Osteoprotegerin and receptor activator of NF-kappaB ligand as biomarkers for bone resorption-related disorders

Abstract

Osteoclastic bone diseases represent a significant health burden, especially in an aging population, due to imbalances in bone homeostasis that result in postmenopausal osteoporosis, inflammatory arthritis, malignancy-induced bone disease and secondary metabolic conditions, which result in fragility fractures, disability and death. Although advances in imaging and pharmacology have enhanced management, existing diagnostic strategies have poor sensitivity in detecting early-stage pathological changes or fracture risk. The NF-kappaB/receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG) pathway is important in bone turnover regulation, which is dysregulated in bone degradation, leading to increased osteoclast numbers and bone degradation. Circular levels of RANKL and OPG, particularly the RANKL/OPG ratio, have shown potential as biomarkers for early diagnosis, monitoring, and evaluating therapeutic response, with the RANKL/OPG ratio reflecting the balance of osteoclastogenic activity in the bone microenvironment and correlating with bone mineral density, fracture risk, and bone pathology. It exhibits lower biological variability and enhanced sensitivity for early disease detection compared with individual markers. This review assesses the experimental, translational and clinical evidence for the predictive and monitoring potential of RANKL and OPG, and correlations between various imaging techniques and circulating biomarkers to gain insight into disease progression. We explore and discuss the limitations with regards to methodological issues, assay variability and biological variability, and the therapeutic implications of modulating the RANKL/OPG axis, highlighting its potential in informing treatment approaches and improving outcomes in bone resorptive diseases.

Key Words: Receptor activator of NF-kappaB; Receptor activator of NF-kappaB/receptor activator of NF-kappaB ligand; Osteoprotegerin axis; Osteoclast; Osteoporosis

Core Tip: The receptor activator of NF-kappaB/receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG) pathway is a pivotal bone remodeling pathway, and its disruption is responsible for osteoclast-driven bone resorption in osteoporosis, arthritis and bone-related cancer. Plasma RANKL and OPG, and their ratio, may be more sensitive indicators for diagnosis, fracture prediction and therapy assessment. While assays are variable, there is evidence that the RANKL/OPG ratio could be a better predictor than either marker alone, with potential to improve diagnosis, treatment and outcomes in bone resorption disease.

INTRODUCTION

Bone remodeling is a dynamic, well-coordinated and highly regulated life-long process essential for maintaining skeletal integrity, structure and metabolism[1]. Throughout life, bone undergoes a continuous bone remodeling process through coordinated cycles of resorption and formation, facilitating the repair of microstructural damage, adaptation to mechanical stress, and regulating calcium and phosphate metabolism[2]. Bone remodeling is tightly regulated by spatiotemporal coordination between osteoblast- and osteoclast-mediated bone formation and resorption activity, respectively, dependent on direct cellular communication[3]. Bone is typically comprised of type 1 collagen fibers, which are intricately woven and arranged together with crystals of calcium hydroxyapatite, conferring strength and flexibility to the bone[4]. Under physiological conditions, the equilibrium between formation and resorption preserves bone mass and microarchitecture. However, even subtle disruptions in this tightly regulated process can progressively impair skeletal integrity, leading to reduced bone mineral density, making it fragile and increasing fracture susceptibility[5]. Significantly, bone degradation often occurs gradually over time and is often undiagnosed until a fracture or deformity[6]. This process is normal during the aging process and occurs in both males and females after peak bone mass[7]. Bone resorption disorders, such as postmenopausal osteoporosis, inflammatory arthritis (IA) and bone diseases in cancer, are a significant global public health burden[8]. Any studies have demonstrated that patients with a low bone mass concentration are at a higher risk of all fracture types, and it was estimated that the number of hip fractures will rise by 240% in women and 310% in men by 2050[9]. These diseases are the major causes of fragility fracture, pain, disability and morbidity[10,11]. Fractures also cause a rise in mortality, loss of independence and a significant global healthcare burden[12]. Osteoporosis is a major global burden affecting more than 200 million people with 8.9 million fractures annually[13]. The clinical and socioeconomic impact is significant. With an aging population around the world, it is expected that these conditions will become more prevalent, putting a greater burden on the healthcare system and highlighting the need for better approaches for early detection, risk assessment and management of these conditions[14].

Despite improvements in diagnostic imaging and drug therapies, existing clinical methods still have limited capacity to detect early pathological changes in bone remodeling[15]. The World Health Organisation considers dual-energy X-ray absorptiometry to be the gold standard method for measuring bone mineral density. However, it measures total bone loss rather than active bone remodeling or molecular events that lead to disease[16]. Furthermore, bone mineral density fails to reflect bone quality, microarchitectural changes and fracture risk, especially in secondary bone diseases and inflammatory conditions. These factors have created a need for molecular biomarkers that may complement diagnostic tools, help in monitoring disease progression, shed light on disease activity, and allow early and accurate intervention[17]. Biomarkers of bone turnover, mostly proteins or their fragments, are released during bone remodeling and are essential to diagnose and treat various metabolic bone diseases[18].

The discovery of the receptor activator of NF-kappaB (RANK) receptor and its ligand RANK ligand (RANKL) in 1999 as a major osteoclastogenic factor and the identification of osteoprotegerin (OPG) as a soluble decoy receptor ushered in a new era in understanding the biology of osteoclast and bone remodeling[19]. These proteins are new members of the tumor necrosis factor (TNF) receptor superfamily and their discovery has greatly facilitated our understanding of how to inhibit osteoclastogenic potential, thus preserving bone from degradation[20]. Bone mesenchymal cells are important players in bone remodeling, as they express RANKL, which controls osteoclastogenesis and bone degradation[21]. Overall, the RANK/RANKL/OPG signaling pathway is the main regulatory pathway that leads to osteoclast differentiation, activation and survival, and provides a common molecular mechanism for the development of bone resorptive diseases[22]. Notably, the RANK/RANKL/OPG system brings together endocrine, immune and mechanical factors, and it sits at the intersection of skeletal and systemic physiology[23]. This pathway has been implicated in a variety of bone-related diseases, including postmenopausal and age-related osteoporosis, IA, and malignant bone erosion, whereas RANKL has been linked with cancers such as bone metastasis, breast cancer, multiple myeloma, urological cancers and lung carcinoma[24].

Unlike traditional molecular markers of bone turnover, which largely signal downstream metabolic end-products of bone matrix formation or resorption, components of the RANK/RANKL/OPG system may offer additional upstream regulatory messages that regulate osteoclastogenesis that could be leveraged for therapeutic monitoring[25]. Therefore, circulating RANKL and OPG serum levels, especially their ratio, have gained attention as promising biomarkers that may more accurately reflect the balance in the bone remodeling process[26]. Increasing evidence suggests that these biomarkers hold potential value not only for disease prediction and risk stratification but also for monitoring disease progression and therapeutic response[27]. Therapeutics that target the RANKL and OPG pathways have been recently studied widely in the context of skeletal disorders[28]. Despite the extensive research already performed on this signaling pathway, the existing literature primarily focuses on its molecular interactions and disease pathology[29].

In contrast to previous studies, this narrative review provides more insight into the prognostic and diagnostic value of these individual biomarkers, as well as their ratio in certain diseases. This review also provides information regarding the comparative value of the RANKL/OPG ratio over individual biomarkers and evaluates the inconsistencies across studies due to variability in methodological sampling techniques. It aims to comprehensively analyze current research and available data on circulating OPG and RANKL as predictive biomarkers in bone resorption disorders, with particular emphasis on serum ratios, correlations with imaging biomarkers, and clinical applications in treatment monitoring[30]. Therefore, this review aims to provide a more detailed understanding of the potential and limitations of these biomarkers in clinical practice. Several narrative reviews have examined the RANK/RANKL/OPG axis from mechanistic, disease-specific, and therapeutic perspectives. However, most lack the integration of circulating biomarkers, different methodological approaches and clinical applications. A comparative table highlighting the differences is presented in Table 1[26,31,32].

Table 1 Comparative literature analysis of the receptor activator of NF-kappaB/receptor activator of NF-kappaB ligand/osteoprotegerin axis.

Ref.	Focus	Limitations	Contribution to present review
Boyce and Xing[26]	Overview of RANK/RANKL/OPG pathway in bone biology	Primarily mechanistic focus with limited discussion of clinical biomarker applications	Extends molecular insights to clinical biomarker relevance across diseases
Khosla[31]	Role of RANKL/OPG in osteoporosis pathophysiology	Focused on osteoporosis with limited multi-disease perspective and no discussion on circulating biomarkers	Provides multi-disease perspective and emphasizes circulating biomarker utility
Di Cicco et al[32]	Role of RANK/RANKL/OPG signaling in osteoarthritis and targeted therapies	Disease-specific focus with limited emphasis on circulating biomarkers and methodological variability	Expands to multi-disease biomarker applications and includes methodological considerations
Present review	Integrated molecular, clinical, and biomarker-focused approach		Combines molecular mechanisms, biomarker evaluation, methodological considerations, and clinical applicability

RANKL: Receptor activator of NF-kappaB ligand; RANK: Receptor activator of NF-kappaB; OPG: Osteoprotegerin.

SEARCH STRATEGY

A search strategy was designed, and a comprehensive literature search was conducted across databases such as PubMed, Web of Science, Google Scholar, and Scopus. Relevant studies were selected for the study using keywords such as “RANKL”, “Osteoprotegerin”, “RANKL/OPG ratio”, “bone remodeling”, “osteoporosis”, and “biomarkers”. Clinical and experimental trials and reviews focused on the role of RANKL, OPG, and their ratio in bone metabolism and disease pathology were considered. The reference list for each article was also considered and reviewed to identify additional studies. Because it is a narrative review, there are no systematic inclusion and exclusion criteria, but only peer-reviewed, relevant, and highly valued articles were included in the study.

MOLECULAR BASIS OF THE RANK/RANKL/OPG AXIS

RANKL expression and osteoclastogenesis

RANKL, encoded by the TNFSF11 gene, is a TNF superfamily cytokine the that serves as a central mediator of osteoclastogenesis and is expressed across multiple bone-related cell types, including hypertrophic chondrocytes, osteoblasts, osteoclasts, and immature osteocytes[33]. Both membrane and soluble RANKL are involved in regulating osteoclast maturation and activation in normal and pathological conditions[34]. It is essential for bone metabolism and is a promising therapeutic target in diseases such as osteoporosis, arthritis and osteolytic metastasis[35]. The membrane-bound form is particularly potent, as it facilitates sustained cell-cell signaling within the bone microenvironment[36]. Under normal conditions, RANKL is predominantly produced by osteoblasts and osteocytes, with osteocytes being the major source of RANKL, now recognized as the principal regulators of osteoclast differentiation in adult bone remodeling[37]. Hence, osteocytes are the key in bone remodeling due to the action of RANKL and their production of sclerostin[38]. However, in pathological states such as IA, bone resorptive lesions, periodontal diseases, and malignancies, immune cells such as T cells, B cells, and synovial fibroblasts become additional sources of RANKL[39,40].

The binding of RANK receptor with its ligand on osteoclast precursors expressed via macrophage-colony stimulating factor stimulation initiates a series of intracellular signaling events responsible for osteoclastogenesis and subsequent bone resorption[41]. Upon activation, RANK mediates intracellular signal transduction via molecules such as tumor necrosis factor receptor-associated factors, resulting in the activation of nuclear factor-κB, mitogen-activated protein kinases, c-Jun N-terminal kinase, and calcium-dependent signaling pathways, that together drive osteoclast differentiation[42,43]. These signals converge on nuclear factor of activated T cells cytoplasmic 1, the master transcription factor governing osteoclastogenesis by regulating several osteoclast-specific genes such as tartrate-resistant acid phosphatase, cathepsin K, calcitonin receptor, and osteoclast-associated receptor through cooperation with microphthalmia-associated transcription factor and c-Fos[44,45]. Nuclear factor of activated T cells cytoplasmic 1 is responsible for the regulation of gene expression required for osteoclast fusion, cytoskeletal reorganization, acid secretion, and bone matrix degradation[46]. Persistent RANKL signaling not only increases osteoclast number but also enhances osteoclast survival, thereby amplifying cumulative bone resorption and contributing to progressive skeletal deterioration in chronic disease states such as malignancy, as shown in Figure 1[47].

Figure 1 Normal bone homeostasis and abnormal osteoclast activation by receptor activator of NF-kappaB ligand from metastatic cancer cells, resulting in bone resorption and increased pro-tumorigenic growth factors. RANKL: Receptor activator of NF-kappaB ligand; RANK: Receptor activator of NF-kappaB; OC: Osteoclast; OB: Osteoblast; IGF: Insulin-like growth factor; TGF: Transforming growth factor. Created in BioRender (Supplementary material).

OPG as an endogenous inhibitor

OPG, encoded by TNFRSF11B, is a soluble glycoprotein and a member of the TNF receptor family[48]. It is secreted primarily by osteoblast lineage cells, endothelial cells, and immune cells, playing a major role in bone remodeling, immunoregulation, vascular function, carcinogenesis, central thermoregulation, and fibrosis[20,49,50]. OPG inhibits osteoclast activation and differentiation by binding to RANKL, preventing its interaction with RANK[51]. Through this mechanism, OPG regulates the bone resorption process, maintaining skeletal integrity[52].

Beyond skeletal regulation, OPG exhibits pleiotropic biological functions by binding to TNF-related apoptosis-inducing ligand, which further binds to its death domain-containing receptors DR4 and DR5, inducing apoptosis[53]. Due to this interaction, OPG has been associated with cardiovascular disorders and is involved in immune regulation, vascular homeostasis, and endothelial biology[54,55]. OPG is associated with accelerating the atherosclerotic process, explaining the underlying reason for poor clinical outcomes in patients with high OPG concentrations[56]. These extra-skeletal effects are clinically relevant when interpreting circulating OPG concentrations, as elevated levels may reflect systemic inflammation or vascular pathology rather than purely skeletal responses[57,58].

Physiological regulation and pathological imbalance

Physiological bone remodeling depends on a finely regulated equilibrium between RANKL and OPG expression[3]. The equilibrium between osteoblastic and osteoclastic activity is essential for maintaining the bone remodeling process that occurs through expression of RANKL, OPG, and certain hormones that occur over weeks[59]. Hormonal regulators, including estrogen, testosterone, parathyroid hormone, glucocorticoids, growth hormone, erythropoietin, insulin-like growth factor 1, vitamin D, and intestinal and adipocyte hormones, modulate this balance at both the transcriptional and post-transcriptional levels[60]. Estrogen deficiency is often associated with accelerated bone loss, enhanced osteoclastogenesis, and fragile bones due to increased RANKL and decreased OPG expression[61]. Parathyroid hormone, insulin-like growth factor 1, and testosterone upregulate bone formation, increasing bone mineral density and preventing bone resorption[62].

This balance is supported by evidence from animal models. Mice lacking OPG suffer from severe osteoporosis and spontaneous fractures, while osteopetrosis is observed in transgenic mice because of increased OPG levels, which inhibits osteoclastogenesis[63,64]. Bone turnover is a highly regulated process controlled by the RANKL/OPG ratio, with a relative increase in RANKL favoring bone degradation[65,66]. In humans, pathological changes in the RANKL/OPG ratio have been reported in several bone-resorptive diseases, suggesting the potential diagnostic use of this ratio as a composite biomarker[32,67].

Analytical and methodological considerations of circulating OPG and RANKL as biomarkers

Circulating OPG and RANKL measurements have gained attention as minimally invasive biomarkers that provide insight into systemic osteoclastogenic activity[68]. The clinical interpretation of circulating RANKL and OPG levels is significantly influenced by methodological variability in their measurements[69,70]. In most studies, enzyme-linked immunosorbent assays (ELISAs) are employed to quantify serum or plasma concentrations[71]. However, all of these commercially available assays vary in their design, including differences in antibody specificity, detection sensitivity, calibration standards, and the ability to distinguish between free and bound RANKL. These variations contribute to wide inter-study variability in reported biomarker levels, hindering the definition of standardized reference ranges suitable for routine clinical use[72-74].

Preanalytical variables further influence biomarker measurements and include sample type, whether serum or plasma was collected, processing time, storage conditions, freeze-thaw cycles, and diurnal variation[75]. In addition, circulating levels of OPG and RANKL may be affected by age, sex, renal function, inflammatory status, and comorbid cardiovascular disease[76,77]. For example, an imbalance has been found in multiple myeloma, where OPG was profoundly decreased and RANKL concentration was significantly higher[78]. Importantly, circulating concentrations do not fully reflect local RANKL and OPG activity within the bone microenvironment, where paracrine and cell-cell interactions predominate and their concentrations vary with pregnancy, age, inflammation, and renal failure[79,80]. This lack of a standardized system and protocol for biomarker detection limits their routine use in clinical application[81]. Despite these limitations, systemic measurements provide valuable integrative information in diseases with generalized skeletal involvement[82]. OPG binds with an affinity to RANKL 500 times stronger than RANK at unique sites, effectively inhibiting osteoclastogenesis[41]. Accumulating evidence indicates that the OPG/RANKL ratio offers greater biological relevance than absolute concentrations alone by capturing the net balance between pro-resorptive and anti-resorptive signaling forces[30,83].

Osteoporosis and age-associated bone loss

Osteoporosis is a prominent skeletal disorder in the elderly population characterized by a progressive decrease in bone mass and alteration in its architectural integrity that requires proper treatment and management[84]. Osteoporosis is more common in elderly females; many studies have reported that 40%-50% of women over 60 years old are affected by osteoporosis[85]. Postmenopausal osteoporosis represents the most extensively studied clinical paradigm of RANK/RANKL/OPG dysregulation and provides a mechanistic framework for understanding age-related bone loss[86,87]. Estrogen is critical in bone remodeling and acts through two major receptors to modulate the RANKL/OPG axis, estrogen receptor-alpha and estrogen receptor-beta[88]. After menopause, estrogen deficiency results in a persistent increase in osteoclast activity and impaired bone remodeling processes due to increased RANKL expression and decreased OPG expression[89]. The dysregulated RANKL/OPG ratio uncouples the bone remodeling processes, causing bone mass and integrity loss[90].

Studies evaluating circulating OPG and RANKL in postmenopausal women have produced variable results due to differences in study populations, disease stage and methodological variations[64,91-93], thus limiting their interpretation into clinical practice and their utility as diagnostic markers. However, when assessed over time or in large populations, there are consistent trends of a decreased OPG/RANKL ratio correlating with lower bone mineral density and incidence of fragility fractures. Measurements of circulating OPG and RANKL have been proposed as clinically useful, non-invasive biomarkers of systemic osteoclastogenesis[68]. However, analytical variations in circulating RANKL and OPG measurements have a profound impact on their clinical interpretations[94-96].

Bone loss associated with rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory bone destruction, focal erosions in the joints and systemic bone fragility[97]. Recent studies have demonstrated that joint and bone erosions in patients with RA result from RANKL overexpression in the synovial tissue of joints[98-100]. Chronic inflammation in the synovium results in the infiltration of activated T cells, macrophages, and fibroblast-like synoviocytes, which increase RANKL production and promote osteoclast differentiation at the bone/pannus junction. This osteoclast activation leads to the formation of marginal erosions seen in RA[101].

In addition to local bone destruction, systemic inflammation in RA results in altered bone metabolism, systemic osteoporosis, and fractures[102]. In patients with RA, increased systemic RANKL levels and decreased OPG/RANKL ratios have been linked to disease activity, radiographic joint damage and systemic bone degradation[103]. TNF-α plays a key role in inflammation and bone erosion in RA. Critical to this process, successful anti-inflammatory treatment with biological agents, especially TNF-α inhibitors, decreases RANKL levels, alleviates symptoms, and partially restores the biomarker profile and slows the rate of progression of damage[104,105]. These observations underscore the utility of RANK/RANKL/OPG biomarkers not only as markers of inflammatory bone destruction but also as tools for monitoring therapeutic response[106]. While these findings suggest a role in disease activity, their utility as reliable clinical biomarkers remain uncertain due to heterogeneity across studies.

Cancer-associated bone disease

Cancer is a rising global burden affecting nearly 12.7 million people worldwide, resulting in mortality of over 7 million people as of 2008[107]. Tumor cells can directly alter the bone microenvironment, favoring bone resorption. Furthermore, anti-tumor therapies can further suppress bone formation and accelerate bone resorption, resulting in cancer-associated bone disease[17]. Cancer-associated bone diseases result in alteration of the microenvironment due to the complex interactions between the different components, resulting in excessive osteoclast activation and skeletal destruction triggered by pro-inflammatory cytokines, which are overactivated in pathological conditions[108,109]. Tumor cells secrete cytokines, growth factors, and extracellular vesicles that stimulate RANKL expression by osteoblasts, stromal cells, and immune cells, thereby promoting osteoclastogenesis, causing further release of growth factors from the bone matrix, such as tumor growth factor β and insulin-like growth factor 1, which in turn stimulates parathyroid hormone-related peptide production and promotes tumor growth[110]. This process establishes a self-perpetuating “vicious cycle” in which tumor growth and survival are established due to growth factors and cytokines released during bone resorption[111].

Clinical studies have frequently demonstrated elevated circulating RANKL levels and reduced OPG concentrations in patients with renal cancer, prostate cancer, oral cancer, breast cancer, and multiple myeloma with bone metastases[112]. Alterations in the RANKL/OPG ratio have been associated with higher risk of skeletal-related pathological states, disease burden, and poor clinical outcomes[83,113]. As a result, this ratio provides integrated results and serves as a potential biomarker that can aid in evaluating disease progression and therapeutic monitoring to antiresorptive therapies such as denosumab and bisphosphonates[26,114]. However, these changes may be affected by several systemic factors and may lack clinical value as a single factor[115]. Overall, there is a clear rationale to include RANK/RANKL/OPG axis profiling in the clinical assessment of patients with oncologic bone disease[116-118].

Secondary and rare bone disorders

The RANK/RANKL/OPG axis plays a key role in bone remodeling and bone homeostasis, and is therefore involved in a wide range of secondary and rare bone diseases[119]. In glucocorticoid-induced osteoporosis, glucocorticoids directly up-regulate RANKL expression, facilitating differentiation of bone-marrow derived macrophages into osteoclasts, stimulating bone degradation while down-regulating OPG expression, resulting in severe and rapid bone degradation[120]. Adverse effects in patients due to prolonged glucocorticoid use include increased fracture risk, cortical porosity and reduced bone mineral density, resulting in an increased burden and reduced quality of life[121,122].

Elevated circulating OPG levels have been reported in chronic kidney disease, which is a mineral and bone disorder that may reflect a complex interplay between skeletal pathology, systemic inflammation, and vascular calcification[123]. Immobilization-induced bone loss following spinal cord injury, stroke, multiple sclerosis, amyotrophic lateral sclerosis, or prolonged bed rest is similarly characterized by increased osteoclast activity and altered RANKL/OPG dynamics, resulting in rapid skeletal deterioration[124,125]. In fibrous dysplasia, excessive RANKL expression drives abnormal osteoclast activation that results in bone resorption by forming resorption pits, making RANKL a potential therapeutic target in this disorder[126,127].

Critical analysis of evidence

Although numerous studies have demonstrated the association between RANKL, OPG, and disease states, the results are still inconsistent. These discrepancies are primarily due to heterogeneity in patient characteristics such as age, sex, ethnicity, and disease severity across studies[96,128]. As discussed above, variations in methodology and differences in study design further introduce heterogeneity[98]. Bone remodeling is a intricately regulated process, influenced strongly by hormones and other systemic conditions that may contribute to these inconsistencies[26]. Hence, though these biomarkers have high clinical utility, more extensive, large-scale, and longitudinal studies need to be performed to fully elucidate their validity.

Imaging biomarkers and correlation with serum markers

While dual-energy X-ray absorptiometry is considered the gold standard for checking bone mineral density, it has limitations, as it does not provide adequate details regarding bone quality and its microarchitecture[129]. Bone mineral density does not provide us with complete information about bone strength, quality, and fragility in the evaluation of bone resorption disorders that may not predict future fracture risk, giving rise to the need for advanced imaging modalities[130]. One such imaging modality is high-resolution peripheral quantitative computed tomography, which can provide detailed assessment of trabecular and cortical bone microarchitecture, helping to integrate the deterioration results in imaging with the biochemical markers in serum for evaluating skeletal disorders[131,132].

Magnetic resonance imaging (MRI) has particular relevance in IA, where it can detect active inflammation and bone marrow edema indicative of active osteitis, which also serves as a strong predictor of subsequent erosion development[133]. MRI is the preferred imaging modality for IA, as it has the capacity to detect early disease and is useful in monitoring treatment efficacy[134]. Significantly, biopsy-proven inflammation at MRI sites is associated with local increases in RANKL, demonstrating a connection between imaging and molecular pathways of osteoclast activation[135]. Combining the latest imaging techniques with serum levels of RANK/RANKL/OPG offers a comprehensive evaluation of disease activity and progression[136]. Yet, more research needs analyze the clinical integration of these imaging modalities and biochemical markers for clinical decision-making.

TREATMENT MONITORING AND THERAPEUTIC IMPLICATIONS

RANKL inhibition

Because of its central role in bone resorption disorders, a human monoclonal antibody that targets RANKL (denosumab) was developed[137]. By functionally mimicking the action of OPG, denosumab potently suppresses osteoclast formation, activity, and survival, increasing bone mass, strength, and mineral density, further reducing the risk of fractures in both osteoporosis and malignancy-associated bone disease[138-140]. Side effects associated with denosumab therapy are low calcium and phosphate in the blood, causing muscle cramps, and rarely cellulitis and numbness, with hypocalcemia being the most common[141]. Because denosumab directly targets the RANK/RANKL/OPG axis, serial monitoring of circulating RANKL and OPG levels reflects pharmacodynamic effects and may assist in evaluating treatment adherence, efficacy, and the risk of rebound bone loss following discontinuation[142,143].

Other antiresorptive and anabolic therapies

Bisphosphonates, selective estrogen receptor modulators, calcitonin, and anabolic agents such as parathyroid hormone analogs exert their skeletal effects through indirect modulation of osteoblast-osteoclast coupling and remodeling dynamics[144]. These therapies influence the RANK/RANKL/OPG axis by altering osteoblast function, inflammatory signaling, and bone turnover rates[145,146]. Biomarker profiling may therefore support individualized therapeutic decision making, optimization of treatment duration, and strategic sequencing of these therapeutic agents, especially in patients with complex or refractory disease[147]. The functional treatment options involved in preventing osteoclast stimulation from the pathological activation of the RANK/RANKL/OPG axis are given in Figure 2.

Figure 2 Management options in preventing osteoclast stimulation from the pathological activation of the receptor activator of NF-kappaB/receptor activator of NF-kappaB ligand/osteoprotegerin axis. RANKL: Receptor activator of NF-kappaB ligand; RANK: Receptor activator of NF-kappaB; OC: Osteoclast; OB: Osteoblast; IGF: Insulin-like growth factor; TGF: Transforming growth factor. Created in BioRender (Supplementary material).

Limitations and challenges

Despite strong biological rationale and emerging clinical evidence, there are inconsistencies in the results due to a wide range of factors that limit the routine clinical application of RANK/RANKL/OPG biomarkers[69,148]. These include the time of sampling, sample storage, assay variability, methods of collection, and confounding effects of age, gender, inflammation, renal dysfunction, and cardiovascular disease[82,149-152]. Additionally, circadian rhythms and seasonal variation may influence circulating biomarker levels, underscoring the need for standardized sampling protocols and careful clinical interpretation[153]. Targeting the RANK/RANKL/OPG pathway has shown significant therapeutic potential, but it still has its limitations where different patients have different responses to the therapies, with some showing suboptimal responses[24]. Discontinuing anti-RANKL therapy is associated with a rebound increase in bone resorption and an increased susceptibility to fractures, posing a great limitation to the therapy[154]. Furthermore, the potential development of resistance in long-term management and the influence of systemic conditions on these therapies affecting the outcome are other limitations that need to be evaluated properly. Hence, an individualized treatment approach is likely required, in addition to biomarkers that can reliably predict therapeutic response[155]. The regulations and limitations of targeting the RANK/RANKL/OPG axis are summarized in Table 2[156,157].

Table 2 Summary of regulations and limitations of markers of receptor activator of NF-kappaB/receptor activator of NF-kappaB ligand/osteoprotegerin axis.

Marker	Type	Clinical use	Variability	Limitations
RANKL	Regulatory	Experimental	High	Assay variability, not standardized
OPG	Regulatory	Experimental	Moderate	Influenced by multiple systemic factors
RANKL/OPG ratio	Integrated marker	Emerging	Moderate	Lack of standardized cut-offs
PINP[156]	Bone formation marker	Established	Low	Reflects slower changes
β-CTX[157]	Bone resorption marker	Established	Moderate	Diurnal variation

RANKL: Receptor activator of NF-kappaB ligand; OPG: Osteoprotegerin; PINP: Procollagen type I N-terminal propeptide; CTX: C-terminal telopeptide of type I collagen.

Future perspectives

Subsequent studies in this area should aim to enhance consistency in laboratory methods and develop robust reference data from large, well-defined populations that reflect differences in age, sex, and ethnicity, amounting to the confounding factors. Future studies should strive to standardize protocols for the assays and establish normal reference ranges for RANKL and OPG. Combining RANK/RANKL/OPG biomarkers with imaging, artificial intelligence-based models, and conventional clinical assessment may enhance the accuracy of predicting early disease onset, fracture risks and disease progression. Knowledge of systemic biology and new tools such as single-cell sequencing may also help us better understand how the various regulatory networks such as hormones, inflammatory factors, and genetic factors work in concert with RANKL/OPG to regulate bone remodeling and help us better define the clinical significance of these biomarkers. Large-scale studies, multicenter and longitudinal studies need to be conducted to validate their use and to translate them to the clinical setting[16,132,134,136,158-160].

CONCLUSION

The RANK/RANKL/OPG pathway plays a critical role in bone turnover, activating and driving osteoclast differentiation. Abnormalities lead to bone homeostasis imbalance, promoting osteoporosis, inflammatory joint conditions, bone destruction in cancer and rare bone diseases. Serum concentrations of RANKL and OPG are variable, but their ratio is more informative for net osteoclastogenic activity because it combines opposing signals. When used alongside innovative imaging technologies, including high-resolution peripheral quantitative computed tomography and MRI, these biomarkers improve evaluation of bone mineral and microarchitecture, fracture risk, and disease progression. While these approaches can enhance assessment of disease staging and response to treatment, as seen with denosumab, which acts on this pathway and results in biomarker changes, significant issues persist. These include assay precision, lack of reference ranges and confounding effects of aging, kidney disease, systemic inflammation and heart disease. To advance these tools, multi-disciplinary translational research is required to enhance assay accuracy, provide reliable reference ranges in a broad population, and combine biomarkers with imaging and clinical measures. This will facilitate risk assessment, early diagnosis and tailored therapies. As bone biology progresses, the RANK/RANKL/OPG pathway will continue to play a pivotal role in the diagnosis and treatment of bone resorption diseases.