The Proinflammatory Role of ANGPTL8 R59W Variant in Modulating Inflammation through NF-κB Signaling Pathway under TNFα Stimulation

Figures

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  Figure 1. Box plots for TNFα, IL6, and IL7 from the discovery cohort. The figure shows the distribution of TNF-alpha (A), IL6 (B), and IL7 (C) levels in two genotype groups: “CC” and “CT+TT” in the discovery cohort. The “CC” group exhibits lower levels of TNF-alpha, IL6, and IL7 compared to the “CT+TT” group. The statistical significance of the difference between the groups was assessed using the Wilcoxon rank-sum test (Mann–Whitney U test), and the resulting p-value was <0.05.

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  Figure 2. The R59W variant activates the NF-κB pathway. (A) Cartoon model for NF-κb reporter plasmid (A1) and luciferase assay illustrating overexpression of R59W variant showing increased activation of NF-κB p65 as compared to the wild type (A2). (B) illustrates increased phosphorylation of P- NF-κB-P65. (C) illustrates increased phosphorylation of P-IKK α/β S176-180. (D) represents image of PCMV6, ANGPTL8 flag, and its variant after transfection by Western blot. The data represent the Mean +/− SEM of three independent experiments, * p < 0.05, ** p < 0.001, as determined using Student’s t-test, n = 3 (original gel images are available in the supplementary).

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  Figure 3. R59W variant activates the NF-κB pathway under TNFα stimulation compared to the wild type. (A–C) illustrate overexpression and TNFα stimulation of R59W further increasing the phosphorylation levels of NF-κB-P65, IKK α/β S176-180, IκBα (Ser32). (D) illustrates further activation of NF-κB p65 in the luciferase reporter assays when stimulated by TNFα compared to the wild type. The data represent the Mean +/− SEM of three independent experiments, * p < 0.05 as determined using student’s t-test, n = 3.

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  Figure 4. (A). Impact of overexpression of the R59W variant with/without TNFα stimulation on IL6 and TNFα, as represented by western blots. The data represent the Mean +/− SEM of three independent experiments, * p < 0.05 as determined using Student’s t-test, n = 3. (B–E). Structural Analysis of ANGPTL8 and IKKβ. (B) depicts the modeled structure of ANGPTL8 with I-TASSER and Swiss-Model; (C) depicts the crystal structure of IKK monomer (PDB ID: 4KIK); D depicts the modeled ANGPTL8 wild type (green) with IKKβ; and (E) depicts the modeled ANGPTL8 W59 (purple) with IKKβ.

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  Figure 5. A proposed model depicting the impact of overexpressing the ANGPTL8 R59W variant in influencing the NF-κB pathway. After the overexpression of the wild type ANGPLT8/ANGPTL8-R59W and under the influence of inflammatory stimuli like TNFα, the activity levels of IKKα/β were enhanced which sequentially phosphorylates the NF-κB inhibitor, IκBα, and triggers its rapid degradation through the proteasomal degradation. This releases the NF-κB heterodimer as it is no longer repressed by IκBα. Subsequently, the NF-κB heterodimer translocates from cytoplasm to nucleus, binds to its downstream target DNA sequence, and induces the expression of proinflammatory proteins. This expression of proinflammatory cytokines is higher in the overexpressed ANGPTL8 R59W variant as compared to the wild type ANGPTL8 under stimulation due to its increased nuclear translocation of NF-κB heterodimers. This model depicts the proinflammatory role of R59W-ANGPTL8 in modulating the inflammation through the NF-κB signaling pathway.