The Beginning of Bone Remodeling
Although bone appears to be a rigid and fixed structure with no change, in reality, it is a dynamic tissue that is constantly and continuously being remodeled for physiological reasons such as replacing micro-damaged or worn bone tissue caused by mechanical stress, or regulating blood calcium levels. What is often overlooked is the fact that this remodeling process always begins with resorption. First, old and damaged bone tissue must be broken down and removed, and the area must be thoroughly cleaned in order for new bone to be efficiently formed in that spot. Therefore, the first step in bone remodeling is the differentiation and activation of osteoclasts, which perform the resorptive work. And for this differentiation, signaling molecules (such as Sclerostin, RANKL) released by osteocytes that sense mechanical load reduction, microdamage, or increased calcium demand in the body are prerequisite. These signals allow bone resorption to proceed safely, strictly, and precisely.
1. Activation Phase
Recruitment and Differentiation of Osteoclast Precursors
In order for hematopoietic stem cells, which can differentiate into various types of blood and immune cells, to eventually differentiate into osteoclasts, two cytokines are required. The first is macrophage colony-stimulating factor (M-CSF), and the second is RANKL (Receptor Activator of Nuclear Factor κB Ligand). M-CSF is a hematopoietic growth factor secreted by osteoblasts and bone marrow stromal cells, and RANKL is mainly expressed by osteoblasts and osteocytes. M-CSF induces hematopoietic stem cells to differentiate into the monocyte/macrophage lineage. During this process, osteoclast precursor cells are formed, and RANK receptors are expressed on their cell membranes. In this state, when the osteoclast precursor cells reach the site in need of remodeling via the bloodstream or adjacent bone marrow, osteoblasts or osteocytes in that area express and secrete RANKL. RANKL binds to RANK receptors on the surface of osteoclast precursor cells. This binding activates intracellular signaling pathways, leading to complete differentiation into mature osteoclasts. This signaling process includes the cell-cell fusion of many osteoclast precursors with single nuclei, transforming into large multinucleated mature osteoclasts.
During differentiation, osteoclasts undergo various changes at the levels of gene expression, cell morphology, and energy metabolism. Because RANKL induces osteoclast differentiation and activation, it was also referred to as Osteoclast Differentiation Factor (ODF).
RANKL-RANK Binding
The binding of RANKL and RANK not only induces differentiation into osteoclasts, but also promotes gene expression necessary for mature osteoclasts to become functionally active and resorb bone. This binding recruits adaptor proteins such as TRAF6 (TNF receptor-associated factor 6), triggering a cascade of downstream signaling pathways. As a result, major transcription factors such as NF-κB, AP-1, and NFATc1 (nuclear factor of activated T cells 1) are sequentially activated. These three transcription factors together regulate the osteoclast-specific gene expression program, leading to the expression of key enzymes essential for bone matrix degradation.
Representative enzymes include TRAP (Tartrate-Resistant Acid Phosphatase), Cathepsin K which degrades collagen, and various proteins required to create an acidic environment. Therefore, if RANKL expression is insufficient, osteoclast function is suppressed, resulting in reduced bone resorption and abnormally increased bone density and thickness, a condition known as osteosclerosis. On the other hand, excessive RANKL expression can lead to osteoporosis due to excessive bone resorption.
The Competitor of RANK: Osteoprotegerin (OPG)
However, osteoblasts secrete not only RANKL, which promotes osteoclast differentiation and bone resorption, but also osteoprotegerin (OPG), which inhibits it. OPG is a glycoprotein that acts like a decoy receptor for RANKL, intercepting and binding RANKL before it can bind to RANK, thereby blocking the differentiation into osteoclasts. As a result, bone resorption is reduced, which is why OPG is also called osteoclastogenesis inhibitory factor (OCIF) or bone-protective ligand.
OPG is closely related to estrogen, a hormone known to affect bone density. Estrogen increases the expression of OPG, suppressing excessive osteoclast formation and activity, thereby preventing bone loss. However, after menopause, when estrogen secretion decreases, this protective effect is reduced, making osteoporosis more likely to occur. Conversely, excessive secretion of OPG can overly suppress osteoclasts, preventing timely removal of old bone tissue, which can lead to osteopetrosis. Osteopetrosis occurs when damaged or old bone is not resorbed but remains, lowering the quality of the bone while making it abnormally dense and brittle. Although the bone may appear dense on the surface, in reality, its durability is reduced, making fractures more likely.
RANKL and OPG Ratio
When RANKL increases and OPG expression decreases, osteoclastogenesis and bone resorption are promoted. Conversely, when RANKL decreases and OPG expression increases, osteoclast formation is suppressed and bone formation is promoted. Therefore, the relative ratio between RANKL and OPG secreted by osteoblasts is a key regulatory factor in determining the balance of bone remodeling. The RANKL:OPG ratio is a very important variable in maintaining bone homeostasis.
Balance Between Bone Resorption and Formation: Coupling of Osteoblasts and Osteoclasts
Maintaining bone homeostasis and normal bone remodeling depends on the precise balance between osteoclasts and osteoblasts. While osteoclasts break down and resorb bone, and osteoblasts build new bone, each cell type not only performs its own function faithfully but also regulates, activates, or suppresses the other through a complex interaction. This mutual regulation ensures that neither bone resorption nor formation becomes excessive, forming the core mechanism of bone remodeling. When the balance between resorption and formation is maintained, bone homeostasis is preserved. However, when this balance breaks down, bone diseases such as osteoporosis can develop. The close interaction between osteoclasts and osteoblasts throughout the remodeling process is referred to as coupling.
This coupling can be observed from the initial stages of remodeling, starting with the recruitment and differentiation of osteoclasts. As shown in the above figure, osteoblast-derived RANKL binds to the RANK receptors on osteoclast precursors recruited from the vasculature or bone marrow, promoting their differentiation into osteoclasts. Interestingly, the same figure suggests a hypothesis in which RANK receptors contained and secreted in vesicles can bind back to RANKL on the osteoblast membrane, promoting osteoblast differentiation. This possibility of bidirectional signaling implies a sophisticated communication between these two cell types.
Let us now explore in detail the various signaling networks and mechanisms through which osteoblasts and osteoclasts maintain this coupling relationship.
EFNB2 and EPHB4
Osteoclasts and osteoblasts communicate bidirectionally through Ephrin-Eph receptor signaling. This bidirectional interaction involves the membrane protein Ephrin B2 (EFNB2) on osteoclasts and the receptor EPHB4 on osteoblasts. When EPHB4 on osteoblasts binds and activates EFNB2 on osteoclasts, it inhibits osteoclast differentiation by blocking the NFATc1 signaling pathway, which is crucial for osteoclastogenesis. Conversely, when EFNB2 on osteoclasts activates EPHB4 on osteoblasts, it promotes osteoblast differentiation and inhibits apoptosis, thus facilitating bone formation. Consequently, higher expression of EPHB4 on osteoblasts enhances their survival and function while suppressing osteoclast differentiation, ultimately increasing bone mass. This EFNB2–EPHB4 signaling pathway is a representative example of osteoblast–osteoclast coupling.
FAS and FASL
FAS, a member of the tumor necrosis factor (TNF) family, contains a death domain and serves as a receptor that triggers programmed cell death (apoptosis) upon binding with its ligand FASL (FAS Ligand). Although the term "cell death" may sound alarming, apoptosis is an essential process in normal cellular activity. It allows damaged or unnecessary cells to be safely and cleanly removed without affecting surrounding cells. For instance, the tail of a sperm that has successfully fertilized an egg or the numerous sperm that failed to fertilize are naturally removed via apoptosis. In contrast, necrosis is a form of uncontrolled and accidental cell death caused by external stimuli or damage, which can result in cell rupture and inflammation.
So, how does estrogen, a hormone closely related to bone health and osteoporosis, interact with osteoclasts? When estrogen binds to estrogen receptors on osteoblasts, it stimulates the production and release of FASL. The FASL then binds to FAS receptors on osteoclasts, inducing their apoptosis. Following ovariectomy or menopause, estrogen levels decline, resulting in decreased FASL expression, reduced osteoclast apoptosis, and increased bone resorption, thus making it easy for osteoporosis to occur. In addition to inducing osteoclast apoptosis, estrogen also inhibits RANKL/M-CSF-induced AP-1-dependent transcription, suppressing osteoclast differentiation.[1] Furthermore, estrogen indirectly suppresses osteoclasts by increasing the production of osteoprotegerin (OPG), a decoy receptor for RANKL. Hence, estrogen can be regarded as a hormone that inhibits bone resorption.
Regarding bone formation, estrogen prolongs osteoblast survival and inhibits apoptosis. Estrogen also acts as an antioxidant by reducing reactive oxygen species (ROS), which are known to inhibit the Wnt/β-catenin pathway crucial for osteoblast differentiation.
In summary, estrogen decreases RANKL expression and increases OPG expression, inhibiting osteoclast differentiation. It also promotes osteoclast apoptosis, thereby reducing bone resorption. Simultaneously, it extends the lifespan of osteoblasts by inhibiting their apoptosis and promotes their differentiation through antioxidant activity. Therefore, when estrogen levels drop sharply after menopause, the balance between bone resorption and formation collapses, leading to accelerated bone loss and progression to osteoporosis. It is important to remember that estrogen does not only affect women; in men, a portion of testosterone is converted to estrogen, and a deficiency of estrogen can also increase the risk of osteoporosis in men.
Sema3A and NRP1
Semaphorin 3A (Sema3A) is a protein originally known for guiding axonal direction via chemorepulsion during neural development. However, it also plays a crucial role in bone remodeling. Sema3A, expressed by osteoblasts, binds to the Neuropilin-1 (NRP1) receptor on osteoclasts, inhibiting their differentiation. At the same time, it activates the Wnt/β-catenin pathway, promoting osteoblast differentiation. Thus, Sema3A functions as a powerful bone-protective factor by decreasing bone resorption and increasing bone formation.
TGF-β1 and IGF-1 Growth Factors
Transforming Growth Factor β1 (TGF-β1), one of the most abundant proteins in the bone matrix, remains inactive within the matrix until released during bone resorption by osteoclasts. Once released, it recruits mesenchymal stem cells to the resorption site, inducing their differentiation into osteoblasts. Insulin-like Growth Factor Type 1 (IGF-1), another growth factor stored in the bone matrix, is activated under the acidic conditions created during bone resorption. Once activated, it stimulates osteoblast lineage cells by activating the mammalian target of rapamycin (mTOR), thereby promoting bone formation.
Wnt/β-catenin Signaling Pathway: Ligands Related to Bone
Wnt proteins are glycoproteins that play critical roles in cell and tissue growth and development. Their signaling mechanisms are divided into β-catenin-dependent (canonical) and β-catenin-independent (non-canonical) pathways. β-catenin is a key messenger that conveys Wnt signals to the nucleus. The goal of the canonical Wnt/β-catenin pathway is to prevent β-catenin degradation by GSK-3 kinase in the cytoplasm, allowing it to translocate to the nucleus and activate target gene expression. This pathway plays a central role in bone formation and skeletal homeostasis.
Several Wnt ligands such as Wnt3a and Wnt1 prevent β-catenin degradation and promote its nuclear translocation, where it binds to transcription factors like TCF/LEF to induce expression of bone-related genes like Runx2 and Osterix. As a result, osteoblast precursors differentiate into mature osteoblasts, enhancing bone formation. This pathway also suppresses RANKL expression and increases OPG expression, inhibiting osteoclast differentiation and tipping the balance toward increased bone mass.
Conversely, Wnt5a functions through the non-canonical pathway, independent of β-catenin. It binds to the Ror2 receptor on osteoclast precursors, amplifying RANKL signaling and activating transcription factors such as NFATc1, thereby promoting osteoclast differentiation and multinucleation. Overexpression of Wnt5a can enhance bone resorption and is associated with osteoporosis.
Wnt16, primarily produced by differentiating osteoblasts, is a protective factor for bone mass. It promotes osteoblast differentiation and suppresses osteoclast formation. Wnt16 is strongly associated with bone mineral density (BMD), cortical thickness, bone strength, and risk of osteoporotic fractures. Wnt16 enhances osteoblast differentiation via the canonical Wnt/β-catenin pathway and suppresses RANKL expression while increasing OPG expression, thereby indirectly inhibiting osteoclastogenesis.
Wnt/β-catenin Inhibitors: Sclerostin and Dkk-1
Two major inhibitors of the Wnt/β-catenin pathway, which regulates osteoblast differentiation and function, are sclerostin and Dkk-1 (Dickkopf-related protein 1). Both are negative regulators that inhibit osteoblast activity and bone formation and are considered therapeutic targets for osteoporosis. They inhibit Wnt signaling by preventing Wnt ligands from binding to the Frizzled–LRP5/6 receptor complex.
Interestingly, decreased mechanical stimulation—such as reduced loading or pressure—leads osteocytes, acting as mechanosensors, to increase sclerostin expression, thereby inhibiting bone formation. Conversely, mechanical loading suppresses sclerostin, activating the Wnt pathway and promoting bone formation. This is explained by Wolff's law, which states that bone adapts its internal structure and shape to the mechanical stress it experiences. This explains why astronauts in zero gravity or bedridden patients experience reduced bone density. For those of us on Earth, regular mechanical stimulation, such as exercise, suppresses sclerostin expression and enhances bone formation, strengthening the skeleton.
Like sclerostin and Dkk-1, oxidative stress and reactive oxygen species (ROS) can also inhibit Wnt signaling. ROS oxidize β-catenin, destabilizing it and promoting its degradation, reducing its nuclear concentration and inhibiting transcription of target genes. Under oxidative stress, β-catenin preferentially binds to FOXO transcription factors instead of TCF/LEF, promoting antioxidant gene expression at the expense of bone-related gene expression, thereby suppressing bone formation.
[References]
[1] Estrogen and the Skeleton
https://pmc.ncbi.nlm.nih.gov/articles/PMC3424385/
