Characterization of the retinoid binding properties of the major fusion products present in acute promyelocytic leukemia cells.

The bcr1- and bcr3- promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha) are the two major fusion proteins expressed in acute promyelocytic leukemia (APL) patients. These proteins, which are present in different lengths of PML (amino acids 1-552 and 1-394, respectively), contain most of the functional domains of PML and RAR alpha, bind all-trans-retinoic acid (t-RA), and act as t-RA-dependent transcription factors. T-RA is an effective inducer of clinical remission only in patients carrying the t(15;17) and expressing the PML/RAR alpha products. However, in APL patients achieving complete remission with t-RA therapy the bcr3-PML/RAR alpha product has been found associated with a poorer prognosis than bcr1-PML/RAR alpha. In the present study we have investigated the structural and functional properties of the bcr3-PML/RAR alpha in comparison to the previously characterized bcr1-PML/RAR alpha. In particular, we have measured the binding properties of the two endogenous ligands t-RA and 9-cis-RA to both of these isoforms. T-RA binding analysis of nuclear and cytosolic extracts prepared from bcr3-PML/RAR alpha APL patients and from bcr3-PML/RAR alpha COS-1 transfected cells indicates that this protein is present only as high-molecular-weight nuclear complexes. Using saturation binding assays and Scatchard analyses we found that t-RA binds with slightly less affinity to the bcr3-PML/RAR alpha receptor than to bcr1-PML/RAR alpha or RAR alpha (Kd = 0.4 nmol/L, 0.13 nmol/L or 0.09 nmol/L, respectively). Moreover, two different high-affinity 9-cis-RA binding sites (Kd = 0.45 and 0.075 nmol/L) were detectable in the bcr3-PML/RAR alpha product but not in the bcr1-PML/RAR alpha product (Kd = 0.77 nmol/L). By competition binding experiments we showed that 9-cis-RA binds with higher specificity to the bcr3-PML/RAR alpha isoform than to the bcr1-PML/RAR alpha or RAR alpha. Consistent with these data, the binding of 9-cis-RA to the bcr3-PML/RAR alpha product resulted in increased transcriptional activation of the RA-responsive element (RARE) TRE, but not of the betaRARE, in transiently transfected COS-1 cells. These results provide evidence indicating that preferential retinoid binding to the different PML/RAR alpha products can be measured.

Effect of vitamin A deficiency on the expression of low affinity glucocorticoid binding site activity and glucocorticoid-dependent induction of CYP3A2 in rat liver.

Maintenance of rats on a vitamin A-deficient diet resulting in undetectable levels of plasma retinol and significant reductions in relative testes weight compared to age-matched controls leads to the loss of liver membrane-bound low affinity glucocorticoid binding site (LAGS) activity without any effects on the levels of constitutively expressed CYP3A2 protein. Subsequent daily administration of retinol acetate to vitamin A-deficient rats results in the re-expression of LAGS activity to control levels by 7 days. To determine any role for the LAGS in the modulation of CYP3A2 expression by glucocorticoids, a single dose of dexamethasone 21-phosphate was administered to vitamin A-deficient rats and vitamin A-deficient rats induced to re-express LAGS by daily retinol acetate treatment. Retinol acetate administration alone induces CYP3A2 protein to apparent maximal levels since dexamethasone 21-phosphate does not further increase the induction response. However, CYP3A2 remains inducible to dexamethasone 21-phosphate in vitamin A-deficient rats. These data suggest that vitamin A status affects the expression of LAGS and CYP3A2 but that glucocorticoids regulate the induction of CYP3A2 by a mechanism(s) independent of their interaction with the LAGS.

A systematic analysis of the AF-2 domain of human retinoic acid receptor alpha reveals amino acids critical for transcriptional activation and conformational integrity.

We previously identified a carboxy-terminal transactivation function termed AF-2 within the last 15 amino acids of the ligand binding domain of the human retinoic acid receptor alpha (hRAR alpha). Truncation of this region abolished transcriptional activity. Here we provide a systematic analysis using alanine scanning mutagenesis of amino acids from Ser405 to Gly419 on a truncated hRAR alpha (delta419) to identify residues within this region that are responsible for transcriptional activity. Whereas mutations in positions 405, 408, 411, and 415-419 have little or no effect on the ability of modified receptors to activate a DR5 response element, mutations in positions 406, 407, 409, 410, and 412-413 modify either the potency or efficacy of all-trans retinoic acid (tRA) -induced gene transcription. Therefore, receptors with mutations in positions 409, 410, 413, and 414 have low transcriptional activity over a wide range of tRA concentrations. Receptors with mutations in positions 406, 407, and 412 exhibit a maximum transcriptional activity similar to wild-type hRAR alpha, but require higher concentrations of tRA. Replacing residues 405-419 on delta419 with the conserved AF-2 domain from the vitamin D3 receptor or the estrogen receptor results in a receptor with wild-type or low transcriptional activity, respectively. A full-length hRAR alpha mutant with an alanine substitution at position 406 (hRAR alpha M406A) binds tRA, but unlike the truncated M406A, which lacks the "F" region, it is not transcriptionally active. Protease mapping experiments detect a consistent difference in the conformation of hRAR alpha M406A compared to wild-type hRAR alpha. These data define amino acids from Ser405 to Gly419 on delta419 that are critical for transcriptional activity and point to the importance of the conformational integrity of receptor domains in maintaining ligand-induced transcriptional activation.

New retinoid X receptor subtypes in zebra fish (Danio rerio) differentially modulate transcription and do not bind 9-cis retinoic acid.

Retinoid X receptors (RXRs), along with retinoic acid (RA) receptors (RARs), mediate the effects of RA on gene expression. Three subtypes of RXRs (alpha, beta, and gamma) which bind to and are activated by the 9-cis stereoisomer of RA have been characterized. They activate gene transcription by binding to specific sites on DNA as homodimers or as heterodimers with RARs and other related nuclear receptors, including the vitamin D receptor, thyroid hormone receptors (TRs), and peroxisome proliferator-activated receptors. Two additional RXR subtypes (delta and epsilon) isolated from zebra fish cDNA libraries are described here; although both subtypes form DNA-binding heterodimers with RARs and TR, neither binds 9-cis RA, and both are transcriptionally inactive on RXR response elements. In cotransfection studies with TR, the delta subtype was found to function in a dominant negative manner, while the epsilon subtype had a slight stimulatory effect on thyroid hormone (T3)-dependent transcriptional activity. The discovery of these two novel receptors in zebra fish expands the functional repertoire of RXRs to include ligand-independent and dominant negative modulation of type II receptor function.

Binding of 9-cis-retinoic acid and all-trans-retinoic acid to retinoic acid receptors alpha, beta, and gamma. Retinoic acid receptor gamma binds all-trans-retinoic acid preferentially over 9-cis-retinoic acid.

Both 9-cis-retinoic acid (RA) and all-trans-RA (t-RA) compete for [3H]9-cis-RA binding to RA receptors (RAR alpha, beta, and gamma) in nucleosol fractions from transiently transfected COS-1 cells with IC50 values of approximately 12 and 5 nM, respectively. Curiously, 9-cis-RA competes for [3H]t-RA binding to mouse RAR alpha, beta, and gamma with IC50 values of 31, 8, and 60 nM, respectively, while t-RA itself does not exhibit such differential competition (IC50 values for RARs, 5 nM). A similar pattern is observed with human retinoic acid receptors (RARs). Differential binding of 9-cis-RA to the RAR beta and gamma receptors is also found following in vitro transcription and translation of these receptors. Displacement assays demonstrate that t-RA exhibits similar off-rates for RAR alpha, beta, and gamma. However, 9-cis-RA is 6-fold more rapidly displaced from RAR gamma than from RAR beta. When RAR-transfected COS-1 cells are incubated with [3H]t-RA, [3H]-9-cis-RA or various mixtures of these two radioligands, high performance liquid chromatography analysis demonstrates that the ligands bound in nucleosol fractions from RAR beta-transfected cells reflect the isomer content of the media. However, in identical whole cell assays, nucleosol fractions from RAR gamma-transfected cells preferentially bind t-RA over 9-cis-RA, consistent with the in vitro data. These binding kinetics in vitro and in whole cells suggest that there could be differences in the interactions of the receptor subtypes with the endogenous retinoic acids under physiologic conditions.

Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.

Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs. This latter observation suggested to us that the two isomers may interact with a common binding site. Here we report that RAR alpha has two distinct but overlapping binding sites for 9-cis RA and t-RA. Truncation of a human RAR alpha to 419 amino acids yields a receptor that binds both t-RA and 9-cis RA with high affinity, but truncation to amino acid 404 yields a mutant receptor that binds only t-RA with high affinity. Remarkably, this region also defines a C-terminal boundary for AF-2, as addition of amino acids 405 to 419 restores receptor-mediated gene activity to a truncated human RAR alpha lacking this region. It is interesting to speculate that binding of retinoid stereoisomers to unique sites within an RAR may function with AF-2 to cause differential activation of retinoid-responsive gene pathways.

Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids.

The binding of endogenous retinoids and stereoisomers of retinoic acid (RA) to the retinoid nuclear receptors, RA receptor (RARs) and retinoid X receptors (RXRs), was characterized using nucleosol preparations from transiently transfected COS-1 cells. Among several stereoisomers of RA tested, including 7-cis-, 9-cis-, 11-cis-, 13-cis-, and all-trans-RA, only 9-cis-RA effectively competes with 9-cis-[3H]RA binding to the RXRs. Additionally, the endogenous retinoid trans-didehydro-RA (t-ddRA) does not interact with RXRs, whereas the 9-cis form of ddRA competes effectively. RXRs (alpha, beta, and gamma) bind 9-cis-RA with dissociation constants (Kd) of 15.7, 18.3, and 14.1 nM, respectively. In contrast to the selectivity of RXRs for 9-cis-RA, RARs bind both t-RA and 9-cis-RA with high affinity, exhibiting Kd values in the 0.2-0.7 nM range for both ligands. Unlike RARs, the cellular RA binding proteins CRABPI or CRABPII bind t-RA but do not bind 9-cis-RA. Consistent with the binding data, 9-cis-RA and 9-cis-ddRA transcriptionally activate both GAL4-RXR and GAL4-RAR chimeric receptors with EC50 values of 3-20 nM for 9-cis-RA and 9-cis-ddRA, whereas t-RA and t-ddRA efficiently activate only GAL4-RAR chimeric receptors. Thus, 9-cis forms of endogenous retinoids can contribute to the pleiotropic effects of retinoids by interacting with both the RARs and RXRs.

Evaluation of the role of germ cells in regulating the route of secretion of immunoactive inhibin from the rat testis.

During normal sexual maturation of the male rat there is a progressive change in the route of secretion of inhibin by the Sertoli cell, from a predominantly basal route of secretion in prepuberty to a predominantly apical route of secretion in adulthood. This change may be monitored by comparing the levels of inhibin in testicular (TV), spermatic and peripheral (PV) venous blood and the levels in testicular interstitial fluid (IF). This study has assessed the role of germ cells in effecting this change by assessing (a) the effect of total germ cell depletion by X-irradiation of the males in utero, and (b) the effect of selective germ cell depletion in adulthood using the testicular toxicant, methoxyacetic acid (MAA). Female rats were X-irradiated on day 20 of gestation to produce male offspring whose testes were germ-cell deficient. Blood and IF samples were collected from groups of these offspring and age-matched controls at 35 and 100 days of age. In blood and IF samples, inhibin concentrations were significantly higher at 35 days of age than at 100 days. The absence of germ cells in X-irradiated animals did not affect the age-related fall in inhibin levels, nor the change in the predominant route of secretion of inhibin from the testis into blood. Testosterone was almost undetectable in 35-day-old controls, but was raised significantly by 100 days of age. In X-irradiated animals, testosterone levels were increased significantly at 35 days of age, and the levels in most samples were increased even more substantially by 100 days of age. However, PV levels of testosterone in 100-day-old X-irradiated animals were significantly lower than in controls. LH and FSH levels were raised in X-irradiated animals compared with their age-matched controls, but FSH levels in X-irradiated animals still fell with age, as in the controls. The role of specific germ cell types in regulating the route of secretion of inhibin from the normal adult testis was studied after depletion (80-100%) of pachytene and later spermatocytes by a single oral administration of MAA (650 mg/kg) to adult rats. At 3 days after MAA treatment, coincident with the loss of pachytene spermatocytes, plasma inhibin levels were increased significantly in blood and IF samples, and this was associated with a dramatic change in the route of secretion of inhibin from the testis, with increased secretion of this peptide via the base of the Sertoli cell into IF and TV blood.(ABSTRACT TRUNCATED AT 400 WORDS)

9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha.

Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.

Evaluation of changes in the secretion of immunoactive inhibin by adult rat seminiferous tubules in vitro as an indicator of early toxicant action on spermatogenesis.

A method for culturing isolated seminiferous tubules (ST) from adult rats for 1-3 days has been developed and optimized rigorously on the basis of the secretion of immunoactive inhibin under basal conditions and after maximal stimulation with rat FSH or dibutyryl cyclic AMP. The effect on these cultures of three known testicular toxicants was assessed. Of these, two are thought to act on the Sertoli cell, meta-dinitrobenzene (mDNB) and nitrobenzene (NB), while the third, methoxy acetic acid (MAA), is thought to act on pachytene spermatocytes. In addition, the effect of a possible testicular toxicant, 3-mononitrotoluene (3-MNT), was investigated. These data were compared with those obtained using cultures of immature rat Sertoli cells (SC) or SC + germ cells and with data on the effect of equivalent doses of the compounds on the secretion of immunoactive inhibin in vivo. In studies designed to optimize conditions for the secretion of immunoactive inhibin by ST in culture, significant effects were found of the type of culture medium used, the duration of culture, the total and individual length of tubules used, etc. All subsequent studies with toxicants utilized optimal conditions. Addition of either mDNB or NB to ST cultures at 10(-5) or 10(-3) M, or MAA at 10(-4) M, stimulated basal secretion of immunoactive inhibin by two- to fourfold on Days 1, 2, or 3 of culture while FSH or dibutyryl cyclic AMP-stimulated secretion of immunoactive inhibin was either unaffected or was enhanced to a small extent. At the same doses, mDNB or NB also enhanced secretion of immunoactive inhibin by SC cultures. although these effects were more variable and of smaller magnitude than the effects on ST cultures. In contrast, addition of up to 10(-3) MAA to cocultures of SC + germ cells had no effect on the secretion of immunoactive inhibin. Exposure of rats in vivo to levels of mDNB, NB, or MAA similar to those which stimulated secretion of immunoactive inhibin in vitro resulted in a two- to fourfold increase in the levels of immunoactive inhibin in testicular interstitial fluid (IF) at 1 and 3 days post-treatment, and this was associated with early impairment of spermatogenesis (as judged by testis weight). In contrast to these effects, addition of 3-MNT to ST or SC cultures had no effect except at 10(-3) M, when the secretion of immunoactive inhibin was increased marginally.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for the control of testicular interstitial fluid volume in the rat by specific germ cell types.

Following on from our recent evidence that Sertoli cells may regulate testicular interstitial fluid (IF) volume, this study has assessed whether depletion of specific germ cell types in vivo is associated with changes in recovered IF volume. Germ cell depletion was induced by either a single oral administration of 650 mg methoxyacetic acid (MAA)/kg or exposure of the testes to local heating (43 degrees C for 30 min). Treatment with MAA induced depletion or loss of most pachytene and later spermatocytes at 1-3 days and, because of maturation depletion, this resulted in the specific depletion of later germ cell types at 7-35 days. Testicular IF volume was unchanged at 1-7 days after MAA treatment but was increased significantly (P less than 0.01) at 14 days and was nearly doubled (P less than 0.001) at 21 days, before returning to control levels at 28-42 days. Serum LH (and FSH) levels were generally higher in MAA-treated rats, especially at 21 and 28 days, but there was no obvious correlation between LH levels and IF volume changes. Similarly, there was no relationship between IF volume changes and testicular weight or IF levels of testosterone. The increase in IF volume at 14-21 days after MAA treatment coincided with specific depletion of the later elongate spermatids (steps 14-19) and, when these cells reappeared in the testis, IF volume normalized. This possible causal association was studied further in rats exposed to local testicular heating which, within 3 days, caused major depletion of pachytene spermatocytes and early (step 1-8) spermatids.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that secretion of immunoactive inhibin by seminiferous tubules from the adult rat testis is regulated by specific germ cell types: correlation between in vivo and in vitro studies.

This study has assessed whether depletion of specific germ cell types is able to alter the secretion of immunoactive inhibin by adult Sertoli cells in vivo and in vitro. Pachytene and later spermatocytes were depleted (80-100%) by a single administration of methoxy acetic acid (MAA; 650 mg/kg) to adult rats. At intervals between 1 and 42 days posttreatment, rats were killed, and the blood levels of FSH, LH, and testosterone were determined together with the levels of immunoactive inhibin in plasma and testicular interstitial fluid (IF). At the same time intervals, seminiferous tubules (ST; 5 x 2 cm) were isolated from control and MAA-treated rats and cultured for 24-72 h in the presence or absence of rat FSH, (Bu)2cAMP, or MAA under rigorously optimized conditions. The hormonal changes observed were related to the presence/absence of specific germ cell types, as determined by assessment of testicular morphology in perfusion-fixed testes from similarly treated rats. One to 3 days after MAA treatment, coincident with the depletion of pachytene spermatocytes, blood levels of FSH were increased significantly compared with controls; FSH returned to control levels at 7-14 days (when early spermatids were depleted), but were increased again at 21-35 days (when late spermatids were depleted). In contrast, while the plasma levels of immunoactive inhibin were increased 2-fold 3 days posttreatment, they were comparable to controls at 7-14 days, but were decreased substantially at 21-28 days. The levels of immunoactive inhibin in testicular IF were more than doubled 1 and 3 days posttreatment, but were comparable to control levels at all other times. Blood levels of LH showed a similar pattern of change to FSH, although only at 21-28 days after MAA treatment was there a significant increase, while blood levels of testosterone were comparable at all times in control and MAA-treated rats. To confirm that the changes observed in vivo after MAA treatment were indicative of changes in Sertoli rather than Leydig cell secretion of immonoactive inhibin, its secretion by isolated ST was assessed, and a pattern of change similar to that in plasma was observed. Thus, when cultured for 24 h under basal conditions, ST from rats 1-3 days after MAA treatment showed a 2- to 3-fold increase in secretion of immunoactive inhibin, which returned to control levels at 7-14 days before being reduced substantially at 21-28 days and then recovering to control levels; similar changes were observed for FSH- and (Bu)2cAMP-stimulated secretion of immunoactive inhibin.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in Sertoli cell function in vitro induced by nitrobenzene.

Nitrobenzene (NB) has been identified as a testicular toxicant in vivo, but its site of action remains unknown. In the present study, the effect of NB on the Sertoli cell was assessed in vitro using Sertoli cell and Sertoli-germ cell cocultures. The parameters measured were the exfoliation of germ cells; the secretion of lactate, pyruvate, and inhibin; and gross cellular morphology. The effect of metadinitrobenzene (mDNB), a related compound which is a known Sertoli cell toxicant, was assessed for comparison. Gross morphological changes including vacuolation of Sertoli cells were observed following treatment of cultures with 10(-3) M NB. Exposure of cocultures to NB also resulted in dose-dependent exfoliation of predominantly viable germ cells. NB (greater than 5 X 10(-4) M) and mDNB at the single dose level used (10(-4) M) stimulated the secretion of lactate and pyruvate significantly by Sertoli cells, an effect that was more marked in the absence of germ cells. Comparable changes were observed in follicle stimulating hormone (FSH)-stimulated cultures. Inhibin secretion by Sertoli cells was also altered by exposure to NB but in a biphasic manner, with low (10(-8) to 10(-6) M) and high (10(-4) to 10(-3) M) doses enhancing inhibin secretion while intermediate (10(-5) M) doses had no effect. These effects were evident in both culture systems but inhibin secretion by Sertoli-germ cell cocultures was always greater than that by Sertoli cell cultures. However, these effects of NB on inhibin secretion were not evident in FSH-stimulated cultures. In contrast to the effects of NB, mDNB had no effect on basal secretion of inhibin but blocked the stimulatory effect of FSH. It is concluded that NB, like mDNB, is probably a Sertoli cell toxicant in view of its similar disruptive effects on various parameters of Sertoli cell function. However, NB is far less toxic than mDNB at equivalent concentrations in vitro. The present study is the first to evaluate the potential of inhibin secretion by Sertoli cells in culture as an additional marker of toxicant action, and concludes that it merits further study in this context.