Osteocrin Human, HEK

What is the molecular structure and characteristics of human Osteocrin?

Human Osteocrin is a single, glycosylated polypeptide chain spanning amino acids 28-133 of the full protein sequence, with a molecular mass of approximately 12.5 kDa. The recombinant form is typically fused to a 6-amino acid His-Tag at the C-terminus for purification purposes . The amino acid sequence of the recombinant protein is: VDVTTTEAFD SGVIDVQSTP TVREEKSATD LTAKLLLLDE LVSLENDVIE TKKKRSFSGF GSPLDRLSAG SVDHKGKQRK VVDHPKRRFG IPMDRIGRNR LSNSRGHHHH HH . The protein undergoes post-translational modifications, including glycosylation, which may be critical for its biological functions. Human Osteocrin proprotein shares 77% and 78% amino acid sequence identity with rat and mouse proteins, respectively .

How should Osteocrin Human be stored and handled for optimal stability?

For optimal stability, Osteocrin Human should be stored at 4°C if the entire vial will be used within 2-4 weeks. For longer storage periods, freezing at -20°C is recommended . To enhance stability during long-term storage, adding a carrier protein (0.1% HSA or BSA) is advised . Multiple freeze-thaw cycles should be strictly avoided as they can significantly compromise protein integrity and biological activity . When working with the lyophilized form, reconstitution should be performed in distilled water at appropriate concentrations, typically maintaining the protein in 20 mM Tris-HCl, 150 mM NaCl at pH 8.0 .

What are the primary biological functions of Osteocrin in different tissue systems?

Osteocrin demonstrates tissue-specific functions across multiple systems:

• Skeletal System: Enhances the anabolic effects of osteoblasts, promoting new bone formation . In zebrafish models, Osteocrin mutants exhibit impaired membranous and chondral bone formation, indicating its crucial role in osteogenesis .

• Cardiovascular System: Functions as an endocrine hormone secreted from cardiomyocytes that regulates blood pressure and cardiac hypertrophy . Recent studies demonstrate that Osteocrin attenuates inflammation, oxidative stress, and apoptosis in doxorubicin-induced cardiac injury models .

• Metabolic Function: May function as an autocrine and paracrine factor linked to glucose metabolism in skeletal muscle .

• Central Nervous System: Emerging evidence suggests a role in cognitive function and neuroprotection, though the precise mechanisms remain under investigation .

How does Osteocrin interact with natriuretic peptide signaling pathways?

Osteocrin's interaction with natriuretic peptide (NP) signaling represents a complex regulatory mechanism. Research indicates that Osteocrin may bind to NPR3 (clearance receptor for NPs), thereby enhancing the binding of natriuretic peptides to NPR1 or NPR2 . In osteoblast models, Osteocrin enhances C-type natriuretic peptide (CNP)-dependent nuclear export of YAP1/WWTR1 even when CNP is at saturable levels, suggesting that Osteocrin might activate unidentified receptors that augment protein kinase G signaling mediated by the CNP-NPR2 axis .

Experimental evidence from zebrafish models demonstrates that the impaired bone formation in Osteocrin mutants can be rescued by cardiomyocyte-specific overexpression of OSTN, confirming its endocrine role in bone development through natriuretic peptide-related signaling . For researchers investigating this pathway, it is advisable to employ functional ELISA with human NPRC to assess binding activity, with effective dose (ED50) typically ≤ 20 ng/ml .

What methodologies are optimal for studying Osteocrin's cardioprotective effects?

To effectively study Osteocrin's cardioprotective effects, several methodological approaches have proven valuable:

In vivo models:

• Cardiac-specific overexpression: Using cardiotropic adeno-associated virus serotype 9 (AAV9) vectors administered via tail vein injection (1 × 10^11 viral genome/mouse) to achieve cardiac-restricted OSTN overexpression .

• Cardiac injury models:

  • Acute injury: Single bolus injection of doxorubicin (15 mg/kg, i.p.)

  • Chronic injury: Weekly administration of doxorubicin (5 mg/kg) for 3 consecutive weeks

  • Diabetic cardiomyopathy: Intraperitoneal injections of streptozotocin (50 mg/kg) for 5 consecutive days

• Assessment parameters:

  • Cardiac function evaluation via echocardiography

  • Measurement of inflammatory markers, oxidative stress indicators, and apoptotic markers

  • Evaluation of PKG-dependent proteasomal activity

In vitro models:

• Cell culture: H9C2 cardiomyocyte cell lines cultured in DMEM with 10% FBS

• Treatment protocols:

  • Recombinant human OSTN (5μg/mL) treatment for 24 hours

  • Doxorubicin (1μmol/L) exposure for 24 hours

  • High glucose stimulation to mimic diabetic conditions

• Gene silencing: siRNA transfection (50 nmol/L) using Lipo6000 for 4 hours

How can researchers effectively measure Osteocrin levels in experimental samples?

Quantifying Osteocrin levels in experimental samples requires specific technical approaches:

• ELISA methodology: For blood samples, centrifuge at 4°C for 20 minutes before applying to pre-coated plates and incubating overnight at 4°C. Use anti-mouse/human OSTN rat antibody (1-hour incubation at room temperature), followed by alkaline phosphatase-conjugated donkey anti-rat antibody incubation. Detection can be performed at 535 nm using CDP-Star™ substrate with Emerald-II™ enhancer .

• Western blotting: For tissue or cell samples, standard Western blot protocols can be employed with specific anti-OSTN antibodies. Typically, samples should be normalized to housekeeping proteins such as GAPDH or β-actin.

• qRT-PCR: For expression analysis at the mRNA level, specific primers targeting OSTN can be used following standard RNA extraction and cDNA synthesis protocols.

• Immunohistochemistry/Immunofluorescence: These techniques can be valuable for localizing OSTN expression in tissue sections, particularly when studying its distribution in cardiac or bone tissues.

What are the key experimental controls needed when studying Osteocrin function?

When designing experiments to investigate Osteocrin function, several critical controls should be incorporated:

• Expression system controls: Since HEK293 cells are commonly used for Osteocrin expression, appropriate empty vector controls should be included to account for any effects of the expression system itself .

• His-tag controls: As recombinant Osteocrin typically contains a His-tag, control experiments with another His-tagged protein of similar size but unrelated function can help distinguish specific Osteocrin effects from potential tag-related effects .

• Signaling pathway validation: When studying PKG-dependent mechanisms, include both positive controls (PKG activators like sildenafil) and negative controls (PKG inhibitors like KT5823) .

• Gene silencing controls: For siRNA experiments targeting OSTN or related pathway components (e.g., PKG), non-targeting siRNA controls must be included to account for non-specific effects of the transfection process .

• Tissue-specific controls: When evaluating Osteocrin's effects across multiple tissues, tissue-specific markers should be monitored to confirm the specificity of observed responses in bone, cardiac, or neural tissues.

How can researchers optimize recombinant Osteocrin Human preparation for functional studies?

Optimizing recombinant Osteocrin preparation requires careful attention to several factors:

• Expression system selection: While E. coli-expressed Osteocrin is more cost-effective, HEK293-expressed protein provides proper glycosylation and post-translational modifications essential for certain functional studies . If specific post-translational modifications are critical to your research question, the HEK293 expression system is strongly recommended.

• Purification approach: Chromatographic techniques should be optimized to ensure high purity (>95% as determined by SDS-PAGE) . For His-tagged Osteocrin, immobilized metal affinity chromatography followed by size exclusion chromatography yields the best results.

• Endotoxin removal: For in vivo applications, ensure endotoxin levels are below 0.1 ng/μg (1 EU/μg) as determined by LAL test to prevent non-specific inflammatory responses .

• Activity verification: Prior to functional studies, verify protein activity using binding assays with NPRC receptors. The ED50 should be ≤20 ng/ml in functional ELISA with Human NPRC .

• Storage optimization: For projects requiring long-term use, aliquot the protein in single-use volumes to avoid repeated freeze-thaw cycles, and add carrier proteins like 0.1% HSA or BSA to enhance stability .

How can researchers address inconsistent results in Osteocrin signaling studies?

Inconsistent results in Osteocrin signaling studies can stem from multiple factors:

• Protein degradation: Osteocrin is sensitive to freeze-thaw cycles and improper storage. Ensure proper aliquoting and storage conditions, and validate protein integrity via SDS-PAGE before experiments .

• Receptor saturation: In natriuretic peptide signaling studies, pre-existing receptor saturation may mask Osteocrin effects. Consider dose-response experiments and temporal dynamics to identify optimal conditions for observing Osteocrin-specific effects .

• Cell type variations: Osteocrin's effects may vary significantly between cell types due to different receptor expression profiles. Characterize receptor expression (especially NPR3, NPR1, and NPR2) in your experimental system before interpreting Osteocrin effects .

• Contextual signaling: Osteocrin functions in complex signaling networks. The PKG pathway, YAP1/WWTR1 nuclear export, and proteasomal activity may all contribute to observed effects . Consider analyzing multiple pathway components simultaneously to obtain a comprehensive understanding.

• Experimental timing: For in vivo studies, particularly with cardiac injury models, the timing of Osteocrin administration relative to injury induction is critical. Pre-treatment, co-treatment, and post-treatment protocols may yield different results, reflecting distinct therapeutic vs. preventive effects .

What are the most effective approaches for studying Osteocrin's role in diabetic cardiomyopathy?

Based on recent research, the following approaches have proven effective for studying Osteocrin's role in diabetic cardiomyopathy:

• Animal model selection: The streptozotocin-induced diabetes model (50 mg/kg for 5 consecutive days) provides a reliable system for studying diabetic cardiomyopathy .

• Therapeutic intervention timing:

  • Prevention protocol: AAV9-OSTN administration prior to diabetes induction

  • Treatment protocol: AAV9-OSTN administration after diabetes establishment to evaluate reversibility of cardiac changes

• Mechanistic studies: Focus on proteasomal activity assessment, as Osteocrin prevents diabetic cardiomyopathy via restoring PKG-dependent proteasomal function . This can be complemented with analyses of:

  • Protein kinase B/forkhead box O1 phosphorylation status

  • PKG activity measurements

  • Ubiquitinated protein accumulation

• Combination therapies: Evaluate synergistic effects of Osteocrin with established cardioprotective agents such as sildenafil, which has shown promising results in combination therapies .

• Translation to human samples: Correlate findings with OSTN expression levels in human diabetic heart samples when available, to establish clinical relevance.

What are the emerging areas of investigation for Osteocrin beyond its established functions?

Several promising research directions for Osteocrin are emerging:

• Neurological applications: Preliminary evidence suggests Osteocrin may play roles in cognitive function and neuroprotection . Investigating its expression and function in neural tissues, particularly in neurodegenerative disease models, represents an exciting frontier.

• Metabolic regulation: Osteocrin's potential role in glucose metabolism in skeletal muscle warrants further investigation, particularly in the context of metabolic disorders like diabetes and obesity .

• Aging-related processes: Given its roles in both cardiovascular and skeletal systems, Osteocrin may have implications for age-related pathologies affecting these systems. Studying its expression and function across the lifespan could reveal new therapeutic opportunities.

• Receptor identification: While Osteocrin is proposed to interact with NPR3, evidence suggests it might activate unidentified receptors . Identifying these novel receptors could significantly advance our understanding of Osteocrin biology.

• Exercise physiology: As a myokine responsive to exercise, Osteocrin may mediate some of exercise's beneficial effects on multiple organ systems. Studying how exercise regimens modulate Osteocrin signaling could provide insights into exercise-based therapeutic interventions.

How might Osteocrin research impact therapeutic development for cardiac and bone disorders?

Osteocrin research has significant therapeutic implications:

• Cardioprotective therapies: Osteocrin's ability to attenuate inflammation, oxidative stress, and apoptosis in cardiac tissue suggests potential applications in treating various cardiac conditions . Development of Osteocrin mimetics or delivery systems for recombinant Osteocrin could provide novel cardioprotective strategies, particularly for:

  • Chemotherapy-induced cardiotoxicity

  • Diabetic cardiomyopathy

  • Heart failure with preserved ejection fraction

• Bone regeneration: Given its role in osteogenesis and chondrogenesis, Osteocrin-based therapies might enhance bone healing and regeneration . Potential applications include:

  • Osteoporosis treatment

  • Fracture healing acceleration

  • Bone tissue engineering

• Combination therapies: The synergistic effects observed between Osteocrin and established medications like sildenafil suggest potential for combination therapies that could enhance efficacy while reducing side effects .

• Diagnostic applications: Circulating Osteocrin levels might serve as biomarkers for certain cardiac or skeletal conditions, potentially aiding in early diagnosis or treatment monitoring.

• Targeted delivery systems: Development of tissue-specific delivery methods for Osteocrin, such as the cardiac-specific AAV9 vectors used in research, could translate into clinical applications with minimized off-target effects .