Retinoid X receptor agonist elevation of serum triglycerides in rats by potentiation of retinoic acid receptor agonist induction or by action as single agents.

Hypertriglyceridemia is a major side-effect of retinoid therapy in humans. We previously reported that agonists for the retinoic acid receptors (RARs), but not the retinoid X receptors (RXRs), elevate serum triglycerides in male Fischer rats, and that, surprisingly, the RAR/RXR pan-agonists 9-cis-retinoic acid and AGN 191659 [(E)-5-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)propen-1-yl]-2-thiophenecarboxylic acid] induce 2- to 3-fold higher levels of serum triglycerides than the RAR-selective agonists alone. We have now demonstrated that hypertriglyceridemia induced by an RAR agonist, AGN 190121 [4-[4-(2',6',6'-trimethylcyclohex-1-enyl)-but-1-yne-3-enyl]benzoic acid], is substantially potentiated by the RXR-selective agonists AGN 191701 [(E) 2-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)propen-1-yl]-4-thiophene-carboxylic acid] and AGN 192849 [(3,5,5,8,8,-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl) (5 carboxypyrid-2-yl)sulfide] in a dose-dependent manner. RXR-specific retinoids, as previously reported, had no independent effect on serum triglycerides when tested at 24 hr after final dosing, but did elicit a reversible hypertriglyceridemia at 2.5 and 5 hr. This induction of serum triglycerides could not be blocked by the potent RAR-specific antagonist AGN 193109 [4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)-ethynyl] benzoic acid]. The RXR ligand-induced hypertriglyceridemia was independent of the effect of feeding or fasting. The relative potencies of RXR-specific retinoids for acute triglyceride elevation (AGN 194204 [3,7-dimethyl-6S,7S-methano-7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl] 2(E),4(E) heptadienoic acid] > AGN 192849 approximately AGN 191701) approximately correlated with potencies in the activation of the RXR receptors. The RAR/RXR pan-agonist effect included >50% inhibition of total heparin-releasable lipase activity in serum, consistent with inhibition of lipase-mediated triglyceride disposal. These data also indicate that RAR and RXR ligands can act synergistically to induce hypertriglyceridemia through distinct mechanisms of action.

Evidence of a lysosomal pathway for apoptosis induced by the synthetic retinoid CD437 in human leukemia HL-60 cells.

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene carboxylic acid (AHPN/CD437) has been proven to be a potent inducer of apoptosis in a variety of tumor cell types. However, the mechanism of its action remains to be elucidated. Recent studies suggest that the lysosomal protease cathepsin D, when released from lysosomes to the cytosol, can initiate apoptosis. In this study, we examined whether cathepsin D and free radicals are involved in the CD437-induced apoptosis. Exposure of human leukemia HL-60 cells to CD437 resulted in rapid induction of apoptosis as indicated by caspase activation, phosphatidylserine exposure, mitochondrial alterations and morphological changes. Addition of the antioxidants alpha-tocopherol acetate effectively inhibited the CD437-induced apoptosis. Measurement of the intracellular free radicals indicated a rise in oxidative stress in CD437-treated cells, which could be attenuated by alpha-tocopherol acetate. Interestingly, pretreatment of cells with the cathepsin D inhibitor pepstatin A blocked the CD437-induced free radical formation and apoptotic effects, suggesting the involvement of cathepsin D. However, Western blotting revealed no difference in cellular quantity of any forms of cathepsin D between control cells and CD437-treated cells, whereas immunofluorescence analysis of the intracellular distribution of cathepsin D showed release of the enzyme from lysosomes to the cytosol. Labeling of lysosomes with lysosomotropic probes confirmed that CD437 could induce lysosomal leakage. The CD437-induced relocation of cathepsin D could not be prevented by alpha-tocopherol acetate, suggesting that the lysosomal leakage precedes free radical formation. Furthermore, a retinoic acid nuclear receptor (RAR) antagonist failed to block these effects of CD437, suggesting that the action of CD437 is RAR-independent. Taken together, these data suggest a novel lysosomal pathway for CD437-induced apoptosis, in which lysosomes are the primary target and cathepsin D and free radicals act as death mediators.

Phenylcyclohexene and phenylcyclohexadiene substituted compounds having retinoid antagonist activity.

Retinoids are natural and synthetic analogues of the hormone retinoic acid. Systemic retinoid agonist therapy is usually associated with toxic side effects, such as mucocutaneous toxicity, which may be alleviated by the use of topical retinoid antagonists. We report the synthesis and biological activity of a new series of potent, RAR-specific antagonists substituted with phenylcyclohexene and phenylcyclohexadiene groups.

Mannose 6-phosphate/insulin-like growth factor II receptor mediates the growth-inhibitory effects of retinoids.

Both retinoids and the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) have been shown to play an important role in controlling cell growth during embryonic development and oncogenesis. Our recent work (Kang et al., Proc. Natl. Acad. Sci. USA, 94: 13671-13676, 1997; Kang et al., Proc. Natl. Acad. Sci. USA, 95: 13687-13691, 1998) revealed a direct biochemical interaction between retinoic acid (RA) and the M6P/IGF2R, thereby leading us to hypothesize that the M6P/IGF2R may mediate a growth-inhibiting effect of RA. To test this hypothesis, cell growth and apoptosis in response to RA and various receptor-selective retinoids were examined in cells that lack or overexpress the M6P/IGF2R. RA and those retinoids capable of binding to the M6P/IGF2R induced a remarkable morphological change with characteristics of round shape and reduced spreading, apoptosis, and growth inhibition in stably transfected mouse P388D1 cells overexpressing the M6P/IGF2R but not in the M6P/IGF2R-deficient P388D1 cells. These effects of RA were neither blocked by a potent RA nuclear receptor (RAR) antagonist (AGN193109), nor mimicked by a selective RAR agonist (TTNPB), suggesting that the observed effects of RA are independent of RARs. Similar effects of the retinoids were observed in cultured neonatal rat cardiac myocytes that have high levels of the M6P/IGF2R. Furthermore, overexpression of the M6P/IGF2R in a RA-resistant cancer cell line (HL-60R) that lacked functional RARs gave the cells a susceptibility to RA-induced apoptosis. These data suggest that the M6P/ IGF2R may play an important role in mediating retinoid-induced apoptosis/growth-inhibition and provide insight into the similar biological effects of RA and the M6P/IGF2R on fetal development and carcinogenesis.

Retinoic acid alters the intracellular trafficking of the mannose-6-phosphate/insulin-like growth factor II receptor and lysosomal enzymes.

Previously, we showed that retinoic acid (RA) binds to the mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) with high affinity, suggesting that M6P/IGF2R may be a receptor for RA. Here, we show that RA, after 2-3 h of incubation with cultured neonatal-rat cardiac fibroblasts, dramatically alters the intracellular distribution of M6P/IGF2R as well as that of cathepsin B (a lysosomal protease bearing M6P). Immunofluorescence techniques indicate that this change in intracellular distribution is characterized by a shift of the proteins from the perinuclear area to cytoplasmic vesicles. The effect of RA was neither blocked by an RA nuclear receptor antagonist (AGN193109) nor mimicked by a selective RA nuclear-receptor agonist (TTNPB). Furthermore, the RA-induced translocation of cathepsin B was not observed in M6P/IGF2R-deficient P388D1 cells but occurred in stably transfected P388D1 cells expressing the receptor, suggesting that the effect of RA might be the result of direct interaction with M6P/IGF2R, rather than the result of binding to the nuclear receptors. These observations not only support the idea that M6P/IGF2R mediates an RA-response pathway but also indicate a role for RA in control of intracellular trafficking of lysosomal enzymes. Therefore, our observations may have important implications for the understanding of the diverse biological effects of retinoids.

Receptor specificity of retinoid-induced epidermal hyperplasia: effect of RXR-selective agonists and correlation with topical irritation.

Retinoid induction of epidermal hyperplasia was investigated in hairless mice with synthetic ligands for the retinoic acid (RAR) and retinoid X (RXR) nuclear receptors. Induction of hyperplasia by all-trans retinoic acid and the RAR-specific retinoids TTNPB, tazarotene and AGN 190121 varied over a wide range (ED50 = 0.2-100 nmol/animal in three daily applications). Potency of induction was not directly correlated to receptor-binding affinity, but specificity of action could be demonstrated by inhibition with the high-affinity antagonist of the RARs, AGN 193109. Although RAR is functionally complexed with RXR in vivo, RXR-selective compounds have only weak potency in induction of hyperplasia. The ED50 value of the RXR-selective AGN 191701 was 600 nmol/animal compared with an ED50 value of 0.2 nmol for the structurally similar RAR-selective TTNPB. SR11237 and SR11217, also RXR-selective, each have an ED50 value of >1000 nmol. Unlike RAR-specific retinoids, RXR-selective retinoids cause only very mild skin flaking at high doses. Relative potencies for cumulative topical irritation (flaking and abrasion) of both RAR and RXR ligands were well correlated with epidermal hyperplasia. These data are consistent with RXR as a silent partner in the RAR-RXR heterodimer in skin.

Mitogenic effect of retinoid X receptor agonists in rat liver.

(E)-2-[2-(5,6,7,8-Tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl) propen-1-yl]-4-thiophenecarboxylic acid (AGN 191701) and other retinoid X receptor (RXR)-selective agonists were observed to cause hepatomegaly in rats. The purpose of the present study was to understand the biochemical basis of RXR agonist-induced hepatomegaly. Male Fischer rats were implanted s.c. with osmotic pumps containing 5-bromo-2'-deoxyuridine (BrdU) and treated by gavage with 0,60, or 180 mumol/kg/day of AGN 191701 for 3 days. AGN 191701 caused dose-dependent hepatomegaly in the absence of hepatic necrosis and necrosis and increased hepatocyte BrdU labeling index (LI). To determine if AGN 191701-induced hepatic hyperplasia was sustained, rats were treated by gavage with 60 mumol/kg of AGN 191701 for up to 7 days and exposed to BrdU via osmotic pump on days 1-3 or on days 6-8. Hepatocyte L1 and mitotic index were increased only in rats exposed to BrdU on days 1-3, indicating that AGN 191701-induced hepatocyte proliferation was transient. The receptor specificity of this mitogenic effect was tested by co-treating rats for 2 days with various retinoids and BrdU. 2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-2-(4-carboxylph enyl)-1,3-dioxolane (SR11237), an RXR-selective agonist, and (E)-5-[2-(5,6,7,8-tetrahydro-3,5,5,8-pentamethyl-2-naphthyl)propen -1-yl]-2-thiophenecarboxylic acid (AGN 191659), a retinoic acid receptor (RAR)/RXR pan-agonist, both increased hepatocyte LI. Two RAR-selective agonist, all-trans-retinoic acid and (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propen -1-yl] benzoic acid (TTNPB), did not affect hepatocyte LI. To determine if RXR agonists have biochemical effects in common with a peroxisome proliferator, various endpoints were measured 24 hr after two daily treatments with AGN 191701, SR11237, or clofibrate. While all three compounds induced hepatic acyl CoA oxidase activity, only clofibrate increased hepatic carnitine acyl transferase activity and lowered serum triglycerides. Taken together, these data show that RXR-selective agonists but not RAR-selective agonists cause hepatomegaly accompanied by hepatocyte mitogenesis in rats. The fact that RXR agonist have some biological effects distinct from RAR agonists and clofibrate suggests that RXR-selective agonists may have unique therapeutic applications.

Retinoid-induced hypertriglyceridemia in rats is mediated by retinoic acid receptors.

Retinoids in clinical use today are known to induce hypertriglyceridemia as one of their major side effects. The purpose of the present study was to determine, in an appropriate animal model, if retinoid-induced hypertriglyceridemia is mediated by retinoic acid receptors (RARs) and/or by retinoid X receptors (RXRs). Oral gavage of male Fischer rats with 13-cis-retinoic acid for 6 days caused a rapid and sustained increase in serum triglycerides that was reversible within 4 days posttreatment. In subsequent experiments, rats were treated by gavage once daily for 3 days with various retinoids, and serum triglyceride levels were determined 24 hr after the last treatment without fasting. All-trans- and 13-cis-retinoic acid, which can be converted to both RAR and RXR agonists, and 9-cis-retinoic acid, an RAR/RXR pan-agonist, caused dose-dependent increases in serum triglycerides at doses that did not cause weight loss or mucocutaneous toxicity. Ro 13-6298 and AGN 190121, two RAR-specific agonists, caused dose-dependent increases in serum triglycerides, although Ro 13-6298 only induced hypertriglyceridemia at weight-suppressive doses. Two RXR-selective agonists, LG100268 and AGN 191701, failed to induce hypertriglyceridemia or weight loss up to the highest doses tested. A structural isomer of AGN 190121 that does not activate RARs or RXRs, AGN 190727, did not induce hypertriglyceridemia. Hypertriglyceridemia induced by AGN 190121 was significantly inhibited by cotreatment with an RAR-selective antagonist, AGN 193109. Taken together, these data provide strong evidence that retinoid-induced hypertriglyceridemia is mediated, at least in part, by RARs. These data also suggest that RXR-specific agonists may have reduced potential to induce hypertriglyceridemia relative to RAR-active retinoids.

Synthesis and structure-activity relationships of retinoid X receptor selective diaryl sulfide analogs of retinoic acid.

Retinoids exert their biological effects by binding to and activating nuclear receptors that interact with responsive elements on DNA to promote gene transcription. There are two families of retinoid receptors, the retinoic acid receptor (RAR) family and the retinoid X receptor (RXR) family, which are each further divided into three subclasses: RAR alpha, beta, gamma and RXR alpha, beta, gamma. Herein we describe the synthesis and structure-activity relationships of a new series of diaryl sulfide retinoid analogs that specifically bind and transactivate the RXRs. Furthermore, the sulfoxide and sulfone derivatives of these analogs are partial agonists which activate the RXRs only at high concentrations. Thus, these compounds possess a potential site of metabolic deactivation and may have less prolonged systemic effects than other compounds with arotinoid-like structures. We show also that these compounds have activity in nontransfected cells as demonstrated by their ability to induce TGase activity in HL-60 cells. Finally, we corroborate our earlier report that RXR-specific agonists may possess reduced teratogenic toxicity compared to RAR-specific agonists since these compounds are much less potent inhibitors of chondrogenesis than RAR-specific agonists such as TTNPB.

Retinoid X receptor-specific retinoids inhibit the ability of retinoic acid receptor-specific retinoids to increase the level of insulin-like growth factor binding protein-3 in human ectocervical epithelial cells.

The hormones derived from vitamin A and related synthetic ligands (retinoids) are important regulators of differentiation and development and have been shown to be therapeutically useful in the treatment of cervical cancer. All-trans-retinoic acid exerts its effects by activation of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers. These heterodimers bind to the retinoic acid response elements of target genes to regulate gene expression. RXR ligands act through RXR homodimers to regulate gene expression. In the present study, we describe the effects of RAR- and RXR-specific ligands on the regulation of insulin-like growth factor binding protein-3 (IGFBP-3) production and cell proliferation in human ectocervical epithelial (ECE) cell lines. Treatment of ECE16-1 cells with a RAR-specific ligand (TTNPB) or a ligand that interacts with both RAR and RXR receptors (9-cis-retinoic acid) increases IGFBP-3 levels and suppresses cell proliferation. In contrast, RXR-specific ligands (AGN191701, SR11217, and SR11237) do not regulate proliferation and slightly suppress the IGFBP-3 level. Cotreatment with increasing concentrations (0.01-1000nm) of RXR-specific ligand antagonizes the growth suppressive and IGFB-3-increasing effects of 1000 nM TTNPB. Similar results are observed in two other ECE cell lines, ECE16-D1 and ECE16-D2. These results indicate that RXR-specific ligands can antagonize RAR responses in these cell lines and suggest that a RAR-specific retinoid may be superior to one with mixed RAR/RXR binding activity for inhibiting cervical cancer cell proliferation. Moreover, the antagonism of RAR-dependent responses by RXR-specific ligands is consistent with a squelching model in which the RXR-specific ligand drives formation of RXR/RXR homodimers at the expense of the more active RAR/RXR heterodimers.

Differential teratogenic response of mouse embryos to receptor selective analogs of retinoic acid.

Early events that initiate teratogenesis by Accutane or other retinoids in mammalian embryos remain unknown. It would be helpful for mechanistic considerations to know whether or not retinoids act through retinoid receptor-dependent pathways, and if they do, which of the two families of receptors (retinoic acid receptors - RARalpha, beta, gamma or retinoid X receptors - RXRalpha, beta, gamma) are more likely involved. We previously used an in vitro bioassay to demonstrate that those retinoid analogs with binding affinity and transactivational activity limited only to the RXRs have a low potential as teratogens. Here, we have extended the study to examine teratogenicity, in pregnant mice, of a number of synthetic retinoids with varying degrees of receptor selectivity. The ability of each compound to induce fetal limb and craniofacial defects after a single exposure on day 11 of gestation was assessed and compared to that of all-trans retinoic acid (RA). The highest dose selected was 100 mg/kg maternal body weight since such a regimen of all-trans RA affects virtually every exposed embryo without any indication of maternal toxicity. We found that although all RAR agonists were strong teratogens, their potencies varied over a wide magnitude. The teratogenic potencies and receptor transactivation profiles of RAR agonists were not directly correlated since compounds with similar receptor activities presented major differences in potencies. Three compounds were exclusively RXR agonists, and these were not teratogenic under our experimental conditions. Two additional compounds which turned out to be non-teratogenic were distinguished by the fact that they activated neither RARs nor RXRs. These data indicate that although RAR-dependent mechanisms are likely involved in retinoid-induced teratogenesis, there are additional factors which determine teratogenic potency. The absence of teratogenic response in the case of RXR agonists suggests that risk-benefit analyses of such receptor-selective compounds may be fruitful in further studies.

Diminished teratogenicity of retinoid X receptor-selective synthetic retinoids.

One feature that contraindicates the wide therapeutic use of retinoids is their teratogenicity. Synthetic retinoids are distinguishable from each other on the basis of their partial or exclusive preference in binding and activation of all-trans retinoic acid receptors (RARs) or retinoid X receptors (RXRs). Using mouse embryo limb bud cells in micromass cultures as a bioassay, we examined the inhibitory activities of a number of standard and novel retinoids on chondrogenic cell differentiation. Transient cotransfection of HeLa cells was used to measure the ability of each retinoid to induce transcription of a reporter gene by activating RAR alpha, RAR beta, RAR gamma, or RXR alpha chimeric constructs. All retinoids in this study that activated RARs to any degree in the cotransfection assay also inhibited chondrogenesis in vitro, whereas retinoids that were either specific for RXR or inactive in the cotransfection assay did not. The activity of RAR-selective agonists and the inactivity of RXR-specific agonists in the cotransfection assay correlated well with the relative teratogenicity of six of the representative retinoids studied when orally administered at day 11 to pregnant ICR mice.

Synthesis and structure-activity relationships of stilbene retinoid analogs substituted with heteroaromatic carboxylic acids.

Retinoids elicit biological responses by activating a series of nuclear receptors. Six retinoid receptors belonging to two families are currently known: retinoic acid receptors (RAR alpha,beta,and gamma) and retinoid X receptors (RXR alpha,beta,and gamma). Stilbene retinoid analogs of retinoic acid (RA), such as (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)prope n-1- yl]benzoic acid (TTNPB, 1) and (E)-4-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)pro pen-1- yl]benzoic acid (3-methyl-TTNPB, 2), display differential RAR and RXR activities, depending on the substituent at C3 of the naphthalene ring. We report here structural modifications of the benzoate moiety of 2 that result in analogs with greater RXR selectivity as well as those with pan-agonist (activate both RAR and RXR receptors) activities, analyze the structural features that impart receptor selectivity, and describe a stereoselective method for the synthesis of these analogs. The biological activities associated with the RAR and RXR receptors were examined by testing representative examples with different receptor activation profiles for their ability to induce tissue transglutaminase (Tgase) activity in a human promyelocytic leukemia cell line (HL-60 cdm-1) and to inhibit tumor-promoter-induced ornithine decarboxylase (ODC) activity in hairless mouse skin. These results suggest that RAR agonists and RXR agonists may have different therapeutic applications. Finally, we show that RXR agonists are significantly reduced in teratogenic potency relative to RAR agonists and may therefore have significant advantages in clinical practice.

Enhancing transfer of training: using role-play to foster teamwork in the cockpit.

Role-plays serve a unique purpose in providing practice and feedback for aircrew team development training in the classroom. When compared with other training strategies, role-play provides targeted practice and feedback of specific behaviors at low cost, with the trade-off being the amount of fidelity it provides. This article presents guidelines for designing role-plays to elicit crew resource management (CRM) behaviors and providing feedback to trainees on their performance. Through careful design procedures, a role-play exercise can provide both targeted practice and feedback and serve a useful purpose in the overall training design of a CRM training program.

Children's expressions of spatial knowledge.

Different expressions of spatial knowledge were examined by having groups of first-, fourth-, and sixth-grade children perform model construction, verbal description, and route reversal tasks after they learned the correct path through a pedestrian maze. Age-related improvement was found in the rate of learning the maze and in the accuracy of verbal descriptions, suggesting that maze learning may be verbally mediated. All children performed well in sequencing intersections in the model but performed poorly in choosing path options in the model. Route reversal after learning was accurate and equivalent across groups. Overall, results suggest that both general and task-specific skills are involved in different products of spatial knowledge.