OSM Human, 209 a.a

What is OSM Human, 209 a.a?

OSM Human, 209 a.a refers to recombinant human Oncostatin M containing 209 amino acids. This multifunctional cytokine belongs to the Interleukin-6 (IL-6) subfamily, which also includes IL-11, leukemia inhibitory factor (LIF), ciliary neurotropic factor, cardiotrophin-1, and novel neurotropin-1. In research contexts, it functions as a growth regulator that can both stimulate and inhibit cell proliferation depending on the target cells. OSM plays roles in regulating neurogenesis, osteogenesis, and hematopoiesis, making it an important target for studying various physiological and pathological processes .

What cellular sources produce OSM in vivo?

In the human body, OSM is primarily produced by activated immune and inflammatory cells. Understanding these natural cellular sources is important when designing experiments to study physiological conditions where OSM might play a regulatory role. The primary sources include:

• Activated T lymphocytes

• Monocytes and macrophages

• Neutrophils

• Endothelial cells

• Kaposi's sarcoma cells

This production pattern suggests OSM plays important roles in immune response coordination and inflammation resolution, which should be considered when designing experimental models.

What are the primary biological functions of OSM?

OSM exhibits pleiotropic effects across multiple biological systems, including:

Growth Regulation:

• Stimulates proliferation of fibroblasts, smooth muscle cells, megakaryocytes, and vascular endothelial cells

• Inhibits proliferation of several tumor cell lines, including solid tissue tumors, lung cancer, melanoma, and breast cancer cells

Cytokine Regulation:

• Regulates production of IL-6, G-CSF, and GM-CSF from endothelial cells

• Coordinates cytokine network responses in inflammatory settings

Developmental Processes:

• Participates in regulation of neurogenesis

• Involved in osteogenesis

• Contributes to hematopoiesis

Other Functions:

• Enhances expression of low-density lipoprotein receptor in hepatoma cells

• Plays roles in inflammatory responses and acute phase protein induction

How should OSM Human, 209 a.a be stored and handled in the laboratory?

Proper storage and handling are critical for maintaining the biological activity of OSM. Researchers should follow these guidelines:

Storage Recommendations:

Handling Guidelines:

• Avoid repeated freeze-thaw cycles as these significantly reduce biological activity

• Allow lyophilized protein to equilibrate to room temperature before opening

• Use sterile techniques when handling the reconstituted protein

• Aliquot reconstituted protein into smaller volumes to minimize freeze-thaw cycles

Following these storage protocols will help ensure consistent experimental outcomes and reproducibility in OSM-related research.

What reconstitution protocols are recommended for OSM Human, 209 a.a?

For optimal reconstitution of lyophilized OSM Human, 209 a.a, follow this step-by-step protocol:

• Allow the lyophilized protein to reach room temperature before opening the vial

• Reconstitute in sterile water or PBS to a concentration of at least 100 μg/ml

• Gently mix by swirling or rotating the vial until completely dissolved (avoid vigorous shaking or vortexing)

• For further dilutions, the reconstituted protein can be diluted in buffers containing carrier proteins

• For long-term storage of reconstituted protein, it is recommended to add a carrier protein (0.1% HSA or BSA)

The solution should be clear after reconstitution. Any particulates or cloudiness may indicate protein degradation or denaturation. If this occurs, the preparation should not be used for experiments as it may yield unreliable results.

How can the biological activity of OSM Human, 209 a.a be assessed?

The biological activity of OSM Human, 209 a.a can be quantitatively assessed through several established methods:

• Cell Proliferation Assays:

  • Using TF-1 human erythroleukemic cell line

  • The ED50 (effective dose inducing 50% maximal response) is typically < 2-10 ng/ml

  • This corresponds to a specific activity of approximately 5×10^5 IU/mg or > 1×10^5 units/mg

• Growth Inhibition Assays:

  • Using cancer cell lines such as A375 melanoma or MCF-7 breast cancer cells

  • Measuring decreased proliferation or cell cycle arrest after OSM treatment

• Cytokine Induction Assays:

  • Measuring IL-6, G-CSF, or GM-CSF production from stimulated endothelial cells

  • Quantification using ELISA or cytometric bead array techniques

• Signaling Pathway Activation:

  • Monitoring phosphorylation of downstream signaling molecules (JAK/STAT, MAPK)

  • Using Western blotting or phospho-specific flow cytometry

These assays provide complementary information about OSM activity and should be selected based on the specific research question being addressed.

What cell lines are commonly used to test OSM activity?

Several established cell lines are particularly useful for studying OSM activity in different research contexts:

When selecting a cell line, researchers should consider receptor expression profiles (gp130, OSMR-β, LIFR-β) and the specific OSM-induced response they aim to study, as OSM effects can vary significantly between different cell types .

How does OSM Human, 209 a.a interact with different receptor complexes?

OSM exhibits a unique receptor binding pattern that distinguishes it from other IL-6 family cytokines:

• Receptor Complexes:

  • OSM can signal through two distinct receptor complexes:
    a) gp130/OSMR-β complex (Type II OSM receptor) - specific to OSM
    b) gp130/LIFR-β complex (Type I OSM receptor) - shared with LIF

• Binding Mechanism:

  • OSM first binds to gp130

  • This binding facilitates recruitment of either OSMR-β or LIFR-β

  • The resulting ternary complex initiates intracellular signaling cascades

• Species Specificity:

  • Human OSM can signal through both Type I and Type II receptors

  • Human OSM can also activate mouse cells, suggesting cross-species activity

  • This has important implications for using human OSM in murine experimental models

Understanding these receptor interactions is crucial for designing experiments targeting specific OSM signaling pathways and for interpreting results in different experimental systems. Researchers should consider receptor expression patterns when selecting cell types for OSM studies.

What signaling pathways are activated by OSM?

OSM activates multiple intracellular signaling pathways that mediate its diverse biological effects:

• JAK/STAT Pathway:

  • Primary pathway activated by OSM

  • JAK1, JAK2, and TYK2 kinases are activated upon receptor dimerization

  • Leads to phosphorylation and activation of STAT1, STAT3, and STAT5

  • STAT3 activation is particularly important for many OSM-mediated responses

• MAPK/ERK Pathway:

  • OSM activates the Ras-Raf-MEK-ERK cascade

  • ERK1/2 phosphorylation leads to activation of various transcription factors

  • Important for OSM-induced proliferative effects

• PI3K/AKT Pathway:

  • OSM can activate PI3K, leading to AKT phosphorylation

  • Contributes to cell survival signals and metabolic regulation

These pathways can be experimentally monitored to assess OSM activity and to understand the mechanisms underlying specific cellular responses. The activation patterns vary depending on cell type, receptor expression levels, and cellular context, contributing to the pleiotropic effects of OSM .

How should researchers distinguish between OSM and other IL-6 family cytokines in experimental design?

OSM shares structural and functional characteristics with other IL-6 family cytokines, but also has unique properties that should be considered in experimental design:

When designing experiments involving multiple IL-6 family cytokines, researchers should:

• Use receptor-specific blocking antibodies to delineate pathway contributions

• Compare dose-response relationships across different cytokines

• Consider cell-type specific receptor expression patterns

• Include appropriate positive and negative controls for each cytokine

What are species-specific considerations when using human OSM in murine experimental models?

Species-specific considerations are crucial when planning cross-species experiments with OSM:

• Receptor Specificity Differences:

  • Human OSM can signal through both Type I (gp130/LIFR-β) and Type II (gp130/OSMR-β) receptors in human cells

  • Human OSM can activate mouse cells but primarily through the Type I receptor complex

  • Mouse OSM cannot effectively bind to human OSMR-β

• Experimental Design Implications:

  • When using human OSM in mouse models, consider that it may not fully recapitulate the physiological effects of mouse OSM

  • For in vitro studies with mouse cells, human OSM may produce results that differ from mouse OSM

  • Dose-response relationships may vary between species due to different receptor affinities

• Control Recommendations:

  • In cross-species experiments, include species-matched controls when possible

  • Consider comparative studies with both human and mouse OSM when using mouse models

  • Validate key findings in species-specific systems to ensure translational relevance

These considerations are essential for accurate interpretation of experimental results and for translating findings between species, particularly in preclinical studies aimed at therapeutic development .

What are common issues when working with OSM in cell culture systems?

Researchers may encounter several challenges when using OSM in experimental systems:

• Loss of Activity:

  • Cause: Protein degradation due to improper storage or handling

  • Solution: Store reconstituted protein at recommended temperatures; avoid repeated freeze-thaw cycles; add carrier proteins (0.1% HSA or BSA) for long-term storage

• Variable Cellular Responses:

  • Cause: Cell passage number, density, or culture conditions affecting receptor expression

  • Solution: Standardize cell culture conditions; use cells within defined passage ranges; check receptor expression levels

• Low Sensitivity in Bioassays:

  • Cause: Reduced receptor expression in target cells; inhibitors in media

  • Solution: Verify receptor expression; optimize cell culture conditions; serum-starve cells before OSM stimulation

• Endotoxin Contamination:

  • Cause: Bacterial endotoxin in recombinant protein preparations

  • Solution: Use endotoxin-tested preparations (<0.2 EU/μg); consider endotoxin removal if necessary

Addressing these common issues requires careful experimental planning and appropriate controls to ensure reliable and reproducible results in OSM research.

How can researchers design dose-response experiments with OSM?

Properly designed dose-response experiments are essential for characterizing OSM activity:

Following these guidelines will help generate reliable and interpretable dose-response data for OSM activity in various experimental systems.

What methods can be used to study OSM-receptor interactions?

Several methodologies are available to study OSM-receptor interactions at different levels:

• Binding Assays:

  • Surface Plasmon Resonance (SPR):

    • Measures real-time binding kinetics

    • Can determine association (kon) and dissociation (koff) rates

    • Calculates equilibrium dissociation constant (KD)

  • Radio-ligand Binding:

    • Uses 125I-labeled OSM

    • Scatchard analysis for receptor number and affinity

    • Competition assays for comparative binding studies

• Receptor Expression Analysis:

  • Flow Cytometry:

    • Quantifies surface receptor levels on intact cells

    • Can be combined with signaling readouts (phospho-flow)

  • Immunoblotting:

    • Measures total receptor protein levels

    • Can detect receptor dimerization using non-reducing conditions

  • qRT-PCR:

    • Quantifies receptor mRNA expression

    • Useful for monitoring regulation of receptor gene expression

• Functional Readouts:

  • STAT3 Phosphorylation:

    • Rapid response (peak at 15-30 minutes)

    • Western blot or ELISA-based detection

    • Dose-response curves can indicate receptor functionality

These methods provide complementary information about OSM-receptor interactions and should be selected based on the specific aspects of receptor biology being investigated.

How can researchers develop and validate OSM antagonists for experimental use?

Developing effective OSM antagonists requires systematic approaches:

• Target Selection:

  • Direct OSM neutralization (antibodies against OSM)

  • Receptor blocking (antibodies against OSMR-β or gp130)

  • Pathway inhibition (JAK/STAT inhibitors)

• In Vitro Validation:

  • Binding Assays:

    • Confirm direct interaction with target

    • Determine binding affinity and specificity

  • Functional Assays:

    • Inhibition of OSM-induced STAT3 phosphorylation

    • Blockade of OSM-dependent cell proliferation

    • Prevention of OSM-induced gene expression

  • Specificity Testing:

    • Cross-reactivity with related cytokines (LIF, IL-6)

    • Effects on other signaling pathways

• Cellular Models:

  • Test in multiple cell types with different receptor expression patterns

  • Evaluate dose-dependent inhibition

  • Assess duration of antagonistic effects

• Controls and Standards:

  • Include established inhibitors as positive controls

  • Use irrelevant antibodies or compounds as negative controls

  • Develop quantitative readouts for standardization

These methodological approaches provide a framework for developing and validating OSM antagonists that can be valuable tools for investigating OSM function in experimental systems.

How is OSM being utilized in cancer research?

OSM is being studied in multiple aspects of cancer biology:

• Tumor Growth Regulation:

  • OSM demonstrates growth inhibition in multiple cancer cell types including melanoma, lung, and breast cancer cells

  • Mechanistic studies show this growth inhibition often involves cell cycle arrest rather than apoptosis

  • Paradoxically, OSM can promote growth in other tumor types, suggesting context-dependent effects

• Metastasis Research:

  • Recent studies investigate OSM's role in promoting epithelial-to-mesenchymal transition (EMT)

  • OSM affects cancer cell migration and invasion through matrix remodeling

  • Research examining OSM production in the tumor microenvironment by infiltrating immune cells

• Experimental Approaches:

  • Using OSM as a growth inhibitor in combination with conventional chemotherapeutics

  • Studying OSM signaling blockade in cancers where it promotes progression

  • Investigating OSM as a biomarker for cancer prognosis or treatment response

These research applications highlight OSM's complex and sometimes contradictory roles in cancer biology, emphasizing the importance of context-specific experimental design.

What experimental models exist for studying OSM in inflammatory conditions?

Several experimental models are employed to study OSM's role in inflammatory processes:

• In Vitro Models:

  • Co-cultures of immune cells and tissue-specific cells (e.g., synoviocytes, chondrocytes)

  • Primary human cell cultures stimulated with recombinant OSM

  • Cell line models for specific inflammatory responses

• Ex Vivo Approaches:

  • Tissue explant cultures from inflammatory disease patients

  • Comparative studies of normal vs. inflamed tissues with OSM treatment

  • Patient-derived cells with genetic or pharmacological OSM pathway modulation

• In Vivo Models:

  • Transgenic mice with tissue-specific OSM overexpression

  • Inflammatory disease models with OSM neutralization or receptor blockade

  • Humanized mouse models for translational studies

• Analytical Methods:

  • Multi-parameter flow cytometry for cellular responses

  • Multiplex cytokine analysis for inflammatory networks

  • Histological assessment of inflammatory infiltration and tissue damage

These models provide complementary approaches to understand OSM's role in different aspects of inflammation, from molecular mechanisms to tissue-level effects and potential therapeutic interventions .

How can researchers study OSM's role in tissue regeneration?

OSM's contribution to tissue repair processes can be investigated through:

• Liver Regeneration Models:

  • Partial hepatectomy with OSM administration or neutralization

  • In vitro studies of hepatocyte proliferation and differentiation

  • Analysis of OSM expression during liver regeneration phases

• Wound Healing Systems:

  • In vitro scratch assays with keratinocytes or fibroblasts

  • Ex vivo human skin models for wound repair assessment

  • In vivo excisional or incisional wound models with OSM modulation

• Bone Remodeling:

  • Osteoblast and osteoclast co-culture systems

  • Bone fracture healing models

  • Micro-CT analysis of bone formation after OSM treatment

• Experimental Approaches:

  • Temporal profiling of OSM expression during repair processes

  • Cell-specific knockout or overexpression of OSM receptors

  • Combinatorial treatments with other growth factors or cytokines

These experimental systems allow researchers to dissect OSM's specific contributions to different aspects of tissue regeneration, potentially informing therapeutic applications for promoting tissue repair .

What methodological approaches are being developed to study OSM signaling specificity?

Investigating OSM signaling specificity requires sophisticated methodological approaches:

• Receptor Engineering:

  • CRISPR-Cas9 modification of receptor expression

  • Domain swapping between OSMR-β and LIFR-β to identify specificity determinants

  • Chimeric receptors to dissect signaling pathway activation

• Pathway Dissection:

  • Small molecule inhibitors targeting specific branches of OSM signaling

  • Phosphoproteomic analysis to identify OSM-specific signaling nodes

  • Temporal analysis of signaling dynamics after OSM stimulation

• Transcriptional Profiling:

  • RNA-seq to identify OSM-specific gene signatures

  • Comparison with other IL-6 family cytokines

  • ChIP-seq to map STAT binding sites after OSM treatment

• Computational Approaches:

  • Systems biology modeling of OSM signaling networks

  • Prediction of pathway cross-talk and feedback mechanisms

  • Integration of multi-omics data for comprehensive pathway analysis

These methodologies enable detailed investigation of how OSM activates specific signaling pathways and gene expression programs, distinct from related cytokines, providing insights into its unique biological functions .