Regulation of Avian Osteoclastic H+-ATPase and Bone Resorption by Tamoxifen and Calmodulin Antagonists

Abstract

We used highly purified avian osteoclasts and isolated membranes from osteoclasts to study effects of tamoxifen, 4-hydroxytamoxifen, calmodulin antagonists, estrogen, diethylstilbestrol, and the anti-estrogen ICI 182780 on cellular degradation of 3 H-labeled bone in vitro and on membrane HCl transport. Bone resorption was reversibly inhibited by tamoxifen, 4-hydroxytamoxifen, and trifluoperazine with IC 50 values of ϳ1 M. Diethylstilbestrol and 17-␤-estradiol had no effects on bone resorption at receptor-saturating concentrations, while ICI 182780 inhibited bone resorption at concentrations greater than 1 M. At these concentrations ICI 182780, like tamoxifen, inhibits calmodulin-stimulated cyclic nucleotide phosphodiesterase activity. Membrane Transport of Ca 2ϩ into and out of bone is critical for maintenance of serum calcium activity. This requires continuous bone turnover at variable rates, which is mediated by the osteoclast. However, skeletal mineral is also structurally vital, so osteoclastic activity is regulated by multiple factors, often acting in opposing directions. Several hormonal signals are involved in this regulation, including peptides and low molecular weight factors (1). Steroids including estrogens have major effects on bone turnover (2), but the receptors and intermediary signaling involved are not established. This study was performed to determine the mechanism of steroid-related effects on central biochemical elements of osteoclastic activity.A limiting biochemical step and the central regulated element of bone turnover is secretion of HCl to dissolve the bone mineral. This is driven by a vacuolar-like H ϩ -ATPase that is highly expressed in a unique osteoclastic organelle, the ruffled membrane (3). Multiple intermediary cell signals influence the activity of acid secretion, but one of critical interest is intracellular calcium activity and the ubiquitous calcium-binding protein, calmodulin. The unique acid-dependent dissolution of calcium salts produces high local extracellular calcium activity (4), which is reflected in an elaborate osteoclastic calcium regulatory mechanism including a calmodulin-dependent calcium ATPase (5), and factors influencing osteoclastic intracellular calcium activity such as matrix attachment (6). The vacuolarlike H ϩ -ATPase driving acid secretion in osteoclasts is also calmodulin-dependent, and osteoclasts concentrate calmodulin at the ruffled membrane (7).The anti-estrogenic compound, tamoxifen, reduces bone turnover (8), suggesting that tamoxifen may be a particularly useful tool to dissect osteoclast control pathways. Tamoxifen, a known calmodulin antagonist (9), is a triphenylethylene derivative with low toxicity and strong antitumor activity, particularly in breast cancer, properties ascribed to its anti-estrogenic activity (10). Tamoxifen may thus regulate osteoclastic activity by either calmodulin or steroid receptor interactions. In contrast to expectations that tamoxifen would cause bone loss because of anti-estrogenic properties (11), it preserves bo...

A Mechanism for Tamoxifen-mediated Inhibition of Acidification

“…The altered membrane properties could shift the voltage dependence of many ion channels. Indeed, tamoxifen has been reported to shift the activity of many ion channels (11)(12)(13)(14)(15)(16). Detailed studies on the model channel, gramicidin, have shown that membrane tension (47) and surface charge (48) are critical determinants of channel activity.…”

“…But it has also been reported to have many pleiotropic effects both in vivo and in vitro that cannot be explained by an interaction with the estrogen receptor (4). For example, tamoxifen has been shown to enhance drug sensitivity of multidrug-resistant cells (5)(6)(7)(8)(9), inhibit bone resorption and osteoporosis both in vivo and in vitro (10), and inhibit a number of channels, including the volume activated chloride channel (11,12) and calcium channels (13)(14)(15)(16). These effects have been attributed to inhibition of P-glycoprotein (17), calmodulin (15), and direct channel interaction (11), respectively.…”

Nitric oxide regulation of cGMP production in osteoclasts

“…A total of 100 µg of 40-to 80-µm bone pieces, ϳ60 dpm [ 3 H]proline/µg bone, were added to ϳ3 ϫ 10 3 osteoclasts in 2-cm 2 tissue culture wells, after Blair et al [1986], but using larger bone pieces to preclude possible effects due to substrate internalization; osteoclasts attached to the substrate are demonstrated by Williams et al [1996a]. Assays were less than 7 days to avoid the effects of pre-osteoclast differentiation [Williams et al, 1996b] in mixed cultures.…”

“…Isolation of osteoclast-rich cell fractions from chicken bone is detailed elsewhere [Blair et al, 1986;Williams et al, 1996a]. Briefly, medullary bone from Gallus domesticus, white leghorn hens, on a Ca 2ϩ -restricted diet, was harvested and cells were removed by vigorous washing and sieved through 110-µm nylon mesh; erythrocytes were lysed in hypotonic media and cells sedimented through 70% serum to recover bone cells.…”

Hyperglycemia Suppresses RANKL-Induced Osteoclast Differentiation through LXRβ Expression in RAW264.7 Cells

“…Monocytes/ macrophages induced by these factors first proliferate actively and become committed preosteoclasts, tartrateresistant acid phosphatase (TRAP)-positive mononuclear cells, which then fuse to one another to become mature multinucleated OCLs (1,4,5). These cells secrete HCl to dissolve bone into its calcium salts (1,6).…”