channels seem to be involved in this induction. Our data suggest PTHrP as a candidate endogenous mediator of the anabolic effects of mechanical force on bone.

Regulatory pathways revealing new approaches to the development of anabolic drugs for osteoporosis

The understanding of cell interactions and genetic controls of bone cells has provided new approaches to drug development for osteoporosis. Current emphasis in the development of new anabolic therapies is directed at modifying the effects of Wnt signalling on osteoblast differentiation and bone formation. Local signalling that results in bone formation during remodelling takes place in several ways. Growth factors released from resorbed bone matrix can contribute to preosteoblast differentiation and bone formation. Osteoclasts in the bone multicellular units (BMUs) might also generate activity that contributes to bone formation. The preosteoblasts themselves, growing in the resorption space, can communicate through cell contact and paracrine signalling mechanisms to differentiate. Osteocytes can sense the need for bone repair by detecting damage and pressure changes, and signalling to surface cells to respond appropriately. These recent insights into cell communication, together with discoveries from human and mouse genetics, have opened new pathways to drug development for osteoporosis. With the anabolic effect of parathyroid hormone on the skeleton having been established, human genetics revealed the major role of Wnt signalling in bone formation, and this has become the target of activity. Current approaches include activation at any of several points in the Wnt pathway, and neutralization of sclerostin, the protein product of the SOST gene that is produced in osteocytes as a powerful inhibitor of bone formation.