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. 2002 May 13:2:13.
doi: 10.1186/1471-2210-2-13.

Selective alteration of gene expression in response to natural and synthetic retinoids

Céline Brand  1 , Pascaline Ségard, Pascal Plouvier, Pierre Formstecher, Pierre-Marie Danzé, Philippe Lefebvre
Affiliations

Selective alteration of gene expression in response to natural and synthetic retinoids

Céline Brand et al. BMC Pharmacol. .

Abstract

Background: 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 (RARalpha,beta, gamma and RXRalpha, beta, gamma). 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.

Results: 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 RARbeta2 and CRABPII gene, showing conclusively that retinoid structure has a dramatic impact on the regulation of endogenous genes.

Conclusions: 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.

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Figures

Figure 1
Figure 1
Expression levels of retinoic acid receptors and of transcriptional intermediary factors in HeLa cells. HeLa cells mRNA was extracted and analyzed by RT-PCR using specific primers to detect (A) hRARα, hRARβ, hRARγ, hRXRα, hRXRβ and hRXRγ transcripts; to confirm the lack of expression of hRARβ, hRARγ and hRXRγ by nested PCR (B) to characterize expression levels of nuclear coactivators (C) and nuclear corepressors (D). E) Western blot analysis of HeLa whole cell extracts. 100 μg of proteins were resolved by 8% SDS-PAGE and blotted onto a nitrocellulose membrane. This membrane was probed with antibodies specific for each indicated receptors, coactivators or corepressor. The left panel shows a silver-stained gel on which 10 μg of cell extract has been separated. Molecular masses are indicated in kDa.
Figure 2
Figure 2
Differential expression of mRNA species in HeLa cells treated by retinoids. Differential display RT-PCR analysis of HeLa transcripts obtained from cells treated with 1μM of the indicated retinoid for 4 hours. Total RNA was extracted and purified from HeLa cells and reverse-transcribed with the H-T11G (AAGCT11G, left panel), H-T11A (AAGCT11A, middle panel) or H-T11C (AAGCT11C, right panel) primers. PCR amplification of cDNAs was carried out using the same 3' primer and the H-AP3 primer (AAGCTTTGGTCAG), the H-AP6 primer (AAGCTTGCACCAT), the H-AP12 primer (AAGCTTGAGTGCT) and the H-AP15 primer (AAGCTTACGCAAC) (from left to right) in the presence of α-[33P] dATP. Amplified cDNA fragments were analyzed on 6% sequencing gels and visualized by autoradiography. Typical lanes are shown, with size markers appearing on the left. Selectively regulated mRNAs are indicated by dots. These materials were extracted from the gel, re-amplified by PCR with the same set of primers, cloned into the pCR-TRAP vector (GenHunter) and sequenced.
Figure 3
Figure 3
Expression of ferritin H mRNA in HeLa cells treated with different ligands of the retinoic acid receptor. HeLa cells were treated with the different ligands for four hours. Total RNA (20 μg) was probed sequentially with fluorescein-labeled partial human ferritin H cDNA and 18S rRNA probes. Blots were quantified using a Storm™ apparatus. Values for the ferritin H transcript were normalized to the 18S rRNA level. A) Upper panel: ferritin H transcript in HeLa cells treated with various retinoids. Lower panel: 18S rRNA. B) Quantification of ferritin H expression. Results are presented as the mean +/- S.E.M. of three different experiments. Cells were treated with 25nM atRA, 30nM CD3106, 80nM TTNPB, 20 nM Am580, 10nM CD367 and 100nM CD2425 for 4 hours.
Figure 4
Figure 4
Expression of TAXREB107 mRNA in HeLa cells treated with different ligands of the retinoic acid receptor. HeLa cells were treated with the different ligands for 4 hours. Total RNA (20 μg) was probed sequentially with fluorescein-labeled partial human TAXREB cDNA and 18S rRNA probes. Blots were quantified using a Storm™ apparatus. Values for the TAXREB transcript were normalized to the 18S rRNA level. A) Upper panel: TAXREB107 transcript in HeLa cells treated with various retinoids. Lower panel: 18S rRNA. B) Quantification of TAXREB107 expression. Results are presented as the mean +/- S.E.M. of three different experiments. Retinoid concentrations were as in Figure 3.
Figure 5
Figure 5
Expression of RARβ and of CRABPII in murine P19 cells treated with different ligands of the retinoic acid receptor. A) Time-course analysis of RARβ transcripts. RARβ transcripts were assayed by Northern blotting (upper panel) and quantified by densitometry. A more rigorous measurement of each time point was carried out by submitting the same sample to real time PCR quantification, using, as for the Northern blot analysis, 18S RNA as an internal standard. All results are expressed relative to RARβ level of expression in non stimulated cells. Representative autoradiograms and PCR quantification are shown here, but have carried out 3 times with similar results. B) Time-course analysis of CRABPII transcripts. Assays were carried out as described in A), setting the reference (non stimulated cells) to 100%. Retinoid concentrations were as in Figure 3.
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