NF-κB p65–Mediated YAP Inactivation Drives Pyroptosis in Ulcerative Colitis

Study Background and Research Question

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by relapsing colonic mucosal inflammation and epithelial injury. While the etiology of UC is multifactorial—encompassing genetic, environmental, microbial, and immune contributions—the precise molecular mechanisms governing the escalation and persistence of mucosal inflammation remain incompletely understood. Recent research has focused on the role of innate immune responses, particularly pyroptosis, a highly inflammatory form of programmed cell death mediated via inflammasome activation and Gasdermin D (GSDMD) cleavage. The Yes-associated protein (YAP), a transcriptional coactivator in the Hippo signaling pathway, is established as a key regulator of tissue regeneration and inflammatory responses, but its specific role in epithelial cell pyroptosis during UC has not been fully elucidated.

The reference study (YAP inactivated by NF-κB p65, can protect against colonic epithelial cell pyroptosis in ulcerative colitis via transcriptionally regulating NLRP3) specifically addresses how inflammatory signaling, mediated by NF-κB p65, intersects with YAP-dependent regulation of NLRP3 inflammasome activity and consequent pyroptosis in the context of UC.

Key Innovation from the Reference Study

The key innovation of this research lies in elucidating a mechanistic axis whereby NF-κB p65 activation not only drives inflammatory gene expression, but also modulates post-translational regulation of YAP via LATS1-mediated phosphorylation. This results in restricted nuclear translocation and reduced functional YAP levels within colonic epithelial cells. As a consequence, transcriptional repression of NLRP3 by YAP is relieved, leading to upregulation of NLRP3 inflammasome activity and increased pyroptosis. This is the first report to mechanistically connect NF-κB–dependent YAP inactivation with exacerbated epithelial pyroptosis in UC, highlighting a previously underappreciated layer of regulation in mucosal inflammation (reference study).

Methods and Experimental Design Insights

The study utilized both in vitro and in vivo models to dissect the interplay between YAP, NF-κB, and pyroptosis:

• In vitro: Human FHC colonic epithelial cells were exposed to LPS and ATP to induce inflammasome-dependent pyroptosis. YAP overexpression was achieved via lentiviral transduction, while molecular interactions were analyzed through nucleoplasmic separation and chromatin immunoprecipitation (ChIP) assays.
• In vivo: Colitis was induced in wild-type and GSDMD knockout (KO) mice using DSS (dextran sulfate sodium). YAP overexpression was achieved through intraperitoneal lentiviral injection. The effects on colonic inflammation, pyroptosis, and molecular markers were subsequently assessed.
• Mechanistic analysis: The study examined post-translational modifications of YAP, particularly LATS1-mediated phosphorylation, in response to NF-κB p65 activation. ChIP analysis determined YAP binding to the NLRP3 promoter.

This multifaceted approach enabled the authors to validate the relevance of their findings across both cellular and animal models, and to link molecular changes to functional outcomes in tissue inflammation and cell death.

Core Findings and Why They Matter

The study demonstrated several important findings:

• Elevated pyroptosis in UC: Both patient biopsy samples and DSS-induced murine colitis models showed increased markers of GSDMD-mediated pyroptosis.
• YAP overexpression suppresses pyroptosis: Augmenting YAP levels in FHC cells or in vivo in mice reduced markers of pyroptosis and ameliorated colonic inflammation.
• YAP represses NLRP3 transcription: ChIP and nucleoplasmic fractionation confirmed that nuclear YAP binds the NLRP3 promoter to inhibit its expression, thereby restraining inflammasome activation.
• NF-κB p65 negatively regulates YAP: Activated NF-κB p65 increases LATS1-mediated YAP phosphorylation, sequestering YAP in the cytoplasm and diminishing its nuclear function. This effect was particularly prominent in inflamed colonic epithelium.
• GSDMD knockout attenuates colitis: Genetic ablation of GSDMD substantially reduced colitis severity, confirming the pathogenic role of pyroptosis.
• YAP’s protective effect is pyroptosis-dependent: In GSDMD-KO mice, YAP overexpression did not confer additional benefit, indicating that YAP’s anti-inflammatory effect in this context is mediated predominantly through regulation of pyroptosis.

Together, these findings reveal a critical axis in which NF-κB–driven YAP inactivation removes transcriptional repression of the NLRP3 inflammasome, promoting pyroptosis and inflammation in UC. This mechanistic clarity may inform development of targeted therapies to disrupt this pathogenic loop.

Comparison with Existing Internal Articles

The internal literature on JSH-23, a small molecule NF-κB inhibitor, provides complementary context for the utility of targeting NF-κB signaling in inflammation research. Existing resources such as "JSH-23: Potent NF-κB Inhibitor for Inflammation Research" and "JSH-23: Precision NF-κB Inhibitor for Inflammation Research" describe how JSH-23 effectively blocks p65 nuclear translocation, thereby inhibiting NF-κB–dependent transcription of pro-inflammatory mediators such as IL-6, IL-1β, and TNF-α. While these articles focus on anti-inflammatory applications in cell-based and animal models—such as cisplatin-induced acute kidney injury—the reference study offers a mechanistic extension to colonic epithelial cells, demonstrating how NF-κB p65–driven processes can be linked to the regulation of programmed cell death pathways, including pyroptosis.

Both the internal articles and the current study reinforce the centrality of NF-κB as a nodal control point in inflammatory signaling. However, the reference study uniquely highlights the intersection of transcriptional regulation, post-translational modification, and inflammasome biology in UC pathogenesis.

Limitations and Transferability

While the study provides compelling evidence for the NF-κB–YAP–NLRP3 axis in UC, there are several limitations to consider:

• The in vitro cellular environment does not fully recapitulate the complex interplay of immune and stromal cells present in the colonic mucosa.
• The DSS-induced colitis model, while widely used, may not capture all aspects of human UC, particularly chronicity and the influence of the microbiome.
• Although YAP and NF-κB modulation were shown to alter pyroptosis and inflammation, off-target or systemic effects of lentiviral overexpression remain possible.
• Further studies are needed to determine the translational potential of targeting this axis in human clinical settings, especially regarding therapeutic safety and specificity.

Despite these limitations, the mechanistic insights are likely transferable to other epithelial-driven inflammatory conditions where NF-κB and pyroptosis are implicated, though direct evidence for such cross-domain applications remains to be established.

Protocol Parameters

• DSS-induced colitis (mouse): 2–3% DSS in drinking water for 5–7 days, followed by water for recovery; suitable for modeling acute colonic inflammation.
• YAP overexpression (in vivo): Lentiviral particles delivered intraperitoneally, typically 1–2 × 10⁸ TU per mouse; confirm transduction efficiency via qPCR or immunoblotting.
• FHC cell pyroptosis induction: Treat with 1 μg/mL LPS for 4–6 hours, followed by 5 mM ATP for 30–60 minutes; monitor cell death and inflammasome activation by LDH release, caspase-1 cleavage, or GSDMD-N detection.
• ChIP and nucleoplasmic separation: Use standard chromatin immunoprecipitation protocols with anti-YAP and anti-NLRP3 antibodies; nucleoplasmic fractionation to assess YAP localization changes upon NF-κB activation.

Research Support Resources

For researchers aiming to dissect the role of NF-κB–mediated inflammation and its downstream effects, validated inhibitors such as JSH-23 (SKU B1645) offer a precise tool to block NF-κB p65 nuclear translocation and transcriptional activity. As described in the internal literature, JSH-23 is suitable for both in vitro and in vivo workflows, including models of epithelial injury and pro-inflammatory cytokine regulation. Detailed compound handling and storage protocols are available via the product information from APExBIO, supporting reproducible workflows in inflammation and NF-κB signaling pathway study.