Matrix regulation of skeletal cell apoptosis III: Mechanism of ion pair‐induced apoptosis

Abstract

Our previous work has demonstrated that while the Ca(2+) and Pi ions acting in concert function as a potent osteoblast apoptogen, the underlying mechanisms by which it activates cell death is not known. We hypothesize that the ion pair causes release of Ca(2+) from intracellular stores ([Ca(2+)]i); the increase in intracellular calcium prompts the mitochondria to uptake more calcium. This accumulation of calcium eventually results in the loss of mitochondrial membrane potential (MMP) and, subsequently, apoptosis. To test this hypothesis, we evaluated apoptosome formation in MC3T3-E1 osteoblast-like cells treated with the ion pair. Western blot analysis indicated migration of cytochrome-c and Smac/DIABLO from mitochondria to the cytoplasm. Inhibition of either the electron transfer chain (with antimycin a and rotenone), or the activation of a MMP transition (with bongkrekic acid) inhibited apoptosis in a dose-dependent manner. Pre-treating osteoblasts with ruthenium red, a Ca(2+) uniporter inhibitor of both mitochondria and the endoplasmic reticulum (ER), also completely abolished Ca(2+.)Pi-induced apoptosis. Moreover, we showed that an increase in [Ca(2+)]i preceded the increase in MMP over the first 45 min of treatment; a mitochondrial membrane permeability transition was evident at 75 min. To determine the role of ER, Ca(2+) stores in the generation of the apoptotic signal by the ion pair, cells were treated with several inhibitors. Apoptosis was inhibited when cells were treated with dantrolene, an inhibitor of ER ryanodine receptors, and 2-aminodiphenylborate, an IP3 Ca(2+) channel inhibitor, but not cyclopiazonic acid, an ER Ca(2)-ATPase inhibitor. Together, these data demonstrate that Ca(2+) Pi-induced osteoblast apoptosis is characterized by the generation of an apoptosome and that Ca(2+) release from ER stores may promote ion pair-dependent cell death.

Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells

“…The apoptosis was assumed to be responsible for the toxic effect of HAP particles. This assumption is supported by the report of Saunders et al [33], who pointed out that increased calcium phosphate in the matrix might trigger apoptosis in osteoblasts during bone resorption, and that bone cell apoptosis induced by high calcium and phosphate exhibited a marked change in mitochondrial function. Recently, our group [34] has proposed a new model in which demineralization reactions actually involve particle-size-dependent critical conditions of energetic control at the molecular level.…”

Cell responses to two kinds of nanohydroxyapatite with different sizes and crystallinities

“…19 As shown in Figure 6B, the control cells showed a network of well formed stress fibers of normal filamentous morphology. The shape of the cells treated with n-HA1 showed no difference compared with controls, with clear longitudinal stress fibers as shown in Figure 6C and D. However, the cells exhibited a polygon-like shape after treatment with n-HA2.…”

Probing the Osteoinductive Effect of Calcium Phosphate by Using an In Vitro Biomimetic Model

“…However, high [P i ] (>5 mM) has been associated with osteoblast apoptosis due to the failure of mitochondrial function, which could be abolished by inhibiting P i transport using Na-P i cotransporter (Pit-1) inhibitors. [62][63][64] However, a critical function of Pit-1 in cellular proliferation, which is independent of its P i transport activity, has been identified. 65 Alkaline phosphatase (ALP) plays an important role in P i homeostasis by converting extracellular pyrophosphate (ePP i , an inhibitor of mineralization) to P i .…”