Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells.

Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenic cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic ß-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/ß-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice.

Regulation of skeletal progenitor differentiation by the BMP and retinoid signaling pathways.

The generation of the paraxial skeleton requires that commitment and differentiation of skeletal progenitors is precisely coordinated during limb outgrowth. Several signaling molecules have been identified that are important in specifying the pattern of these skeletal primordia. Very little is known, however, about the mechanisms regulating the differentiation of limb mesenchyme into chondrocytes. Overexpression of RARalpha in transgenic animals interferes with chondrogenesis and leads to appendicular skeletal defects (Cash, D.E., C.B. Bock, K. Schughart, E. Linney, and T.M. Underhill. 1997. J. Cell Biol. 136:445-457). Further analysis of these animals shows that expression of the transgene in chondroprogenitors maintains a prechondrogenic phenotype and prevents chondroblast differentiation even in the presence of BMPs, which are known stimulators of cartilage formation. Moreover, an RAR antagonist accelerates chondroblast differentiation as demonstrated by the emergence of collagen type II-expressing cells much earlier than in control or BMP-treated cultures. Addition of Noggin to limb mesenchyme cultures inhibits cartilage formation and the appearance of precartilaginous condensations. In contrast, abrogation of retinoid signaling is sufficient to induce the expression of the chondroblastic phenotype in the presence of Noggin. These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation. Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.

Retinoid X receptor (RXR) agonist-induced activation of dominant-negative RXR-retinoic acid receptor alpha403 heterodimers is developmentally regulated during myeloid differentiation.

The multiple biologic activities of retinoic acid (RA) are mediated through RAR and retinoid X receptor (RXR) nuclear receptors that interact with specific DNA target sequences as heterodimers (RXR-RAR) or homodimers (RXR-RXR). RA receptor activation appears critical to regulating important aspects of hematopoiesis, since transducing a COOH-terminally truncated RARalpha exhibiting dominant-negative activity (RARalpha403) into normal mouse bone marrow generates hematopoietic growth factor-dependent cell lines frozen at the multipotent progenitor (EML) or committed promyelocyte (MPRO) stages. Nevertheless, relatively high, pharmacological concentrations of RA (1 to 10 microM) overcome these differentiation blocks and induce terminal granulocytic differentiation of the MPRO promyelocytes while potentiating interleukin-3 (IL-3)-induced commitment of EML cells to the granulocyte/monocyte lineage. In the present study, we utilized RXR- and RAR-specific agonists and antagonists to determine how RA overcomes the dominant-negative activity of the truncated RARalpha in these different myeloid developmental stages. Unexpectedly, we observed that an RXR-specific, rather than an RAR-specific, agonist induces terminal granulocytic differentiation of MPRO promyelocytes, and this differentiation is associated with activation of DNA response elements corresponding to RAR-RXR heterodimers rather than RXR-RXR homodimers. This RXR agonist activity is blocked by RAR-specific antagonists, suggesting extensive cross-talk between the partners of the RXR-RARalpha403 heterodimer. In contrast, in the more immature, multipotent EML cells we observed that this RXR-specific agonist is inactive either in potentiating IL-3-mediated commitment of EML cells to the granulocyte lineage or in transactivating RAR-RXR response elements. RA-triggered GALdbd-RARalpha hybrid activity in these cells indicates that the multipotent EML cells harbor substantial nuclear hormone receptor coactivator activity. However, the histone deacetylase (HDAC) inhibitor trichostatin A readily activates an RXR-RAR reporter construct in the multipotent EML cells but not in the committed MPRO promyelocytes, indicating that differences in HDAC-containing repressor complexes in these two closely related but distinct hematopoietic lineages might account for the differential activation of the RXR-RARalpha403 heterodimers that we observed at these different stages of myeloid development.

Retinoid antagonism of NF-IL6: insight into the mechanism of antiproliferative effects of retinoids in Kaposi's sarcoma.

All-trans-retinoic acid (RA) is active in the treatment of Kaposi's sarcoma (KS), and retinoids inhibit KS cell growth in vitro. To understand the mechanism of retinoid action in KS, we studied the expression of autocrine growth factors of KS cells after RA treatment. We demonstrate that RA and its synthetic analogs inhibit the proliferation of KS cells by inhibiting the mRNA and protein levels of interleukin-6 (IL-6), an autocrine growth factor for KS cells. We further demonstrate that nuclear retinoid receptors (RA receptors [RARs] and retinoid X receptors [RXRs]) inhibit IL-6 promoter action by antagonizing the enhancer action of NF-IL6, a basic domain leucine zipper transcription factor belonging to the family of CAAT enhancer binding proteins. Furthermore, RARs and RXRs do not bind in vitro to an NF-IL6 binding site. However, the secondary folded structure of the DNA binding domain of RAR and RXR is obligatory for inhibiting NF-IL6 activity. Thus, NF-IL6 is a potential therapeutic target for the treatment of KS. Finally, using receptor-selective synthetic retinoids, we demonstrate that NF-IL6 antagonism and transactivation are separable functions of RAR alpha, thus indicating that synthetic retinoids with properties of NF-IL6 antagonism but lacking transactivation capabilities can be synthesized. Such retinoids might increase therapeutic potential in KS.

Activation of retinoid X receptors induces apoptosis in HL-60 cell lines.

Retinoids induce myeloblastic leukemia (HL-60) cells to differentiate into granulocytes, which subsequently die by apoptosis. Retinoid action is mediated through at least two classes of nuclear receptors: retinoic acid receptors, which bind both all-trans retinoic acid and 9-cis retinoic acid, and retinoid X receptors, which bind only 9-cis retinoic acid. Using receptor-selective synthetic retinoids and HL-60 cell sublines with different retinoid responsiveness, we have investigated the contribution that each class of receptors makes to the processes of cellular differentiation and death. Our results demonstrate that ligand activation of retinoic acid receptors is sufficient to induce differentiation, whereas ligand activation of retinoid X receptors is essential for the induction of apoptosis in HL-60 cell lines.

Retinoic acid receptor alpha expression correlates with retinoid-induced growth inhibition of human breast cancer cells regardless of estrogen receptor status.

Retinoic acid receptor (RAR) alpha has been shown to play a role in retinoid-induced growth inhibition of human breast cancer cell lines that express the estrogen receptor (ER). The dogma in the field has been that ER-positive breast cancer cell lines respond to retinoid treatment because they express RAR alpha, whereas ER-negative breast cancer cell lines are refractory to retinoid treatment and have been thought to express little or no RAR alpha. We set out to test several ER-negative breast cancer cell lines for expression of RAR alpha protein and responsiveness to retinoids in growth inhibition assays. Of six ER-negative breast cancer cell lines that were tested, one (SK-BR-3) had high levels of RAR alpha protein as measured by ligand-binding immunoprecipitation (approximately 55 fmol/mg protein) and also displayed sensitivity to growth inhibition by retinoids (9-cis-retinoic acid; EC50, approximately 3 nM). These cells were more sensitive than an ER-positive cell line, T-47D, which expressed approximately 35 fmol RAR alpha/mg total protein (9-cis retinoic acid; EC50, approximately 50-100 nM). Another ER-negative cell line, Hs578T, also expressed RAR alpha (approximately 23 fmol/mg) and was sensitive to retinoid-induced growth inhibition, albeit to a lesser extent than SK-BR-3 or T-47D cells. In contrast, the other ER-negative cell lines tested expressed low (<10 fmol/mg) or no detectable levels of RAR alpha protein and also did not respond to retinoids in growth inhibition assays. A RAR alpha agonist displayed 100 times greater potency than a RARgamma agonist in growth inhibition of both T-47D and SK-BR-3 cells, suggesting RAR alpha involvement in the process. Furthermore, a RAR alpha antagonist completely abolished the growth inhibition induced by RAR agonists, implying that the activity of the agonists is exerted solely through RAR alpha, not RARgamma, which is also expressed in both cell lines. Additionally, although retinoid X receptor (RXR) compounds are weakly active in growth inhibition of the RAR alpha-positive cell lines, they markedly increased the growth-inhibitory activity of RAR ligands. RXR compounds also potentiated the action of the antiestrogen 4-hydroxytamoxifen to inhibit the growth of T-47D cells. These findings have clinical ramifications in that patients with ER-negative tumors that are RAR alpha positive may be candidates for retinoid therapy. Additionally, combinations of RXR ligands with RAR ligands (especially RAR alpha agonists) and/or antiestrogens may have utility in the treatment of breast cancer.

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.

Retinoic acid increases tyrosine phosphorylation of focal adhesion kinase and paxillin in MCF-7 human breast cancer cells.

Treatment of estrogen receptor (ER)-positive MCF-7 human breast cancer cells with retinoic acid (RA) inhibited cell growth and increased cell adhesion to fibronectin. In contrast, ER- MDA-MB-231 cells failed to respond. Western blot analysis showed that tyrosine phosphorylation of two major bands at Mr 125,000 and Mr 68,000 was induced by RA in ER+ MCF-7 human breast carcinoma cells. However, this induction was a late phenomenon detectable at 12 and 24 h, but not within 3 h. A similar increase of tyrosine phosphorylation by RA was observed in ER+ human breast cancer cell lines T-47D and ZR-75-1, but not in the ER- cell lines MDA-MB-231, MDA-MB-453, and MDA-MB-468. Focal adhesion kinase and paxillin, which localize in focal adhesion plaques and may play important roles in the integrin signaling pathway, were identified as the major proteins showing RA-induced tyrosine phosphorylation. The retinoid X receptor-selective compound SR11237 failed to induce tyrosine phosphorylation, indicating that retinoid X receptor activation is not involved in this phenomenon. In contrast, stable overexpression of a truncated RA receptor (RAR) alpha cDNA, RARalpha403, with strong RAR dominant negative activity prevented the increase in tyrosine phosphate, suggesting that RAR signaling is involved in RA-induced tyrosine phosphorylation. Tyrosine phosphorylation was induced the most by the RAR-alpha (193836), followed by RAR-gamma (194433), but was not significantly induced by RAR-gamma (193174)-selective retinoids. This study demonstrates a coordinated albeit relatively late effect of RA on cell adhesion and tyrosine phosphorylation in ER+ human breast cancer cells and suggests RAR-alpha as the major responsible retinoid receptor.

Differential effects of synthetic nuclear retinoid receptor-selective retinoids on the growth of human non-small cell lung carcinoma cells.

Retinoids are promising agents for cancer chemoprevention and therapy. Nuclear retinoic acid receptors (RARs; RARalpha, -beta, and -gamma) and retinoid X receptors (RXRs; RXRalpha, -beta, and -gamma) are thought to mediate most of retinoids' effects on cell growth and differentiation. Because the majority of human non-small cell lung carcinoma (NSCLC) cell lines are resistant to all-trans-retinoic acid, we searched for more potent retinoids. Therefore, we examined the effects of 37 natural and synthetic retinoids that exhibit specific binding to and transactivation of individual RARs or RXRs on the proliferation of eight human NSCLC cell lines. All of these cells expressed mRNAs of the three RXRs; however, they expressed varying levels of RARalpha and RARgamma, and only three of the eight cell lines expressed RARbeta mRNA. Cellular retinoic acid-binding proteins (CRABPs) I and II were detected in one and three of the eight cell lines, respectively. Only 8 of the 37 retinoids exhibited growth-inhibitory activity (IC50, < 10 microM) against at least two of the eight NSCLC cell lines. The active retinoids included one (TD550) of five RARalpha-selective, one (Ch55) of three RARbeta-selective, three (CD437, CD2325, and SR11364) of six RARgamma-selective, and one (CD271) of four RARbeta/gamma-selective retinoids. The potency of these retinoids was low (IC50, > 1 microM), except for CD437, which was very potent (IC50, 0.1-0.5 microM). The six RXR-selective retinoids were mostly inactive even at 10 microM. However, combinations of RAR-selective and RXR-selective retinoids exhibited additive effects. There appeared to be no simple correlation among the histological type of the NSCLC (adeno- or squamous), the levels of nuclear receptors or CRABPs, and the response of the cells to the growth-inhibitory effects of retinoids. Nevertheless, in contrast with former studies with natural retinoids, these results suggest that several synthetic retinoids do exhibit inhibitory activity against NSCLC cells, and some of them may be useful clinically.

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.

Retinoids inhibit proliferation of human coronary smooth muscle cells by modulating cell cycle regulators.

Retinoids inhibit rat vascular smooth muscle cell (VSMC) proliferation in vitro and intimal hyperplasia in vivo. We examined the mechanism of the antiproliferative effect of retinoids on human coronary artery smooth muscle cells (human CASMCs). The RAR ligands all-trans-retinoic acid (atRA) and ethyl-p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-l-propenyl]-benzoic acid (TTNPB); a pan-RXR/RAR agonist, 9-cis-retinoic acid (9cRA); and the RXR-selective ligand AGN4204 all inhibited DNA synthesis stimulated with platelet-derived growth factor and insulin (IC(50): TTNPB 63 nmol/L, atRA 120 nmol/L, AGN4204 460 nmol/L, 9cRA 1.5 micromol/L). All retinoids blocked cell cycle progression as determined by flow cytometry and inhibited retinoblastoma protein (Rb) phosphorylation. TTNPB, atRA, and AGN4204 inhibited the mitogenic induction of cyclin D1, whereas 9cRA had no effect. None of the retinoids affected the expression of CDK 2, 4, or 6 or cyclin E. All retinoids attenuated mitogen-induced downregulation of CDKI p27(Kip1), a major negative regulator of Rb phosphorylation, partly through stabilizing p27(Kip1) turnover. These data demonstrate that retinoids have antiproliferative activity by modulating G(1) --> S cell cycle regulators in human CASMCs through inhibition of Rb phosphorylation and elevation of p27(Kip1) levels.

Retinoic acid receptor antagonism in vivo expands the numbers of precursor cells during granulopoiesis.

The role/s of retinoids in granulopoiesis has been recognised for many years, being powerful differentiation inducers. The physiological role/s of retinoic acid receptor (RAR)-mediated signalling during adult haemopoiesis has by contrast proved more elusive. The recent generation of highly specific pan-RAR antagonists has now made possible an assessment of the specific physiological role/s of RAR signalling, allowing the separation for the first time of the RAR and RXR pathways. Mice were treated with AGN194310, a synthetic retinoid that antagonises the physiological function of the three RAR isotypes (alpha, beta, gamma) but does not interact with RXRs. Analyses of the granulocytic lineage using Gr-1, c-Kit and CD11b antibodies, demonstrated that granulocyte numbers were strikingly increased across haemopoietic compartments in all AGN194310-treated mice. A significant increase in the frequency of progenitor cells containing granulocytes was observed in the bone marrow of mice following treatment with AGN194310. In contrast we were not able to detect any differences in cell death of either mature granulocytes or granulocytic progenitors from AGN194310-treated mice compared with control animals. These data demonstrate an essential role for RAR signalling in regulating the numbers of granulocytic precursors in vivo.

Differential responses of normal, premalignant, and malignant human bronchial epithelial cells to receptor-selective retinoids.

Using an in vitro lung carcinogenesis model consisting of normal, premalignant, and malignant human bronchial epithelial (HBE) cells, we analyzed the growth inhibitory effects of 26 novel synthetic retinoic acid receptor (RAR)- and retinoid X receptor (RXR)-selective retinoids. RAR-selective retinoids such as CD271, CD437, CD2325, and SR11364 showed potent activity in inhibiting the growth of either normal or premalignant and malignant HBE cells (IC50s mostly <1 microM) and were much more potent than RXR-selective retinoids. Nonetheless, the combination of RAR- and RXR-selective retinoids exhibited additive effects in HBE cells. As the HBE cells became progressively more malignant, they exhibited decreased or lost sensitivity to many retinoids. The activity of the RAR-selective retinoids, with the exception of the most potent retinoid, CD437, could be suppressed by an RAR panantagonist. These results suggest that: (a) RAR/RXR heterodimers play an important role in mediating the growth inhibitory effects of most retinoids in HBE cells; (b) CD437 may act through an RAR-independent pathway; (c) some of the RAR-selective retinoids may have the potential to be used in the clinic as chemopreventive and chemotherapeutic agents for lung cancer; and (d) early stages of lung carcinogenesis may be responsive targets for chemoprevention by retinoids, as opposed to later stages.

Identification of receptor-selective retinoids that are potent inhibitors of the growth of human head and neck squamous cell carcinoma cells.

Retinoids modulate the growth and differentiation of cancer cells presumably by activating gene transcription via the nuclear retinoic acid receptor (RAR) alpha, beta, and gamma and retinoid X receptor (RXR) alpha, beta, and gamma. We analyzed the effects of 38 RAR-selective and RXR-selective retinoids on the proliferation of 10 human head and neck squamous cell carcinoma (HNSCC) cell lines. All of these cell lines expressed constitutively all of the receptor subtypes except RARbeta, which was detected in only two of them. Most of the RAR-selective retinoids inhibited the growth of HNSCC cells to varying degrees, whereas the RXR-selective retinoids showed very weak or no inhibitory effects. Three RAR antagonists suppressed growth inhibition by RAR-selective agonists, as well as by RAR/RXR panagonists such as 9-cis-retinoic acid. Combinations of RXR-selective and RAR-selective retinoids exhibited additive growth-inhibitory effects. Furthermore, we found that CD437, the most potent growth-inhibitory retinoid induced apoptosis and up-regulated the expression of several apoptosis-related genes in HNSCC cells. These results indicate that: (a) retinoid receptors are involved in the growth-inhibitory effects of retinoids; (b) RXR-RAR heterodimers rather than RXR-RXR homodimer are the major mediators of growth inhibition by retinoids in HNSCC cells; and (c) induction of apoptosis can account for one mechanism by which retinoids such as CD437 inhibit the growth of HNSCC cells. Finally, these studies identified several synthetic retinoids, which are much more effective than the natural RAs and can be good candidates for chemoprevention and therapy of head and neck cancers.

Expression of a retinoid-inducible tumor suppressor, Tazarotene-inducible gene-3, is decreased in psoriasis and skin cancer.

Tazarotene-induced gene-3 (TIG-3), isolated from human keratinocytes treated with the retinoic acid receptor-selective retinoid Tazarotene, is homologous to H-rev, a class II tumor suppressor. TIG-3 gene localized to chromosome 11q23, a site of loss of heterozygosity in several malignancies. Retinoids influence epidermal differentiation and are used to treat and prevent skin cancer. Therefore, we studied TIG-3 mRNA expression in psoriasis and in basal and SCCs by in situ hybridization and a quantitative QT-RT-PCR assay. Psoriasis lesions had significantly lower staining (median, 3) than paired normal control skin (median, 4; P = 0.012). TIG-3 mRNA was significantly higher in normal control skin (P = 0.001), in paired adjacent skin (median, 3; P = 0.007), and in overlying epidermis (median, 3.0; P = 0.0001) than in 21 SCC specimens as a group (median, 1.5).

The use of a retinoid receptor antagonist in a new model to study vitamin A-dependent developmental events.

Multiple fetal anomalies occur in vitamin A deficient animals as well as in retinoic acid receptor gene 'knockout' mice, indicating that retinoic acid (an active metabolite of vitamin A) performs some essential functions in normal development. Additional approaches are needed to probe directly the stages and sites in the embryo where a presence of endogenous retinoic acid is indispensable. We have employed a new strategy for this purpose which involved an intervention in retinoic acid receptor (RAR)-dependent functions at specific developmental stages by means of a highly effective RAR antagonist, AGN 193109. We report that in an in vitro cell differentiation bioassay, AGN 193109 completely reversed the inhibitory action of a potent RAR agonist, AGN 190121. In pregnant mice, a single oral 1 mg/kg dose of the antagonist given on 8 day post coitum (dpc) produced a severe craniofacial anomaly (median cleft face or frontonasal dysplasia) and eye malformations in virtually all exposed fetuses. On the other hand, treatment on 11 dpc, a time in development when RARs are strategically expressed in the limb bud primordium, no limb anomalies could be induced by the antagonist. Even after a high dose of 100 mg/kg, limb development progressed normally in spite of the fact that measurable concentrations of the antagonist were present. Because retinoids are long known to influence skin morphology, we next monitored the effects of the antagonist on skin development. When given late in gestation, on 14 dpc, we found that the antagonist delayed differentiation and maturation of the fetal skin and hair follicles. We conclude that this model provides a convenient and pertinent system which enables us to seek and clarify true functions of retinoic acid and its cognate receptors in embryogenesis and in adult animals.

Retinoic acid receptor-beta: an endogenous inhibitor of the perinatal formation of pulmonary alveoli.

Pulmonary alveoli are formed, in part, by subdivision (septation) of the gas-exchange saccules of the immature lung. Septation is developmentally regulated, and failure to septate at the appropriate time is not followed by delayed spontaneous septation. We report retinoic acid receptor (RAR) beta knockout mice exhibit premature septation; in addition, they form alveoli twice as fast as wild-type mice during the period of septation but at the same rate as wild-type mice thereafter. Consistent with the perinatal effect of RARbeta knockout, RARbeta agonist treatment of newborn rats impairs septation. These results 1) identify RARbeta as the first recognized endogenous signaling that inhibits septation, 2) demonstrate premature onset of septation may be induced, and 3) show the molecular signaling regulating alveolus formation differs during and after the period of septation. Suppressing perinatal RARbeta signaling by RARbeta antagonists may offer a novel, nonsurgical, means of preventing, or remediating, failed septation in prematurely born children.

Tazarotene-induced gene 1 (TIG1), a novel retinoic acid receptor-responsive gene in skin.

Retinoids exert their effect through ligand-dependent transcription factors, retinoic acid receptors (RARalpha, beta, and gamma) and retinoid X receptor (RXRalpha, beta, and gamma), which belong to the superfamily of steroid/thyroid/vitamin D3, nuclear receptors. Using a subtraction hybridization approach, we have identified a cDNA sequence, Tazarotene Induced Gene 1 (TIG1), which is highly upregulated in skin raft cultures by an RARbeta/gamma -selective retinoid AGN 190168 (tazarotene/ethyl 6-[2-(4,4-dimethylthiochroman-6-yl)-ethynyl]nicotinate), which is effective in the treatment of psoriasis. The retinoid-mediated upregulation in the expression of TIG1 was confirmed by Southern and Northern analyses. Upon sequencing, TIG1 was found to be a novel cDNA which encodes a protein of 228 amino acids whose sequence suggests that is a transmembrane protein with a small N-terminal intracellular region, a single membrane-spanning hydrophobic region, and a large C-terminal extracellular region containing a glycosylation signal. We demonstrate that TIG1 is also upregulated by AGN 190168 in skin raft cultures prepared from psoriatic fibroblasts and normal keratinocytes and in primary fibroblast and keratinocyte cultures. We also show that TIG1 is upregulated by retinoic acid receptor but not by retinoid X receptor-specific synthetic retinoids. Finally, we demonstrate that TIG1 is induced by AGN 190168 in psoriatic lesions during the course of clinical treatment of the disease.

Tazarotene-induced gene 2 (TIG2), a novel retinoid-responsive gene in skin.

Retinoids exert their biologic effects through two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which belong to the superfamily of steroid/thyroid hormone nuclear receptors. By using a subtraction hybridization approach, we have identified a cDNA sequence TIG2 (Tazarotene-induced gene 2), whose expression is up-regulated by the treatment of skin raft cultures by an RAR beta/gamma-selective anti-psoriatic synthetic retinoid tazarotene [AGN 190168/ethyl 6-[2-(4,4-dimethylthiochroman-6-yl)-ethynyl] nicotinate]. The retinoid-mediated up-regulation in the expression of TIG2 was confirmed by Northern blot analysis. Upon sequencing, TIG2 was found to be a cDNA whose complete sequence was not in the GenBank and EMBL data bases. The TIG2 cDNA is 830 bp long and encodes a putative protein product of 164 amino acids. TIG2 is neither expressed nor induced by tazarotene in primary keratinocyte and fibroblast cultures. Thus, TIG2 is expressed and induced by tazarotene only when keratinocytes and fibroblasts form a tissue-like 3-dimensional structure. We further demonstrate that RAR-specific retinoids increase TIG2 mRNA levels. In contrast, neither RXR-specific retinoids nor 1,25-dihydroxyvitamin D3 increased TIG2 levels. Finally, we demonstrate that TIG2 is expressed at high levels in nonlesional psoriatic skin but at lower levels in the psoriatic lesion and that its expression is up-regulated in psoriatic lesions after topical application of tazarotene.

13-cis retinoic acid exerts its specific activity on human sebocytes through selective intracellular isomerization to all-trans retinoic acid and binding to retinoid acid receptors.

Despite its potent biologic effect on human sebocytes, 13-cis retinoic acid exhibits low binding affinity for cellular retinoic acid binding proteins and nuclear retinoid receptors. Hence, 13-cis retinoic acid may represent a pro-drug possibly acting through all-trans isomerization. In this study, marked isomerization of 13-cis retinoic acid has been confirmed in cultured SZ95 sebocytes showing 2- to 15-fold higher levels of all-trans retinoic acid at 12-72 h, as measured by high performance liquid chromatography. In contrast, only low amounts of all-trans retinoic acid were converted intracellularly to its 13-cis isoform. 9-cis retinoic acid was not detected after either 13-cis retinoic acid or all-trans retinoic acid treatment. The rapid isomerization of 13-cis retinoic acid to high levels of all-trans retinoic acid was a sebocyte-specific event, as no significant isomerization of 13-cis retinoic acid to all-trans retinoic acid occurred in HaCaT keratinocytes. De novo mRNA expression of cytochrome P450 1A1, a major xenobiotic metabolizing enzyme, in SZ95 sebocytes was induced by all-trans retinoic acid, but not by 13-cis retinoic acid. In addition, mRNA levels of cellular retinoic acid-binding protein II, which is supposed to regulate the concentration of intracellular all-trans retinoic acid, rapidly increased under all-trans retinoic acid treatment (30 min-6 h), whereas the 13-cis retinoic acid effect was markedly weaker and delayed. Both 13-cis retinoic acid and all-trans retinoic acid suppressed mRNA expression of cytochrome P450 1A2. In parallel experiments, 13-cis retinoic acid significantly reduced SZ95 sebocyte proliferation at 10-7 M, show- ing 30-40% inhibition after 9 d. All-trans retinoic acid and 9-cis retinoic acid exhibited similar anti-proliferative effects. AGN 193109, a pan-antagonist of the retinoic acid receptors, antagonized the anti-proliferative activity of all retinoic acid isomers tested in a concentration-dependent manner with complete abolishment at ratios of 1:10 13-cis retinoic acid and 1:1 all-trans retinoic acid. Coincubation of SZ95 sebocytes with 13-cis retinoic acid and AGN 193109 did not alter the intracellular concentration of 13-cis retinoic acid and its isomerization profile. In contrast, the retinoid X receptor antagonist CD 3507 did not affect the inhibition of SZ95 sebocyte proliferation induced by retinoic acids. Our findings indicate: (i) a selective 13-cis retinoic acid isomerization to all-trans retinoic acid in the intracellular compartment of SZ95 sebocytes; (ii) a reduced all-trans retinoic acid inactivation process after 13-cis retinoic acid treatment as compared with treatment with all-trans retinoic acid; and (iii) a retinoic acid receptor-mediated inhibition of SZ95 sebocyte proliferation. These data explain the sebocyte-specific activity of 13-cis retinoic acid and support a pro-drug/drug relation between 13-cis retinoic acid and all-trans retinoic acid.