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Human enteric α-defensin 5 is essential for intestinal barrier integrity by promoting focal adhesions via P2Y11-Rac1 activation

ABSTRACT: Besides its well-documented antimicrobial functions, human enteric α-defensins 5 (HD5) has been recently found to act as a "double-edged sword" in intestinal defense by enhancing Shigella infection through inducing filopodial-like extensions via activating P2Y11 receptor in human colonic epithelial cells. Nonetheless, the physiological implications of HD5-P2Y11 interaction, beyond its involvement in Shigella infection, have not been comprehensively elaborated. Here, by using primary human colonic epithelial cells, human colonoid organoids, and a gut-on-a-chip model, we showed that HD5 plays an essential roles in establishing and restoring the human intestinal barrier by promoting cell adhesion in suspension or detached cells. Mechanistic investigation demonstrates that although P2Y11 still serves as the responsible receptor mediating the the barrier-protecting effect of HD5, a cell state-dependent alternative downstream signaling cascade was uncovered. In well-adhered cells, HD5-P2Y11 signaling preferentially engages the cAMP-PKA pathway to induce exploratory protrusions like filopodia. Conversely, in suspension cells or detached cells where adhesion is compromised, the signaling shifts priority to the FAK pathway to rapidly reinforce cell adhesion. This dynamic switching allows HD5 to precisely orchestrate epithelial barrier maintenance and repair. This pathway involves the phosphorylation and activation of FAK at key tyrosine residues (Tyr397, Tyr576, Tyr925), which in turn activates the small GTPase Rac1. This HD5-P2Y11-FAK-Rac1 axis enhances barrier function by promoting the formation and stabilization of focal adhesions, accelerating cell attachment, and facilitating epithelial migration for efficient repair. By delineating this novel pathway and its context-dependent regulation, the study fills the knowledge gap to understand the paradoxical roles of HD5 in human intestinal physiology and more importantly, provides a new perspective to understand the physiological significance of this human-specific defensin, extending its role beyond direct antimicrobial action to a key regulator of cytoskeletal dynamics and intestinal homeostasis.

ORGANISM(S): Homo Sapiens

SUBMITTER: Dan Xu

PROVIDER: PXD070617 | iProX | Wed Nov 12 00:00:00 GMT 2025

REPOSITORIES: iProX

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Project description:Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) dramatically limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME thus rendering tumors more responsive to immunotherapy. We treated KP orthotopic tumor-bearing mice with vehicle and FAK inhibitor (FAKi) for 14 days, then extracted total RNA from tumor tissues.

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Human enteric α-defensin 5 is essential for intestinal barrier integrity by promoting focal adhesions via P2Y11-Rac1 activation

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