P71 Bcl11b forms mutual connections with NF-κB1 and NF-κB2 in keratinocytes

Abstract Psoriasis is a chronic inflammatory skin condition characterized by complex molecular interactions. Our earlier data showed that Bcl11b expression is downregulated in psoriasis, and that proinflammatory cytokines act differently on Bcl11b associated activity of the NF-κB pathways. Given the critical role of NF-κB1 in the canonical pathway and NF-κB2 in the non-canonical pathway, it prompted us to elucidate the mutual regulatory connections between Bcl11b, NF-κB1, and NF-κB2, which are critical in the inflammatory response in psoriasis. To investigate the regulatory interplay between Bcl11b, NF-κB1, NF-κB2 and other key genes in the NF-κB pathways. We employed a series of knockout (KO) experiments using CRISPR–Cas9 technology to target Bcl11b, NF-κB1 and NF-κB2 in HaCaT keratinocytes. Gene and protein expression levels were assessed using qPCR and western blot, respectively. Specifically, we examined the impact of Bcl11b KO on NF-κB1 and NF-κB2 expression, followed by evaluating the reciprocal effects of NF-κB1 KO on NF-κB2 and BCL11B and the effect of NF-κB2 KO on NF-κB1 and Bcl11b. Additionally, we investigated the expression of related genes, including RelA, TNF-α, TAK1 RelB, NIK, TRAF2, TRAF3 and TRAF6. Bcl11b KO leads to a downregulation of both NF-κB1 and NF-κB2 at the RNA and protein levels, along with a downregulation of related pathway genes such as, RelA, TNF-α, RelB, and NIK. Conversely, both NF-kB1 KO and NF-kB2 KO conditions also demonstrated a mutual connection among Bcl11b and these two transcription factors. To gain a deeper understanding of the role of Bcl11b in the regulation of the NF-κB pathways, we evaluated the expression levels of several upstream regulators. Our study indicates an upregulation of TRAF2 and TRAF3 in Bcl11b KO conditions, corroborating the downregulation of the non-canonical pathway via NIK. Additionally, we observed a downregulation of TRAF6 under Bcl11b KO conditions, explaining the inactivation of the canonical NF-κB pathway. Interestingly, TAK1 has upregulated in Bcl11b KO cells, indicating a complex regulatory role of Bcl11b within the NF-κB signalling pathway. These data suggest a regulatory network where Bcl11b plays a crucial role in modulating both the canonical and non-canonical NF-κB pathways via TRAF2, TRAF3, and TRAF6. The mutual regulation between Bcl11b, NF-κB1 and NF-κB2 highlights potential therapeutic targets for modulating inflammatory responses in psoriasis.