RARalpha Protein Research Articles

Retinoic acid receptor alpha: One of plasma biomarkers associated with exacerbation of chronic obstructive pulmonary disease

Background: Exacerbation of COPD is a major risk factor for bad prognosis of COPD. A few plasma proteins have been discovered to associate with hospital admission due to exacerbation up to date. We tried to find new plasma biomarkers to predict the exacerbation of COPD. Methods: We examined the plasma of normal control (n = 8) and COPD stable (n = 8) and exacerbation (n = 8) using 2-Dimentional Electrophoresis. The differentially expressed protein spots were identified by MALDI-TOF. ELISA were performed for quantitative measurement of RARα in plasma from normal control (n = 37) and COPD (n = 35). Results: 17 proteins were differentially expressed in plasma between stable and exacerbation state in the subjects with COPD. Identification using MALDI-TOF showed that retinoic acid receptor alpha, ninein, isoform CRA_a, alpha-1 antitrypsin, fibrinogen gamma, tyrosyl-DNA phosphodiesterase 2, and T cell receptor delta chain were increased in exacerbation of COPD, while fibrin beta, Crystal Structure Of An Autoimmune Complex Between A Human Igm RF* And Igg1 Fc, transferrin, serpin peptidase inhibitor member 6, complement factor B preproprotein, Chain B, Crig Bound To C3c, and WD repeat-containing protein 1 isoform 1 were decreased. Quantitative measurement showed that RARα plasma levels significantly increased in exacerbation state compared to stable state of COPD (n = 14). In the plasma of stable state, the COPD subjects (n = 14) having more than 0.4 time/yr of admission had very high levels of RAR alpha protein and those (n = 11) having less than 0.4 times/yr of admission had the intermediate levels compared to those having no exacerbation (n = 10). ROC analysis of RAR alpha levels to frequency of admission showed an area under the curve of 0.844. A cut-off of 0.154 ng/ml of RAR alpha predicted hospital admission with a sensitivity of 71.4% and a specificity of 92.8%. Conclusion: The proteomic analysis of plasma indicates that alteration of several proteins may be associated with admission of COPD. Among them, plasma RAR alpha level may predict hospital admission with a sensitivity of 71.4% and a specificity of 92.8%.

Changes in Expression and Localization of GPRC5B and RAR.ALPHA. in the Placenta and Yolk Sac During Middle to Late Gestation in Mice

The mRNA expression of GPRC5B, an orphan G protein-coupled receptor, is induced by retinoic acid (RA). Because RA plays critical roles in embryonic development, reproductive functions, metabolism and homeostasis, GPRC5B is also considered crucial in these physiological events. We investigated the changes in expression of GPRC5B and RA receptor (RAR) alpha mRNAs and immunohistochemical localization of their proteins in the murine placenta and yolk sac at 13.5, 15.5 and 17.5 days post coitus. Stable levels of GPRC5B and RARalpha mRNAs were detected in the placenta and yolk sac. In the placenta, GPRC5B was present in maternal and fetal vascular endothelial cells, stromal cells, fibroblast-like cells and glycogen cells. A strong reaction to RARalpha was detected in maternal and fetal vascular endothelial cells and stromal cells. The levels of GPRC5B and RARalpha proteins in maternal and fetal vascular endothelial cells decreased with gestation. In the yolk sac, GPRC5B and RARalpha proteins were detected in vascular endothelial cells, but their levels did not change during the gestation period. These findings indicate that GPRC5B is involved in RA-dependent morphogenesis/angiogenesis and regulation of extracellular matrix synthesis in the murine placenta and yolk sac.

Role of retinoic acid receptors in squamous-cell carcinoma in human esophagus

BackgroundWorldwide, cancer in the esophagus ranks among the 10 most common cancers. Alterations of retinoic acid receptors (e.g. RARα, β, γ, and RXRα, β, γ) expression is considered to play an important role in development of squamous-cell carcinoma (SCC), which is the most common esophageal cancer. Alcohol consumption and smoking, which can alter retinoic acid receptor levels, have been identified as key risk factors in the development of carcinoma in the aero-digestive tract. Therefore, the aim of the present study was to evaluate protein levels of retinoic acid receptors (i.e. RARα, β, γ, and RXRβ) in esophageal SCC and surrounding normal tissue of patients with untreated SCC and controls.MethodsAll study participants completed a questionnaire concerning smoking and alcohol drinking habits as well as anthropometrical parameters. Protein levels of RARα, β, γ, and RXRβ were determined by Western Blot in normal esophageal tissue and tissue obtained from SCC of 21 patients with newly diagnosed esophageal SCC and normal esophageal tissue of 10 controls.ResultsProtein levels of RARγ were significantly lower by ~68% in SCC compared to normal surrounding tissue in patients with SCC that smoked and/or consumed elevated amounts of alcohol. Furthermore, RARα protein levels were significantly lower (~- 45%) in SCC in comparison to normal esophageal mucosa in patients with elevated alcohol intake. When comparing protein levels of retinoic acid receptors between normal tissue of patients with SCC and controls, RARγ protein levels were found to be significantly higher (~2.7-fold) in normal esophageal tissue of SCC patients than in esophageal tissue obtained from controls. No differences were found for RARα, β, and RXRβ protein levels between normal esophageal tissue of patients and that of controls.ConclusionIn conclusion, results of the present study suggest that alterations of retinoic acid receptors protein may contribute in the development of SCC in esophagus and that in some patients life style (e.g. smoking and alcohol consumption) may be a critical component in the alteration of retinoic acid receptor levels in esophagus.

Breast cancer progression in MCF10A series of cell lines is associated with alterations in retinoic acid and retinoid X receptors and with differential response to retinoids.

In most breast carcinomas and in breast cancer cell lines, retinoic acid receptor beta (RARbeta) is lost or down-regulated, whereas retinoic acid receptor alpha and gamma (RARalpha, gamma) and retinoid X receptors (RXRalpha, beta, gamma) are variably expressed. Little is known about alterations of the above receptors in hyperplastic and premalignant stages of breast cancer development. In this study, we employed the MCF10A series of breast epithelial cell lines (the parental and benign MCF10A, premalignant MCF10AT, and malignant MCF10CA1a) to assess whether in the course of their malignant transformation specific alterations in RARalpha, beta, gamma and RXRalpha, beta, gamma expression occur and whether they may affect the sensitivity of cells to retinoids. Malignant properties of the above cell lines were estimated by the nude mice xenograft transplantation assay. Among the above receptors most significant alterations occurred in RARbeta2, which was detected in the normal breast epithelial cells both, at mRNA and protein levels, but expressed in the MCF10A cell lines at mRNA level only. The transformation of benign MCF10A cells into premalignant MCF10AT and malignant MCF10CA1a was also associated with increase in RARalpha, RARgamma, RXRalpha, and RXRbeta proteins expression. All-trans retinoic acid (atRA), 9-cis retinoic acid (9cRA), and 4-(hydroxyphenyl) retinamide (4-HPR) induced RARbeta2 protein expression exclusively in the benign MCF10A cells and the former two retinoids, mRNA expression in MCF10A and MCF10AT cells, but not in malignant, MCF10CA1a cells, suggesting that the loss of inducible RARbeta expression is associated with the progression and malignant transformation of MCF10A cells. Retinoids also variable decreased the RARalpha, RARgamma and RXRalpha protein expression preferentially in the premalignant and malignant, but not in benign MCF10A cells. Among the above retinoids, 4-HPR was most efficacious in inhibiting the growth of the three cell lines and this apparently was not dependent on the levels of the RARbeta2 transcriptional activation. Thus, our data support the hypothesis that breast epithelial cells in the course of their progression and malignant transformation may differentially respond to retinoids and that not only RARbeta, but RARalpha, gamma and/or RXRalpha, beta may also serve as potential targets for retinoids in breast cancer prevention and therapy trials.

Ubiquitinated or sumoylated retinoic acid receptor alpha determines its characteristic and interacting model with retinoid X receptor alpha in gastric and breast cancer cells

Retinoic acid receptor alpha (RARalpha) plays an important role in mediating all-trans retinoic acid (ATRA) signals. In this study, we found that ATRA up-regulated RARalpha mRNA and protein expression in gastric cancer BGC-823 cells. However, in breast cancer MCF-7 cells it down-regulated RARalpha protein expression with no effect on its RARalpha mRNA. Immunoprecipitation/Western blot analysis showed that, although sumoylated and ubiquitinated RARalpha existed simultaneously in both cancer cell lines, ATRA exerted different regulatory effects on sumoylation and ubiquitination of RARalpha. In MCF-7 cells, ATRA treatment enhanced the ubiquitination of RARalpha and the subsequent degradation of RARalpha through the ubiquitin/proteasome pathway. This resulted in a reduction in the DNA binding activity of RARalpha/retinoid X receptor alpha (RXRalpha) heterodimer, the separation of RXRalpha from RARalpha and the translocation of RXRalpha from the nucleus to the cytoplasm. By contrast, in BGC-823 cells, ATRA augmented sumoylation, not ubiquitination, of RARalpha. The stability of sumoylated RARalpha was significantly stronger than in non-sumoylated RARalpha. These results also showed an increase in the DNA binding activity of the RARalpha/RXRalpha heterodimer and the stability of nuclear localization of this heterodimer, which normally facilitates the ATRA signal transduction. In conclusion, our results reveal a novel mechanism for the regulation of RARalpha-dependent signal transduction through the ubiquitin/proteasome pathway in breast cancer cells and the sumoylation pathway in gastric cancer cells.

Retinoic acid receptor-alpha as a prognostic indicator in oral squamous cell carcinoma.

Retinoids reverse potentially malignant lesions and inhibit the development of second primary tumors in oral cancer patients by binding to nuclear retinoid receptors. Alterations in the expression of retinoid receptor-alpha are implicated in tumor progression. Herein, we hypothesized that increased expression of RARalpha protein in oral squamous cell carcinoma (SCC) is associated with a poor clinical outcome and thus may serve as a prognostic factor. Retrospective immunohistochemical analysis of RARalpha protein expression was carried out in paraffin-embedded tissue sections from 115 patients with completely resected oral SCCs for whom clinical follow-up data were available. Increased expression of RARalpha protein was observed in 67/115 (58%) oral SCCs (weakly positive in 38 patients and strongly positive in 29 patients). Kaplan-Meier analysis showed that patients with RARalpha positivity had significantly shorter disease-free survival time (median time 40 months vs. 86 months, p = 0.0229). Furthermore, disease-free survival time of the 29 patients with strongly positive RARalpha was significantly worse than for the 86 patients with weak or undetectable levels of RARalpha (p = 0.0328). Strong RARalpha expression in oral SCCs was associated with a significantly worse disease-free survival, suggesting that RARalpha may serve as a prognostic indicator of poor clinical outcome. Further studies are warranted to determine its utility in identifying the subset of patients who would benefit from use of retinoids as adjuvant in chemotherapy or chemopreventive approaches.

Her2/neu induces all-trans retinoic acid (ATRA) resistance in breast cancer cells.

We observed that all-trans retinoic acid (ATRA) inhibited the growth of MCF-7 breast cancer cells, but not those transfected with HER2/NEU or its transactivating ligand HEREGULIN. This suggests that Her2/neu causes breast cancer cells to be resistant to the growth inhibitory effects of ATRA. To confirm this observation, MDA-MB-453 and BT-474 cells, which have high levels of Her2/neu and are resistant to ATRA, were incubated with the trastuzumab (Herceptin) antibody so that we could determine whether inhibition of the expression and function of Her2/neu would resensitize these cells to ATRA. Indeed, we found that MDA-MB-453 and BT-474 cells treated with trastuzumab were growth inhibitory by ATRA. We then determined whether Her2/neu uses Grb2 and Akt proteins to induce ATRA resistance. Liposome-incorporated Grb2 antisense oligonucleotides (L-Grb2) and a dominant negative (DN) AKT mutant were used to down-regulate Grb2 expression and inhibit Akt activity, respectively. When incubated with L-Grb2 or transfected with the DN AKT mutant, ATRA-resistant, Her2/neu-overexpressing cells became sensitive to ATRA. Our results indicate that Her2/neu utilizes Grb2 and Akt proteins to induce ATRA resistance in breast cancer cells. ATRA sensitivity was also correlated with RARalpha protein levels since higher RARalpha protein levels were observed in cells in which the Her2/neu pathway was inhibited.

High levels of cellular retinol binding protein-1 expression in leiomyosarcoma: possible implications for diagnostic evaluation

Retinoid bioavailability is regulated by the activity of specific cytoplasmic receptors. High levels of cellular retinol binding protein-1 (CRBP-1) have been documented during experimental arterial and wound-healing processes, but data concerning neoplastic smooth muscle tissues are scarce and/or controversial. This study reports that the expression of CRBP-1 is markedly higher in uterine and gastrointestinal leiomyosarcomas than in leiomyomas and normal myometrium; CRBP-1 was practically absent in normal gastrointestinal smooth muscle tissue. CRBP-1 positivity was particularly elevated in the epithelioid variant of leiomyosarcoma; it was associated with increased proliferative and apoptotic rates and inversely related to smooth muscle differentiation evaluated by alpha- and gamma-smooth muscle actin and desmin expression. Western blotting and reverse-transcription polymerase chain reaction confirmed the observations concerning CRBP-1 and actin isoform expression and revealed higher NF-kappa-Bp65 and RAR alpha and lower Bax protein levels in leiomyosarcoma than in the other conditions. These findings document that a high CRBP-1 expression is associated with smooth muscle malignancy and suggest that CRBP-1 expression represents a new useful marker for the classification of unusual variants of smooth muscle tumors.

Oncogenes and tumor suppressors in the molecular pathogenesis of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is associated with reciprocal chromosomal translocations always involving the retinoic acid receptor alpha (RARalpha) gene on chromosome 17 and variable partner genes (X genes) on distinct chromosomes. RARalpha fuses to the PML gene in the vast majority of APL cases, and in a few cases to the PLZF, NPM, NuMA and Stat5b genes, respectively, leading to the generation of RARalpha-X: and X:-RARalpha fusion genes. Both fusion proteins can exert oncogenic functions through their ability to interfere with the activities of X and RARalpha proteins. Here, it will be discussed in detail how an extensive biochemical analysis as well as a systematic in vivo genetic approach in the mouse has allowed the definition of the multiple oncogenic activities of PML-RARalpha, and how it has become apparent that this oncoprotein is able to impair RARalpha at the transcription level and the tumor suppressive function of the PML protein.

Maintenance of retinoic acid receptor alpha pools by granulocyte colony-stimulating factor and lithium chloride in all-trans retinoic acid–treated WEHI-3B leukemia cells: relevance to the synergistic induction of terminal differentiation

Previous studies have demonstrated that combinations of all-trans retinoic acid (ATRA) with either granulocyte-colony stimulating factor (G-CSF) or lithium chloride (LiCl) produced synergistic terminal differentiation of WEHI-3B myelomonocytic leukemia (D(+)) cells. It was found that steady-state retinoic acid receptor alpha (RARalpha) protein levels were markedly reduced in these cells after exposure to ATRA. Because the presence of receptors for a hormone ligand is required for its action, differentiation therapy with ATRA may be self-limiting. The combination of G-CSF with ATRA significantly attenuated the loss of RARalpha protein, and synergistic terminal differentiation occurred. LiCl was more effective than G-CSF in preserving RARalpha pools and synergized with ATRA more strongly than G-CSF. These findings suggested that the prevention of RARalpha protein loss by G-CSF or LiCl in ATRA-treated cells functioned to extend the differentiation response to the retinoid and was responsible, at least in part, for the observed synergism. D(+) cells transfected with an expression plasmid containing RARalpha cDNA had a 6- to 8-fold increase in steady-state RARalpha mRNA compared with vector-transfected cells and showed a 2- to 3-fold increase in RARalpha protein. ATRA caused a reduction, but not a complete loss, of RARalpha protein in these transfectants, which were considerably more responsive than parental D(+) cells to ATRA as a single agent, supporting the concept that the protection of RARalpha pools results in a heightened differentiation response to ATRA.

Follicle-stimulating Hormone Inhibits All-trans-retinoic Acid-induced Retinoic Acid Receptor α Nuclear Localization and Transcriptional Activation in Mouse Sertoli Cell Lines

The regulation of retinoic acid receptor alpha (RARalpha) signal transduction has not been well characterized. In this study, we determined whether all-trans-retinoic acid (tRA) and follicle-stimulating hormone (FSH) modulate RARalpha receptor subcellular localization, leading to changes in its transcriptional activity and protein expression in mouse Sertoli cell lines. We found that tRA induced the nuclear localization of RARalpha within 30 min and that longer term exposure increased the receptor transcriptional activity and RARalpha protein expression. Conversely, FSH suppressed the tRA-induced nuclear localization, transcriptional transactivation, and protein expression of RARalpha. Treatment with two different protein kinase A-selective antagonists reversed the inhibitory actions of FSH on tRA-dependent RARalpha nuclear localization and transcriptional activity. These results are consistent with the involvement of protein kinase A in mediating the inhibitory effects of FSH. For the first time, we demonstrate a unique signaling convergence between the RARalpha and the FSH-mediated signaling pathways, which may have significant implications in the testis because both are critical regulators of testis physiology.

Expression of RARalpha and RARbeta in human oral potentially malignant and neoplastic lesions.

Retinoids reverse potentially malignant lesions and inhibit the development of second primary cancers in patients with head-and-neck cancer. Many of the effects of retinoids result from modulation of gene expression by 2 distinct classes of nuclear receptor, RARs and RXRs; alterations in their expression can lead to tumorigenesis. To determine whether aberrations in expression of the receptors are related to the development of betel- and tobacco-related oral cancer, we used specific monoclonal antibodies against RARalpha and RARbeta to detect expression of these proteins in 30 histopathologically normal tissues, 45 potentially malignant lesions (leukoplakia) with histological evidence of either hyperplasia (31 cases) or dysplasia (14 cases) and 64 oral squamous-cell carcinomas (SCCs) by immunohistochemistry. Of the 30 normal oral tissues analysed, 8 cases showed detectable levels of RARalpha protein, while 10 cases did not show detectable RARbeta immunoreactivity. Immunostaining for RARalpha protein was observed in 12/31 (39%) hyperplastic lesions, 6/14 (43%) dysplastic lesions and 43/64 (67%) oral SCCs. Expression of RARalpha in oral SCC was significantly associated with the histological differentiation status of tumours (p = 0.016). In contrast, lack of detectable immunoreactivity was observed in 19/31 (61%) hyperplastic lesions, 8/14 (57%) dysplastic lesions and 21/64 (33%) oral SCCs. The hallmark of the study was the significant increase in RARalpha immunopositivity in oral SCCs compared to normal tissue (p = 0.0005) and hyperplastic lesions (p = 0.016). One intriguing feature was the significant decrease in RARbeta immunopositivity in hyperplastic lesions compared with normal oral mucosa (p = 0.05) as well as in oral SCCs compared with normal tissues (p = 0.0008).

Immunohistochemical detection of retinoic acid receptor-alpha in prostate carcinoma: correlation with proliferative activity and tumor grade.

The use of retinoids as differentiation therapy is a novel approach to prostate cancer. Retinoids act via their own nuclear receptors, RARs and RXRs, modulating gene activity, cell growth and differentiation. This study provides new data on the content and cellular distribution of RARalpha protein in prostate cancer specimens, in correlation to tumor grade and proliferative activity. A total of 84 cases of primary prostate carcinoma, divided into 3 subgroups according to tumor grade, were immunohistochemically evaluated for retinoic acid receptor-alpha (RARalpha) and Ki67 using the streptavidin/peroxidase method on formalin fixed, paraffin embedded tissues. RARalpha positivity was detected in all cases of prostatic carcinoma, with a more profound expression in well differentiated cancers. A statistically significant correlation between RARalpha staining and tumor grade was found (ANOVA, p < 0.031). Ki67 immunoreactivity was present in 35.7% of cases, but no correlation with tumor grade was found. When RARalpha staining was correlated with Ki67 positivity, a statistically significant correlation was present (unpaired t-test, p < 0.003). These findings indicate that RARalpha expression is correlated to some extent with tumor grade and its presence is more profound in highly proliferative tumors. Further studies are needed to establish the possible clinical value of the immunohistochemical evaluation of RARalpha content in tumor specimens.

In acute promyelocytic leukemia NB4 cells, the synthetic retinoid CD437 induces contemporaneously apoptosis, a caspase-3-mediated degradation of PML/RARalpha protein and the PML retargeting on PML-nuclear bodies.

CD437-induced apoptosis has been investigated in NB4, a human t(15;17) acute promyelocytic leukemia (APL) cell line, and in the retinoic acid (RA)-resistant NB4-R1 derivative subclone. Both NB4 and NB4-R1 cells underwent rapid apoptosis in response to low doses of CD437 (10(-7)M). This apoptosis did not require the activation of classical retinoid receptors and like arsenic (As)-induced apoptosis was preceded by the rapid activation of a caspase-3-like enzymatic activity as indicated by the increase of DEVD-pNA hydrolytic activity, by the processing of procaspase-3 protein and by the cleavage of poly(ADP-ribose) polymerase (PARP). Furthermore, it was demonstrated that the caspase-3-like proteolytic activity is responsible for the degradation of both the PML/RARalpha oncogenic protein and the normal RARalpha proteins. In CD437-treated cells, PML proteins were not degraded and PML relocalization on PMLNBs occurred in all the cells before death. CD437-induced apoptosis and receptor degradation were proteasome independent and not influenced either by inhibitors of protein tyrosine kinases (PTK), protein tyrosine phosphatases (PTPases) and serine proteases or by glutathione levels. Moreover, our data suggested that as for As2O3-induced apoptosis Bc12 modulation is not significant for CD437-induced apoptosis of NB4 cells. Since CD437 induces in vitro the rapid apoptosis of both RA-sensitive and -resistant APL cells, it could represent the first retinoid potentially able to eradicate in vivo malignant leukemia blasts.

Ligand-dependent regulation of retinoic acid receptor alpha in rat testis: in vivo response to depletion and repletion of vitamin A.

Male animals are sterile due to testicular degeneration in the absence of retinoic acid (RA) or functional retinoic acid receptor-alpha (RAR alpha). This degeneration can be reversed by injecting retinol, a precursor of RA, into vitamin A-deficient (VAD) rats. To determine the relationship between this ligand-dependent testicular degeneration and regeneration and the expression levels of RAR alpha messenger RNA and protein, testes were depleted and then replenished with retinol in vivo. Results showed that RAR alpha messenger RNA and protein levels declined to VAD amounts after 7 weeks on a VAD diet. This decline was due to decreased RAR alpha levels in early meiotic spermatocytes and the loss of advanced germ cells. Interestingly, the advanced germ cells still contained RAR alpha, but the protein was primarily cytoplasmic instead of nuclear, indicating inactivity as a transcription factor. In VAD testis, RAR alpha levels were low and then increased primarily in Sertoli cells after retinol replenishment. TUNEL analyses showed that most germ cells at the basal aspect of seminiferous tubules were undergoing apoptosis during degeneration. These results indicate that RAR alpha is either down-regulated or inactivated in RA-deficient testis and coincident with that, testes degenerate by apoptosis or selective loss of germ cells.

Retinoic acid downregulates growth, fibronectin and RAR alpha in 3T3 cells: Ha-ras blocks this response and RA metabolism.

Retinoic acid (RA) reduced growth, fibronectin, and retinoic acid receptor (RAR alpha) in NIH 3T3 cells but not in cells transformed by the Ha-ras oncogene. RA lowered RAR alpha transcript and protein, increased RAR beta transcripts, and had no effect on RAR gamma. H-ras transformation downregulated RAR expression and abolished responsiveness to RA. Ha-ras-transformed cells were as active as normal NIH-3T3 cells in RA uptake but were unable to degrade it to medium oxidation product, so that, paradoxically, the resistant cells accumulated 20-30-fold as much RA as the sensitive cells. RA sensitivity/insensitivity correlated with RA metabolism/lack thereof in 15 cell lines in serum-free medium. These data suggest a relationship between RA inhibition of cell growth and intracellular RA metabolism.

Retinoic acid stimulates growth hormone synthesis in human somatotrophic adenoma cells: Characterization of its nuclear receptors

In order to gain a better understanding on the possible role of retinoic acid (RA) on human GH secretion, we have characterized the expression of its nuclear receptors in somatotropic adenoma cell extracts. By immunoblotting with rabbit polyclonal antibodies directed against RAR alpha, beta, and gamma and RXR alpha and beta, we could only detect the presence of RAR alpha and RXR alpha proteins. The predominant expression of RXR alpha was confirmed at the mRNA level by Northern and slot-blot analysis. We then investigated the effect of RA on GH synthesis in cell culture of adenomatous somatotrophs. In cultured cells, RA (1 microM) stimulated GH secretion, increased intracellular GH content and GH mRNA levels within 72 h, suggesting a modulation of GH synthesis by RA.

Potentiation of VD-induced monocytic leukemia cell differentiation by retinoids involves both RAR and RXR signaling pathways.

Retinoids and vitamin D (VD) cooperate to induce the differentiation and inhibit the proliferation of human myelomonocytic leukemia cells. Two classes of retinoids receptors, the RARs and RXRs, respectively, can mediate these effects. RXR forms heterodimers with a variety of nuclear receptors, including RAR and the VD receptor. We have previously found that VD treatment increases RXR alpha levels in myelomonocytic leukemia cells. By immunoanalysis, we observed in the present work that the RAR alpha protein is expressed in proliferating U937, HL-60 and THP-1 human leukemia cells and that VD treatment induces alterations of its electrophoretic pattern, although with large differences between cell lines. In the three cell lines, 9-cis RA, an agonist of both RARs and RXRs, cooperated with VD more efficiently than all-trans RA and RAR-specific synthetic ligands, thus suggesting an involvement of both RAR and RXR pathways in cell differentiation. Using U937 cells as a model, we delineated the relative contributions of RAR and RXR by assessing the effects of receptor-selective synthetic retinoids. The synergy between VD and all-trans RA or RAR-specific agonists (TTNPB and Ro 40-6055) was abrogated by a RAR alpha-specific antagonist (Ro 41-5253), confirming an involvement of RAR alpha. However, the cooperation between VD and 9-cis RA, although reduced, was not suppressed by the antagonist, suggesting also an involvement of the RXR pathway. The role of RXR as a ligand-activated receptor was confirmed using RXR-specific agonists (CD2608 and LGD1069), which also proved able to cooperate with VD. Finally, while each synthetic agonist alone was significantly less potent than 9-cis RA, combinations of the RAR and RXR selective agonists TTNPB and LGD1069 appeared to be as effective as the pan agonist 9-cis-RA. These results confirm that various retinoids can cooperate with VD and demonstrate that, at a whole cell level, optimal effects require the activation of both RAR and RXR receptors.

Expression of retinoid-X receptors (-alpha,-beta,-gamma) and retinoic acid receptors (-alpha,-beta,-gamma) in normal human skin: an immunohistological evaluation.

Increasing evidence suggests that the retinoid-X receptors (RXR-alpha,-beta,-gamma) play a crucial role in regulating the transcriptional activity of several steroid hormone receptors, including the receptors for retinoic acid (RAR-alpha,-beta,-gamma), 1,25-dihydroxyvitamin D3 and thyroid hormone. We investigated the localization of the different types of RXR-alpha,-beta,-gamma and RAR-alpha,-beta,-gamma proteins in frozen sections of normal human skin (n = 12) in situ, applying recently raised corresponding specific monoclonal antibodies and an immunohistochemical technique that we established for the detection of these nuclear receptors. Our findings indicate that RXR-alpha,-beta,-gamma and RAR-alpha,-beta,-gamma proteins can be detected by immunohistochemistry in normal human skin. In contrast to RXR-alpha,-beta,-gamma as well as RAR-alpha and RAR-gamma proteins that were consistently detected in cell layers of the viable epidermis, RAR-beta was only focally demonstrated in single epidermal cells in three out of 12 biopsies analysed. Immunohistochemical labelling of RAR-alpha,-beta,-gamma and RXR-alpha,-beta,-gamma proteins in epidermal nuclei was also pronounced in the stratum granulosum, suggesting a function of RXR and RAR proteins in the transition from proliferation to differatiation in epidermal keratinocytes. Expression of RXRs and RARs in hair follicles, sebaceous glands and endothelial cell points to a biological function from these nuclear receptors to hair growth as well as to the physiology of sebaceous glands and endothelial cells.

Estrogen and estrogen receptor antagonists stimulate transcription from the human retinoic acid receptor-alpha 1 promoter via a novel sequence.

We and others previously reported that up-regulation of retinoic acid receptor-alpha (RAR alpha) RNA and protein levels is elicited by estrogen in human breast cancer cells. We set out to determine the mechanism by which estrogen up-regulates RAR alpha. Cloning of 500 bp of the human (h) RAR alpha 1 promoter has been reported previously; we obtained this 500-bp DNA sequence by PCR techniques from human genomic DNA and tested its activity in the context of a luciferase-containing reporter vector in Hep G2 cell contransactivation assays. Estradiol elicited a 6- to 8-fold increase in luciferase activity from the reporter vector driven by hRAR alpha promoter sequence between -491 and +36 bp that was dependent on the presence of contransfected estrogen receptor (ER). Analysis of various truncated versions of this promoter sequence indicated that two regions of the sequence are sensitive to estrogen stimulation. The first resides in the region -49 to -79 bp upstream from the transcription start site and conferred approximately 2-fold activation by estrogen. This region does not contain a consensus estrogen response element, and ER binding to this DNA sequence was not observed. The second responsive sequence lies at -455 to -491 bp and conferred in additional 4- to 6-fold activation by estrogen. This upstream sequence contains two A/TGGTCA half-sites; however, direct binding of ER to this sequence was not observed. Additionally, ER DNA-binding domain mutants that are not capable of binding to DNA were just as effective as wild type ER in their ability to confer estrogen responsiveness to the RAR alpha promoter, implying that ER DNA-binding ability is not required for the estrogen-induced increase in transcriptional activity. Mutation of either half-site or of an additional immediate downstream sequence in the context of the -491 to +36 bp construct reduced the luciferase activity induction by estrogen from 6-fold to 1.5- to 2-fold. Placement of the region between -455 to -491 bp upstream of an SV40 promoter-driven luciferase vector conferred approximately 20- to 30-fold stimulation of luciferase activity by estrogen in an ER-dependent manner. The ER antagonists, 4-hydroxy-tamoxifen, keoxifene, and ICI 164384, each acted as weak agonist via the hRAR alpha promoter in contransactivation assays, exhibiting 20-30% of the efficacy that was demonstrated by estradiol. Interestingly, upon treatment of MCF7 cells with estradiol or the ER antagonists, increased levels of RAR alpha RNA and protein were observed with the antagonists as well as with estrogen.