4-Oxoretinol, a new natural ligand and transactivator of the retinoic acid receptors.

All-trans-retinoic acid (at-RA) induces cell differentiation in a wide variety of cell types, including F9 embryonic teratocarcinoma cells, and can influence axial pattern formation during embryonic development. We now identify a novel retinoid synthetic pathway in differentiating F9 cells that results in the intracellular production of 4-oxoretinol (4-oxo-ROL) from retinol (vitamin A). Approximately 10-15% of the total retinol in the culture is metabolized to 4-hydroxyretinol and 4-oxo-ROL by the at-RA-treated, differentiating F9 cells over an 18-hr period, but no detectable metabolism of all-trans-retinol to at-RA or 9-cis-retinoic acid is observed in these cells. Remarkably, we show that 4-oxo-ROL can bind and activate transcription of the retinoic acid receptors whereas all-trans-retinol shows neither activity. Low doses of 4-oxo-ROL (e.g., 10(-9) or 10(-10 M) can activate the retinoic acid receptors even though, unlike at-RA, 4-oxo-ROL does not contain an acid moiety at the carbon 15 position. 4-oxo-ROL does not bind or transcriptionally activate the retinoid X receptors. Treatment of F9 cells with 4-oxo-ROL induces differentiation without conversion to the acid and 4-oxo-ROL is active in causing axial truncation when administered to Xenopus embryos at the blastula stage. Thus, 4-oxo-ROL is a natural, biologically active retinoid that is present in differentiated F9 cells. Our data suggest that 4-oxo-ROL may be a novel signaling molecule and regulator of cell differentiation.

Targeted disruption of retinoic acid receptor alpha (RAR alpha) and RAR gamma results in receptor-specific alterations in retinoic acid-mediated differentiation and retinoic acid metabolism.

F9 embryonic teratocarcinoma stem cells differentiate into an epithelial cell type called extraembryonic endoderm when treated with retinoic acid (RA), a derivative of retinol (vitamin A). This differentiation is presumably mediated through the actions of retinoid receptors, the RARs and RXRs. To delineate the functions of each of the different retinoid receptors in this model system, we have generated F9 cell lines in which both copies of either the RAR alpha gene or the RAR gamma gene are disrupted by homologous recombination. The absence of RAR alpha is associated with a reduction in the RA-induced expression of both the CRABP-II and Hoxb-1 (formerly 2.9) genes. The absence of RAR gamma is associated with a loss of the RA-inducible expression of the Hoxa-1 (formerly Hox-1.6), Hoxa-3 (formerly Hox-1.5), laminin B1, collagen IV (alpha 1), GATA-4, and BMP-2 genes. Furthermore, the loss of RAR gamma is associated with a reduction in the metabolism of all-trans-RA to more polar derivatives, while the loss of RAR alpha is associated with an increase in metabolism of RA relative to wild-type F9 cells. Thus, each of these RARs exhibits some specificity with respect to the regulation of differentiation-specific gene expression. These results provide an explanation for the expression of multiple RAR types within one cell type and suggest that each RAR has specific functions.

9-Cis retinoic acid inhibits growth of breast cancer cells and down-regulates estrogen receptor RNA and protein.

All-trans retinoic acid (tRA) inhibits growth of estrogen receptor-positive (ER+) breast cancer cells in vitro, and a variety of retinoids inhibit development of breast cancer in animal models. 9-cis retinoic acid (9-cis RA) is a naturally occurring high affinity ligand for the retinoid X receptors, as well as the retinoic acid receptors (RARs). Whether 9-cis RA has a different spectrum of biological activity from tRA, which only binds RARs with high affinity, is largely unknown. We studied the effects of 9-cis RA on growth and gene expression in ER+ and ER- human breast cancer cells. 9-cis RA inhibited the growth in monolayer culture of several ER+, but not ER-, cell lines in a dose-dependent manner. Growth inhibition and morphological changes by 9-cis RA were similar to those of tRA, suggesting that the ability to bind both RAR and retinoid X receptors did not significantly augment growth inhibition or confer sensitivity to tRA-resistant lines. MCF-7 cells exposed to 9-cis RA showed a dose-dependent accumulation in G1. Northern analyses showed that RAR-alpha and RAR-beta were not significantly regulated, while RAR-gamma was up-regulated and retinoid X receptor alpha was down-regulated by 9-cis RA. Since interactions between tRA and ER-dependent transcription have recently been reported, we investigated whether these retinoids regulate expression of ER itself or estrogen-responsive genes. Both 9-cis RA and tRA induce down-regulation of ER mRNA and protein in MCF-7 cells. 9-cis RA down-regulates expression of the estrogen-responsive genes PR and pS2 in MCF-7 cells as reported previously for tRA. In several ER-positive subclones, we found that the degree of ER expression and regulation, but not always estrogen-sensitivity, correlates with the growth-inhibitory effects of 9-cis RA. Further, in an ER-, retinoid-unresponsive breast cancer cell line, induced ER expression confers responsiveness to retinoid growth inhibition. These data, combined with reports of additive growth inhibition of tRA and tamoxifen in vitro, suggest that 9-cis RA might augment the ability of tamoxifen to inhibit growth of ER+ breast cancer cells in vivo.

Conversion of anterior limb bud cells to ZPA signaling cells in vitro and in vivo.

Following a graft of posterior (zone of polarizing activity or ZPA) cells into the anterior margin of the developing chick wing bud, anterior cells are induced to alter their developmental fate and form structures that are normally composed of posterior cells. When anterior cells are cultured under microdissociation conditions they develop ZPA signaling ability within 24 hr. ZPA signaling in these cultures is transient and once established the level of ZPA signaling declines with time in culture. ZPA signaling in anterior cells is sensitive to treatment with fibroblast growth factor-2 (FGF-2); the development of ZPA signaling is inhibited when nonsignaling anterior cells are cultured in the presence of FGF-2. Conversely, when anterior cells that have developed ZPA signaling are treated with FGF-2, ZPA signaling levels are maintained. Thus, our results suggest that FGF-2 maintains or stabilizes the positional character of anterior (nonsignaling) cells, as well as anterior ZPA signaling converted cells, and posterior (ZPA signaling) limb bud cells in vitro (R. Anderson, M. Landry, and K. Muneoka (1993) Development 117, 1421-1433). In addition, anterior cells will convert to ZPA signaling cells in vivo following apical ectodermal ridge (AER) removal, suggesting that a factor(s) localized to the AER prevents anterior cells from developing ZPA signaling capability during limb outgrowth. These findings indicate that nonsignaling anterior limb bud cells have the potential to become ZPA signaling cells and that FGF-2, or a related factor, functions in the maintenance of positional states in the developing limb.

Transcriptional regulation of cathepsin B expression in B16 melanomas of varying metastatic potential.

The highly metastatic B16a melanoma has been shown to express higher levels of cathepsin B (CB) mRNA when compared to the less metastatic variants, B16-F1 and B16-F10, and with normal mouse tissues. This increased expression is now shown to be due to increased gene transcription by nuclear run-off assays and measurements of mRNA stability. Transient expression assays, using promoter fragments from the mouse and human CB genes, demonstrated that both promoters were more active in B16a than in the less metastatic melanomas, B16-F1 and B16-F10. The differential gene expression did not depend on the presence of multiple Sp1 sites in both promoters. A Gel shift assay revealed a specific CB promoter binding protein whose levels are correlated with CB expression and the metastatic potential of the three B16 melanoma variants. These results indicate that the increased expression of CB in the B16a melanoma is due to a specific increase in the amount or activity of a transcriptional activator of the CB gene. The ability of the human CB promoter to activate gene expression in B16a melanoma cells suggests similarities in the regulation of CB expression in tumors from humans and mice.

Enzymatic conversion of retinaldehyde to retinoic acid by cloned murine cytosolic and mitochondrial aldehyde dehydrogenases.

It has previously been reported that retinaldehyde can be converted to retinoic acid by cytosolic aldehyde dehydrogenase (AHD-2) in liver extracts [Biochem. Pharmacol. 42: 1279-1285 (1991)]. To determine which enzyme(s) carried out this reaction in murine embryonic stem cells, two aldehyde dehydrogenases were cloned; the AHD-2 gene was cloned from a liver cDNA library, and a closely related gene, AHD-M1, was cloned from an embryonic F9 cell cDNA library by conserved oligonucleotide sequence screening. AHD-M1 contained an open reading frame of 1554 base pairs, which encoded 517 amino acids. The AHD-M1 gene encoded a protein with a putative amino acid sequence that was 94% and 97% identical to the mitochondrial aldehyde dehydrogenases of human and rat, respectively, and thus we have cloned the murine cDNA for this enzyme for the first time. The AHD-M1 cDNA was only 64% identical to AHD-2. Northern analysis showed that AHD-M1 mRNA was constitutively expressed in F9 and P19 embryonic teratocarcinoma stem cells and in AB1 embryonic stem cells. There was a 3-5-fold retinoic acid-associated increase in the amount of this mRNA during the differentiation of F9 cells into parietal endoderm. In contrast, we could not detect the expression of AHD-2 mRNA in AB1, P19, or F9 cells, even though the F9 cells could convert retinaldehyde to retinoic acid. When the AHD-M1 and AHD-2 cDNAs were inserted into the expression vector pSG5 and transfected into cultured COS cells, 3-5-fold and 100-fold increases, respectively, in the conversion of [3H]retinaldehyde to [3H]retinoic acid could be detected by high performance liquid chromatographic assay. We conclude that both enzymes are capable of converting retinaldehyde to retinoic acid in intact COS cells. AHD-2 is more active than AHD-M1 in this conversion, but AHD-2 is not the enzyme responsible for this conversion in F9 embryonic stem cells.

Differences in the pharmacokinetic properties of orally administered all-trans-retinoic acid and 9-cis-retinoic acid in the plasma of nude mice.

All trans-retinoic acid (tr-RA) has been used to induce leukemic cell differentiation in patients with acute promyelocytic leukemia (APL). However, the duration of remission is brief and is associated with a progressive decrease in peak plasma concentrations following chronic dosing. 9-Cis-retinoic acid (9-cis-RA) has the potential to elicit the same effects as tr-RA, because it can bind and activate the same family of nuclear receptors. It is not known whether the pharmacokinetics of this novel compound resemble those of tr-RA. In this study, we report major differences in the uptake and pharmacokinetics between orally administered tr-RA and 9-cis-RA in the plasma of nude mice. Following a single initial oral administration of either isomer, the plasma peak time of 9-cis-RA (15-30 min) occurred earlier than that of tr-RA (60-180 min), but with lower plasma concentrations and area under the concentration-time curve (AUC) value. A decrease in the AUC of plasma tr-RA was seen in animals that were given a second dose 2 days after the first dose. In contrast, an increase in the AUC of plasma 9-cis-RA was seen in animals that were given a second dose 2 days after the first dose. This increase was due to the appearance of a second 9-cis-RA peak at 180 min. When liarozole, an inhibitor of tr-RA metabolism, was coadministered with the initial tr-RA dose or a second tr-RA dose 2 weeks later, the AUC of plasma tr-RA was increased relative to tr-RA alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in targeting and secretion of cathepsins B and L by BALB/3T3 fibroblasts and Moloney murine sarcoma virus-transformed BALB/3T3 fibroblasts.

BALB/3T3 fibroblasts (3T3) were observed to secrete latent, pepsin-activatable forms of cathepsin B and cathepsin L as well as an active form of beta-glucuronidase when cultured in the absence of serum. The secretion of these proteins was stimulated by the cation ionophore monensin: cathepsin B, 4.3-fold; cathepsin L, 7.2-fold; and beta-glucuronidase, 3.1-fold. These increases were accompanied by a 50% decline in cellular levels of the active forms of these enzymes and by the cellular accumulation of latent forms of cathepsin B and cathepsin L. Latent forms of beta-glucuronidase were not detected. In contrast, Moloney murine sarcoma virus-transformed BALB/3T3 fibroblasts (MMSV) secreted greatly increased amounts of latent cathepsin B (17-fold) and latent cathepsin L (27-fold), and moderately increased amounts of active beta-glucuronidase (2-fold) in a manner which was not further increased by monensin. The increased monensin-insensitive secretion of these lysosomal enzymes by MMSV cells may be due to a transformation-induced decrease in mannose 6-phosphate receptors. Thus, 3T3 cells bound the neoglycoconjugate pentamannosyl 6-phosphate-bovine serum albumin at 4 degrees C in a pentamannosyl 6 phosphate and mannose 6-phosphate-inhibitable manner, whereas MMSV cells showed no measurable cell surface mannose 6-phosphate receptor binding activity.