Limits...
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.

Show MeSH
Androgen has a minor and negative effect on hTGP mRNA expression while AR knockdown has a negative effect on its expression. (A) Relative hTGp mRNA expression, in LNCaP and PC346C cells treated with vehicle or increasing concentrations of the synthetic androgen R1881. (B) RT–PCR detection of hTGp, PSA and GAPDH in LNCaP and PC346C cells treated with atRA (500 nM), androgen (0.1 or 10 nM) or a combination for 24 h. (C) AR mRNA (left) and protein knockdown (right) in LNCaP cells. (D) hTGp mRNA expression in LNCaP cells after AR knockdown with or without R1881 treatment for 24 h. Statistically significant t-test differences are denoted with the asterisk symbol (P < 0.05). **Note the lower molecular weight band in the hTGP RT–PCR amplification is a known splice variant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3367184&req=5

gks143-F3: Androgen has a minor and negative effect on hTGP mRNA expression while AR knockdown has a negative effect on its expression. (A) Relative hTGp mRNA expression, in LNCaP and PC346C cells treated with vehicle or increasing concentrations of the synthetic androgen R1881. (B) RT–PCR detection of hTGp, PSA and GAPDH in LNCaP and PC346C cells treated with atRA (500 nM), androgen (0.1 or 10 nM) or a combination for 24 h. (C) AR mRNA (left) and protein knockdown (right) in LNCaP cells. (D) hTGp mRNA expression in LNCaP cells after AR knockdown with or without R1881 treatment for 24 h. Statistically significant t-test differences are denoted with the asterisk symbol (P < 0.05). **Note the lower molecular weight band in the hTGP RT–PCR amplification is a known splice variant.

Mentions: Next, to test whether androgen affects hTGP expression within a more physiological 24 h treatment period than in the previous studies, LNCaP and PC346C cells were treated with increasing concentrations of the synthetic androgen R1881. Surprisingly, hTGP expression decreased slightly after R1881 treatments in LNCaP, while in PC346C, hTGP expression decreased significantly down to 0.65-fold (P < 0.05) in cells treated with 0.1, 1 and 10 nM R1881 (Figure 3A). LNCaP and PC346C cells were treated with R1881 0.1 or 10 nM and/or atRA 500 nM for 24 h to assess whether the effects of R1881 and atRA were antagonistic. LNCaP and PC346C cells treated with atRA or R1881 showed an increase and decrease of hTGP expression respectively and co-treatment with 0.1 nM R1881 and 500 nM atRA decreased atRA-dependent hTGP expression in LNCaP but not in PC346C cells (Figure 3B, left panel). However co-treatment of 10 nM R1881 and 500 nM atRA resulted in complete abrogation of atRA-induced hTGP expression in LNCaP and a small decrease in PC346C cells (Figure 3B, right panel). To test whether AR knockdown could rescue R1881-dependent hTGP down-regulation, LNCaP cells were transfected with siRNA targeting the AR. AR was successfully knocked-down and mRNA levels remained low even 24 h after R1881 treatment (Figure 3C). Surprisingly, AR knockdown not only failed to rescue hTGP down-regulation after R1881 treatment but also had a significant (P < 0.05) negative effect on basal hTGP expression (Figure 3D).Figure 3.


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)

Androgen has a minor and negative effect on hTGP mRNA expression while AR knockdown has a negative effect on its expression. (A) Relative hTGp mRNA expression, in LNCaP and PC346C cells treated with vehicle or increasing concentrations of the synthetic androgen R1881. (B) RT–PCR detection of hTGp, PSA and GAPDH in LNCaP and PC346C cells treated with atRA (500 nM), androgen (0.1 or 10 nM) or a combination for 24 h. (C) AR mRNA (left) and protein knockdown (right) in LNCaP cells. (D) hTGp mRNA expression in LNCaP cells after AR knockdown with or without R1881 treatment for 24 h. Statistically significant t-test differences are denoted with the asterisk symbol (P < 0.05). **Note the lower molecular weight band in the hTGP RT–PCR amplification is a known splice variant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks143-F3: Androgen has a minor and negative effect on hTGP mRNA expression while AR knockdown has a negative effect on its expression. (A) Relative hTGp mRNA expression, in LNCaP and PC346C cells treated with vehicle or increasing concentrations of the synthetic androgen R1881. (B) RT–PCR detection of hTGp, PSA and GAPDH in LNCaP and PC346C cells treated with atRA (500 nM), androgen (0.1 or 10 nM) or a combination for 24 h. (C) AR mRNA (left) and protein knockdown (right) in LNCaP cells. (D) hTGp mRNA expression in LNCaP cells after AR knockdown with or without R1881 treatment for 24 h. Statistically significant t-test differences are denoted with the asterisk symbol (P < 0.05). **Note the lower molecular weight band in the hTGP RT–PCR amplification is a known splice variant.
Mentions: Next, to test whether androgen affects hTGP expression within a more physiological 24 h treatment period than in the previous studies, LNCaP and PC346C cells were treated with increasing concentrations of the synthetic androgen R1881. Surprisingly, hTGP expression decreased slightly after R1881 treatments in LNCaP, while in PC346C, hTGP expression decreased significantly down to 0.65-fold (P < 0.05) in cells treated with 0.1, 1 and 10 nM R1881 (Figure 3A). LNCaP and PC346C cells were treated with R1881 0.1 or 10 nM and/or atRA 500 nM for 24 h to assess whether the effects of R1881 and atRA were antagonistic. LNCaP and PC346C cells treated with atRA or R1881 showed an increase and decrease of hTGP expression respectively and co-treatment with 0.1 nM R1881 and 500 nM atRA decreased atRA-dependent hTGP expression in LNCaP but not in PC346C cells (Figure 3B, left panel). However co-treatment of 10 nM R1881 and 500 nM atRA resulted in complete abrogation of atRA-induced hTGP expression in LNCaP and a small decrease in PC346C cells (Figure 3B, right panel). To test whether AR knockdown could rescue R1881-dependent hTGP down-regulation, LNCaP cells were transfected with siRNA targeting the AR. AR was successfully knocked-down and mRNA levels remained low even 24 h after R1881 treatment (Figure 3C). Surprisingly, AR knockdown not only failed to rescue hTGP down-regulation after R1881 treatment but also had a significant (P < 0.05) negative effect on basal hTGP expression (Figure 3D).Figure 3.

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