Selective alteration of gene expression in response to natural and synthetic retinoids.
© Brand et al; licensee BioMed Central Ltd. 2002
Received: 14 February 2002
Accepted: 13 May 2002
Published: 13 May 2002
Retinoids are very potent inducers of cellular differentiation and apoptosis, and are efficient anti-tumoral agents. Synthetic retinoids are designed to restrict their toxicity and side effects, mostly by increasing their selectivity toward each isotype of retinoic acids receptors (RARα,β, γ and RXRα, β, γ). We however previously showed that retinoids displayed very different abilities to activate retinoid-inducible reporter genes, and that these differential properties were correlated to the ability of a given ligand to promote SRC-1 recruitment by DNA-bound RXR:RAR heterodimers. This suggested that gene-selective modulation could be achieved by structurally distinct retinoids.
Using the differential display mRNA technique, we identified several genes on the basis of their differential induction by natural or synthetic retinoids in human cervix adenocarcinoma cells. Furthermore, this differential ability to regulate promoter activities was also observed in murine P19 cells for the RARβ2 and CRABPII gene, showing conclusively that retinoid structure has a dramatic impact on the regulation of endogenous genes.
Our findings therefore show that some degree of selective induction or repression of gene expression may be achieved when using appropriately designed ligands for retinoic acid receptors, extending the concept of selective modulators from estrogen and peroxisome proliferator activated receptors to the class of retinoid receptors.
Retinoic acids exert profound effects on cellular differentiation and proliferation. In many cases, retinoids display anti-tumoral activities [1, 2] which are characterized by a retinoid-induced cell cycle arrest in the G0/G1 transition phase  These biological properties are either due to transcriptional upregulation of target genes through a well defined mechanism [reviewed in ] or/and mediated through the ability of retinoids to interfere with the activation of transcription factors controlling proliferative responses of cells to mitogenic stimuli such as AP-1. Transcriptional activation by retinoids is mediated through two families of nuclear receptors, all-trans retinoic acid (RARs) and 9-cis retinoic acid receptors (RXRs), whereas interference with AP-1 is likely to be due to the inhibition of signalling pathways controlled by membrane receptors [5, 6] or to protein:protein interactions [7, 8] Modification of the all-trans retinoic acid structure to improve the specificity and/or the potency of naturally occuring molecules led to the synthesis of a number of compounds characterized by the cyclization of the polyenic chain of all-trans retinoic acid and the addition of various groups at different positions. These conformationally restricted retinoids are now used to achieve selective activation of RAR isotypes α, β or γ in-vitro and in-vivo, which reduces side-effects in therapeutical applications. Synthetic retinoids mimic some of all-trans retinoic acid biological effects in-vivo, but interact differently with the ligand binding domain of RARα and induce distinct structural transitions of the receptor [9, 10]. We have demonstrated that RAR-selective ligands have distinct quantitative activation properties which are reflected by their ability to promote interaction of DNA-bound hRXRα/hRARα heterodimers with the nuclear receptor coactivator (NCoA) SRC-1in-vitro The hormone response element core motifs spacing has a determining influence of RXR:RAR DNA-binding activity, by defining the relative affinity of liganded heterodimers for NCoAs. hRXRα AF2 was critical to confer hRARα full responsiveness, but not differential sensitivity of hRARα to natural or synthetic retinoids. These findings suggested that the use of physically distinct NCoA binding interfaces may be important in controlling specific genes by conformationally restricted ligands and may affect the overall activity of synthetic retinoids vs natural molecules.
Previous studies have demonstrated that synthetic retinoids can not only be isotype-selective, but also display a certain degree of selectivity toward defined receptor-RARE combinations [12, 13]. The role of the ligand structure is emphasized by our recent observations , which suggested that further refinement in gene selectivity could be achieved by altering NCoA interaction surfaces. Selective recruitment of p300 or CBP has indeed been shown to be required for selective activation of p21Cip1 and of p27Kip1 genes respectively . Since transcriptional activation is the end result of multiple interactions between the receptor, its dimerization partner, DNA and ligand, one may speculate that conformationally restricted retinoids with highly selective biological activities may be designed. Beside the tremendous interest for therapeutical applications, this raised the possibility that such retinoids display distinctive abilities to activate endogenous target genes. To further test this hypothesis, we have used the differential display technique as described by Liang and Pardee  to investigate the differential regulation of genes by natural and synthetic retinoids in a human cervical carcinoma cell line (HeLa). A first screening allowed to isolate and to clone 140 ESTs that were differentially induced or repressed by retinoids. In this paper, we report the characterization of two genes which are down-regulated by retinoids, and show that differential regulation is observed in different cell types.
Expression of retinoic acid receptors and of nuclear corepressors and coactivators in HeLa cells
Thus HeLa cells express most of coactivators described so far, and SMRT was found to be expressed at significant levels. Retinoic acid receptors hRARα, hRXRα and hRXRβ were predominantly expressed, with trace amounts of hRARβ. We note that the expression of this receptor was barely inducible upon atRA treatment (data not shown), suggesting that cell-specific features may condition the responsiveness of endogenous genes to retinoids. However, we also observed that transiently transfected reporter genes bearing consensus retinoic acid response elements (RARE) are fully inducible in this cell line , raising the possibility that chromatin assembly on DNA templates strongly regulates retinoid responsiveness . The expression level of SMRT and of several coactivators was also confirmed by western blot analysis of HeLa whole cell extracts (Figure 1E). This analysis confirmed that most of coactivators are coexpressed in this cell line, as well as very low amount of RARα and detectable amounts of RXRs (available antibodies are not isotype-selective).
Identification of genes differentially expressed in response to retinoid treatment
Thus modulation of gene expression by RAR-specific retinoids may be expected to be mostly dependent upon ligand binding to hRARα and dimerization with either RXRα or RXRβ
Summary of newly identified ESTs and of known ESTs potentially regulated by retinoids.
A) New sequences
GenBank Accession Numbers
SRIG 23*, SRIG 28*, SRIG 33.1*, SRIG 53*, SRIG 56*, SRIG 61, SRIG 74, SRIG 81, SRIG 86, SRIG 89, SRIG 90, SRIG 102, SRIG 105, SRIG 107, SRIG 118*, SRIG 119, SRIG 124, SRIG 131, SRIG 144, SRIG 145, SRIG 148, SRIG 154, SRIG 160, SRIG 164, SRIG 178, SRIG 179, SRIG 181, SRIG 185-1.
No significant homology
AI374463*, AI374461*, AI374438*, AI376338*, AI374462*, AI374456, AI374464, AI374455, AI376315, AI374453, AI37441, AI374452, AI374451, AI374450, AI374449*, AI374448, AI374447, AI374446, AI376322, AI376323, AI376324, AI374443, AF096777, AI376330, AI374465, AI376335, AI374444, AI374445
No signal*, other clones were not tested.
B) Identified ESTs
100% with EST AA931835
AI376308 (Unigene Hs 181165)
100% with EST AA027854
AI376309 (Unigene Hs 8117)
100% with EST AA534569
AI376310 (Unigene Hs 13836)
100% with KIAA0043 gene
74% with EST AA699895
AI376312 (Unigene Hs 117353)
90% with EST AA826918
89% with EST AA568770
97% with EST R02820
AI376317 (Unigene Hs 31921)
99% with EST AA449652
AI376318 (Unigene Hs 11803)
80% with EST AA205076
AI374442 (Unigene Hs 17872)
94% with EST C75518
AI376319 (Unigene Hs 61184)
96% with EST AA983976
AI376320 (Unigene Hs 127105)
96% with EST AA093075
AI376321 (Unigene Hs 49015)
100% with EST AA768579
AI376326 (Unigene Hs 22549)
97% with EST AA768579
AI376327 (Unigene Hs 112227)
100% with EST AA505468
AI376329 (Unigene Hs 58609)
99% with EST AI097038
AI376331 (Unigene Hs 156103)
99% with EST AA838424
AI376333 (Unigene Hs 110978)
Summary of genes with known functions as potential targets for retinoid modulation.
100% homology with ZNF beta
99% homology with human ribosomal protein L27a
98% homology with human nuclear protein 55
No differential regulation
100% homology with human fibrilline-2
100% homology with cytochrome oxydase II
No differential regulation
100% homology with human ubiquitin conjugating enzyme
No differential regulation
100% homology with human thymidilate synthase
No differential regulation
91% homology with human phosphate cyclase
Brd3-human bromodomain-containing protein 3 (RING3-like protein)
97% homology with human initiation factor 4B
77% homology with human spermine/spermidine acetyl transferase
97% homology with human 5T4 oncofetal antigen
98% homology with human histone H2B.2
No differential regulation
79% homology with human TRIP7
99% homology with epilepsy holoproencephaly candidate protein-1
97% avec protein phosphatase
97% with human NaCl electroneutral thiazide-sensitive transporter
98% human 60S ribosomal protein
99% homology with EST similar to human TRAM protein
98% homology with human aspartyl beta hydroxylase
100% with human protein kinase C binding protein Nel
96% homology with human enolase
85%homology with human carbamyl phosphate synthase
95% homology with human apoferritine H
99% homology with human cytochrome B
No differential regulation
96% homology with human CG1
100% homology with human duplicate spinal muscular atrophy
99% homology with spermidine acyl transferase
99% homology with human TAXREB 107
98% homology with human plasminogen activator
Characterization of genes differentially regulated in response to retinoid treatment
Homeostasis of iron is mostly dependent on two cellular proteins, ferritins H and L. Ferritin L has been shown to be regulated in HeLa cells by iron, while ferritin H is not regulated by this metal . Northern blot analysis of HeLa cells RNA showed that the SRIG 157 clone, identified as ferritin H in our screen, was clearly down-regulated (Figure 3) by some retinoids. The most active were Am580, CD367, and the RXR-selective ligand CD2425. Thus these retinoids, which do not share receptor binding properties, repressed apoferritin H expression, in contrast to atRA which was inactive in this test.
TAXREB107 binds to tax-responsive elements and thus have functions in the context of HIV infection, by mediating the DNA binding of the HTLV-1 transactivator Tax. It was also identified as a ribosomal protein, and we noted that the ribosomal protein L27a was also identified several times in our screening procedure (Table 2 and data not shown). Again, this mRNA was submitted to a reproducible, selective down-regulation in the presence of TTNPB, CD367 and CD2425 (Figure 4). Thus although no prediction could be drawn from the selectivity of a given ligand for a RAR isotype, this result establishes again that retinoids have distinct abilities to modulate the rate of expression of several cellular genes.
The RARβ2 and CRABPII promoters respond differentially to retinoids in murine embryonal carcinoma cells
Ranking of retinoids for their potency is therefore similar when assessing both RARβ2 and CRABPII mRNA accumulation at 48 hours (TTNPB>CD367>Am580>atRA). However, this order of potency may considerably vary when considering earlier time points: a 2-hours induction, more likely to reflect transcriptional processes, yields the following ranking for the RARβ2 gene: CD367>atRA>TTNPB>Am580, and a 8-hours induction for the CRABPII gene gives the following ranking: atRA>CD367>TTNBP=Am580.
Pursuant to our discovery that natural and synthetic retinoids possess distinct abilities to activate a reporter gene in an identical cellular background, and irrespective of their affinity for their cognate receptor , we set up a differential display approach to extend this observation to the regulation of chromatin-organized, endogenous genes. Selective induction or repression of tens of mRNAs species was observed during the initial screening, revealing that genes involved in multiple aspects of cellular regulation could be potentially regulated specifically by one or several retinoids. Only a few genes are known to be regulated by retinoids, and identification of new targets for these molecules is critical for a better understanding of the pharmacology of retinoids.
As stated by a number of investigators, the mRNA differential display method yields false positives and also identified cDNAs which were not detectable by northern blotting. However, it is worth noting that we restricted our study to genes that are induced very early by retinoids by using induction times of 4 hours, and this may be an explanation for not reaching an intracellular concentration allowing further detection. In addition, intracellular retinoid uptake and metabolic transformation of each compound may vary, and thus introduce variations which are not related directly to transcriptional regulation. Indeed, retinoid-regulated genes follow various kinetics of induction and reaching a steady state may necessitate up to 24–48 hours in P19 cells, as noted very clearly for the CRABPII gene ( and Figure 5). Within this cellular context, we further show that retinoids have also differential abilities to promote both RARβ and CRABPII gene transcription, giving support to our working hypothesis. This regulation appeared to vary according to the promoter.
Interestingly, we identified several genes that were down-regulated by retinoids. Apoferritin H and TAXREB107 were clearly inhibited by a specific set of retinoids (40–50% inhibition), to an extent which is considered to be highly significant in pathological states. This may also be relevant in normal conditions, when considering that a biological response is very likely to be part of an integrated signaling pathway, in which a decrease of 50% of a given step may strongly alter the end result of the activation of this pathway. For example, vascular endothelial growth factor (VEGF) has been shown to be down-regulated by retinoids by two-fold in human keratinocytes and this may be related to the therapeutic effects of retinoids in diseases such as psoriasis and Kaposi' sarcoma .
Retinoids have numerous side effects which severely limit dosage in clinical trials. They include skin irritation and inflammation, elevation of serum triglycerides, hypothyroidism and others such as headache (reviewed in ). Given the potential of retinoids in treating various disorders such as skin hyperproliferation and photoaging, cancer therapy and in metabolic disorders, it is of interest to identify systematically target genes in altered tissues. Our approach identified such candidates genes, and the avaibility of DNA microarrays and of the human genome sequence allows now a genome-wide search of directly or indirectly regulated genes. Combined with appropriate structure-activity relationships studies, retinoids define a very promising field in medicinal chemistry. Used alone or in combination with other powerful molecules such as histone deacetylase inhibitors, one may think of achieving a high degree of selectivity, and thereby reduce toxicity and other side effects of these promising therapeutic agents.
Materials and methods
All-trans retinoic acid was purchased from Sigma (Saint Quentin Fallavier, France). Synthetic retinoids CD3106, TTNPB, Am580, CD367 and CD2425 were obtained from Galderma Inc. (Sophia-Antipolis, France). 10 mM stock solutions were prepared in DMSO and stored at -20°C in the dark. Dulbecco's modified Eagle's medium, fetal calf serum and penicillin/streptomycin mix were purchased from Biowhittaker (BioWhittaker, Verviers, Belgium). Oligonucleotides were purchased from Eurogentec (Le Sart-Tilman, Belgium).
Human HeLa cells were grown in DMEM medium supplemented with 10% fetal calf serum and 1000 U/mL of penicillin and 10 μg/mL of streptomycin. Cells were treated with the indicated retinoic acid receptors ligands for 4 hours. Total RNA was prepared using RNAble reagent (Eurobio, Les Ulis, France) according to the manufacturer's protocol. Total RNA (50 μg) was then treated with 10 U RNase-free DNaseI (Genhunter, Nashville, TN, USA) for 1 hour at 37°C to digest genomic DNA. The purified RNA was adjusted to 1 μg/μl and checked for integrity by standard agarose gel electrophoresis.
Differential display PCR
The differential display reaction was performed using the RNAimage™ kit as indicated by the manufacturer (Genhunter, Nashville, TN, USA). Briefly, reverse transcription was performed using 1 μg RNA and an oligodT anchored primer. The PCR reaction was carried out with the anchored oligodT primer used in all possible combinations with sixteen different arbitrary primers (HAP-1/HAP-16) in presence of [α-33P]dATP (2000 Ci/mmol, Amersham, Les Ulis, France). Reactions mixes were submitted to 40 cycles of PCR as follows: 94°C for15 sec, 40°C for 2 min and 72°C for 30 sec followed by an elongation step at 72°C for 5 min. PCR products were then fractionated on a 8 M urea-6% polyacrylamide gel and visualized by autoradiography. Differentially expressed cDNAs were extracted, purified and reamplified under similar PCR conditions with radioinert deoxyribonucleotides. Amplified cDNAs were then cloned into the pCR-TRAP vector (Genhunter, Nashville, TN, USA) as indicated by the manufacturer. The size of the cloned insert was checked from 3 to 4 colonies and sequenced. Sequence homologies were established using Basic Local Alignement Tool against the GenBank databases. Identified ESTs were then searched against the UniGene (NCBI) database.
Western blotting and antibodies: Whole cell extracts were prepared as follows: 5.106 cells cells were grown, and monolayers were scraped rapidly in ice-cold 1× Phosphate Buffered Saline (PBS). Cells were lysed in one volume of SDS-PAGE loading buffer and briefly sonicated. Western blotting was carried out as described . The DRIP205, 130 and 150 anti sera were a gift from Drs C. Rachez and L.P. Freedman. Peroxidase-coupled anti-mouse, anti-goat or anti-rabbit IgGs were from Sigma. All other antibodies were purchased from SantaCruz Biotechnology (SantaCruz, CA.).
Reverse transcription and amplification (RT-PCR) of retinoic acid receptors, coactivators and corepressors mRNAs in HeLa cells
RNA was extracted and submitted to reverse transcription as described above. Primers were designed to amplify cDNAs fragments ranging in size from 300 to 600 bp and were as follows: hRARα, 5'-CCATTGAGACCCAGAGCAGC-3' and 5'-TGTGTCCATGTGGCGTGGGC-3'; hRARβ, 5'-CAATTGAAACACAGAGCACC-3' and 5'-CCACCAAGTGGTGACTGACTG-3'; hRARγ, 5'-TGGAGACACAGAGCACCAGC-3' and 5'-GTCAGTCTGCTGCCTGAAGC-3'; hRXRα, 5'-CTCCTCAAGCAAGCACTATG-3' and 5'-AGAGCTTAGCGAACCTTCCC-3'; hRXRβ, 5'-TCAGGCAAACACTACGGGGT-3' and 5'-GCATACACTTTCTCCCGCAG-3'; hRXRγ, 5'-CTCAGGAAAGCACTACGGGG-3' and 5'-CCGGATACTTCTGCTTGGTG-3'; AIB1, 5'-GAGCCGACAGGCACTTGAAT-3' and 5'-CCACTGCTGCCATTCATGTG-3'; CBP, 5'-CGCTCAGATGGGACAGCTTG-3' and 5'-ACTTCTCTAGCGTGTCCCCC-3'; p300, 5'-TGGGGTCCCCTGTTCAGC-3' and 5'-GTTATCGGTGCTGAGTCCCAGG-3'; p/CIP, 5'-AAGCCCCTCCACAACAGTTT-3' and 5'-CAGCAGTATTTCTGATCGGG-3'; RAC3, 5'-CCAGATCCAGCCTTTGGTCG-3' and 5'-ATGCCAGACATGGGCATGGG-3'; RIP140, 5'-TCAGCCCAGCAGTTGCATGG-3' and 5'-TCCATTTGCGCTGTGTGGGC-3'; SRC1, 5'-AATGTGTTCAGTCAAGCTGTCCAG-3' and 5'-TGGTTATTCAGTCAGTAGCTGCTG-3'; TIF1, 5'-CCAATGAGGACTGGTGTGCAG-3' and 5'-GCTTTTGAGGCGTTTCTTCCG-3'; TIF2, 5'-CTGAACCAGCATCTTCGAACA-3' and 5'-ATTTCCGTGTTGTGTCTCCC-3'; TRIP1, 5'-GGCTGTGGCTCATCATACGG-3' and 5'-TGAGTGACATGGACTCGCCG-3'; SMRT, 5'-TGACCTATAGAAGCCAGGC-3' and 5'-GAGAGTGTCTCGTACTGCG-3'; N-CoR, 5'-GATCATGGTGTTGTCATGTCC-3' and 5'-AGACAGTGTCTCATACTGCGC-3'. Actin primers were, 5'-ATCATGTTTGAGACCTTCAA-3' and 5'-CATCTCTTGCTCGAAGTCCA-3'. Linearity was assessed as described above.
Reverse transcription and amplification (RT-PCR) of CRABPII and RARβ2 transcripts in P19 cells
Reverse transcription was performed using oligodT primers as recommended by the manufacturer (Promega, Charbonnières, France). Primers were designed as follows:RARβ 5'-AAGTGGTAGGAAGTGAGCTG-3' and 5'-CTACATTGAGCAGTATGCCG-3 and CRABPII 5'-CCAGGTGGAAGGATCTGTTC-3' and 5'-ATTGGTCAGTTCTCGGCTCC-3'. PCR conditions were 40 cycles of 30 sec at 94°C, 1 min at 58°C and 1 min 30 sec at 72°C followed by an elongation step at 72°C for 7 min (RARβ) and 30 cycles of 30 sec at 94°C, 1 min at 56°C and 1 min 30 sec at 72°C followed by an elongation step at 72°C for 7 min (CRABPII).
Northern Blot analysis
20 μg of total RNA were separated by electrophoresis through a 1% agarose gel containing 0.62 M formaldehyde. RNA was then transferred to a Hybond-N+ membrane (Amersham, Les Ulis, France). Membranes were probed sequentially with cDNA of interest and an 18S rRNA probe. Probes were labeled with Fluorescein-dUTP using the Random Prime labelling module (Amersham, Les Ulis, France). Prehybridization and overnight hybridization at 65°C were performed in 5X SSC (1X SSC is 0.15 M NaCl, 15 mM sodium citrate, pH 7), 0.1% SDS, 5% dextran sulfate and 5% liquid block (Amersham, Les Ulis, France). After hybridization, membranes were washed at 65°C successively in 2X SSC, 0.1% SDS, 1X SSC, 0.1% SDS, 0.5X SSC, 0.1% SDS, and 0.1X SSC, 0.1% SDS.
Hybridized probes were revealed using the ECF amplification system (Amersham, Les Ulis, France), and visualized using a Storm™ phosphofluoroimager (Molecular Dynamics, Sunnyvale, CA). Bands intensities were quantified using the ImageQuant™ software (Molecular Dynamics, Sunnyvale, CA). Values for mRNA of interest were normalized to values for the 18S rRNA.
After purification of RNAs and reverse-transcription as described above, the synthesized cDNAs were analyzed by PCR amplification using the TaqMan PCR master mix (Applied Biosysytems, Foster City, CA.) and the appropriate mix of primers. Typically, a mix of 18S mRARβ promoter primers was used. 18S primers were purchased from Applied Biosystems. The FAM/TAMRA probe, forward and reverse primers for the mRARβ transcript were CAGCACCGGCATACTGCTCAA, TCAGTGGATTCACCCAGGC (RAR468F) and TCGGGACGAGCTCCTCAG (RAR557B). Reactions (40 cycles) and data analysis were carried out on a ABI Prism 7700 (Perkin-Elmer).
We would like to thank. Dr U. Reichert (Galderma) for providing us with retinoids and Drs L.P Freedman and C. Rachez for anti-DRIP antibodies. We thank Mrs B. Masselot for technical help. INSERM U459 is part of IFR 22 (INSERM, C.H. et U. de Lille, C.O.L. and University of Lille 2). This work was supported by grants from I.N.S.E.R.M., A.R.E.R.S., Association pour la Recherche sur le Cancer et la Ligue Nationale contre le Cancer.
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