N4BP3 Activates TLR4-NF-κB Pathway in Inflammatory Bowel Disease by Promoting K48-Linked IκBα Ubiquitination

Area of Science:

• Molecular Biology
• Immunology
• Gastroenterology

Background:

• N4BP3 is a ubiquitination-related gene implicated in neurological and neoplastic diseases.
• Previous studies linked N4BP3 to enhanced inflammatory responses via the NOD2 signaling pathway.
• The role of N4BP3 in inflammatory bowel disease (IBD) through the TLR4 signaling pathway remains unexplored.

Purpose of the Study:

• To investigate the role of N4BP3 in regulating inflammatory bowel disease (IBD) via the TLR4 signaling pathway.
• To elucidate the underlying molecular mechanisms by which N4BP3 influences IBD pathogenesis.

Main Methods:

• Utilized THP-1/Caco-2 cell lines with N4BP3 overexpression or knockdown, stimulated with lipopolysaccharides (LPS) to activate the TLR4 pathway.
• Established dextran sodium sulfate (DSS)-induced colitis mouse models and N4BP3-knockdown mouse models using adeno-associated virus (AAV).
• Analyzed inflammatory marker expression (TNF-α, IL-1β, IL-6, IL-8) and NF-κB pathway components (phospho-NF-κB p65, IκBα) via mRNA and protein levels, and assessed K48-linked ubiquitination.

Main Results:

• LPS stimulation significantly upregulated N4BP3 expression in THP-1/Caco-2 cells.
• N4BP3 overexpression amplified LPS-induced inflammatory cytokine production and NF-κB activation, while N4BP3 knockdown attenuated these effects.
• In vivo, N4BP3 knockdown in DSS-induced colitis mice reduced phospho-NF-κB p65 and increased IκBα expression, indicating suppressed NF-κB signaling.
• N4BP3 was found to interact with IκBα, promoting its K48-linked ubiquitination.

Conclusions:

• LPS upregulates N4BP3, which enhances IκBα ubiquitination, leading to NF-κB pathway activation and exacerbation of IBD.
• N4BP3 emerges as a critical mediator in TLR4-driven inflammation relevant to IBD.
• N4BP3 represents a potential therapeutic target for novel IBD treatments.