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Bone Homeostasis

Physiology

The bone shaft is composed of cortical (aka compact) bone while the ends are composed of trabecular (aka spongy) bone.

Cortical bone is arranged in units called osteons (aka Havesian systems), a circular network of concentric calcified matrix rings (lamellae) alternating with small spaces (lacunae) which contain osteocytes, i.e. bone cells. The osteocytes extent slender processes from one lacunae to the next through tiny channels called canaculi.

Spongy bone does not contain true osteons but consists of lamellae arranged in an irregular lattice of thin columns (trabeculae) separated by macroscopic spaces filled with red bone marrow.

Within the cortical bone is the bone marrow, a source of blood and bone cell progenitors and fat storage. The endosteon is a membrane containing bone-forming cells that lines the medullary cavity.

There are four types of bone cells: osteoprogenitor cells, osteoblasts, osteocytes and osteaoclasts.

Osteoprogenitor cells give rise to osteoblast and osteocytes. They have no no specific markers but are highly proliferative/committed bone cells. Osteoprogenitor cells are located in the marrow space and express parathyroid hormone (PTH) receptors.

Osteoblasts are bone matrix producing cells that differentiate from mesenchymal stem cells by the action of Runx2 (runt-related transcription factor 2, aka Osf2 and Cbfa1). Runx2 belongs to the RUnt family of transcription factors, in whic the Runt domain binds DNA at response elements called OSE2. Runx2/Osf2/Cbfa1 knockout mice have no bones.

Osteoblasts are plump and basophilic, line bone matrix, and express PTH receptors. Osteoblasts that become trap in the mineralized matrix become osteocytes. Osteocytes have fibrotic morphology (?) and express PTH receptors. Osteocytes communicates with each other through canaliculi in the bone matrix and gap junctions in their filapodial processes.

Osteoclasts are large multinuclear cells (5-50 nuclei) derived from the fusion of blood-derived monocytes (mononuclear phagocyte system). They are bone resorbers which release acid phosphatase and hyaluronic acid. Local decreases in pH caused by osteoclasts favor hydroxyapatite solubilization and release of calcium and phosphate ions from the collagen matrix.

Osteoclast do not have PTH receptors and receive most important signals from osteoblasts. Positive regulators include vitamin D, PTH, PGE2, IL-6, IL-11, and M-CSF. Negative regulators include TGF-b, IL-4, IL-10, IL-13, IL-18, and interferon.

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Calcium Regulation

Calcium in the diet varies: therefore internal levels must be regulated. The skeletal pool of calcium is larger than in soft tissue, which is larger than the ffluid pool. In the blood, about 50% exists as free Ca and 50% bound to albumins. Calcium gain in an adult roughly equals loss, while in a child gain exceeds loss. In summary, PTH and Vitamin D hormone increase calcium in blood, while calcitonin decreases calcium in blood. Estrogens also play a role.

PTH is a 84 amino acid polypeptide hormone produced by the parathyroid glands. It is derived from a precursor and the fully active region is in amino acids 1-34.

The PTH receptor is coupled to cAMP and Ca /IP3 pathways. cAMP is the primary pathway for stimulation of the "osteoblast genes", e.g. collagenase gene (also requires induction of c-fos as a tertiary messenger). Chronic or high doses of PTH downregulate collagen transcription, while low, intermittent doses upregulate collagen transcription. PTH also induces Runx2/Osf2/Cbfa1 mRNA in osteoblasts, the "master" regulator of bone formation. PTH-mediated bone anabolic effects are reversed by co-treatment with Runx2/Cbfa1 antisense oligonucleotide.

PTH acts on bone, gut and kidney: promotes vitamin D-mediated uptake of Ca from the gut to the blood; mobilization of Ca and phosphate from bone to the blood; and vitamin D-mediated retention of Ca by the kidneys. Removal of the parathyroid glands without PTH supplementation leads to plasma calcium decrease and death.

PTH mediates both bone reabsorption at higher doses and bone formation at lower doses. Although the cellular basis for the anabolic response has not be fully elucidated, osteoprogenitors and osteoblasts are targets for the anabolic action. PTH visibly alters osteoblast cellular and nuclear structure and this plays a role in gene expression.

The pharmaceutical industry has developed PTH as a drug for osteoporosis (Lilly's rhPTH(1-34), known as teriparatide or FORTEO; Approved Nov 2002). Given in continuous dose it breaks down bone (catabolic) but give in intermittent doses it builds bone (anabolic). Aged female rats were given vehicle or PTH low dose by injection every 24 hr for 15 days (i.e. an intermittent dose). At day 15-18, a dye (calcein) that marks newly forming bone was injected. Animals were sacrificed at day 20, and the vertebrae were sectioned and examined. There was an increase in bone formation (Dr. Janet Hock and colleagues at Eli Lilly).

Vitamin D hormone is an ancient biological seco-sterol molecule found in ocean plankton and important in animals with ossified endoskeletons. I has been considered a vitamin since the 1920's and a hormone since the 1970's.

Parent (pre-) Vitamin D is known as calciferol, which is metabolized to calcitriol (1,25-dihydroxyvitamin D3), the final active hormone. Humans can produce calciferol from photoisomerization of 7-dehydrocholesterol in the skin. A second photoisomerization reaction produces vitamin D3 (cholecalciferol), which is transported in the circulation bound to transcalciferin. Vitamin D3 can also be obtained in the diet from cod liver. Plants and yeasts contain vitamin D2 (Ergocalciferol).

Both vitamin D3 and D2 can be converted to calcitrol in a two step process. First they are hydroxylated by 25-hydroxylase in the liver. The final step in their converrsion to calcitrol occurs in the kidney and is catalyzed by 1a-hydroxylase.

PTH promotes kidney 1a-hydroxylase activity. Free Ca negatively feeds back to inhibit PTH synthesis, thus inhibiting 1a-hydroxylase activity. Free phosphate negatively feeds back to inhibit kidney 1a-hydroxylase activity.

Vitamin D receptors are important in the intestine, bone, and kidney. They are also found in blood cells, brain, skin, muscle, and pituitary. Most likely they work as VDR/RXR heterodimers. VDRs may be mostly nuclear, even in absence of ligand. AN example of a gene regulated by VDR/RXR is 24-hydroxylase, therefore this is a regulatory feedback mechanism.

Vitamin D regulates mineral homeostasis by promoting uptake of Ca to the blood from gut, mobilization of Ca2+ & phosphate from bone to blood and retention of Ca by he kidneys. It regulates bone remodeling (both osteoclastogenesis and osteoblast function, immunomodulation, cell differentiation, and suppression of malignant phenotype.

Calcitonin is produced by the clear cells of the tyroid gland. It plays a role in satiety, regulates bone mineral metabolism (decreases calcium) and vitamin D action. Plasma calcitonin is higher in pregnant and lactating women, and is inverselly proportional to plasma PTH. Calcitonin activity increases in response to a rise in serum calcium or to the GI hormone gastrin.

Calcitonin is a product of the calcitonin/CGRP gene, which undergoes tissue-specific, alternate RNA splicing. While calcitonin is the main product of the gene in the thyroid, CGRP, a neuropeptide involved in hypertension, is the main product in the brain. The half-life of human calcitonin is about 5 minutes. Salmon calcitonin is ten times more potent than human calcitonin: it has a longer half-time and a higher affinity for the calcitonin receptor (CT-R).

The calcitonin receptor (CT-R) is found in skeletal tissue, kidney, Leydig cells, and other tisues. CT-R signaling is complex and may depend on the cell cycle. CT-R is a G-coupled receptor related to the PTH/secretin receptors that increases cAMP in the cell by coupling with Gs. It may also couple with Gi to decrease intracellular cAMP levels and with Gp to activate PKC.

The general effect of calcitonin in bone is deposition of calcium. Calcitonin inhibits mitosis of precursor cells, increases osteoblast activity, and signals osteocytes for bone formation. In the kidney it inhibits calcium reabsorption and in the GI inhibit absorption.

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Bone Formation and Remodeling

Most bones are formed by endochondrial ossification. Mesenchymeal cells differentiate into chondroblasts that produce cartillage. As the area of cartilage grows (proliferative zone), chondroblasts that remain buried in the matrix (chondrocytes) hypertrophy and burst, changing the matix pH and thus triggering calcification. As chondrocytes die, lacunae form.

As arteries extend into the calcifying matrix, the blood flow stimulates osteoprogenitor cells to differentiate into osteoblasts. The osteoblasts then deposit bone matrix over the calcified cartilage, forming spongy bone (ossification zone).

Cells beneth the outer membrane of the bone (periosteum) secrete a thin shell of compact bone (periosteal bone). Osteoblasts in the deeper layers of the fibrous periosteum synthesize and release collagen. Individual collagen molecules become oriented in a specific molecular architecture to form an extracellular matrix around osteoblasts.

Calcium and phosphate ions are stereochemically bound to specific sites on the collagen matrix where they are precipitated as Ca10(PO4)6(OH)2 (hydroxyapatite). Precipitation of calcium phosphate depends on the concentration of calcium and phosphate ions (exceed the solubility product at the local site of ossification). Precipitation of bone mineral may be enhanced under conditions of local alkanization resulting from osteoblastic activity.

As ossification proceeds towards the end of the bone, osteoclasts break down newlly formed trabeculae leaving a cavity for bone marrow.

Cartilage remains between the end spongy bone and the bone shaft, known as the epiphyseal plate, that is responsible for bone growth before adulthood.

In the adult, bone remodeling is the process by which catabolic effects (bone resorption) of the osteoclasts are balanced by the anabolic effects (bone formation) of the osteoblasts in a highly regulated cycle. Osteoclasts adhere to bone and remove it by acidification/proteolysis; then osteoblasts enter the site and secrete osteoid (a matrix of collagen and other proteins) which is calcified into new bone.

Balanced bone turnover is required for normal bone density. While balanced, bone cell activity is not always equal. Trabecular bone volume increases through childhood and reaches a peak in early adulthood, to slowly decline during the rest of adult life. Many factors contribute to either bone formation or resorption. Intermitent PTH, androgens and exercise increase bone formation. Estrogens, calcitonin and vitamin D decrease bone resorption. Chronic PTH, low calcium levels and immobilization increase bone resorption.

Osteoclast activity is a function of both activation (function increased) and recruitment (progenitors make more osteoclasts). Since osteoclasts do not have PTH receptors, they depend on activating and recruiting signals from osteoblasts. Osteoclast progenitor cells have RANK (receptor activator of NF-kB) receptors that respond to RANKL (RANK ligand), a juxtacrine ligand produced by osteoblasts in response to pro-resorptive and calciotropic factors like vitamin D and PTH. Activation of the RANK receptor leads to differentiation of osteoclasts. RANKL signals through the Ras/MEKK/JNK/Jun pathway.

Osteoblast also produce a paracrine ligand, OPG, that binds RANKL and prevents its binding to RANK, in response to anabolic and resorptive factors like estrogens and calcitonin.

Osteoprotegerin (OPG) is an inhibitor of osteoclast formation and function.OPG-/- mice are born with osteoporosis, while OPG overexpression leads to osteopetrosis. Small molecules stimulate OPG production (?).

Estrogens decrease osteoclast formation by down-regulating RANKL-induced activation of JNK and subsequent phosphorylation (and activation) the Jun transcription factor. This occurs within 5 minutes of estrogen treatment in vitro and is reversed by treatment with estrogen antagonist. Estrogens also by lower Fos levels (Fos is a partner of Jun - together they make the AP-1 heterodimeric transcription factor).

In women taking hormone replacement therapy (HRT) or a selective estrogen receptor modulator (SERM), the incidence of hip fractures decreased by 34% and 47%, respectivelly. HRT, a combination of conjugated equine estrogens (CEE) and medroxyprogesterone acetate (MPA), may have unwanted side effects not seen after SERM treatment.

ERa also mediates bone formation induced by mechanical loading (excersise). This may work through the CBFA1/Runx2/Osf2 bone “master” transcription factor.

The physiological osteoprotective effects of estrogens are eliminated by deletion of both estrogen receptors (ERa and ERb). while trabevcular bone mass decreases in either wild type, ERa-/- or ERb-/- mice after overectomy, ERab-/- mice already have a decreased trabecular bone mass relative to wild type, and ovarectomy does not significantly change it.

Another important protein in osteoblasts is the LDL receptor-related protein 5 (LRP5). LRP5 interats with Wnt at the membrane to activate the Wnt/Fz/GSK3/b-catenin pathway. Increased b-catenin levels in the nucleus leads to more bone mineral density. A mutation in LRP5 that prevents interaction with its inhibitor (Dkk) leads to high bone mass in humans and rodents.

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Diseases

Runx2/Osf2/Cbfa1+/- mice have a generalized defect in bone formation, inclusding hypoplastic clavicles and incomplete closure of fontanelles. Humans with heterozygous deletions , insertions or missense mutations of the Runx2/Osf2/Cbfa1 gene suffer cleidocranial displasia

Paget's disease is a disorder of bone remodeling probably due to viral causes. There is an increased osteoclast-mediated bone resorption, and the resulting bone is expanded in size, less compact, more vascular (bone warmth) and susceptible to deformity (very painful). Common "Pagetic" sites include the skull, femur and pelvis. Salmon calcitonin or bisphosphonates are used as treatment. Bisphosphonates are organic compounds that bind to hydroxyapatite crystals in bone and reduce their absorption by osteoclasts.

Rickets is a bone disease due to Vitamin D deficiency. It first became a public health problem during the Industrial Revolution, when children started living in dark, crowded cities. In some cities of the late 1800s about 90% of the children were affected. Weaken bones (rachitic cartilage in epiphyseal plates, no calcification of the osteoid) lead to high infant and maternal mortality (during childbirth). The traditional therapy was exposure to sunlight or cod liver oil (fat soluble vitamin = vitamin D). Now Vitamin D is added to milk.

In adults, osteomalacia and leads to softening of bones and may be due to either vitamin D deficiency or resistance. Vitamin D resistance occurs when there are vitamin D metabolism defects (e.g. renal hydroxylase defect) or vitamin D receptor mutants.

Osteoporosis is the most common metabolic bone disease and is characterized by an asymptomatic reduction in the quantity of bone, a decrease in both bone matrix and mineralization. It is a multifactorial disorder leading to uncoupling of the remodeling cycle and net loss of bone mass. In the U.S. osteoporosis is the cause of about 500,000 vertebral fractures and 250,000 hip fractures per year.

Osteoporosis is hard to diagnose using conventional X-rays, since only more than ~30% bone loss can be seen in the film. DEXA (dual energy x-ray absorptiometry) can measure bone density more accurately, and is a non-invasive, quick, low radiation absorption method. A bone mineral mass (BMD) over 2.5 standard deviations (S.D.) below the mean is considered osteoporosis. Severe osteoporosis is when BMD is more than 2.5 S.D. below mean and there are already fractures. A patient with BMD between 1-2.5 S.D. below the mean is considered to have osteopenia low bone mass). A normal bone mass density is less than 1 S.D. below mean.

Risk factors for osteoporosis include older age, being females (especially post-menopause), being Caucasians or Asians, lean (increased compartment for sex steroid aromatization; gravity makes bones stronger), and alchohol or tobbaco use. Calcium intake of 1000 - 1500 mg/day is recommended for prevention of osteoporosis, but the average U.S. women intake is 500 mg/day.

Prevention is the best treatment, since it is hard to regain bone mass. Other treatments include calcium supplements (inhibits PTH), calcitonin, bisphosphonates, estrogen/hormonal supplements, and maybe PTH.

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