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

Brand C, Ségard P, Plouvier P, Formstecher P, Danzé PM, Lefebvre P - BMC Pharmacol. (2002)

Bottom Line: This suggested that gene-selective modulation could be achieved by structurally distinct retinoids.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U 459 and Ligue nationale contre le Cancer, Faculté de Médecine Henri Warembourg, 1, place de Verdun, 59045 Lille cedex, France. brand@lille.inserm.fr

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC113761&req=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.

Mentions: Murine pluripotent P19 cells can differentiate into endodermal and mesodermal cells after retinoic acid treatment (reviewed in [36]) and have a number of characteristics which make them suitable for analysis of RA-mediated gene induction [37]. Retinoic acids receptors RARα, RARγ and RXRγ are constitutively expressed in this cell line (data not shown), whereas RARβ is induced upon atRA treatment (Figure 5A). The cellular retinoic acid binding protein type II (CRABPII) gene expression is also regulated by retinoids (Figure 5B and [38]). To further investigate the possibility that retinoids have differential abilities to stimulate gene expression in a similar cellular background, we monitored RARβ and CRABPII expression by Northern blotting and RT-PCR upon stimulation by limited concentrations (2x Kd) of several natural or synthetic retinoids. atRA turned out to be a good inducer of the RARβ promoter, with transcripts becoming detectable after 4 hours of induction. mRNA accumulation reached a plateau after 8–10 hours of induction, after which a clear, specific down-regulation was observed. (Figure 5A). Other retinoids could be distinguished on the basis of the rate of induction and of their efficiencies to promote RARβ mRNA accumulation. TTNPB was a strong inducer in our system, as well as CD367. Am580 caused a time course of mRNA accumulation slower when compared to that observed with atRA and other retinoids, and yielded maximal mRNA levels increased by 3-fold when compared to atRA. The CRABPII gene transcriptional regulation by retinoids exhibited a very different time-course, characterzed by a very slow accumulation of mRNA at early time points (<8 hours) with synthetic retinoids (Figure 5B). atRA, on the contrary, induced a moderate but rapid induction of the CRABPII mRNA synthesis, reaching a plateau in less than 8 hours, whereas all other agonists were weak inducers at early time points, and were much more potent at 24 and 48 hours.


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

Brand C, Ségard P, Plouvier P, Formstecher P, Danzé PM, Lefebvre P - BMC Pharmacol. (2002)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC113761&req=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.
Mentions: Murine pluripotent P19 cells can differentiate into endodermal and mesodermal cells after retinoic acid treatment (reviewed in [36]) and have a number of characteristics which make them suitable for analysis of RA-mediated gene induction [37]. Retinoic acids receptors RARα, RARγ and RXRγ are constitutively expressed in this cell line (data not shown), whereas RARβ is induced upon atRA treatment (Figure 5A). The cellular retinoic acid binding protein type II (CRABPII) gene expression is also regulated by retinoids (Figure 5B and [38]). To further investigate the possibility that retinoids have differential abilities to stimulate gene expression in a similar cellular background, we monitored RARβ and CRABPII expression by Northern blotting and RT-PCR upon stimulation by limited concentrations (2x Kd) of several natural or synthetic retinoids. atRA turned out to be a good inducer of the RARβ promoter, with transcripts becoming detectable after 4 hours of induction. mRNA accumulation reached a plateau after 8–10 hours of induction, after which a clear, specific down-regulation was observed. (Figure 5A). Other retinoids could be distinguished on the basis of the rate of induction and of their efficiencies to promote RARβ mRNA accumulation. TTNPB was a strong inducer in our system, as well as CD367. Am580 caused a time course of mRNA accumulation slower when compared to that observed with atRA and other retinoids, and yielded maximal mRNA levels increased by 3-fold when compared to atRA. The CRABPII gene transcriptional regulation by retinoids exhibited a very different time-course, characterzed by a very slow accumulation of mRNA at early time points (<8 hours) with synthetic retinoids (Figure 5B). atRA, on the contrary, induced a moderate but rapid induction of the CRABPII mRNA synthesis, reaching a plateau in less than 8 hours, whereas all other agonists were weak inducers at early time points, and were much more potent at 24 and 48 hours.

Bottom Line: This suggested that gene-selective modulation could be achieved by structurally distinct retinoids.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U 459 and Ligue nationale contre le Cancer, Faculté de Médecine Henri Warembourg, 1, place de Verdun, 59045 Lille cedex, France. brand@lille.inserm.fr

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.

Show MeSH
Related in: MedlinePlus