OPG Fc Human

What is the molecular structure of human OPG-Fc?

Human OPG-Fc is a disulfide-linked homodimeric protein. Each monomer contains 380 residues from mature OPG and 243 residues from the Fc protein and linker. The OPG portion includes four TNF receptor (TNFR)-like domains (RANKL binding sites), two death domains (TRAIL binding sites), and a highly basic heparin-binding domain. Due to glycosylation, recombinant human OPG-Fc migrates as a 77 kDa protein in SDS-PAGE under reducing conditions, while the calculated molecular mass is 71 kDa . The mature OPG-Fc is produced through expression systems such as insect cells, yeast, or mammalian cells .

How does OPG-Fc differ structurally and functionally from native OPG?

Native OPG is a secreted 55-60 kDa glycoprotein that exists as a disulfide-linked homodimer (120 kDa) or as a monomer (60 kDa), with the dimer being more bioactive than the monomer . OPG-Fc retains the RANKL binding capability of native OPG but has significantly enhanced stability and circulating half-life due to the Fc fusion. The full-length OPG-Fc protein contains all functional domains of OPG, allowing it to bind not only RANKL but also TRAIL through its death domains and to interact with proteoglycans via its heparin-binding domain . This contrasts with truncated OPG-Fc versions that contain only the RANKL binding domains .

What are the physiological roles of OPG in tissue homeostasis?

OPG plays diverse roles across multiple tissue systems:

• Bone: OPG acts as a soluble decoy receptor for RANKL, preventing RANKL from binding to RANK and thus inhibiting osteoclastogenesis and bone resorption . The RANKL/OPG ratio is critical for bone modeling and remodeling, with changes in this ratio potentially leading to skeletal disorders .

• Muscle: OPG secreted by type II fast-twitch myofibers exerts anti-inflammatory effects and protects against muscle weakness and atrophy. Mice lacking OPG exhibit selective muscle atrophy of fast-twitch myofibers .

• Vascular system: OPG prevents vascular calcification, with OPG-deficient mice showing calcification of the aorta and renal arteries .

• Immune system: OPG regulates B cell maturation and development, with OPG-deficient mice showing accumulation of type 1 transitional B cells and isotype class switch defects .

How does the RANKL/OPG ratio affect biological functions?

The RANKL/OPG ratio is a critical determinant of bone homeostasis and various pathological conditions:

At normal physiological conditions, OPG and RANKL are in balance, linking bone resorption and formation. This balance can be disrupted by estrogen deficiency, inflammatory cytokines, and changes in hormone levels (glucocorticoids, thyroid hormones, parathyroid hormone, calcitriol) . Any modification in the RANKL/OPG ratio can induce either excessive bone resorption or formation, potentially leading to pathological conditions .

What are the optimal dosing strategies for OPG-Fc in different experimental models?

Optimal dosing of OPG-Fc varies by research model and target tissue:

When designing dosing strategies, researchers should consider that both high-dose (20 mg/kg twice weekly) and low-dose (1 mg/kg/week) OPG-Fc treatment resulted in osteopetrotic changes in infant mice , suggesting special caution is needed when studying OPG-Fc in developmental contexts.

What methods are most effective for detecting and quantifying OPG-Fc in experimental samples?

Several analytical approaches can be employed depending on research needs:

Human Osteoprotegerin ELISA:

• Sensitivity: Limit of Detection (LOD) = 0.03 pmol/l

• Specificity: No detectable cross-reactivity with human sRANKL and TRAIL at 120 pmol/l

• Cross-reactivity: Approximately 1% with recombinant mouse OPG, less than 0.06% with recombinant human CD40, rec. human sTNF RI and sTNF RII

• Performance metrics:

Sample Matrix Considerations:
Different anticoagulants affect measured OPG levels:

Freezing/thawing effects: No significant decline was observed in OPG concentration after repeated (5x) freeze/thaw cycles, though unnecessary repeated freezing/thawing should be avoided .

How can researchers distinguish between OPG-Fc effects on RANKL versus TRAIL pathways?

To differentiate between OPG-Fc effects on RANKL versus TRAIL signaling:

• Use modified OPG variants:

  • Truncated OPG-Fc (TR-OPG-Fc) containing only RANKL binding domains

  • OPG-Fc without the heparin-binding domain

  • Engineered OPG that selectively binds RANKL but not TRAIL

• Employ pathway-specific readouts:

  • For RANKL effects: Measure osteoclast numbers, TRACP-5b activity, bone morphometric parameters

  • For TRAIL effects: Assess apoptosis markers, caspase activation, cell viability in TRAIL-sensitive cell lines

• Conduct comparative studies with specific inhibitors:

  • Include RANK-Fc (specific RANKL inhibitor) as comparison

  • Use specific TRAIL inhibitors to determine which pathway mediates observed effects

• Analyze cell type-specific responses:

  • RANKL effects predominantly manifest in bone cells and immune cells

  • TRAIL effects are prominent in cells susceptible to TRAIL-induced apoptosis, including cancer cells

This methodological distinction is particularly important in cancer research, where the dual binding capability of OPG-Fc may produce complex effects on tumor progression .

What mechanisms regulate OPG expression and activity in different tissues?

OPG expression and activity are regulated through multiple mechanisms:

Interestingly, OPG-Fc treatment itself influences endogenous OPG expression. In the cortical compartment, OPG-Fc treatment reduced the proportion of OPG-expressing bone surface cells by 40% while increasing RANKL expression, suggesting a compensatory feedback mechanism .

How effective is human OPG-Fc in models of muscle injury and repair?

Human full-length OPG-Fc (hFL-OPG-Fc) demonstrates significant efficacy in muscle injury models:

Key findings from cardiotoxin (CTX)-induced muscle injury model:

• A 7-day hFL-OPG-Fc treatment improved force production of soleus muscle

• Enhanced muscle integrity and regeneration through multiple mechanisms:

  • Increased satellite cell density and fiber cross-sectional area

  • Attenuated neutrophil inflammatory cell infiltration at 3 and 7 days post-CTX injury

  • Increased anti-inflammatory M2 macrophages 7 days post-CTX injury

  • Favored M2 over M1 macrophage phenotypic polarization in vitro

In vitro effects:

• Improved myotube maturation and fusion

• Reduced cytotoxicity and cell apoptosis

These findings demonstrate that hFL-OPG-Fc has therapeutic potential for muscle diseases in which repair and regeneration are impaired, operating through mechanisms that involve both modulation of inflammatory responses and direct effects on muscle cell survival and differentiation .

What evidence supports OPG-Fc efficacy in preventing vascular calcification?

OPG-Fc demonstrates significant protective effects against vascular calcification:

Experimental evidence:

• In ldlr(-/-) mice fed an atherogenic diet, Fc-OPG treatment significantly reduced calcified lesion area without affecting atherosclerotic lesion size or number

• OPG-deficient mice exhibit marked calcification of the aorta and renal arteries, suggesting OPG normally protects against this process

• OPG regulates insulin-like growth factor 1 receptor (IGF1R) expression and activity, which can modulate vascular smooth muscle cell calcification in vitro

Clinical correlations:

• Elevated serum OPG levels are associated with cardiovascular complications in patients with type 1 diabetes

• Increased serum OPG levels are detected in patients with carotid calcification

These findings suggest that while elevated OPG in patients with cardiovascular disease may represent a compensatory response rather than a causative factor, therapeutic administration of OPG-Fc could potentially prevent or reduce vascular calcification .

How does OPG-Fc treatment affect bone modeling and remodeling in different age groups?

OPG-Fc effects on bone exhibit pronounced age-dependent differences:

In infant mice:

• Both high-dose (20 mg/kg twice weekly) and low-dose (1 mg/kg/week) OPG-Fc treatment resulted in radiographic and histologic osteopetrosis

• No evidence of bone modeling

• Negative tartrate-resistant acid phosphatase staining (indicating absence of osteoclasts)

• Root dentin abnormalities

• TRACP-5b activity suppression

In adult mice:

• OPG-Fc treatment induced a significant increase in bone mineral density (BMD) and trabecular bone volume

• Increased trabecular thickness and decreased trabecular separation

• BMD peaked at week 8 post-treatment initiation due to prolonged half-life of OPG-Fc

• After treatment withdrawal, BMD gradually decreased to vehicle levels by week 13

Cellular and molecular responses:

• OPG-Fc treatment significantly upregulated RANKL expression in trabecular bone surface cells compared to vehicle

• Percentage of RANKL-positive bone surface cells was significantly higher than in osteocytes and proximate marrow cells in OPG-Fc treated mice

• OPG-Fc treatment reduced the proportion of OPG-expressing cells in the primary spongiosa

This age-dependent difference in response highlights the importance of considering developmental stage when designing studies or therapeutic applications involving RANKL inhibitors.

What is the potential role of OPG-Fc in cancer research and therapy?

OPG-Fc has complex roles in cancer with both potential benefits and challenges:

Potential therapeutic applications:

• Prevention of bone metastasis and skeletal-related events by inhibiting RANKL-mediated osteoclastogenesis

• Modulation of tumor microenvironment through effects on immune cells and inflammatory processes

• Potential anti-angiogenic effects in certain contexts

Challenges and concerns:

• OPG binds to TRAIL and can inhibit TRAIL-induced apoptosis of cancer cells, potentially promoting tumor survival

• The effects of OPG in cancer are context-dependent and sometimes contradictory across different cancer types

• Elevated OPG expression correlates with worse outcomes in several cancers, including pancreatic cancer and oral squamous cell carcinoma

Advanced approaches:

• Development of engineered OPG variants that selectively bind RANKL but not TRAIL to avoid anti-apoptotic effects

• Potential replacement with more specific RANKL inhibitors like denosumab (AMG 162), which specifically targets RANKL without affecting TRAIL signaling

OPG is involved in multiple hallmarks of cancer, including tumor survival, epithelial-to-mesenchymal transition (EMT), neo-angiogenesis, and invasion , making it both a potential therapeutic target and a complex regulator of tumor biology.

How does OPG-Fc treatment alter the RANKL/OPG expression patterns in bone?

OPG-Fc treatment induces significant changes in RANKL and OPG expression patterns:

In trabecular bone:

• OPG-Fc significantly increased the percentage of RANKL-positive cells among bone surface cells compared to vehicle

• RANKL expression was significantly higher in bone surface cells compared to osteocytes and proximate marrow cells in OPG-Fc treated mice

• OPG expression was reduced in bone surface cells upon OPG-Fc treatment

In cortical bone:

• The percentage of RANKL-positive cells did not differ between vehicle and OPG-Fc-treated mice

• OPG-Fc treatment resulted in a 40% reduction in OPG-positive bone surface cells

• RANKL staining intensity in bone surface cells was increased by approximately 1.5-fold compared to vehicle

In growth plate region:

• OPG-Fc treatment significantly reduced the proportion of OPG-positive cells in the primary spongiosa

• The RANKL/OPG cell ratio significantly increased in cells in the primary spongiosa

• RANKL staining intensity was enhanced by OPG-Fc treatment in the primary spongiosa

These changes reflect a compensatory feedback mechanism where exogenous OPG-Fc suppresses endogenous OPG production while triggering increased RANKL expression, particularly in bone surface cells, likely as an attempt to restore bone remodeling homeostasis .

What explains the different responses to OPG-Fc between infant and adult animal models?

Multiple factors contribute to age-dependent differences in response to OPG-Fc:

• Developmental bone modeling: Infant skeletons undergo extensive modeling and rapid growth, making them more sensitive to disruptions in the RANKL/RANK/OPG axis. OPG-Fc treatment in infants resulted in severe osteopetrotic changes not seen in adults .

• Growth plate activity: OPG-Fc particularly affects the RANKL/OPG balance in the primary spongiosa of growth plates, with studies showing a more than 4-fold increase in the RANKL/OPG staining intensity ratio in this region .

• Higher baseline osteoclast activity: Growing skeletons have higher rates of bone turnover, amplifying the effects of osteoclast inhibition.

• Compensatory mechanisms: Adult animals may have more robust compensatory mechanisms to maintain bone homeostasis when the RANKL/OPG system is perturbed.

• Tooth development effects: OPG-Fc treatment in infant mice resulted in root dentin abnormalities, indicating effects on tooth development that would not occur in adults with fully formed dentition .

These findings suggest that RANKL inhibitors require careful evaluation before consideration for use in pediatric populations, as effects may be significantly different from those observed in adults .

How do we reconcile the apparent contradictions between OPG's roles in vascular health and disease?

The seemingly contradictory roles of OPG in vascular biology can be explained by several factors:

Dual roles of OPG in vascular biology:

• Protection against vascular calcification:

  • OPG-deficient mice develop aortic and renal artery calcification

  • Fc-OPG treatment in ldlr(-/-) mice reduced calcified lesion area

  • OPG regulates IGF1R expression and activity in vascular smooth muscle cells

• Association with cardiovascular disease:

  • Elevated serum OPG levels correlate with cardiovascular complications in type 1 diabetes

  • Increased OPG levels are seen in patients with carotid calcification

  • Serum OPG predicts mortality and cardiovascular events in patients with heart failure after myocardial infarction

Reconciliation framework:

• Compensatory response hypothesis: Elevated OPG in cardiovascular disease may represent a protective response rather than a causative factor

• Temporal progression: OPG may have different roles during initiation versus progression of vascular disease

• Mechanistic separation: OPG's effect on calcification may operate through different mechanisms than those affecting atherosclerosis progression

This is supported by research showing that Fc-OPG reduced vascular calcification without affecting atherosclerotic lesion number or size in ldlr(-/-) mice, suggesting OPG is an inhibitor of vascular calcification and potentially a compensatory response to atherosclerosis .

What molecular mechanisms underlie OPG-Fc effects beyond RANKL inhibition?

OPG-Fc exerts effects through multiple molecular pathways beyond simple RANKL inhibition:

• TRAIL binding and anti-apoptotic effects:

  • Through its death domains, OPG-Fc binds TRAIL and can protect against TRAIL-induced apoptosis

  • This creates potential concerns in cancer applications where TRAIL-induced apoptosis may be beneficial

• Heparin/proteoglycan interactions:

  • The heparin-binding domain of OPG interacts with cell surface proteoglycans and extracellular matrix components

  • OPG can bind to von Willebrand factor (vWF) and is stored within Weibel-Palade bodies in endothelial cells

  • Heparan sulfate proteoglycans on myeloma cell lines can bind OPG, leading to its internalization and degradation

• Inflammatory modulation:

  • OPG-Fc favors M2 over M1 macrophage phenotypic polarization in vitro

  • OPG activates Akt/PI3K signaling pathway in endothelial cells, leading to monocyte recruitment and adhesion

  • OPG influences B cell maturation and development

• Direct effects on muscle cells:

  • OPG-Fc improves myotube maturation and fusion in vitro

  • OPG secreted by type II fast-twitch myofibers protects pancreatic β cells against TNF-α-induced insulin resistance

These diverse mechanisms help explain the broad tissue effects of OPG-Fc beyond bone homeostasis and highlight the complexity of potential therapeutic applications.

What modifications to OPG-Fc have been developed to enhance specificity or functionality?

Several strategic modifications to OPG-Fc have been developed:

Particularly noteworthy is the development of engineered MSCs overexpressing OPG that selectively bind RANKL but not TRAIL, which significantly suppressed osteoclast activity induced by tumor cells without interfering with TRAIL-induced apoptosis .

What are the important considerations for translating OPG-Fc research to clinical applications?

Several critical factors must be considered for potential clinical translation:

• Age-dependent effects:

  • Both high and low doses of OPG-Fc caused osteopetrotic changes in infant mice

  • Special caution needed for pediatric applications

• Alternative approaches:

  • Denosumab (AMG 162), a human monoclonal antibody that specifically inhibits RANKL, has largely replaced OPG-Fc in clinical development

  • Denosumab avoids potential complications from TRAIL binding

• Treatment withdrawal effects:

  • After OPG-Fc discontinuation, compensatory increases in RANKL expression may cause rapid bone loss

  • Studies show signs of recovery appearing 4-8 weeks post-treatment in mice

• Target tissue specificity:

  • OPG-Fc affects multiple tissue systems beyond bone

  • Potential for off-target effects in vascular, immune, and other systems

• Cancer-specific considerations:

  • Dual roles in cancer progression through RANKL inhibition (potentially beneficial) and TRAIL inhibition (potentially detrimental)

  • Need for engineered variants with selective binding properties for cancer applications

The development of more targeted RANKL inhibitors like denosumab represents an example of how research insights from OPG-Fc studies have contributed to improved therapeutic approaches.

How do species differences affect research with human OPG-Fc in animal models?

Species differences create important considerations when using human OPG-Fc in animal studies:

Cross-species homology:

• Human OPG shares 85% amino acid identity with mouse OPG, 86% with rat OPG

• Sequence alignment shows that 85% of amino acids are identical, 6% are similar, and 9% are different between mouse and human OPG

Immunological considerations:

• The human Fc portion may elicit an immune response in animals, potentially limiting long-term studies

• Human OPG ELISA shows approximately 1% cross-reactivity with recombinant mouse OPG

Cross-reactivity in detection assays:

• Human OPG ELISA does not cross-react with bovine, cat, dog, goat, hamster, horse, mouse, pig, rabbit, or sheep sera

• Positive cross-reactivity is observed with monkey sera

Functional conservation:

• Despite sequence differences, human OPG-Fc shows functional activity in mouse models

• Both mouse and human OPG-Fc demonstrate beneficial effects in dystrophic muscle models

These species considerations are important for experimental design, data interpretation, and appropriate translation of findings to human applications.