The
venom protein, s-
echistatin, originally derived from the saw-scaled viper Echis carinatus, was found to be a potent inhibitor of
bone resorption by isolated osteoclasts. This Arg24-Gly25-Asp26-(RGD)-containing
protein inhibited the excavation of bone slices by rat osteoclasts (IC50 = 0.1 nM). It also inhibited the release of [3H]
proline from labeled bone particles by chicken osteoclasts (IC50 = 100 nM). By comparison, the tetrapeptide
Arg-Gly-Asp-Ser (RGDS) inhibited resorption by rat or chicken osteoclasts with an IC50 of 0.1 mM while ala24-echistatin was inactive. Video microscopy showed that rat osteoclast attachment to substrate was more sensitive to s-
echistatin than was the attachment of mononuclear cells or chicken osteoclasts. The difference in sensitivity of rat and chicken osteoclasts to s-
echistatin may be due to differences between receptors on rat and chicken osteoclasts for s-
echistatin. Antibody localization of
echistatin on these cells showed much greater
echistatin binding to rat osteoclasts than to chicken osteoclasts.
Laser scanning confocal microscopy after immunohistochemical staining showed that s-
echistatin binds to osteoclasts, that s-
echistatin receptors are most abundant at the osteoclast/glass interface, and that s-
echistatin colocalizes with
vinculin. Confocal interference reflection microscopy of osteoclasts incubated with s-
echistatin, demonstrated colocalization of s-
echistatin with the outer edges of clusters of grey contacts at the
tips of some lamellipodia. Identification of the
echistatin receptor as an
integrin was confirmed by colocalization of
echistatin fluorescence with staining for an alpha-like subunit. Attachment of bone particles labeled with [3H]
proline to chicken osteoclasts confirmed that the mechanism of action of
echistatin was to inhibit osteoclast binding to bone presumably by disrupting adhesion structures. These data demonstrate that osteoclasts bind to bone via an RGD-sequence as an obligatory step in
bone resorption, that this RGD-binding
integrin is at adhesion structures, and that it colocalizes with
vinculin and has an alpha-like subunit.