Bone Metabolism Antibodies and Antigens

Overview

Bone is a solid structure undergoing perpetual renewal with crucial functions such as kinematic support, visceral protection, and regulation of hematopoiesis and mineral balance. Maintenance of these functions depends on normal bone mass and strength. Skeletal diseases, such as osteoporosis, are usually accompanied by abnormal bone remodeling, in which osteoclast-mediated bone resorption preponderates over osteoblast-mediated bone formation, leading to decreased bone mass, deteriorated microstructure, and increased fragile fracture risk.

Bone formation or modeling

Bone needs to be remodeled to maintain integrity with osteoblasts, which are bone-forming cells, and osteoclasts, which are bone-degrading cells.

Osteoblasts
Osteoblasts are specialized cells of mesenchymal origin that are implicated in the synthesis of bone matrix and the regulation of bone mineralization. Osteoblast activities start after their differentiation from their parental mesenchymal lineage, a process mediated by RUNX2 (runt-related transcription factor 2) and osterix.

Osteoclasts
Osteoclasts originate from monocytes/macrophages, which adhere to the bone matrix and cause the degradation of bone matrix. The differentiation of osteoclasts from their parental lineage is achieved upon the stimulation of RANKL and the M-CSF. The binding of M-CSF to receptor c-Fms ensures the survival of osteoclasts for the precursor and mature cells. While, RANKL has been identified as the primary osteoclast differentiation factor, regulating the expression of genes involved in osteoclast synthesis.

Fig. 1: An overview of intracellular regulation of osteoclastogenesis and resorption activities

The major factors in bone formation include fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), wingless-type (Wnt) genes, runt-related transcription factor 2 (RUNX2), and osteoblast-specific transcription factor (osterix or OSX). In addition, metabolic functions of bone largely involve the homeostasis of calcium and phosphate. Release of calcium, or absorption of calcium by bone, is largely regulated by hormones (including PTH, Calcitonin, Sex hormones, Growth hormone, and Thyroid hormones) and, less so, by steroids (Vitamin D, Glucocorticosteroids).

Major molecular signaling of bone remodeling

The maintenance of bone homeostasis is regulated by a cascade of signaling pathways. The major pathways associated with bone remodeling and pathogenesis of most bone diseases including RANKL/M-CSF/OPG, MAPKs/JNK/NF-κB, BMP2-Wnt/β-catenin, PI3K/Akt/GSK3β, and other signaling pathways. The binding of RANKL and M-CSF to their respective receptors, RANK and c-Fms, leads to upregulation of TRAF6 that activates (ERK)/JNK/MAPK signaling, NF-ĸB/nuclear factor of activated T Cells 1 (NFATc1) signaling, Ca2+ signaling, and the PI3K/Akt/GSK3β signaling pathways, leading to bone components’ degradation. The BMP-2/Wnt/β-catenin signaling pathway regulates RUNX2 activity, and BMP activates the movement of β-catenin into the nucleus to initiate osteoblast development via the canonical Wnt/β-catenin signaling pathway that elevates osteoblastogenesis (OBG) and bone production. The nuclear activity of β-catenin includes the expression of osteogenic proteins such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein (BSP).

Fig. 2: Role of Wnt signaling in osteoblasts

Bone biomarkers

Various biomarkers are now available for specific and sensitive assessment of the rate for bone formation and bone resorption. For example, the bone formation biomarkers are total alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BALP), osteocalcin (OC), procollagen type 1 N-terminal propeptide (P1NP) and procollagen type 1 C-terminal propeptide (P1CP). The bone resorption biomarkers are hydroxyproline (HYP), hydroxylysine (HYL), deoxypyridinoline (DPD), pyridinoline (PYD), bone sialoprotein (BSP), osteopontin (OP), tartrate-resistant acid phosphatase 5b (TRAP 5b), carboxy-terminal crosslinked telopeptide of type 1 collagen (CTX-1), amino-terminal crosslinked telopeptide of type 1 collagen (NTX-1) and cathepsin K (CTSK). The regulators of bone turnover are receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), dickkopf-1 (DDK-1) and sclerostin. These biomarkers are useful to provide the early assessment of bone diseases when the BMD measurement of DXA does not offer enough information to make the diagnosis.

Fig. 3: Biochemical biomarkers of bone turnover

Major targets

BMP family
Bone morphogenic proteins are members of the transforming growth factor-β (TGF-β) superfamily and have various roles in the development and regulation of bone formation. BMP2, BMP6, BMP7, and BMP9 can stimulate osteoblastic differentiation in MSCs. BMP3 and BMP13, inhibit bone formation. BMPs in pre-osteoblastic cell lines increase alkaline phosphatase (ALP) activity, as well as osteocalcin and osteopontin. Recently, it was shown that BMP2 can induce activation of ALP, an early marker of osteoblast differentiation. Bone morphogenic protein 2 also regulates the expression of the critical osteogenic regulator RUNX2.

Osteocalcin (BGLAP)
It is specifically produced by osteoblasts, and is the most abundant non-collagenous protein in bone, and is often used as a marker for the bone formation process. Research has also suggested that osteocalcin can be related to male fertility as it may enhance the synthesis of testosterone.

Collagen
It is the main protein that makes up the bone matrix and provides bone strength and stability. Type I collagen: Measuring bone type collagen levels can be used to assess bone density and the risk of osteoporosis.

Osteonectin
Osteonectin regulates the interaction between collagen and mineralization to promote bone formation and stability.

Osteopontin, OPN
OPN Regulates Bone Development and Maintenance of Bone Mass.

Osteoprotegerin, OPG
OPG protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from interacting with RANK. The RANKL/OPG ratio in bone marrow is thus an important determinant of bone mass in normal and disease states.

Sclerostin
Sclerostin is expressed in the bone cell interstitium and on the surface of bone cells, and it inhibits the canonical Wnt-signaling pathway, and through this action, it controls bone formation by osteoblasts.

Bone-specific alkaline phosphatase, BALP
Bone Specific Alkaline Phosphatase is used to diagnose and assess the severity of metabolic bone disease including Paget disease, osteomalacia, and other states of high bone turnover. The test can monitor efficacy of antiresorptive therapies including postmenopausal osteoporosis treatment.

N-terminal propeptide of type I collagen, PINP
PINP is a degradation product of collagen I, and its level can reflect bone formation activity.

C-terminal telopeptide of type I collagen, CTX
CTX is a degradation product of collagen I, and its level can reflect the activity of bone resorption.

Wnt
Wnt family members like Wnt10a, Wnt10b, and Wnt6 have been found to regulate the fate of MSC differentiation into osteoblasts. The overexpression of Wnt10a and Wnt10b greatly stabilizes β-catenin and stimulates osteoblastogenesis through a canonical β-catenin-dependent pathway.

β-catenin
β catenin is involved in the regulation of both osteoblasts and osteoclasts, thus controlling overall bone formation.

RANK/RANKL
RANKL/RANK signaling regulates processes such as bone cell proliferation, differentiation, and bone matrix synthesis and degradation.

Sclerostin
Sclerostin inhibits the canonical Wnt-signaling pathway, and through this action, it controls bone formation by osteoblasts.

Cathepsin K (CatK)
CatK is a part of the family of cysteine proteases involved in the degradation activity of collagen 1 and elastin in bone resorption. Changes in levels of CatK are associated with various pathological conditions, primarily related to bone and cartilage degradation, such as pycnodysostosis (associated with CatK deficiency), osteoporosis, and osteoarthritis (associated with CatK overexpression).

Bone Sialoprotein (BSP)
BSP is a major non-collagenous protein in bone, and calcified cartilage tissues, and is involved in regulating hydroxyapatite crystal formation in bones.

Katacalcin/PDN-21
Katacalcin is a potent plasma calcium lowering peptide, that belongs to the calcitonin family, that can cause a rapid but short-lived drop in the level of calcium and phosphate in the blood.

Myostatin/Growth Differentiation Factor 8
Myostatin (GDF-8) is a member of the transforming growth factor-beta (TGF-beta) superfamily that is highly expressed in skeletal muscle.

Osteoactivin
Osteoactivin is a positive regulator of bone formation.

Osteo inductive Factor (OIF)
Osteoinductive factor (OIF) is a glycoprotein in bone that induces ectopic bone formation.

Vitamin D
Vitamin D maintains normal serum calcium levels and promotes the mineralization of the osteoid matrix. 1,25-(OH)2-vitamin D3 stimulates terminal differentiation of osteoclasts and promotes the mineralization of osteoid matrix produced by osteoblasts.

PTH
PHT regulates blood calcium concentration and promotes bone resorption and bone formation.

Calcium-sensing receptor, CaSR
CaSR is capable of sensing blood calcium levels and regulating PTH secretion and bone metabolism.

Calcium-binding protein
Calcium-binding protein plays an important role in regulating calcium ion balance and bone metabolism.

Calcitonin
Calcitonin inhibits osteoclastic bone resorption by decreasing the number and activity of osteoclasts.