Identification and Functional Separation of Retinoic Acid Receptor Neutral Antagonists and Inverse Agonists

Abstract

Inverse agonists are ligands that are capable of repressing basal receptor activity in the absence of an agonist. We have designed a series of C-1-substituted acetylenic retinoids that exhibit potent antagonism of retinoic acid receptor (RAR)-mediated transactivation. Comparison of these related retinoid antagonists for their ability to repress basal RAR transcriptional activity demonstrates that the identity of the C-1 substituent differentiates these ligands into two groups: RAR inverse agonists and neutral antagonists. We show that treatment of cultured human keratinocytes with a RAR inverse agonist, but not a RAR neutral antagonist, leads to the repression of the serum-induced differentiation marker MRP-8. While RAR-selective agonists also repress expression of MRP-8, cotreatment with a RAR inverse agonist and a RAR agonist results in a mutual repression of their individual inhibitory activities, indicating the distinct modes of action of these two disparate retinoids in modulating MRP-8 expression. Our data indicate that RARs, like beta2-adrenoreceptors, are sensitive to inverse agonists and that this new class of retinoids will provide insight into the molecular mechanisms of RAR function in skin and other responsive tissues.

Casein Kinase 1α Interacts with Retinoid X Receptor and Interferes with Agonist-induced Apoptosis

“…The pRXRE-Luc is the report plasmid that contains five tandem repeats of a 35-base pair sequence (DR-1) from the promoter of the mouse CRBP-II gene (21) inserted immediately upstream of thymidine kinase-luciferase. pRARE-Luc contains three copies of DR-5 (22). 24 h after transfection, the cells were incubated for another 16 h in medium containing 0.5% charcoal-treated serum along with the ligand and then harvested for the measurement of ␤-galactosidase activity and luciferase activity using a system and protocol from Promega.…”

Active repression by RARγ signaling is required for vertebrate axial elongation

“…The mutant receptor recognizes a mutant TK-luc reporter, (RXRE 1/2 -GRE 1/2 )×4 TK-luc, to which endogenous RARs do not bind (Klein et al, 1996). In transient transfection assays, 4647 selectively activated RARγ at doses below 0.1 µM (supplementary material Fig.…”

“…pCDG1-xRarβ2 and pCMX-GAL4-xRarβ cloning primers are listed in supplementary material Table S2. pCDG1-xCyp26a1 and pCDG1-c-smrt were constructed by PCR amplification of xCyp26a1 coding regions (Hollemann et al, 1998) or Xl c-smrt (37b−, 41+) (Chen et al, 1996;Malartre et al, 2004) EGCKG→GSCKV and xRarγ2 EGCKG→GSCKV were designed according to Klein et al (1996), constructed by two-fragment PCR, and cloned into pCDG1 (primer sequences are provided in supplementary material Table S3). Four copies of RXRE 1/2 -GRE 1/2 (GGAAGGGTTCACCGAA-AGAACACTCGC) were cloned upstream of the TK-luciferase reporter.…”

A Retinoid-Related Molecule that Does Not Bind to Classical Retinoid Receptors Potently Induces Apoptosis in Human Prostate Cancer Cells through Rapid Caspase Activation

“…1; Ref. 44). After 5 h, cells were fed with DMEM containing 10% charcoal-treated fetal bovine serum (FBS, Life Technologies, Inc.).…”

“…For RAR transactivation assays, the ERE-tk-Luc that contains the binding site for estrogen receptors was cotransfected with chimeric estrogen receptor-RARs, in which the DNA-binding domain of RAR was substituted with that of the estrogen receptor. Antagonism of ATRA transactivation of RARs by AGN193198 or AGN194310 was assayed using RARs-progesterone/RXR␣ heterodimers, in which the P-box sequence of RAR had been changed into the sequence (GSCKV) of glucocorticoid receptor, so called RARs-progesterone (44). RARs-progesterone/RXR␣ heterodimers recognize the half-site binding motif of glucocorticoid receptor and the half-site binding motif of RXR.…”