Toward understanding the cellular control of vertebrate mineralization: The potential role of mitochondria

Abstract

This review examines the possible role of mitochondria in maintaining calcium and phosphate ion homeostasis and participating in the mineralization of bone, cartilage and other vertebrate hard tissues. The paper builds on the known structural features of mitochondria and the documented observations in these tissues that the organelles contain calcium phosphate granules. Such deposits in mitochondria putatively form to buffer excessively high cytosolic calcium ion concentrations and prevent metabolic deficits and even cell death. While mitochondria protect cytosolic enzyme systems through this buffering capacity, the accumulation of calcium ions by mitochondria promotes the activity of enzymes of the tricarboxylic acid (TCA/Krebs) cycle, increases oxidative phosphorylation and ATP synthesis, and leads to changes in intramitochondrial pH. These pH alterations influence ion solubility and possibly the transitions and composition in the mineral phase structure of the granules. Based on these considerations, mitochondria are proposed to support the mineralization process by providing a mobile store of calcium and phosphate ions, in smaller cluster or larger granule form, while maintaining critical cellular activities. The rise in the mitochondrial calcium level also increases the generation of citrate and other TCA cycle intermediates that contribute to cell function and the development of extracellular mineral. This paper suggests that another key role of the mitochondrion, along with the effects just noted, is to supply phosphate ions, derived from the breakdown of ATP, to endolysosomes and autophagic vesicles originating in the endoplasmic reticulum and Golgi and at the plasma membrane. These many separate but interdependent mitochondrial functions emphasize the critical importance of this organelle in the cellular control of vertebrate mineralization.

Keywords: ATP; Calcium; Endoplasmic reticulum; Mineralization; Mitochondria; Mitochondrial granules; Phosphate.

Figure 1.

Schematic representation of energy generation in mitochondria. The inset (top left) shows a cross section of a mitochondrion with a segment of the organelle enlarged to illustrate its characteristic membrane structure as well as components of the Krebs/tricarboxylic acid (TCA) cycle and electron transport chain (ETC). Catalyzed by enzymes of the Krebs cycle, mitochondria generate NADH and FADH₂, which interact with specific molecules of the ETC. The ETC is comprised of the multienzyme complexes (complexes I-IV) together with cytochrome C (Cyto C) and ubiquinone (Q). Complex I (NADH-ubiquinone oxidoreductase) catalyzes the transfer of electrons (e-) from NADH to ubiquinone. Following acceptance of a pair of high energy electrons from NADH, Complex I undergoes a redox-related conformational change that promotes the pumping of 4 protons (H⁺) across the inner mitochondrial membrane (IMM) and its infoldings (cristae). Complex II (succinate-ubiquinone oxidoreductase) receives reducing equivalents (FADH₂) from succinate dehydrogenase. Although this conformational change generates less energy than that of Complex I, it is sufficient to reduce ubiquinone, which can then interact with Complex III. Complex III (ubiquinone-cytochrome C reductase) allows the stepwise flow of two electrons from Complex I and II and shuttles other electrons to Cyto C, a protein located partly in the mitochondrial intermembranous space. Complex IV (cytochrome oxidase) is the final electron acceptor molecule in the ETC. Complex IV has two functions. First, it transfers electrons to oxygen, which react with protons to form water. Second, undergoing a conformational change, Complex IV causes the movement of two protons from the mitochondrial matrix to the intermembranous space. Pumping of these and the other protons from Complexes I and III across the inner membrane of the mitochondrion creates a proton motive force (ΔΨm, 180 – 200 mV; dashed arrows) that drives F₁F₀ATP synthase to phosphorylate ADP to form ATP. Additional protons resulting from ETC reactions involving Complexes I, III and IV are utilized with oxygen to change Cyto C from a reduced state (Cyto C_r) to an oxidized state (Cyto C_o) with the generation of water. Citrate, an important molecule in mineralization and other cellular processes, is a product of the Krebs cycle. ADP: adenosine diphosphate; ATP: adenosine triphosphate; P_i: inorganic phosphate; GDP: guanosine diphosphate; GTP: guanosine triphosphate; NAD⁺/NADH: reduced and oxidized forms of nicotinamide adenine dinucleotide, respectively; FAD/FADH₂: reduced and oxidized forms of flavin adenine dinucleotide, respectively; OMM: outer mitochondrial membrane.

Figure 2.

Schematic of calcium and phosphate ion uptake and granule formation in mitochondria of cells of vertebrate mineralized tissues. A segment of a mitochondrion, a neighboring region of endoplasmic reticulum (ER), and aspects of the intracellular and intramitochondrial matrices are depicted in the diagram. Following depletion of calcium ions from the ER, STIM1 and associated proteins at the plasma membrane (not shown) promote the entry of extracellular calcium ions (Ca²⁺, red dots) into the cytosol (intracellular matrix) as well as the saccules, tubules and lamellae of the ER. Calcium ions enter mitochondria from the intracellular matrix and endoplasmic reticulum-mitochondria attachment complexes (mitochondria associated membranes, MAM). Calcium ion transfer from the ER to mitochondria is mediated by activation of inositol 1,4,5-trisphosphate (IP3R) and ryanodine receptors (RyR) in the ER membrane and voltage-dependent anion channel (VDAC) activity in the outer membrane of the mitochondria (OMM). The inner mitochondrial membrane (IMM) contains a mitochondrial calcium uniporter (MCU), which transfers calcium ions into the mitochondrial matrix. GRP75 enhances the stability of the MAM complex and increases the efficiency of ion transfer. Phosphate ion (Pi, blue dots) entry into mitochondria is mediated by a solute carrier protein, SLC253A3, and a mitochondrial proton/phosphate symporter (PiC). Other calcium ion mitochondrial transporters include voltage-operated calcium channels (VOC), receptor-operated calcium channels (ROC), and members of the transient receptor potential family of channels (TRP). Within the mitochondrial matrix, calcium and phosphate ions may cluster and form calcium phosphate mitochondrial granules (red/blue). A rise in intramitochondrial levels of calcium ions increases TCA cycle enzyme activity, whose initial metabolic reaction is conversion of pyruvate into acetyl CoA [Figure 1]. The subsequent synthesis of citrate both promotes TCA cycle activity and provides a source of this anion for apatite crystallite interactions [Figure 1]. The increase in mitochondrial metabolic activity influences oxidative phosphorylation and ATP synthase function [Figure 1]. With the upregulation in oxidative phosphorylation activity and the release of protons, there is an increase in the mitochondrial pH, enhancing granule formation.

Figure 3.

Schematic of possible pathways in which mitochondria may be involved in the efflux of calcium and phosphate to the extracellular matrix for subsequent mineralization events. A segment of a mitochondrion, a portion of a cell and its plasma membrane (PM), and aspects of the mitochondrial, intracellular and extracellular matrices are shown in the diagram. Mitochondrial granules in the intramitochondrial matrix may liberate calcium (Ca²⁺) and phosphate (Pi) ions with changes in the mitochondrial pH. Ca²⁺ ions exit mitochondria through NCLX/VDAC channels embedded in the inner (IMM) and outer (OMM) mitochondrial membrane, respectively. Pi exits mitochondria by unknown means (dashed line across the IMM and OMM). Intact mitochondrial granules may interact with the IMM and OMM and disrupt the membranes, an event leading to mitophagy. This process initially involves the formation of mitophagic vesicles enveloping the damaged mitochondria, their Ca²⁺ and Pi ions and any other compromised cellular components. Vesicle contents may be enzymatically digested and recycled in the cell or exported to the extracellular matrix. Ca²⁺ and Pi ions passed through the mitochondrial membrane to the intracellular matrix may follow different pathways that include uptake by endosomes, autophagosomes (APS), and lysosomes. Other Ca²⁺ and Pi ions may be released to the extracellular matrix by passage respectively through NCLX or XPR1 channels located in the PM of the cell. Endosomes and APS may form amphisomes carrying the Ca²⁺ and Pi ions as well as their small aggregates (Ca-P). Amphisomes traverse the PM and may make their way into the extracellular as mineralizing exosomes or mineralizing vesicles (matrix vesicles). Lysosomes and APS form autophagolysosomes (APLS) enclosing Ca-P and these organelles may pass the PM through lysosomal exocytosis to enter the extracellular matrix. Pi in the extracellular matrix appears in the form of HPO₄ ²⁻ and H₂PO₄ ⁻.