Peter J. R. Bevis

Stimulation of osteoclastic bone resorption by hydrogen peroxide

The molecular mechanisms underlying the pathophysiology of bone destruction still remain poorly understood. We have found that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is a potent stimulator of osteoclastic bone resorption and cell motility. A marked enhancement of bone resorption was noted when rat osteoclasts, cultured on devitalised bovine cortical bone, were exposed to 10 nM [H2O2]. Apart from exposing osteoclasts to a low extracellular pH, which is known to enhance osteoclastic bone resorption, we provide first evidence for a molecule that stimulates osteoclastic bone resorption in osteoclast cultures that do not respond to parathyroid hormone and 1, 25 dihydroxyvitamin D3. We envisage that both basic biological and practical clinical implications may eventually follow from these studies.

A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis.

Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.

Forty years of calcitonin—where are we now? A tribute to the work of Iain Macintyre, FRS

Calcitonin was discovered as a hypocalcemic principal that was initially thought to originate from the parathyroid gland. This view was corrected subsequently, and an origin from the thyroid C cells was documented. The purification and sequencing of various calcitonins soon followed. Calcitonin is a 32-amino-acid-long peptide with an N-terminal disulfide bridge and a C-terminal prolineamide residue. The peptide was shown to potently inhibit bone resorption; however, a direct osteoclastic action of the peptide was confirmed only in the early 1980s. Several osteoclast calcitonin receptors have subsequently been cloned and sequenced. Specific regions of the receptor necessary for ligand binding and intracellular signaling through cyclic AMP and calcium have been identified through systematic deletion mutagenesis and chimeric receptor studies. Calcitonins potent antiresorptive effect has led to its use in treating Pagets disease of bone, osteoporosis, and hypercalcemia. This review retraces key aspects of the synthesis and structure of calcitonin, its cellular and molecular actions, and its therapeutic uses as they have emerged over the 40 years since its discovery. The review also examines the implications of these findings for future clinical applications as a tribute to early workers to whom credit must be given for creation of an important and expanding field. Notable are the new approaches currently being used to enhance calcitonin action, including novel allosteric activators of the calcitonin receptor, modulation of the release of endogenous calcitonin by calcimimetic agents, as well as the development of oral calcitonins.

The calcitonin gene peptides: biology and clinical relevance

The calcitonin/CGRP multigene complex encodes a family of peptides: calcitonin, its C-terminal flanking peptide, katacalcin, and a third novel peptide, calcitonin gene-related peptide (CGRP). The 32-amino acid peptide calcitonin inhibits the osteoclast, thereby conserving skeletal mass during periods of potential calcium lack, such as pregnancy, growth, and lactation. This hormonal role is emphasized by observations that lower circulating calcitonin levels are associated with bone loss and that calcitonin replacement prevents further bone loss. Structurally, CGRP resembles calcitonin and has been implicated in neuromodulation and in the physiological regulation of blood flow. Here we review the molecular genetics, structure, and function of the calcitonin-gene peptides as analyzed in the laboratory and focus on more recent clinical studies relating to disorders and therapeutics.

Extracellular Ca2+ sensing by the osteoclast

An increasing number of cell types appear to detect changes in the extracellular Ca2+ concentration and and accordingly modify their function. We review recent evidence for the existence and function of such a mechanism in the osteoclast. Elevated external [Ca2+] in the mM range reduces bone resorption and results in motile changes in the cells. These changes may partly result from elevations of cytosolic [Ca2+] triggered through activation of a surface Ca2+ receptor. Closer analyses of the increases in cytosolic [Ca2+] associated with receptor activation are hindered by the action of this ion both as extracellular agonist and intracellular second messenger. Variations in the peak cytosolic [Ca2+] response to external Ca2+ with changes in cell membrane potential by K+ and valinomycin establish a contribution from extracellular Ca2+. Use of CIO4-, Ni2+ and Cd2+ as surrogate activators in low extracellular [Ca2+] indicate a contribution from Ca2+ release from intracellular stores as well. Such agonists also modify Ca2+ redistribution in other systems, such as skeletal muscle. Thus, we may gain insights into osteoclast extracellular Ca2+ detection and transduction from known features of more well-characterised cell systems.

CELLULAR BIOLOGY OF BONE RESORPTION

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged.

Amylin‐amide: a new bone‐conserving peptide from the pancreas

Amylin‐amide is a new member of the family of peptides encoded by the calcitonin multigene complex. In the present study, we have compared directly, the hypocalcaemic potency and duration of action of human amylin‐amide and human calcitonin in an in vivo rat bioassay and an in vitro osteoclast bone resorption assay. Amylin‐amide was found to have a potency approximately 40‐fold lower than human calcitonin, whilst both peptides followed the same time course. This suggests that amylin‐amide is the most potent non‐calcitonin hypocalcaemic peptide so far reported. An important physiological implication follows. It would seem that amylin‐amide can play a central role in the maintenance of the skeleton by virtue of its inhibitory influence on osteoclastic function.

Disordered osteoclast formation and function in a CD38 (ADP-ribosyl cyclase)-deficient mouse establishes an essential role for CD38 in bone resorption

We have evaluated the role of the ADP‐ribosyl cyclase, CD38, in bone remodeling, a process by which the skeleton is being renewed constantly through the coordinated activity of osteoclasts and osteoblasts. CD38 catalyzes the cyclization of its substrate, NAD+, to the Ca2+‐releasing second messenger, cyclic ADP‐ribose (cADPr). We have shown previously that CD38 is expressed both in osteoblasts and osteoclasts. Its activation in the osteoclast triggers Ca2+ release through ryanodine receptors (RyRs), stimulation of interleukin‐6 (IL‐6), and an inhibition of bone resorption. Here, we have examined the consequences of deleting the CD38 gene in mice on skeletal remodeling. We report that CD38−/− mice displayed a markedly reduced bone mineral density (BMD) at the femur, tibia, and lumbar spine at 3 months and at the lumbar spine at 4 months, with full normalization of the BMD at all sites at 5 months. The osteoporosis at 3 months was accompanied by a reduction in primary spongiosa and increased osteoclast surfaces on histomorphometric analysis. Hematopoetic stem cells isolated ex vivo from CD38−/− mice showed a dramatic ~fourfold increase in osteoclast formation in response to incubation for 6 days with RANK‐L and M‐CSF. The osteoclasts so formed in these cultures showed a ~2.5‐fold increase in resorptive activity compared with wild‐type cells. However, when adherent bone marrow stromal cells were allowed to mature into alkaline phosphatase‐positive colony‐forming units (CFU‐Fs), those derived from CD38−/− mice showed a significant reduction in differentiation compared with wild‐type cells. Real‐time RT‐PCR on mRNA isolated from osteoclasts at day 6 showed a significant reduction in IL‐6 and IL‐6 receptor mRNA, together with significant decreases in the expression of all calcineurin A isoforms, α, β, and γ. These findings establish a critical role for CD38 in osteoclast formation and bone resorption. We speculate that CD38 functions as a cellular NAD+ “sensor,” particularly during periods of active motility and secretion.—Sun, L., Iqbal, J., Dolgilevich, S., Yuen, T., Wu, X.‐B., Moonga, B. S., Adebanjo, O. A., Bevis, P. J. R., Lund, F., Huang, C. L. H., Blair, H. C., Abe, E., Zaidi, M. Disordered osteoclast formation and function in a CD38 (ADP‐ribosyl cyclase) ‐deficient mouse establishes an essential role for CD38 in bone resorption. FASEB J. 17, 369–375 (2003)

A novel mechanism for coupling cellular intermediary metabolism to cytosolic Ca2+ signaling via CD38/ADP-ribosyl cyclase, a putative intracellular NAD+ sensor

CD38 is an ectocyclase that converts NAD+ to the Ca2+‐releasing second messenger cyclic ADP‐ribose (cADPr). Here we report that in addition to CD38 ecto‐catalysis, intracellularly expressed CD38 may catalyze NAD+→cADPr conversion to cause cytosolic Ca2+ release. High levels of CD38 were found in the plasma membranes, endoplasmic reticulum, and nuclear membranes of osteoblastic MC3T3‐E1 cells. More important, intracellular CD38 was colocalized with target ryanodine receptors. The cyclase also converted a NAD+ surrogate, NGD+, to its fluorescent product, cGDPr (Km ~5.13 μM). NAD+ also triggered a cytosolic Ca2+ signal. Similar results were obtained with NIH3T3 cells, which overexpressed a CD38‐EGFP fusion protein. The Δ‐49‐CD38‐EGFP mutant with a deleted amino‐terminal tail and transmembrane domain appeared mainly in the mitochondria with an expected loss of its membrane localization, but the NAD+‐induced cytosolic Ca2+ signal was preserved. Likewise, Ca2+ release persisted in cells transfected with the Myr‐Δ‐49‐CD38‐EGFP or Δ‐49‐CD38‐EGFP‐Fan mutants, both directed to the plasma membrane but in an opposite topology to the full‐length CD38‐EGFP. Finally, ryanodine inhibited Ca2+ signaling, indicating the downstream activation of ryanodine receptors by cADPr. We conclude that intracellularly expressed CD38 might link cellular NAD+ production to cytosolic Ca2+ signaling.—Sun, L., Adebanjo, O. A., Koval, A., Anandatheerthavarada, H. K., Iqbal, J., Wu, X. Y., Moonga, B. S., Wu, X. B., Biswas, G., Bevis, P. J. R., Kumegawa, M., Epstein, S., Huang, C. L.‐H., Avadhani, N. G., Abe, E., Zaidi, M. A novel mechanism for coupling cellular intermediary metabolism to cytosolic Ca2+ signaling via CD38/ADP‐ribosyl cyclase, a putative intracellular NAD+ sensor. FASEB J. 16, 302–314 (2002)

Osteoclast function and its control

Bone resorption appears to be dependent on a range of processes. It requires an adequate number of osteoclasts to access bone mineral. These osteoclasts must be activated by a mechanism which is dependent upon prior osteoblastic stimulation. A range of factors then contribute to the formation of a functionally effective resorptive hemivacuole. These entail osteoclast adhesion to the bone surface leading to the formation of a sealing zone. Only then can subsequent processes such as H+ ion transport, enzyme secretion and matrix digestion become effective. Thus, any one process is potentially limiting to resorption and is a potential target for regulation. Long‐range regulation takes place through the action of hormones, of which the mode of action of calcitonin has been the subject of recent investigations in isolated osteoclasts. Such studies have shown a possible involvement of distinguishable receptor subtypes, the occupancy of which may activate at least two types of triggering mechanism. It is likely that an eventual influence on motility properties through G protein mediation accounts for the actions of this hormone and of related peptides such as amylin and CGRP at the cellular level. Similar pathways may contribute to shorter range modulation of osteoclast activity by increases in ambient Ca2+. Finally, there is recent evidence for a contribution of endothelial cell‐derived product to osteoclast regulation.