New p65 iso5 isoforms as dexamethasone-binding proteins: novel potential therapeutic targets for inflammatory diseases.

Introduction: The transcription factor NF-κB is a central regulator of immune and inflammatory responses whose activity is tightly controlled by IκB proteins and glucocorticoid receptor (GR)-mediated repression. However, the diversity of NF-κB subunit variants and their contribution to glucocorticoid signaling remain incompletely understood. Methods: Human peripheral blood mononuclear cells (PBMCs) exposed to pro- and anti-inflammatory stimuli were analyzed to identify splice variants of the NF-κB subunit p65 iso5. mRNA expression was evaluated under different stimuli. Protein interactions with the synthetic glucocorticoid dexamethasone (Dex) and GR were assessed, together with nuclear translocation dynamics. Functional transcriptional activity was examined using NF-κB and IL-2 responsive promoter assays. Expression profiling was also performed in disease contexts, including COVID-19 and liver cirrhosis. Results: We identified and characterized two previously unrecognized splice variants of p65 iso5, named p65 iso5 Δ6/7 and p65 iso5 Δ10. Their mRNA expression was differentially regulated depending on the stimulus. Both isoforms unexpectedly bound dexamethasone, formed nuclear complexes with Dex-activated GR, and translocated to the nucleus independently of IκBα. Functional assays revealed distinct transcriptional activities on NF-κB and IL-2 responsive promoters, indicating that these isoforms act as noncanonical modulators of glucocorticoid signaling. Expression profiling showed disease-specific regulation, with reciprocal modulation of the two isoforms in COVID-19 and selective upregulation of p65 iso5 Δ6/7 in liver cirrhosis. Discussion: These findings uncover a previously unrecognized layer of NF-κB/GR crosstalk and identify a new class of dexamethasone-binding proteins outside the nuclear receptor superfamily. Our data highlight their glucocorticoid-interacting properties and provide mechanistic insight into the diversity of glucocorticoid responses, with potential implications for inflammation-related pathologies.