The interactions of derivatives of lumisterol (L3) and vitamin D3 (D3) with liver X receptors (LXRs) were investigated. Molecular docking using crystal structures of the ligand binding domains (LBDs) of LXRα and β revealed high docking scores for L3 and D3 hydroxymetabolites, similar to those of the natural ligands, predicting good binding to the receptor. RNA sequencing of murine dermal fibroblasts stimulated with D3-hydroxyderivatives revealed LXR as the second nuclear receptor pathway for several D3-hydroxyderivatives, including 1,25(OH)2D3. This was validated by their induction of genes downstream of LXR. L3 and D3-derivatives activated an LXR-response element (LXRE)-driven reporter in CHO cells and human keratinocytes, and by enhanced expression of LXR target genes. L3 and D3 derivatives showed high affinity binding to the LBD of the LXRα and β in LanthaScreen TR-FRET LXRα and β coactivator assays. The majority of metabolites functioned as LXRα/β agonists; however, 1,20,25(OH)3D3, 1,25(OH)2D3, 1,20(OH)2D3 and 25(OH)D3 acted as inverse agonists of LXRα, but as agonists of LXRβ. Molecular dynamics simulations for the selected compounds, including 1,25(OH)2D3, 1,20(OH)2D3, 25(OH)D3, 20(OH)D3, 20(OH)L3 and 20,22(OH)2L3, showed different but overlapping interactions with LXRs. Identification of D3 and L3 derivatives as ligands for LXRs suggests a new mechanism of action for these compounds.

Since its discovery, over 30 years ago, CD5 has been used as a marker to identify T cells, B1‐a cells, and B cell chronic lymphocytic leukemia cells. Throughout the years, many studies have described the functional relevance of CD5 as a modulator of T and B cell receptor signaling. However, it has not been until recent years that CD5 has emerged as a functional receptor in other areas of the immune system. Here, we review some of the most important aspects of CD5 as a modulator of TCR and BCR signaling, cell survival receptor both in T and B cells during health and disease, as well as the newly discovered roles of this receptor in thymocyte selection, T cell effector differentiation, and immune tolerance. CD5 was found to promote T cell survival by protecting autoreactive T cell from activation‐induced cell death, to promote de novo induction of regulatory T cells in the periphery, to modulate Th17 and Th2 differentiation, and to modulate immune responses by modulating dendritic cell functions. CD5 is overexpressed in Tregs and Bregs, which are fundamental to maintain immune homeostasis. The newly established roles of CD5 in modulating different aspects of immune responses identify this receptor as an immune checkpoint modulator, and therefore it could be used as a target for immune intervention in different pathologies such as cancer, autoimmune diseases or infections.

CD5 is well recognized for its importance in thymic selection. Although this property of CD5 has been attributed to its ITIM-domain dependent regulation of TCR-signal strength, the mechanism has not been established. A second major signaling domain within the cytoplasmic tail of CD5 is a CK2 binding/activation domain (CD5-CK2BD). Using a gene-targeted mouse in which the CD5-CK2BD is selectively ablated (CD5-ΔCK2BD), we determined that loss of function of CD5-CK2 signaling in a MHC-II selecting TCR transgenic (OT-II) mouse resulted in decrease in double positive (DP) thymocytes, which correlated with enhanced apoptosis. Remarkably, DP cells expressing high levels of CD5 and CD69 and single positive (CD4+SP) thymocytes were increased in CD5-ΔCK2BD mice indicating that CD5-CK2 signaling regulates positive selection and promotes survival. Consistent with this possibility, we determined that the activation and nuclear localization of ERK as well as apoptosis was greater in thymic populations from OTII CD5-ΔCK2BD mice than OTII CD5-WT mice following injection of OVA323-339-peptide. The mobilization of Ca2+, an early event of TCR activation, was not altered by the loss of CD5-CK2 signaling. Collectively, these data demonstrate that the CD5-CK2 signaling axis regulates positive selection by modulating activation of ERK and promoting survival independent of proximal TCR signals.

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New pathways of vitamin D3 (D3) activation initiated by CYP11A1 and involving other CYPs have been discovered. At least 15 hydroxyderivatives, including 20(OH)D3 as the major product, are generated by these pathways (1,2) with some being present in human serum, epidermis, and pig adrenals. CYP11A1 can also metabolize 7-dehydrocholesterol to produce 7-dehydropregnenolone, which can be further modified by steroidogenic enzymes generating Δ7-steroids (1,2). Lastly, CYP11A1 and CYP27A1 act on lumisterol (L3) producing at least 9 biologically active derivatives (1,2). Thus, new pathways generating a large number of biologically active secosteroids and lumisterol-derivatives have now been described. These compounds interact with the vitamin D receptor (VDR), retinoic acid receptors (RORs) α and γ, and the aryl hydrocarbon receptor (AhR)(1). These findings challenge dogmas that lumisterol is biologically inactive and that 1,25(OH)2D3 is the only active form of D3 exerting its effects exclusively through interaction with the VDR. In view of the above and since liver X receptors (LXRs) can be activated by oxysterols, we investigated the interactions of novel products of L3 and D3 metabolism with LXRs. Molecular docking, using crystal structures of the ligand binding domains (LBDs) of LXRα and β, revealed high docking scores for L3 and D3 hydroxymetabolites, like those of the natural ligands, predicting good receptor binding. RNA sequencing of murine dermal fibroblasts stimulated with D3-hydroxyderivatives revealed LXR as the second major nuclear receptor signaling pathway for several D3-hydroxyderivatives, including 1,25(OH)2D3. The involvement of LXRs was validated by the induction of several genes downstream of LXR. Furthermore, L3 and D3-hydroxyderivatives activated an LXR-response element (LXRE)-driven reporter in CHO cells and human keratinocytes. For keratinocytes, enhanced expression of LXR target genes was also observed supporting the involvement of LXR. Importantly, L3 and D3 derivatives showed high affinity binding to the LBD of the LXRα and β in LanthaScreen TR-FRET LXRα and β coactivator assays. The majority of metabolites functioned as LXRα/β agonists; however, 1,20,25(OH)3D3, 1,25(OH)2D3, 1,20(OH)2D3 and 25(OH)D3 acted as inverse agonists of LXRα, but as agonists of LXRβ. Molecular dynamics simulations performed for selected compounds, including 1,25(OH)2D3, 1,20(OH)2D3, 25(OH)D3, 20(OH)D3, 20(OH)L3 and 20,22(OH)2L3, showed overlapping and different interactions with LXRs. Identification of D3 and L3 derivatives as ligands for LXRs changes the accepted paradigms on their biological role and mechanism of action. 1. Cell Biochem Biophys. 2020;78(2):165-180. 2. J Steroid Biochem Mol Biol. 2019;186:4-21.