Protein tyrosine kinase inhibitors increase cytosolic calcium and inhibit actin organization as resorbing activity in rat osteoclasts

Abstract

Although there is evidence that protein tyrosine kinase inhibitors (PTKIs) suppress bone resorption activity, the mechanism of action of these compounds on osteoclastic bone resorption remains obscure. In the present study, we investigated the effect of PTKIs on cytosolic Ca(2+) concentration ([Ca(2+)](i)) and on the cytoskeleton in rat osteoclasts. The PTKIs, genistein and herbimycin A, reversibly elevated [Ca(2+)](i) measured by fura-2 microfluorimetry. The PTKI-induced increase was abolished by omission of extracellular Ca(2+), but was not attenuated by depletion of Ca(2+) stores. The PTKI-induced increase was inhibited by addition of La(3+) and Ni(2+), but not abolished by dihydropyridine (DHP) Ca(2+) channel blockers. Genistin, an inactive analogue of genistein, had no effect on [Ca(2+)](i). In the cytoskeleton assay, genistein rapidly disrupted the actin ring formation that serves as a marker for the resorbing state of osteoclasts. Disruption of the actin ring formation was also diminished in Ca(2+)-free extracellular solution. These results suggest that PTKIs in rat osteoclasts elevate [Ca(2+)](i) via activation of a DHP-insensitive, nonspecific Ca(2+) entry pathway and disrupt the formation of actin rings, resulting in suppression of bone resorption activity. The regulation of this Ca(2+)-influx by PTKIs is likely to contribute to inhibition of bone resorption by these compounds.

Mechanisms of calcium disposal from osteoclastic resorption hemivacuole

“…Similar considerations apply to the stimulators, e.g. PTH, where our experimental data show a rise, fall or no effect on the osteoclast cytosolic Ca 2+ , the variety of responses produced being determined by the extent and kinetics of stimulation of efflux and influx by the particular agent (Zaidi et al 1990, MacIntyre et al 1991, Bizzari et al 1994, Rathod et al 1994, Parkinson et al 1998, Kajiya et al 2000, Berger et al 2001. The above arguments are valid and applicable both for bulk transcytosis as well as to selective uptake involving specialised Ca 2+ channels.…”

“…A number of diverse agents, such as calcitonin (CT), interleukin-4 (IL-4), protein tyrosine kinase inhibitors and elevated extracellular [Ca 2+ ], which acutely increase [Ca 2+ ] i , inhibit the osteoclast (Bizzari et al 1994, Rathod et al 1995, Kajiya et al 2000, Berger et al 2001. Indeed, an increase in [Ca 2+ ] i has been generally accepted as a potential mechanism by which various agents inhibit osteoclastic activity.…”

“…PTH has been shown to increase, decrease or have no effect on cytosolic calcium (Rathod et al 1995, Miyauchi et al 1990, Berger et al 2001. A closer scrutiny of the published literature has revealed an unusual association between [Ca 2+ ] i and osteoclastic activity, namely that an elevation of [Ca 2+ ] i in osteoclasts can be stimulatory, inhibitory or without effect (Miyauchi et al 1990, Zaidi et al 1990, MacIntyre et al 1991, Bizzari et al 1994, Rathod et al 1994, Parkinson et al 1998, Kajiya et al 2000, Berger et al 2001. This implies the lack of a simple general association between acute changes in osteoclast functional modalities and elevation in cytosolic calcium.…”

Effects of Dietary Genistein on Nutrient Use and Mineral Status in Heat-Stressed Quails

“…It has been documented that soy isoflavones can increase bone mineral content and bone density in humans [15,17]. Genistein has been shown to increase proliferation and differentiation of osteoblast-like cells and collagen synthesis [9] and decrease intracellular Ca concentrations in osteoclasts in animals [16,23]. Yamaguchi and Gao [39] reported that genistein has an anabolic effect on bone mineralization in the tissue culture system.…”

“…One of the major soy isoflavones, genistein has been shown to increase osteoblast proliferation/ differentiation, alkaline phosphatase (ALP) activity and collagen synthesis, but inhibits the recruitment and function of osteoclasts [15]. Genistein also causes elevation in intracellular calcium concentrations and decreases phosphorylation in osteoclasts [16,23].…”