Bone Metabolism : Regulation (Part 2)

Calcium and phosphate balance

Soluble Ca²⁺, hydroxyapatite and calcitonin 

As calcium (Ca²⁺) is one of the main components of our bones, large amounts are present

in our body. At the same time, comparatively low extracellular concentrations of Ca²⁺ are fine-tuned to regulate important functions, not to speak of even far lower intracellular concentrations. This dichotomy is possible due to the low solubility product of Ca²⁺ and phosphate (PO₄^3-): if one ion is added to a solution of the other, most of it precipitates as calcium phosphate.

In the bone, the two ions combine with hydroxide (OH-) to form hydroxyapatite Ca₁₀(PO4)₆(OH)₂  a hard mineral forming hexagonal crystals. Up to 70% of the weight of bone is due to hydroxyapatite. Dental enamel consists almost exclusively of the mineral, accounting for its mechanical resistance. The disadvantage to this solution is that hydroxyapatite is sensitive to acidity. Low pH attacks enamel via the same mechanism that osteoclasts use to resorb bone. Citric acid from an orange, or lactic acid produced by bacteria metabolizing sugar in dental plaque make protons come into contact with the enamel surface. A proton H+ pulls out the hydroxide ion OH- from Ca₅(PO4)₃(OH) to form a H2O water molecule, with the rest disintegrating into 5 Ca²⁺ und 3 PO43- ions. The hydroxyapatite complex dissolves, ultimately leading to caries.

Plasma Ca²⁺ concentration is physiologically maintained in a small window between 2.2 and 2.7 mM. This measured Ca²⁺ is the sum of three forms: Ca²⁺ bound to plasma proteins (about 45%), Ca²⁺ complexed with small organic anions (10%) and free ionized Ca2+ (about 45%). Hence, total Ca²⁺ depends on plasma protein concentration. The biologically relevant, regulated parameter is free Ca²⁺.

Ca²⁺ balance is basically maintained by two hormones: parathyroid hormone (PTH) and calcitriol (1,25-dihydroxyvitamin D). PTH regulates short-term plasma Ca²⁺ concentrations by dipping into bone reserves. Vitamin D strategically maintains the total Ca²⁺ pool of the body.

A third Ca²⁺ regulating hormone, calcitonin, is of minor importance in humans. It is secreted by parafollicular C cells in the thyroid gland and lowers plasma Ca²⁺ levels for a short time by directly inhibiting osteoclast activity, with the system quickly swinging back to a neutral position. Neither a total loss of calcitonin-producing cells (e. g., by thyroidectomy), nor massive overproduction by rare tumors lastingly interfere with Ca²⁺ balance. Probably, calcitonin is a remnant from evolution. Animals such as salmon, which switch from fresh water to calcium-rich sea water, seem to rely on calcitonin to cope with massive differences in Ca²⁺ intake.

Parathyroid hormone

Parathyroid hormone (PTH) is named for the four parathyroid glands producing it, tiny epitheloid bodies located right behind the thyroid. An increase in the concentration of free Ca2+ activates the calcium-sensing receptor (CaSR) located at the membrane of their chief cells. The cells react by decreasing PTH production. A second means to lower PTH secretion is a high concentration of 1,25 dihydroxyvitamin D. The message of Vitamin D seems to be: "Stop cannibalizing our bones, I'll organize more Ca²⁺ from outside in a minute!" PTH is a small protein of 84 amino acids and has an extremely short half-life of about four minutes. PTH increases Ca²⁺ concentration via two main mechanisms: by liberating it from bone and by influencing the kidneys.

PTH's net effect in bone is an increase in resorption by activation of osteoclasts. This is achieved via a detour, as osteoclasts do not express PTH receptors. PTH is sensed by osteoblasts, which react by producing IL-1, IL-6 and other cytokines to activate osteoclasts. In addition, PTH increases osteoblast production of the two molecules that induce differentiation and proliferation of more osteoclasts: M-CSF (macrophage colony-stimulating factor) and RANKL.

RANK-ligand (RANKL) is a molecule from the TNF-superfamily. It acts as a trimer, either on the surface of osteoblasts, or, "cut off", as a soluble signaling molecule. In the bone marrow, M-CSF and RANKL encounter precursor cells of the hematopoietic lineage leading to macrophages and neutrophil granulocytes. These precursor cells express RANK (receptor-activator of NFκB), a transmembrane protein of the TNF receptor superfamily. RANK functions as receptor for RANKL. As precursor cell RANK is trimerized by osteoblast-emitted RANKL, the precursor cells are activated to differentiate first to mononucleated osteoclast precursors that subsequently fuse to mature polynucleated osteoclasts. Osteoblasts secrete a further protein, osteoprotegerin (OPG), that looks like a soluble receptor for RANKL. This is called a decoy receptor; by neutralizing RANKL, it acts as its inhibitor. Thus, the formation rate of osteoclasts depends on the relative amounts of RANKL and OPG produced by osteoblasts. While PTH induces expression of M-CSF and RANKL, it inhibits production of OPG, cranking up the generation of osteoclasts.

If PTH just mobilized Ca²⁺, not much would be gained: due to the low solubility product with phosphate, it would soon reprecipitate. Therefore, PTH simultaneously lowers phosphate levels by inhibiting renal reabsorption in both the proximal and distal tubule. This is achieved by removing the Na/phosphate cotransporter from the luminal membrane and parking it in vesicles below. Apart from inducing phosphaturia, PTH increases reabsorption of Ca²⁺ in the distal tubule, further reducing the already minimal loss of Ca²⁺ in the urine. The third renal function of PTH is to stimulate hydroxylation of carbon atom 1 of vitamin D: this is the last and rate-limiting step in its activation. From there, 1,25 dihydroxyvitamin D sets out to refill the Ca²⁺ pool.

Vitamin D

Lipid-soluble vitamin D3 can be taken up with animal foods, especially fatty fish species (cod liver oil, mackerel, salmon), or can be produced in our own skin from 7-dehydrocholesterol. This requires sunlight to open the second ring of the cholesterol backbone. This UV B-dependent synthesis is probably the cause of Caucasians' pale complexion. Until the first wave of homo sapiens left Africa about 60,000 years ago, all modern humans probably had dark skin. The further north the people migrated, the less ultraviolet light they absorbed. Those with lighter complexions obtained a selective advantage, as they were better able to synthesize vitamin D. Ethnic groups using the sea as their primary food source, like the Inuit, took up enough vitamin D3 with their food and were thus able to retain a higher level of pigmentation than people living off the land. The causal relationship between a lack of sunlight and rickets was only recognized in the late 19th century.

Two successive hydroxylation steps are required to metabolize D3 and D2, which already contain one hydroxyl group, to their active form, calcitriol. The first hydroxyl group is added at position 25, the end of the side chain, in the liver. The second hydroxylation occurs in the kidney, at position 1 of the first ring of the erstwhile cholesterol structure. This decisive, last activation step is performed in the proximal tubule under tightly regulated conditions. PTH stimulates hydroxylation, while the end product calcitriol as well as increased levels of phosphate act inhibitory. 1,25-dihydroxyvitamin D (calcitriol) equilibrates over the entire body and binds to the vitamin D receptor (VDR), a member of the nuclear receptor superfamily. As a ligand-dependent transcription factor, one of its functions is the induction of genes that are necessary to maintain Ca²⁺ reserves.

The central target organ in this respect is the duodenum. Here, calcitriol induces several proteins that in concert enhance absorption of Ca²⁺ from food. While Ca²⁺ concentrations in the lumen of the gut and in blood are in the nanomolar range, they are much lower inside the cell; too much free Ca²⁺ in the cytosol would be dangerous. At the luminal side of the duodenal epithelial cell, vitamin D induces a channel, allowing Ca²⁺ to trickle in passively. In the cytosol, the Ca²⁺-affine protein calbindin is increased to neutralize passaging Ca²⁺. At the basolateral membrane, an ATP-driven Ca²⁺-H⁺-antiporter as well as a Na+-driven Ca²⁺-Na⁺-antiporter are induced to pump Ca2+ into the blood against a steep concentration gradient. Calcitriol also enhances phosphate absorption in the small intestine.

In the kidney, the action of vitamin D parallels that of PTH by increasing reabsorption of Ca²⁺ in the distal tubule, although its effect is much weaker. Contrary to PTH, vitamin D also enhances reabsorption of phosphate: both ions are required to promote bone mineralization.

Together, these effects of vitamin D raise Ca²⁺ and phosphate concentrations above their solubility product, inducing their precipitation in osteoid, an effect that is enhanced by vitamin D-stimulated transcription of the osteocalcin gene in osteoblasts. This predominant, indirect effect outweighs an opposite direct, receptor-mediated activation of osteoblast and osteoclast precursors that enhances bone turnover and Ca²⁺ mobilization.

(Credit: Arno Helmberg)