Recombinant Rat Ninjurin-1 (Ninj1)

What is the molecular structure and basic properties of rat Ninjurin-1?

Rat Ninjurin-1 (NINJ1) is a transmembrane protein belonging to the Ninjurin family. The recombinant form typically comprises the extracellular domain (Met 1-Pro 79) with various tags (commonly Fc tag). The protein has a calculated molecular weight of approximately 37.0 kDa, though the observed molecular weight is typically around 50 kDa due to post-translational modifications . The protein contains a homophilic binding domain in its extracellular region (amino acids 1-50), which is critical for its adhesive and signaling functions .

How is recombinant rat Ninjurin-1 typically produced and purified?

Recombinant rat Ninjurin-1 is commonly expressed in mammalian expression systems, particularly HEK293 cells, to ensure proper folding and post-translational modifications . The protein is typically tagged (e.g., with N-hFc) to facilitate purification and detection. Purification is achieved through affinity chromatography, yielding protein with >95% purity as determined by reducing SDS-PAGE . The purified protein is often lyophilized from sterile PBS (pH 7.4) with protectants such as 5-8% trehalose, mannitol, and 0.01% Tween 80 to maintain stability during storage and shipping .

What is the expression pattern of Ninjurin-1 in normal rat tissues?

Ninjurin-1 is expressed in various adult and embryonic tissues in rats, predominantly in epithelial cells . It shows significant expression in:

Expression is dynamically regulated, with significant upregulation observed following nerve injury .

How should recombinant rat Ninjurin-1 be reconstituted and stored for optimal activity?

Recombinant rat Ninjurin-1 should be reconstituted according to the manufacturer's protocol, typically using sterile PBS or similar buffer. After reconstitution, the protein solution can be stored at 4-8°C for short-term use (2-7 days) . For longer storage, it is recommended to prepare aliquots and store them at temperatures below -20°C, where they remain stable for approximately 3 months .

For lyophilized protein, storage at -20°C to -80°C generally maintains stability for up to 12 months . It's crucial to avoid repeated freeze-thaw cycles as they can compromise protein integrity and activity. When working with the protein, maintain sterile conditions and use low-protein binding tubes to prevent significant loss due to adsorption.

What are the proven methods for detecting Ninjurin-1 expression in tissue samples?

Several validated methods can detect Ninjurin-1 expression in tissue samples:

• Western Blot Analysis: Effective for quantitative assessment of Ninjurin-1 protein levels. Custom antibodies generated against specific epitopes (such as residues 1-15) have been successfully used . A band at approximately 22 kDa represents the native protein, while recombinant tagged versions appear at higher molecular weights (e.g., 50 kDa for Fc-tagged versions) .

• Immunohistochemistry/Immunofluorescence: These techniques allow visualization of Ninjurin-1 expression patterns in different cell types within tissue sections. Particularly useful for analyzing expression in pathological conditions such as spinal cord injury, where Ninjurin-1 immunoreactivity increases sharply post-injury .

• qRT-PCR: For quantifying Ninjurin-1 mRNA expression levels, particularly useful for comparing expression under different conditions or treatments.

For optimal results, it is recommended to use positive and negative controls, including tissues from Ninjurin-1 knockout mice as negative controls .

How does recombinant Ninjurin-1 (particularly the 1-50 amino acid fragment) affect macrophage activation?

The recombinant extracellular domain of Ninjurin-1 (amino acids 1-50) has been demonstrated to directly activate macrophages through homophilic binding interactions. When wild-type macrophages are exposed to rmNinj1 1-50, they exhibit:

• Significant elevation in phosphorylation of NF-κB signaling protein p65 within 1 hour of exposure

• Increased mRNA expression of pro-inflammatory cytokines such as IL-1β

• Enhanced production of pro-fibrotic factors

This activation is Ninjurin-1-dependent, as macrophages from Ninjurin-1 knockout mice show only transient and minimal responses to rmNinj1 1-50 treatment . The conditioned medium from rmNinj1 1-50-treated wild-type macrophages can induce:

• Phosphorylation of SMAD3 in fibroblasts

• Nuclear localization of SMAD3

• Increased expression of α-SMA

• Enhanced fibroblast migration

These effects are significantly reduced when using conditioned medium from similarly treated Ninjurin-1 knockout macrophages . This demonstrates that the homophilic binding activity of Ninjurin-1's extracellular domain is crucial for macrophage activation and subsequent promotion of fibrotic processes.

What is the role of Ninjurin-1 in pulmonary fibrosis and what experimental models are available to study this?

Ninjurin-1 plays a crucial role in the development of pulmonary fibrosis by enhancing inflammatory responses and promoting macrophage-epithelial cell interactions. Experimental evidence suggests the following mechanisms:

• Ninjurin-1 expression is upregulated in lung specimens from idiopathic pulmonary fibrosis patients and in mice with bleomycin-induced pulmonary fibrosis .

• Ninjurin-1 knockout mice exhibit milder fibrotic phenotypes following bleomycin treatment compared to wild-type controls .

• Mechanistically, Ninjurin-1 promotes contact-dependent activation of macrophages when they interact with alveolar epithelial cells (AECs) that have elevated Ninjurin-1 expression due to injury .

The most established experimental model to study Ninjurin-1's role in pulmonary fibrosis is the bleomycin-induced model in mice, comparing wild-type and Ninjurin-1 knockout animals. This model reveals that while the number of infiltrated inflammatory cells may not differ significantly between genotypes, the expression of pro-inflammatory and pro-fibrotic mediators is diminished in Ninjurin-1 knockout mice .

How does Ninjurin-1 expression change during the progression of spinal cord injury?

Ninjurin-1 expression follows a distinct temporal pattern after spinal cord injury (SCI), making it a potential biomarker for monitoring injury progression. Western blot analyses reveal:

Immunohistochemistry confirms this pattern, showing sharp increases in Ninjurin-1 immunoreactivity on days 1 and 4 post-injury, followed by a decrease on days 7 and 21 . While control tissues show weak Ninjurin-1 immunostaining in vascular endothelial cells, ependymal cells, and some glial cells, injured tissues exhibit strong staining in macrophages, microglia, and reactive astrocytes . This temporal expression pattern suggests Ninjurin-1 actively participates in the inflammatory and regenerative processes following SCI.

What are the optimal techniques for genetic manipulation of Ninjurin-1 in experimental models?

Several approaches have been validated for effective genetic manipulation of Ninjurin-1:

• Global Knockout Models: Generation of Ninjurin-1 knockout mice by removing exon 1 of the four exons encoding Ninjurin-1 located on chromosome 13 using homologous recombination . Genotyping can be performed using specific primers:

  • Wild type (forward): 5′-GAG ATA GAG GGA GCA CGA CG-3′

  • Neo (forward): 5′-ACG CGT CAC CTT AAT ATG CG-3′

  • Reverse primer: 5′-CGG GTT GTT GAG GTC ATA CTT G-3′

• RNAi Knockdown: Small interfering RNAs (siRNAs) targeting mouse Ninjurin-1 (NM_013610) have been effectively used to achieve temporary reduction of expression in cell lines such as Raw264.7 .

• Overexpression Systems:

  • Transient overexpression can be achieved using Myc-tagged Ninjurin-1 vectors (Myc-mNinj1) transfected into cells using Nucleofector technology .

  • Stable overexpression cell lines can be created by transfecting GFP-mNinj1 plasmids (full-length cDNA encoding mouse Ninjurin-1 cloned into pEGFP C3 backbone plasmid) and selecting with G418 (500 μg/ml) .

For optimal results, it's essential to verify expression changes using multiple techniques such as Western blotting, qRT-PCR, and immunostaining to confirm successful manipulation of Ninjurin-1 levels.

What experimental approaches are most effective for studying Ninjurin-1-mediated cell-cell interactions?

Several complementary approaches have proven effective for investigating Ninjurin-1-mediated cell-cell interactions:

• Co-culture Systems: Establishing co-cultures of macrophages with alveolar epithelial cells or other relevant cell types allows observation of contact-dependent activation . This approach can be enhanced by using cells from Ninjurin-1 knockout animals or cells with Ninjurin-1 knockdown to confirm specificity.

• Recombinant Protein Treatment: Application of recombinant Ninjurin-1 fragments (particularly the extracellular domain amino acids 1-50) to target cells can mimic homophilic binding and trigger downstream signaling . This approach allows precise control of dosage and timing.

• Time-lapse Imaging: Specialized microscopy setups (e.g., Carl Zeiss Axiovert M200 and LSM 700) equipped with computer-driven cooled CCD cameras, humidified CO2 chambers, and autofocus systems enable real-time visualization of cell motility and membrane dynamics .

• Conditioned Medium Experiments: Collecting conditioned medium from Ninjurin-1-activated cells and applying it to secondary cell types allows assessment of paracrine signaling effects . This approach helps distinguish direct cell-cell contact effects from secreted factor effects.

• Transendothelial Migration Assays: These assays are particularly valuable for studying Ninjurin-1's role in leukocyte trafficking during inflammation .

How is Ninjurin-1 implicated in cancer progression, particularly hepatocellular carcinoma?

Ninjurin-1 shows elevated expression in liver tissues of patients with hepatocellular carcinoma (HCC), particularly in cases associated with cirrhosis and chronic viral hepatitis . Its role in cancer appears complex, with evidence suggesting both pro-tumorigenic and anti-tumorigenic functions:

• Cellular Senescence Induction: Ninjurin-1 has been reported to increase p21 expression and induce cellular senescence in human hepatoma cells, which could represent a tumor-suppressive function .

• Inflammatory Microenvironment: Given Ninjurin-1's established role in promoting inflammatory responses, it may contribute to the inflammatory microenvironment that facilitates HCC development and progression.

• Cell Adhesion Modulation: As a mediator of homophilic adhesion, alterations in Ninjurin-1 expression could affect cancer cell migration and invasion properties.

The specific mechanisms by which Ninjurin-1 influences HCC progression warrant further investigation, particularly regarding its potential as a biomarker or therapeutic target. Researchers should consider examining Ninjurin-1 expression in patient samples correlated with clinical outcomes, as well as functional studies in HCC cell lines and animal models with manipulated Ninjurin-1 expression.

What is the relationship between Ninjurin-1 and inflammatory diseases beyond pulmonary fibrosis?

Ninjurin-1's involvement extends to various inflammatory conditions beyond pulmonary fibrosis, with several key mechanisms identified:

• Leukocyte Trafficking: Ninjurin-1 promotes leukocyte trafficking during inflammation through homotypic binding, affecting immune cell infiltration at inflammatory sites .

• Membrane Dynamics and Motility: Changes in Ninjurin-1 expression regulate protrusive membrane dynamics and cell motility, facilitating transendothelial migration of inflammatory cells .

• Neuroinflammation: In models of spinal cord injury, Ninjurin-1 expression is upregulated in macrophages, microglia, and reactive astrocytes, suggesting a role in neuroinflammatory processes .

• B-cell Related Disorders: Ninjurin-1 is expressed in CD19+ CD10+ B-cell progenitor cells and at higher levels in B-lineage acute lymphoblastic leukemia cells, implicating it in hematological disorders .

Given these diverse roles, Ninjurin-1 represents a potential therapeutic target for treating inflammatory conditions. Advanced research should focus on developing specific inhibitors of Ninjurin-1 homophilic interactions or modulating its expression to attenuate inflammatory responses in various disease contexts.