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Retinoic acid and androgen receptors combine to achieve tissue specific control of human prostatic transglutaminase expression: a novel regulatory network with broader significance.

Rivera-Gonzalez GC, Droop AP, Rippon HJ, Tiemann K, Pellacani D, Georgopoulos LJ, Maitland NJ - Nucleic Acids Res. (2012)

Bottom Line: RA and androgen responsive elements (RARE and ARE) were mapped to the hTGP promoter by chromatin immunoprecipitation (ChIP), which also indicated that the active ARE and RARE sites were adjacent, suggesting that the antagonistic effect of androgen and RA is related to the relative position of binding sites.Publicly available AR and RAR ChIP-seq data was used to find gene potentially regulated by AR and RAR.Four of these genes (CDCA7L, CDK6, BTG1 and SAMD3) were tested for RAR and AR binding and two of them (CDCA7L and CDK6) proved to be antagonistically regulated by androgens and RA confirming that this regulation is not particular of hTGP.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Yorkshire Cancer Research Unit, University of York, Heslington, York YO10 5DD, UK.

ABSTRACT
In the human prostate, expression of prostate-specific genes is known to be directly regulated by the androgen-induced stimulation of the androgen receptor (AR). However, less is known about the expression control of the prostate-restricted TGM4 (hTGP) gene. In the present study we demonstrate that the regulation of the hTGP gene depends mainly on retinoic acid (RA). We provide evidence that the retinoic acid receptor gamma (RAR-G) plays a major role in the regulation of the hTGP gene and that presence of the AR, but not its transcriptional transactivation activity, is critical for hTGP transcription. RA and androgen responsive elements (RARE and ARE) were mapped to the hTGP promoter by chromatin immunoprecipitation (ChIP), which also indicated that the active ARE and RARE sites were adjacent, suggesting that the antagonistic effect of androgen and RA is related to the relative position of binding sites. Publicly available AR and RAR ChIP-seq data was used to find gene potentially regulated by AR and RAR. Four of these genes (CDCA7L, CDK6, BTG1 and SAMD3) were tested for RAR and AR binding and two of them (CDCA7L and CDK6) proved to be antagonistically regulated by androgens and RA confirming that this regulation is not particular of hTGP.

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hTGP expression is up-regulated by retinoic acid. (A) Schematic representation of a 4.5-kb hTGp promoter showing putative AREs and retinoic acid responsive elements (RARE). (B) hTGp relative expression in LNCaP, PC346C, PNT1A and PNT2C2 cells treated with vehicle or atRA (500 nM) for 24 h. Statistically significant T-test differences are denoted with the asterisk symbol (P < 0.05) (C) RAR protein expression in LNCaP, PC346C, PNT1A and PNT2C2 whole cell lysates, B-actin was used as a loading control. (D) mRNA expression of RAR-A, RAR-B and RAR-G in LNCaP, PC346C, PNT1A and PNT2C2 cells. (E) atRA responsiveness of each cell line was measured by co-transfecting a reporter plasmid where the firefly luciferase gene is under the control of a tandem of RAREs and a reporter plasmid where the Renilla luciferase expression is controlled by the constitutive CMV promoter. The negative control consisted in the same luciferase reporter gene construct as the test plasmid but lacking the RAREs elements. After transfection cells were treated with increasing concentrations of atRA for 18 h and luciferase activity was measured. All firefly luciferase values were normalized against Renilla luciferase expression.
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gks143-F2: hTGP expression is up-regulated by retinoic acid. (A) Schematic representation of a 4.5-kb hTGp promoter showing putative AREs and retinoic acid responsive elements (RARE). (B) hTGp relative expression in LNCaP, PC346C, PNT1A and PNT2C2 cells treated with vehicle or atRA (500 nM) for 24 h. Statistically significant T-test differences are denoted with the asterisk symbol (P < 0.05) (C) RAR protein expression in LNCaP, PC346C, PNT1A and PNT2C2 whole cell lysates, B-actin was used as a loading control. (D) mRNA expression of RAR-A, RAR-B and RAR-G in LNCaP, PC346C, PNT1A and PNT2C2 cells. (E) atRA responsiveness of each cell line was measured by co-transfecting a reporter plasmid where the firefly luciferase gene is under the control of a tandem of RAREs and a reporter plasmid where the Renilla luciferase expression is controlled by the constitutive CMV promoter. The negative control consisted in the same luciferase reporter gene construct as the test plasmid but lacking the RAREs elements. After transfection cells were treated with increasing concentrations of atRA for 18 h and luciferase activity was measured. All firefly luciferase values were normalized against Renilla luciferase expression.

Mentions: Previous studies indicated that hTGP expression was controlled by androgen over long periods of androgen exposure (7–10 days) (21), but paradoxically no AREs could be found in a 2.1-kb region of the proximal promoter (29) which suggests an indirect androgen regulation of the hTGP gene. Bioinformatic re-analysis of a longer section of the hTGP promoter indicated the presence of a H3K4me2 region between −4898 and −3698. H3K4me2 is a histone mark associated with the presence of enhancers or transcription start sites (37,38). Since this was the only region within the hTGP promoter and gene with this feature, it was decided to find putative binding sites for transcription factors within this 5.5-kb upstream of the hTGP transcriptional start site. Bioinformatic analysis located not only AREs but also RAREs (Figure 2A). In the light of these results, it was decided to re-examine the effects of both hormones on hTGP expression in cell lines from normal and malignant prostate. First, to explore RA regulation, the prostate cell lines LNCaP, PC346C, PNT1A and PNT2C2 were treated with 500 nM all-trans retinoic acid (atRA), which is within the range of commonly used atRA concentrations (35,39,40), for 24 h to test whether this compound could regulate hTGP expression. LNCaP and PC346C are prostate cancer cell lines with luminal characteristics; they express PSA, AR and are responsive to androgen. PNT1A and PNT2C2 are benign prostate cell lines less differentiated than LNCaP and PC346C. PNT1A and PNT2C2 do not express AR and are not affected by the presence or absence of androgens (41). While LNCaP and PC346C cells treated with atRA showed a marked increase in hTGP expression, the normal, less-differentiated PNT1A and PNT2C2 cells showed a small decrease (Figure 2B). As the difference in hTGP regulation after atRA treatment could be the result of differential RAR expression in the different cell lines, total RAR protein and mRNA was measured in LNCaP, PC346C, PNT1A and PNT2C2 cells. PNT1A and PNT2C2 showed higher levels of total RAR protein in comparison to LNCaP and PC346C while mRNA for the different RARs did not show a clear tendency that could explain the lack of hTGP up-regulation after atRA treatment in PNT1A and PNT2C2 cells (Figure 2C and D). To functionally test the extent to which each cell line could sustain atRA-dependent gene activation, the cells were co-transfected with a reporter plasmid containing a tandem of RAREs controlling the expression of the firefly luciferase gene and a control construct where the CMV promoter controlled expression of the Renilla luciferase gene. Twenty-four hours after transfection cells were treated with increasing concentrations of atRA and luciferase activity was measured after a further 18 h. The results showed that LNCaP and PC346C cells could sustain atRA-dependent gene expression to a higher extent (up to 10.6- and 17.9-fold increase respectively) in comparison to PNT1A and PNT2C2 cells (up to 5.4- and 4.53-fold increase respectively) (Figure 2E). The greater extent to which LNCaP and PC346C sustain atRA-dependent transcription explains the lack of hTGP up-regulation observed in PNT1A and PNT2C2 cells.Figure 2.


Retinoic acid and androgen receptors combine to achieve tissue specific control of human prostatic transglutaminase expression: a novel regulatory network with broader significance.

Rivera-Gonzalez GC, Droop AP, Rippon HJ, Tiemann K, Pellacani D, Georgopoulos LJ, Maitland NJ - Nucleic Acids Res. (2012)

hTGP expression is up-regulated by retinoic acid. (A) Schematic representation of a 4.5-kb hTGp promoter showing putative AREs and retinoic acid responsive elements (RARE). (B) hTGp relative expression in LNCaP, PC346C, PNT1A and PNT2C2 cells treated with vehicle or atRA (500 nM) for 24 h. Statistically significant T-test differences are denoted with the asterisk symbol (P < 0.05) (C) RAR protein expression in LNCaP, PC346C, PNT1A and PNT2C2 whole cell lysates, B-actin was used as a loading control. (D) mRNA expression of RAR-A, RAR-B and RAR-G in LNCaP, PC346C, PNT1A and PNT2C2 cells. (E) atRA responsiveness of each cell line was measured by co-transfecting a reporter plasmid where the firefly luciferase gene is under the control of a tandem of RAREs and a reporter plasmid where the Renilla luciferase expression is controlled by the constitutive CMV promoter. The negative control consisted in the same luciferase reporter gene construct as the test plasmid but lacking the RAREs elements. After transfection cells were treated with increasing concentrations of atRA for 18 h and luciferase activity was measured. All firefly luciferase values were normalized against Renilla luciferase expression.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3367184&req=5

gks143-F2: hTGP expression is up-regulated by retinoic acid. (A) Schematic representation of a 4.5-kb hTGp promoter showing putative AREs and retinoic acid responsive elements (RARE). (B) hTGp relative expression in LNCaP, PC346C, PNT1A and PNT2C2 cells treated with vehicle or atRA (500 nM) for 24 h. Statistically significant T-test differences are denoted with the asterisk symbol (P < 0.05) (C) RAR protein expression in LNCaP, PC346C, PNT1A and PNT2C2 whole cell lysates, B-actin was used as a loading control. (D) mRNA expression of RAR-A, RAR-B and RAR-G in LNCaP, PC346C, PNT1A and PNT2C2 cells. (E) atRA responsiveness of each cell line was measured by co-transfecting a reporter plasmid where the firefly luciferase gene is under the control of a tandem of RAREs and a reporter plasmid where the Renilla luciferase expression is controlled by the constitutive CMV promoter. The negative control consisted in the same luciferase reporter gene construct as the test plasmid but lacking the RAREs elements. After transfection cells were treated with increasing concentrations of atRA for 18 h and luciferase activity was measured. All firefly luciferase values were normalized against Renilla luciferase expression.
Mentions: Previous studies indicated that hTGP expression was controlled by androgen over long periods of androgen exposure (7–10 days) (21), but paradoxically no AREs could be found in a 2.1-kb region of the proximal promoter (29) which suggests an indirect androgen regulation of the hTGP gene. Bioinformatic re-analysis of a longer section of the hTGP promoter indicated the presence of a H3K4me2 region between −4898 and −3698. H3K4me2 is a histone mark associated with the presence of enhancers or transcription start sites (37,38). Since this was the only region within the hTGP promoter and gene with this feature, it was decided to find putative binding sites for transcription factors within this 5.5-kb upstream of the hTGP transcriptional start site. Bioinformatic analysis located not only AREs but also RAREs (Figure 2A). In the light of these results, it was decided to re-examine the effects of both hormones on hTGP expression in cell lines from normal and malignant prostate. First, to explore RA regulation, the prostate cell lines LNCaP, PC346C, PNT1A and PNT2C2 were treated with 500 nM all-trans retinoic acid (atRA), which is within the range of commonly used atRA concentrations (35,39,40), for 24 h to test whether this compound could regulate hTGP expression. LNCaP and PC346C are prostate cancer cell lines with luminal characteristics; they express PSA, AR and are responsive to androgen. PNT1A and PNT2C2 are benign prostate cell lines less differentiated than LNCaP and PC346C. PNT1A and PNT2C2 do not express AR and are not affected by the presence or absence of androgens (41). While LNCaP and PC346C cells treated with atRA showed a marked increase in hTGP expression, the normal, less-differentiated PNT1A and PNT2C2 cells showed a small decrease (Figure 2B). As the difference in hTGP regulation after atRA treatment could be the result of differential RAR expression in the different cell lines, total RAR protein and mRNA was measured in LNCaP, PC346C, PNT1A and PNT2C2 cells. PNT1A and PNT2C2 showed higher levels of total RAR protein in comparison to LNCaP and PC346C while mRNA for the different RARs did not show a clear tendency that could explain the lack of hTGP up-regulation after atRA treatment in PNT1A and PNT2C2 cells (Figure 2C and D). To functionally test the extent to which each cell line could sustain atRA-dependent gene activation, the cells were co-transfected with a reporter plasmid containing a tandem of RAREs controlling the expression of the firefly luciferase gene and a control construct where the CMV promoter controlled expression of the Renilla luciferase gene. Twenty-four hours after transfection cells were treated with increasing concentrations of atRA and luciferase activity was measured after a further 18 h. The results showed that LNCaP and PC346C cells could sustain atRA-dependent gene expression to a higher extent (up to 10.6- and 17.9-fold increase respectively) in comparison to PNT1A and PNT2C2 cells (up to 5.4- and 4.53-fold increase respectively) (Figure 2E). The greater extent to which LNCaP and PC346C sustain atRA-dependent transcription explains the lack of hTGP up-regulation observed in PNT1A and PNT2C2 cells.Figure 2.

Bottom Line: RA and androgen responsive elements (RARE and ARE) were mapped to the hTGP promoter by chromatin immunoprecipitation (ChIP), which also indicated that the active ARE and RARE sites were adjacent, suggesting that the antagonistic effect of androgen and RA is related to the relative position of binding sites.Publicly available AR and RAR ChIP-seq data was used to find gene potentially regulated by AR and RAR.Four of these genes (CDCA7L, CDK6, BTG1 and SAMD3) were tested for RAR and AR binding and two of them (CDCA7L and CDK6) proved to be antagonistically regulated by androgens and RA confirming that this regulation is not particular of hTGP.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Yorkshire Cancer Research Unit, University of York, Heslington, York YO10 5DD, UK.

ABSTRACT
In the human prostate, expression of prostate-specific genes is known to be directly regulated by the androgen-induced stimulation of the androgen receptor (AR). However, less is known about the expression control of the prostate-restricted TGM4 (hTGP) gene. In the present study we demonstrate that the regulation of the hTGP gene depends mainly on retinoic acid (RA). We provide evidence that the retinoic acid receptor gamma (RAR-G) plays a major role in the regulation of the hTGP gene and that presence of the AR, but not its transcriptional transactivation activity, is critical for hTGP transcription. RA and androgen responsive elements (RARE and ARE) were mapped to the hTGP promoter by chromatin immunoprecipitation (ChIP), which also indicated that the active ARE and RARE sites were adjacent, suggesting that the antagonistic effect of androgen and RA is related to the relative position of binding sites. Publicly available AR and RAR ChIP-seq data was used to find gene potentially regulated by AR and RAR. Four of these genes (CDCA7L, CDK6, BTG1 and SAMD3) were tested for RAR and AR binding and two of them (CDCA7L and CDK6) proved to be antagonistically regulated by androgens and RA confirming that this regulation is not particular of hTGP.

Show MeSH