Calcium & Phosphorus Metabolism Continued

Bone Formation and Resorption

Bone Formation

Bone formation is initiated under a variety of circumstances. It occurs during development, disease, and normal bone homeostasis. Three types of bone formation are described: intramembranous, perichondrial, and endochondrial.¹²⁸ For the purpose of this discussion, aspects of all three processes will be integrated into one generalization that may, or may not, always apply. As a practical matter, the first stage of bone formation involves the recruitment/creation/generation of osteoblasts. It is suggested that they are either pre-existing or migrate to sites of osteogenesis, or they form from resident precursors under the influence of various osteoblast inducers.^129-132 The following is a potential model for general osteogenesis. First, it is assumed that blood/extracellular fluid Ca²⁺ levels are elevated, and that there is a need for a repository for excess Ca²⁺. Such a circumstance might well occur during aggressive hormone-mediated Ca²⁺ absorption. Elevated circulating Ca²⁺ in bone may act on macrophages and induce BMP-2 production.¹³³ BMP-2 is known to promote a mesenchymal cell-to-osteoblast transition.^132,133 If VitD is the active agent promoting hypercalcemia through increased Ca²⁺ absorption, VitD may also contribute to the generation of pre-osteoblasts.^3,7,132-134 Once formed, pre-osteoblasts will either align themselves along the surface of previous resorption areas, or arrange themselves in a pattern that facilitates mineralization.^128,129 Once placed, pre-osteoblasts will divide, secrete collagen type I and osteonectin, synthesize alkaline phosphatase, and express PTH1R, as well as alpha_v, alpha₅, beta₃, and beta₅ integrins.¹²⁸ Collagen I is the predominant organic fiber in bone. It is suggested by many to be the only collagen in bone.¹³⁵ Alkaline phosphatase is a GPI-linked membrane protein that generates P_i for internal transport. This ultimately complexes with ionic Ca²⁺ to form a CaP crystal around which hydroxyapatite may form.^137-139 Osteonectin is a 40 kDa glycoprotein that may modulate (interfere) with mineralization.^135,139

Following the generation of extracellular P_i and the synthesis of collagen, pre-osteoblasts mature into osteoblasts. These cells are characterized by the expression of collagen I, BSP-1 (bone sialoprotein-1), alkaline phosphatase, PTH1R, E11/gp36, and osteocalcin.¹²⁸ At this stage, BSP-1 and osteocalcin are of particular interest. BSP-1 is an 85 kDa, sulfated glycoprotein that shows an affinity for hydroxyapatite.^{6,129,135,140} It represents approximately 10% of non-collagenous protein in bone and is characterized by the presence of multiple glutamic acid residues. These residues, by virtue of their R-COO^- anionic form, are able to bind up to 83 Ca²⁺ ions. Overall, crystal growth and regulation is not well understood, but in the case of BSP-1, Ca²⁺ binding is believed to anchor BSP-1 to hydroxyapatite and contribute to organized CaP_i formation.^140,141 Osteocalcin (or bone Gla protein), by contrast, is a small 6 kDa, 49 aa residue polypeptide with unclear function. It is one of the many vitamin-dependent proteins associated with bone. Vitamin C is necessary for hydroxylation at Pro9, VitD initiates gene expression, and vitamin K is a cofactor for vitamin K-dependent carboxylase. The latter enzyme adds a gamma-carboxyl residue to the molecule.^55,144,145 This creates two gamma-carboxyl residues that serve as a binding site for exogenous Ca²⁺. Osteocalcin has been described as both a promoter and inhibitor of mineralization.^143-145 PTH/PTH1R activity in the osteoblast downregulates osteocalcin expression. Since PTH is associated with bone demineralization, it may be more important in bone formation. Yet low doses of osteocalcin block the transition of CaHPO₄•2H₂O to Ca₁₀(PO₄)₆(OH)₂ (i.e.-hydroxyapatite), so the issue is undecided.

Once mineralization commences, that is, osteoid is laid down, osteoblasts begin a two phase process for Ca²⁺ deposition (Figure 6). It starts with the generation of 100 nm matrix vesicles. These are cytoplasmic membrane-bound vesicles that are created near the osteoid-osteoblast interface. These matrix vesicles contain a variety of substances, including annexin V (on the inner membrane surface), alkaline phosphatase (GPI-linked), calbindin-D_9K, pyrophosphatases, carbonic anhydrase, AMPase, BSP-1, osteonectin, and osteocalcin. The matrix vesicle membrane is enriched in cholesterol, phosphatidylserine, and sphingomyelin.^138,139 The matrix vesicles are extruded from the osteoblast in an annexin-dependent process where they attach to the underlying matrix.¹⁴⁶ The attachment in bone is not well understood. In cartilage, the matrix vesicle attaches to cartilage matrix collagen types II and X. In bone, there appears to be only type I collagen. Both alkaline phosphatase and annexin are reported to bind to type I collagen, and these molecules, if expressed on the outer surface of the matrix vesicle, may provide the anchor for osteoblast-derived matrix vesicles.¹⁴⁷ Once externalized, the matrix vesicle undergoes physical changes. Both Ca²⁺ and P_i are transported into the vesicle. Annexins form channels that transmit Ca²⁺. This is accompanied by phosphate uptake, which is tied to external P_i generation via alkaline phosphatase activity.^136,146,148 Uptake of P_i occurs via NPT3/Pit1 Na/P_i cotransporters.^{91,136,146-149} Once internalized, Ca²⁺ and P_i form rudimentary (immature?) CaP crystal (amorphous CaP). Early formation is predicated upon initial Ca²⁺ and P_i interaction with phosphatidylserine.^{142,148,150-152} The first crystal(s) formed is referred to as octa-CaP_i-like mineral and does not yet qualify as organized hydroxyapatite.^150,151 In the presence of elevated intra-vesicle Ca²⁺ and P_i, crystal growth occurs internally on the Octa-CaP_i-like complex, generating early hydroxyapatite. Undoubtedly, some of the gamma-carboxy-expressing molecules regulate these initial phases of the process. Events to this point constitute the first phase of hydroxyapatite formation.¹³⁸

Figure 6. Phase I of the mineralization process takes place in 100 nm matrix vesicles released from adjacent osteoblasts. Ca2+ and Pi are internalized, forming non-crystalline, amorphous CaP. Amorphous CaP is then converted to octa-CaP crystals, and finally into insoluble hydroxyapatite crystals (phase I). During phase II of the mineralization process, phospholipases mediate penetration of the hydroxyapatite through the vesicle membrane. The levels of extra-vesicular Ca2+, Pi, and H+, as well as Ca2+-binding proteins including BSP-1, osteocalcin, and osteopontin regulate the continued nucleation of hydroxyapatite crystals.

In the second phase, the enlarging CaP_i complex actually begins to penetrate the matrix vesicle membrane. Complete matrix vesicle rupture does not seem to occur until later. Rather, phospholipases punch holes in the plasma membrane, exposing the nascent hydroxyapatite to the extracellular fluid. Factors that regulate the continuing growth of the hydroxyapatite include Ca²⁺ and P_i concentrations, local pH, and the presence of non-collagenous matrix proteins.¹³⁸ At a minimum, these proteins include BSP-1, osteocalcin, and osteonectin. They also include osteopontin, which would seem to play a major, if not crucial, role in large-scale hydroxyapatite formation. Osteopontin is a secreted phosphoglycoprotein that is induced by VitD.^{6,135,142,143} This molecule has an RGD sequence that may provide anchorage for cells. It is also variably phosphorylated, the degree to which apparently impacts its function. When 40% of its phosphorylation sites are filled, it blocks mineralization. When 95% of its sites are filled, it appears to promote hydroxyapatite formation.¹⁴³ Whether 95% phosphorylated osteopontin occurs regularly in bone is unknown.^6,143 Osteopontin and osteocalcin are suggested to interact and expose Ca²⁺-binding nucleation sites on osteopontin necessary for hydroxyapatite formation. ¹⁴³

Bone Resorption

The process of bone resorption is relegated to the osteoclast, a multi-nucleated, monocyte-derived cell that responds to both low pH and PTH/low extracellular Ca²⁺ (Figure 7). Although cytokine-induction of osteoclasts has been a favorite topic of experimental biologists over the last decade, acidosis and the response to acidosis by osteoclasts is perhaps just as important. Osteoclasts express a proton-sensing G protein-coupled receptor termed OGR1/GPCR1. In the presence of low pH, this receptor is activated and increases intracellular Ca²⁺. This begins the process of bone resorption with demineralization. Hydroxyapatite [(Ca₁₀(PO₄)₆(OH)₂] contains a considerable amount of stored OH- ion. During metabolic acidosis, where renal and respiratory homeostatic mechanisms are inadequate, bone is broken down to provide buffering capacity to the blood/extracellular fluid.¹⁵³

Figure 7. The activity of bone-resorbing osteoclasts is sensitive to extracellular pH due to their expression of OGR1 H+ receptors. H+ is extruded by transporters and proton pumps, dissolving mineral in the sealed space between osteoclast and bone, while enzymes including Cathepsin K and TRAP digest the organic matrix. Negative feedback mechanisms to bone resorption involve osteoclast apoptosis triggered by Ca2+ influx through TRPV channels. In addition, PDGF-BB and/or VitD enhance osteoblast secretion of OPG, potentially limiting RANK L/RANK-mediated osteoclast development. IFN-beta may also inhibit osteoclast development.

Osteoclasts are generated from the actions of M-CSF, RANK ligand (RANK L/TRANCE), and likely, IL-1 on osteoclast precursors. M-CSF is secreted by immature osteoblasts and promotes the formation and proliferation of osteoclast precursors from CFU-M.^154,155 In the presence of elevated VitD, IL-11 or a hypocalcemic condition that induces PTH release, osteoblasts will be stimulated to express RANK L on their surface.^155,156 RANK L is a 45 kDa, type II transmembrane glycoprotein that belongs to the TNF superfamily. When expressed, it binds to either constitutively expressed, or M-CSF-induced, RANK on the surface of osteoclast precursors.¹⁵⁷ In synergy with M-CSF, RANK L induces differentiation to "pre-fusion" osteoclasts that ultimately fuse under the influence of M-CSF, IL-1, and RANK L. Fusion forms a mature, multi-nucleated osteoclast.^155,156 Finally, RANK L and IL-1 activate the osteoclast, initiating bone resorption. It should be noted that pro-inflammatory cytokines such as IL-1 beta and TNF-alpha will also induce RANK L expression. Although this expression is on T cells rather than osteoblasts, the effect is the same; osteoclast development.^158,159 The osteoclast first generates a sub-osteoclastic space between the osteoclast and underlying bone. A proton pump/transporter (V-ATPase and/or Na⁺/H⁺) linked to Cl- transporters/channels (KCC1 and CLC7) facilitates the release of H⁺ into the sealed space or lacuna.¹⁶⁰ This dissolves the underlying mineral and generates high local levels of Ca²⁺ and P_i. Tartrate-resistant acid phosphatase (TRAP) and cathepsin K are also secreted, and these enzymes digest the accompanying organic matrix.^155,156

There appear to be at least three negative-feedback mechanisms associated with osteoclast activity. The first involves released Ca²⁺, which is suggested to enter the osteoclast via TRPV channels.¹⁵⁷ What opens the channels is not clear. Osteoclasts have CaSRs associated with ryanodine receptor 2 (RyR2), and this complex may initiate Ca²⁺ transit through the osteoclast into the extracellular fluid. Regardless, Ca²⁺-influx is reported to induce apoptosis in osteoclasts, potentially limiting the time the bone is exposed to osteolytic agents. Clearly, intracellular Ca²⁺-binding agents such as calbindin may modulate the effects of any increased cytoplasmic Ca²⁺ levels.^161-165 A second potential negative-feedback mechanism involves PDGF-BB. In this scenario, RANK L on osteoblasts will bind to RANK on osteoclast precursors, generating functional osteoclasts. Osteoclasts now secrete a PDGF-BB homodimer that has a down-modulating effect on osteoblast activity. This includes a reduction in type I collagen production and osteocalcin secretion. Perhaps most importantly, PDGF-BB induces osteoblasts to secrete OPG (osteoprotegerin), a natural soluble receptor for RANK L.¹⁶⁶ This blocks RANK L activity on osteoclast precursors and limits osteoclast development.¹⁶¹ The effect is amplified by the presence of VitD (a bone anabolic agent), which also promotes OPG secretion by osteoblasts.¹⁶⁷ A final mechanism for osteoclast down-regulation involves osteoclast production of IFN-beta, an autocrine inhibitor of osteoclast development. IFN-beta, acting through the type I interferon receptor, reduces c-fos, a transcription factor needed for continued osteoclast activity.¹⁵⁵

Significance of Phosphorus Homeostasis

Although the above provides some insight into the homeostatic processes associated with Ca²⁺ and P_i metabolism, it does not address the consequences of unregulated or dysregulated metabolism. Recently, the discovery of the phosphatonins has focused attention on phosphorus resorption. When hypophosphatemia (low phosphorus) is present, muscle weakness, rhabdomyolysis (skeletal muscle death), bone pain and rickets (failure to form and maintain bone) may present. These can be accompanied by lethargy, confusion and impaired sphincter control. One particular problem in severe cases is a generalized impairment of energy metabolism. Limiting P_i means limited ATP formation and reduced oxidative phosphorylation. In the blood, this has (or may have) material consequences. In white cells, a reduction in ATP results in an impairment of bactericidal activity and predisposition to infection. In red cells, 2,3-diphosphoglycerate (DPG) is reduced, which shifts the oxygen dissociation curve, making it more difficult to release bound oxygen. Hypophosphatemia occurs during alkalosis, when cells take up circulating P_i and the kidneys do not resorb enough to account for the shortfall. It may also occur in hospitals during cytokine and cancer therapy and when the administration of IV glucose is the only energy source.

It is not uncommon for renal insufficiency to be accompanied by hyperphosphatemia, when the glomerular filtration rate is low and P_i does not appear in urine (for either excretion or resorption). Once present, hyperphosphatemia induces metabolic acidosis. Symptoms of the condition include a prolonged Q-T interval in an EKG, muscle cramps, tetany, and the suppression of VitD formation. One specific danger involves the abnormal deposition of CaP complexes in non-osseous tissue. This is particularly troublesome in blood vessels.²

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