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

Abstract

Purpose: N4BP3 is a ubiquitination-related gene that plays a pivotal role in neurology and neoplasia. Studies have demonstrated its essential function in axonal and dendritic branching, promoting hepatocellular carcinoma and breast cancer. Our previous research reveals that N4BP3 enhances inflammatory responses by modulating the NOD2 signaling pathway. It is crucial to investigate whether N4BP3 regulates inflammatory bowel disease (IBD) through the TLR4 signaling pathway and to elucidate the underlying mechanisms.

Methods: Lipopolysaccharides (LPS) were used to activate the TLR4 pathway in THP-1/Caco-2 cells. THP-1/Caco-2 cells were transfected with either N4BP3 overexpression or knockdown plasmids, generating N4BP3-overexpressing or N4BP3-deficient cell lines. For in vivo studies, colitis was induced in mice using dextran sodium sulfate (DSS). Additionally, negative control and N4BP3-knockdown C57BL/6 mouse models were established via intraperitoneal injection of control or N4BP3-targeting adeno-associated virus (AAV).

Results: LPS stimulation significantly upregulated N4BP3 expression in THP-1/Caco-2 cells compared to sterile water treatment (P < 0.05). In N4BP3-overexpressing cells, LPS induction led to significantly higher expression of TNF-α, IL-1β, IL-6, and IL-8 mRNA, as well as phospho-NF-κB p65 protein, compared to wild-type THP-1/Caco-2 cells (P < 0.05). Conversely, these inflammatory markers were markedly downregulated in N4BP3-knockdown THP-1 cells following LPS stimulation (P < 0.05). In DSS-induced colitis models, N4BP3-knockdown mice showed decreased phospho-NF-κB p65 but increased IκBα protein expression in colonic tissues compared to DSS-treated control mice (P < 0.05). Furthermore, we observed interaction between N4BP3 and IκBα, with N4BP3-overexpressing THP-1 cells demonstrating significantly elevated K48-linked ubiquitination levels versus controls.

Conclusion: LPS upregulates N4BP3 expression, which subsequently enhances K48-linked ubiquitination of IκBα, leading to NF-κB pathway activation, and exacerbating IBD progression. These findings suggest N4BP3 as a potential therapeutic target for developing novel IBD treatments.

Keywords: IκBα; N4BP3; NF-κB; TLR4; inflammatory bowel disease; ubiquitination.

Figure 1

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Figure 1

Changes in N4BP3 expression after inducing THP-1/Caco2 cells with LPS. (A and B) Changes in the expression of N4BP3 mRNA (A) and protein (B) after inducing THP-1 cells with LPS (1 μg/mL) for different times. (C and D) Changes in the expression of N4BP3 mRNA (C) and protein (D) after inducing THP-1 cells with different concentrations of LPS for 3 h. (E and F) Changes in the expression of N4BP3 mRNA (E) and protein (F) after inducing Caco2 cells with LPS (10 μg/mL) for different times. (G and H) Changes in the expression of N4BP3 mRNA (G) and protein (H) after inducing Caco2 cells with different concentrations of LPS for 24 h. Statistical analysis using paired t-test. *P < 0.05 represents a significant difference between the group below the asterisk (*) indicator and the control group (the first group). Indicator “ns” representing no significant difference between the group below the indicator “ns” and the control group (the first group). The error bar represents the mean ± SD. (I and J) Changes in the expression of N4BP3 mRNA (I) and protein (J) after inducing THP-1 cells with U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), SB203580 (P38 inhibitor), and BAY11-7082 (NF-κB inhibitor), respectively. Statistical analysis using One-way ANOVA. * P < 0.05, representing a significant difference between the groups at either end of the horizontal line. Indicator “ns” representing no significant difference between the groups at either end of the horizontal line. The error bar represents the mean ± SD.

Figure 2

Changes in LPS-induced mRNA expression of cytokines after overexpressing N4BP3 in THP-1/Caco2 cells. (A–D) Changes in LPS-induced mRNA expression of TNFα (B), IL-6 (C), and IL-8 (D) after overexpressing N4BP3 (A) in THP-1 cells. (E–H) Changes in LPS-induced mRNA expression of IL-1β (F), IL-6 (G), and IL-8 (H) after overexpressing N4BP3 (E) in Caco2 cells. Statistical analysis using One-way ANOVA. * P < 0.05, representing a significant difference between the data. The error bar represents the mean ± SD.

Figure 3

Changes in LPS-induced expression of cytokines after knocking down N4BP3 in THP-1 cells. (A) Changes in N4BP3 protein expression after transfection of plasmids containing different knockdown N4BP3 sequences into THP-1 cells. (B–E) Changes in LPS-induced mRNA expression of TNFα (C), IL-6 (D), and IL-8 (E) after knocking down N4BP3 (B) in THP-1 cells. (F) Changes in LPS-induced protein expression of TNFα and IL-1β after knocking down N4BP3 in THP-1 cells. Statistical analysis using One-way ANOVA. * P < 0.05, representing a significant difference between the groups at either end of the horizontal line. Indicator “ns” representing no significant difference between the groups at either end of the horizontal line. The error bar represents the mean ± SD.

Figure 4

Changes in LPS-induced protein expression of signaling molecules after overexpressing N4BP3 in THP-1 cells. Changes in LPS-induced protein expression of TLR4, MyD88, IRAK1, IRAK4, TRAF6, p-TAK1, NEMO, IκBα, p-P65, p-ERK, p-JNK, and p-P38 after overexpressing N4BP3 in THP-1 cells. Statistical analysis using One-way ANOVA. *P < 0.05, representing a significant difference between the groups at either end of the horizontal line. Indicator “ns” representing no significant difference between the groups at either end of the horizontal line. The error bar represents the mean ± SD.

Figure 5

Expression of IκBα protein and p-P65 protein in colonic tissues of mice. (A) Expression of IκBα protein in colon tissues of negative control mice. (B) Expression of IκBα protein in colon tissues of N4BP3 knockdown mice. (C) Relative expression of IκBα protein in colon tissues of negative control mice and N4BP3 knockdown mice. (D) Expression of p-P65 protein in colon tissues of negative control mice. (E) Expression of p-P65 protein in colon tissues of N4BP3 knockdown mice. (F) Relative expression of p-P65 protein in colon tissues of negative control mice and N4BP3 knockdown mice. Statistical analysis using paired t-test. *P < 0.05, representing a significant difference between the data. The error bar represents the mean ± SD.

Figure 6

Target protein and ubiquitination modification assays for N4BP3 interactions. (A and B) Expression of different proteins detected by immunoblotting after immunoprecipitating LPS-induced THP-1 cells overexpressing N4BP3 with the N4BP3 antibody (A) and the IκBα antibody (B). (C) Expression of different proteins detected by immunoblotting after immunoprecipitating LPS-induced control THP-1 cells and THP-1 cells overexpressing N4BP3 with the IκBα antibody.

Figure 7

Schematic diagram of the mechanism by which N4BP3 activates TLR4-NF-κB signaling. In inflammatory bowel disease, the activation of TLR4 in macrophages promotes the expression of N4BP3 via the NF-κB pathway. N4BP3 can further promote the ubiquitination of IκBα K48 linkages, facilitate the entry of NF-κB into the nucleus, and promote the production and release of cytokine. This figure was created with BioGDP.com.