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. 2023 Jan;299(1):102746.
doi: 10.1016/j.jbc.2022.102746. Epub 2022 Nov 24.

Next-generation retinoid X receptor agonists increase ATRA signaling in organotypic epithelium cultures and have distinct effects on receptor dynamics

Nathalia Melo  1 , Olga V Belyaeva  1 , Wilhelm K Berger  2 , Laszlo Halasz  2 , Jianshi Yu  3 , Nagesh Pilli  3 , Zhengrong Yang  1 , Alla V Klyuyeva  4 , Craig A Elmets  5 , Venkatram Atigadda  6 , Donald D Muccio  1 , Maureen A Kane  3 , Laszlo Nagy  2 , Natalia Y Kedishvili  7 , Matthew B Renfrow  8
Affiliations

Next-generation retinoid X receptor agonists increase ATRA signaling in organotypic epithelium cultures and have distinct effects on receptor dynamics

Nathalia Melo et al. J Biol Chem. 2023 Jan.

Abstract

Retinoid X receptors (RXRs) are nuclear transcription factors that partner with other nuclear receptors to regulate numerous physiological processes. Although RXR represents a valid therapeutic target, only a few RXR-specific ligands (rexinoids) have been identified, in part due to the lack of clarity on how rexinoids selectively modulate RXR response. Previously, we showed that rexinoid UAB30 potentiates all-trans-retinoic acid (ATRA) signaling in human keratinocytes, in part by stimulating ATRA biosynthesis. Here, we examined the mechanism of action of next-generation rexinoids UAB110 and UAB111 that are more potent in vitro than UAB30 and the FDA-approved Targretin. Both UAB110 and UAB111 enhanced ATRA signaling in human organotypic epithelium at a 50-fold lower concentration than UAB30. This was consistent with the 2- to 5- fold greater increase in ATRA in organotypic epidermis treated with UAB110/111 versus UAB30. Furthermore, at 0.2 μM, UAB110/111 increased the expression of ATRA genes up to 16-fold stronger than Targretin. The less toxic and more potent UAB110 also induced more changes in differential gene expression than Targretin. Additionally, our hydrogen deuterium exchange mass spectrometry analysis showed that both ligands reduced the dynamics of the ligand-binding pocket but also induced unique dynamic responses that were indicative of higher affinity binding relative to UAB30, especially for Helix 3. UAB110 binding also showed increased dynamics towards the dimer interface through the Helix 8 and Helix 9 regions. These data suggest that UAB110 and UAB111 are potent activators of RXR-RAR signaling pathways but accomplish activation through different molecular responses to ligand binding.

Keywords: Targretin; UAB110; UAB111; UAB30; nuclear receptor; organotypic epidermis; retinoic acid; retinoid X receptor; rexinoid agonists.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
RXR homodimer ligand-binding domain bound to UAB110.A, crystal structure of RXR-LBD in complex with UAB110 (magenta). B, structures of rexinoids UAB110, UAB111, UAB30, and Targretin. LBD, ligand-binding domain; RXR, retinoid X receptor.
Figure 2
Figure 2
H&E staining of skin raft cultures treated with rexinoid agonists. (A) DMSO, solvent control, human organotypic skin raft cultures were treated with rexinoid agonists: (B) 0.2 μM Targretin, (C) 0.2 μM UAB30, (D) 10 μM UAB30, (E) 0.2 μM UAB110, and (F) 0.2 μM UAB111. The rexinoids were added to the culture medium at the onset of organotypic skin raft formation, and the rafts were allowed to grow for 11 days. Colored lines at the left side of each panel demarcate the layers of the epidermis: cornified (red), granular (blue), spinous (green), and basal (black).
Figure 3
Figure 3
QPCR analysis of gene expression in skin rafts treated with rexinoids.A–E, quantitative PCR analyses of gene expression in human skin raft cultures were done in triplicate. Conditions used were as follows: DMSO (black circle), UAB30 (hollow blue circle), Targretin (red triangle), UAB110 (magenta downward triangle), UAB111 (green diamond); n = 3 independent skin rafts, Ω p < 0.05 compared to DMSO control.
Figure 4
Figure 4
RNA-seq analysis of skin rafts treated with rexinoids. RNA-seq analysis of skin raft cultures treated with rexinoids for 11 days. A–C, volcano plots showing significantly changing genes (CPM > 5, FDR < 0.05, FC ≥ 1.5). (A) 1 μM UAB30, (B) 0.2 μM Targretin, (C) 0.2 μM UAB110. D, UpSet plot showing the overlaps of significantly changing genes (CPM > 5, FDR < 0.05). Intersection size represents the number of genes unique to the overlap of the rexinoid treatments. Set size represents total size of the significantly changing genes for each rexinoid treatment. E–G, heatmaps of top 20 upregulated and downregulated genes (CPM > 5, FDR < 0.05, FC ≥ 1.5). (E) 1 μM UAB30, (F) 0.2 μM Targretin, (G) 0.2 μM UAB110. H–J, boxplots of individual gene examples of normalized logCPM expression. (H) MAL, (I) ACER1, (J) ABCG1. CPM, Counts Per Million.
Figure 5
Figure 5
Quantification of retinoic acid, retinol, and retinyl esters. Retinoic acid (RA), retinol (ROL), and retinyl esters (RE) levels were assessed by liquid chromatography with multiple reaction monitoring mass spectrometry. Treatments of skin rafts were as follows: DMSO (black circle), UAB30 (hollow blue circle), UAB110 (magenta downward triangle), UAB111 (green diamond). RE represent the storage form of ROL. ROL is the metabolic precursor of RA. A, steady state levels of RA were significantly elevated in skin rafts treated with UAB110 or UAB111. B, skin rafts treated with UAB110 or UAB111 displayed significant reduction of retinol. C, the content of RE was similar in skin rafts treated by different rexinoids (n = 8 to 10 of skin rafts, ∗p < 0.05; ∗∗∗∗p< 0.0001 compared to DMSO control.
Figure 6
Figure 6
Deuterium incorporation difference maps of RXRα-LBD in complex with different rexinoids. HDX MS analysis of four rexinoids with six on-exchange time points (15, 30, 60, 300, 900, and 3600 s) organized into a difference map. Results are reported as an increase (shades of red) or decrease (shades of blue) in deuterium in different regions of the RXR–LBD–rexinoid complex relative to apo-RXR-LBD. All four complexes displayed reduced dynamics in regions of the LBP with UAB111 resulting in the greatest reduction of dynamics in H3. RXR-UAB110 and RXR-Targretin complexes revealed increased dynamics in the C-terminal end of H3, H8, and H9. Significance was established via volcano plots (Fig. S5). LBD, ligand-binding domain; LBP, ligand-binding pocket; RXR, retinoid X receptor.
Figure 7
Figure 7
RXRα-LBD ligand-binding pocket Helix 3 demonstrates the highest extent of HDX MS variation when bound to different rexinoids. H3 is involved in both the ligand-binding pocket and the formation of the AF-2 coactivator binding site. A, Deuterium uptake versus time for the H3 peptide A271ADKQLFTL2792+ when bound to UAB30 (blue), Targretin (red), UAB110 (magenta), and UAB111 (green) relative to apo-RXRα-LBD. UAB111 binding lowers the deuterium uptake for this region the most. B, the HDX MS raw spectral data for the H3 peptide for the 0, 15, 300, and 3600 s time points. As the deuterium is incorporated over time, the isotopic envelope increases in mass. For UAB30 bound, the population of peptide ions shifts higher as a whole. When the other rexinoids are bound, a separation of the H3 peptide ion populations can be seen at the 15 s time point that indicates two populations of tightly bound versus more deuterium accessible forms of the RXR-LBD in this region. This separation in the peptide ion populations is still visible in the UAB110- and UAB111-bound raw data at the 300 s time points with both ligands still showing less overall deuterium incorporation at 3600 s (broader isotopic envelope). These HDX MS bimodal distributions for UAB110 and UAB111 demonstrate the differential interactions of the various rexinoids with H3 and H3’s “sensitivity” to differential ligand binding. Other ligand-binding pocket regions of the RXR-LBD do not show this distinction in the data when the various rexinoids are bound. A similar plot of a Helix 7 peptide D347 RVLTEL353 (1+) (also part of the ligand-binding pocket) is provided (Fig. S8). HDX MS, hydrogen deuterium exchange mass spectrometry; LBD, ligand-binding domain; RXR, retinoid X receptor.
Figure 8
Figure 8
HDX MS analysis of RXR ternary complexes demonstrates potency of UAB110 and UAB111.A, the HDX MS difference map of RXRα-LBD in complex with UAB110 or UAB111 and GRIP-1 coactivator peptide. When the HDX MS results for the two potent rexinoids are painted onto X-ray crystal structures, the patterns of deuterium uptake can be visualized and shown how UAB110 binding (B, magenta) and UAB111 binding (D, green) to RXRα-LBD result in different patterns of dynamics. The regions shaded in blue show the extent of decreased dynamics for regions that are part of RXR ligand-binding pocket. There are positive dynamics observed in H3 and H8/H9 (shades of red) when UAB110 is bound. When UAB111 alone is bound (D) to the RXRα-LBD, the extent of decreased dynamics nearly matches the HDX MS results for the ternary complexes of UAB110 (C) or UAB111(E) bound plus the GRIP-1 coactivator peptide (black). Each potent rexinoid induces unique in solution dynamics for the RXRα-LBD. HDX MS, hydrogen deuterium exchange mass spectrometry; LBD, ligand-binding domain; RXR, retinoid X receptor.
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