Molecular evidence for 9CDHRA selective activation of RXRs.

<p>(a) Significant overlap between global transcriptional changes induced by R-9CDHRA (10^{<b><i>-5</i></b>}M), 9CRA (10^{<b><i>-6</i></b>}M) or a synthetic RXR agonist (LG268; 10^{<b><i>-7</i></b>}M) revealed by DNA microarray analyses in differentiating monocyte-derived human dendritic cells. (b) Scatter plot comparison of fold-changes for transcripts altered by 9CRA and R-9CDHRA treatments. (c) S- and R-9CDHRA, similarly to 9CRA and/or LG268 (see “RXR” columns), induced the expression of genes (see rows) identified previously as direct transcriptional targets of LXR-RXR, PPAR-RXR and RAR-RXR. Corresponding transcripts were also induced by agonists of respective RXR-heterodimer partners (see “partners” column and arrowheads): GW3965 (LXRα/β; 10^{<b><i>-6</i></b>}M), RSG (PPARγ; 10^{<b><i>-6</i></b>}M), GW1516 (PPARδ; 10^{<b><i>-6</i></b>}M) and AM580 (RAR; 10^{<b><i>-7</i></b>}M).</p

9CDHRA binds and transactivates RXR <i>in vitro</i>, and displays RXR agonist-like activities <i>in vivo</i>.

<p>(a) ESI mass spectra of hRXRα LBD protein after incubation with a 5-fold molar excess of 9CRA, R-9CDHRA and S-9CDHRA. (b) Distribution plot of the percentage of bound hRXRα. (c) Close-up view showing the ligand-binding pocket of RXRα bound to R-9CDHRA (in grey). Residues in close contacts (<3.5 Å) are labelled. Dashed lines indicate hydrogen bonds and the red sphere a water molecule. (d) Superposition of the RXRalpha ligand-binding pocket bound to R-9CDHRA (grey) and 9CRA (cyan, PDB code: 1XDK). Superposition was made on the protein. Hydrogen bonds with R-9CDHRA or 9CRA are shown by grey and cyan dashed lines, respectively. Water molecules are shown by spheres (red for RXR-R-9CDHRA and cyan for RXR-9CRA). (e) Transcriptional activation of RXRα by R-9CDHRA and S-9CDHRA in comparison to 9CRA (10^{<b><i>–5</i></b>}–10^{<b><i>-9</i></b>}M) in RXR-reporter COS1 cells. (f) 9CRA (10^{<b><i>-7</i></b>}M) and both 9CDHRA enantiomers (10^{<b><i>-5</i></b>}M) induced RXRα-mediated signaling. RXR-antagonist LG101208 (LG; 10^{<b><i>-6</i></b>}M) diminished activity of both 9CDHRA enantiomers (10^{<b><i>-5</i></b>}M). (g) Transcriptional activation of RAR-RXR heterodimers by R- and S-9CDHRA in comparison to ATRA in RARα-RXRα-reporter COS1 cells. (h) Increasing doses of R-9CDHRA reversed working memory deficits in <i>Rbp1</i>^{<b><i>-/-</i></b>} mice and showed pro-mnemonic activity in WT mice (n = 8/group) in DNMTP task when tested at minimal ITI, at which mice performed at chance level (50%) and which was 6min for the <i>Rbp1-/-</i> or 12min for WT mice. ttt: ATRA at concentration 10^{<b><i>–5</i></b>} M was cytotoxic. *, p<0.05. #, p<0.05; ##, p<0.01 as compared to vehicle treatment in the same group; $, p<0.05;$$, p<0.01; one group student t-test for comparison with performance at chance level of 50%. All the error bars represent S.E.M.</p

9CDHRA is present in mouse serum, brain and liver.

<p>(a) Elution profiles of WT mouse serum (n = 7) and brain (n = 3) samples in comparison with three retinoic acid standards (all-<i>trans</i> [ATRA], 13-<i>cis</i> [13CRA] and 9<i>-cis</i> retinoic acid [9CRA] with retention times 10.4, 9.6, and 9.9 min, respectively) allow identification of ATRA but not 9CRA. Boxes represent areas with comparable retention times to identify co-eluting peaks. (b) 9-<i>cis</i>-13,14-dihydroretinoic acid (9CDHRA; retention time 9.4 min highlighted by dotted-line box) is present in mouse serum (n = 7), brain (n = 3) and liver (n = 7) samples as determined by co-elution with a mixture of 9CDHRA and all-<i>trans</i>-13,14-dihydroretinoic acid (ATDHRA; retention time 9.9 min, highlighted by continuous-line box) standard solution and confirmed by MS-MS (303->207 m/z) and DAD (290 nm) detection. (c) Significant reduction of 9CDHRA, but not ATDHRA levels in serum, brain and liver of <i>Rbp1</i>^{<b><i>-/-</i></b>} animals (n = 8, n = 3 for brain) as compared to WT mice (n = 8, n = 3 for brain). All the error bars represent S.E.M.</p