Effect of three types of bisphosphonates on molecules regulating the differentiation and activity of clastic cells cultured over different substrates

Abstract

The function of clastic cells is to degrade mineralized tissues in physiological or pathological conditions. To perform their function, they develop podosomes that contain actin filaments arranged in a ring-shaped manner in order to attach to their substrate. The adhesion is mediated by ±v²3 integrins in the plasma membrane to the RGD sequence of non-collagenous matrix proteins. In the ruffled membrane are secreted enzymes such as tartrate-resistant acid phosphatase (TRAP) and Cl- and H+ ions. The vacuolar ATPase (V-ATPase) promotes the secretion of H+ ions, and its subunit B2 binds to actin filaments when targeted to the plasma membrane of activated clastic cells. The bisphosphonates are drugs with well known capacity to inhibit the resorption of mineralized tissues by clastic cells. The non-nitrogenated bisphosphonates like etidronate induce the formation of toxic ATP analogues. The nitrogenated bisphosphonates like alendronate and zoledronic acid inhibit the prenylation of GTPases that are essential to clastic cells activity. The aim of the present project is to analyze the effect of the bisphosphonates etidronate, alendronate and zoledronic acid on the expression of RANK, c-FMS, TRAP, cathepsin K and DC-STAMP by clastic cells in primary culture obtained from mice bone marrow and the expression of RANKL and OPG by the undifferentiated cells by Real Time PCR. We will also evaluate the targeting of the B2 V-ATPase subunit to the plasma membrane of cultured cells, from which the cytoplasm proteins will be separated from the membrane proteins by ultracentrifugation followed by Western Blotting analysis. These methods will be allied to a morphological approach of the adhesion process and activity of clastic cells derived from mice bone marrow and cultured over different substracts (bovine compact bone, dentin and enamel) utilizing light and transmission electron microscopy and immunofluorescence. The resorbed areas will be analyzed by scanning electron microscopy. Based on the methods proposed, we aim to verify the function of clastic cells facing different substracts. By employing bisphosphonates, we also aim to obtain better comprehension of the resorption mechanism of clastic cells, as well as the mechanism of inhibition of their activity.