␤-catenin is a component of stable cell adherent complexes whereas its free form functions as a transcription factor that regulate genes involved in oncogenesis and metastasis. Free ␤-catenin is eliminated by two adenomatous polyposis coli (APC)-dependent proteasomal degradation pathways regulated by glycogen synthase kinase 3␤ (GSK3␤) or p53-inducible Siah-1. Dysregulation of ␤-catenin turnover consequent to mutations in critical genes of the APC-dependent pathways is implicated in cancers such as colorectal cancer. We have identified a novel retinoid X receptor (RXR)-mediated APC-independent pathway in the regulation of ␤-catenin. In this proteasomal pathway, RXR agonists induce degradation of ␤-catenin and RXR␣ and repress ␤-catenin-mediated transcription. In vivo, ␤-catenin interacts with RXR␣ in the absence of ligand, but RXR agonists enhanced the interaction. RXR agonist action was not impaired by GSK3␤ inhibitors or deletion of the GSK3␤-targeted sequence from ␤-catenin. In APC-and p53-mutated colorectal cancer cells, RXR agonists still inactivated endogenous ␤-catenin via RXR␣. Interestingly, deletion of the RXR␣ A/B region abolished ligand-induced ␤-catenin degradation but not RXR␣-mediated transactivation. RXR␣-mediated inactivation of oncogenic ␤-catenin paralleled a reduction in cell proliferation. These results suggest a potential role for RXR and its agonists in the regulation of ␤-catenin turnover and related biological events.

The findings indicate that bimatoprost interacts with a prostamide receptor in the trabecular meshwork to increase outflow facility.

Balding causes widespread psychological distress but is poorly controlled. The commonest treatment, minoxidil, was originally an antihypertensive drug that promoted unwanted hair. We hypothesized that another serendipitous discovery, increased eyelash growth side-effects of prostamide F2α-related eyedrops for glaucoma, may be relevant for scalp alopecias. Eyelash hairs and follicles are highly specialized and remain unaffected by androgens that inhibit scalp follicles and stimulate many others. Therefore, we investigated whether non-eyelash follicles could respond to bimatoprost, a prostamide F2α analog recently licensed for eyelash hypotrichosis. Bimatoprost, at pharmacologically selective concentrations, increased hair synthesis in scalp follicle organ culture and advanced mouse pelage hair regrowth in vivo compared to vehicle alone. A prostamide receptor antagonist blocked isolated follicle growth, confirming a direct, receptor-mediated mechanism within follicles; RT-PCR analysis identified 3 relevant receptor genes in scalp follicles in vivo. Receptors were located in the key follicle regulator, the dermal papilla, by analyzing individual follicular structures and immunohistochemistry. Thus, bimatoprost stimulates human scalp follicles in culture and rodent pelage follicles in vivo, mirroring eyelash behavior, and scalp follicles contain bimatoprost-sensitive prostamide receptors in vivo. This highlights a new follicular signaling system and confirms that bimatoprost offers a novel, low-risk therapeutic approach for scalp alopecias.—Khidhir, K. G., Woodward, D. F., Farjo, N. P., Farjo, B. K., Tang, E. S., Wang, J. W., Picksley, S. M., and Randall, V. A. The prostamide-related glaucoma therapy, bimatoprost, offers a novel approach for treating scalp alopecias.

Ligand activation of retinoic acid receptors (RARs) involves coordinated changes in their interaction with coregulatory molecules. Binding of the agonist all-transretinoic acid to the RAR results in increased interaction with coactivator molecules as well as a decreased interaction with corepressor molecules. Thus, an all-transretinoic acid antagonist might function either by preventing agonist induction of such events or, additionally, by actively increasing repression via corepressor recruitment. We demonstrate that the repression of the transcriptional activity of a constitutively active RAR␥-VP-16 chimeric receptor by the inverse agonist AGN193109 requires a functional Co-R box and that binding of this ligand to RAR␥ leads to an increased interaction with the corepressor N-CoR both in glutathione S-transferase pull-down and yeast two-hybrid analyses. Detection of nuclear receptor corepressor (N-CoR) association with RAR␥ was greatly facilitated by inclusion of a RARE oligonucleotide in coimmunoprecipitation analyses, a result of an increase in association of the ternary complex consisting of RAR, RXR, and DNA. Similarly, this DNA-dependent increase in heterodimer formation likewise resulted in an increase in agonistmediated recruitment efficiency of the coactivator SRC-1. Under conditions which favor ternary complex formation, a RAR neutral antagonist is distinguished from an inverse agonist with respect to corepressor recruitment as is a RAR partial agonist distinguished from an agonist with respect to coactivator recruitment. These results indicate that it is possible to design RAR ligands with distinct recruitment capabilities for coregulators, both coactivators as well as corepressors. In addition, using this recruitment assay, we show that SRC-1 and the related coactivator molecule ACTR associate with the ternary complex via utilization of different helical motifs within their conserved receptor interaction domains.

A polypharmacologic approach to prostanoid based anti-inflammatory therapeutics was undertaken in order to exploit both the anti- and proinflammatory properties attributed to the various prostanoid receptors. Multitargeting of selected prostanoid receptors yielded a prototype compound, compound 1 (AGN 211377), that antagonizes prostaglandin D2 receptors (DPs) DP1 (49) and DP2 (558), prostaglandin E2 receptors (EPs) EP1 (266) and EP4 (117), prostaglandin F2α receptor (FP) (61), and thromboxane A2 receptor (TP) (11) while sparing EP2, EP3, and prostaglandin I2 receptors (IPs); Kb values (in nanomoles) are given in parentheses. Compound 1 evoked a pronounced inhibition of cytokine/chemokine secretion from lipopolysaccharide or TNF-α stimulated primary human macrophages. These cytokine/chemokines included cluster of designation 40 receptor (CD40), epithelial-derived neutrophil-activating protein 78 (ENA-78), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), IL-8, IL-18, monocyte chemotactic protein-1 (CCL2) (MCP-1), tissue plasminogen activator inhibitor (PAI-1), and regulated on activation, normal T cell expressed and secreted (RANTES). In contrast, the inhibitory effects of most antagonists selective for a single receptor were modest or absent, and selective EP2 receptor blockade increased cytokine release in some instances. Compound 1 also showed clear superiority to the cyclooxygenase inhibitors diclofenac and rofecoxib. These findings reveal that blockade of multiple prostanoid receptors, with absent antagonism of EP2 and IP, may provide more effective anti-inflammatory activity than global suppression of prostanoid synthesis or highly selective prostanoid receptor blockade. These investigations demonstrate the first working example of prostanoid receptor polypharmacology for potentially safer and more effective anti-inflammatory therapeutics by blocking multiple proinflammatory receptors while sparing those with anti-inflammatory activity.

Taken together, results suggest that bimatoprost specifically activates receptors in both cell types of the human conventional outflow pathway to modify intraocular pressure. However, only TM cell monolayers appear to have autocrine, or agonist-independent, receptor signaling that is sensitive to a prostamide receptor antagonist.

Cell impedance positively correlates with cellular stiffness/contractility. Because EP2/4 receptors caused decreased cell stiffness in SC, but not in TM cells, both receptors appear to mediate IOP lowering via changes in SC cell stiffness in the conventional outflow pathway.

2-Arachidonoylglycerol is oxygenated by cyclooxygenase-2 to form prostaglandin glyceryl esters. Previous work in this laboratory has suggested that PGE2-G activates a novel G protein-coupled receptor in a murine macrophage-like cell line, RAW 264.7. To probe the structural determinants for the putative receptor in RAW 264.7 cells, a panel of 10 analogs was tested for their ability to increase intracellular calcium. These analogs included PGE2- and PGF2α– ethanolamide, 4 PGE2 glyceryl ester analogs, and 4 PGF2α glyceryl ester analogs. The glyceryl ester analogs differed in the positioning of the hydroxyl groups in the glycerol moiety and the type of linker (ester, amide, or thioester) of the prostaglandin to the glycerol moiety. Compounds were also evaluated in a human non-small cell lung cancer cell line (H1819). The glycerol moiety was required for the calcium response. All glyceryl ester analogs but not ethanolamides caused a concentration-dependent increase in calcium levels in both RAW 264.7 and H1819 cells. An amide or ester linkage was preferable to a thioester linkage. The EC50 values did not significantly change when the positioning of the hydroxyls was varied. This calcium response induced by the glyceryl ester analogs appears to be independent of the putative hydrolysis products, PGE2 and PGF2α, and appears to represent a novel signaling pathway.