Limits...
Poly (A) Binding Protein Cytoplasmic 1 Is a Novel Co-Regulator of the Androgen Receptor.

Eisermann K, Dar JA, Dong J, Wang D, Masoodi KZ, Wang Z - PLoS ONE (2015)

Bottom Line: Mass spectrometry analysis of the pulled down proteins identified poly (A) binding protein cytoplasmic 1 (PABPC1) interaction with this region of the AR.Knockdown of PABPC1 decreased nuclear AR protein levels and inhibited androgen activation of the AR target PSA in LNCaP and C4-2 cells.These findings suggest that PABPC1 is a novel co-regulator of the AR and may be a potential target for blocking activation of the AR in CRPC.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
The androgen receptor (AR) is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner. The NTD of the AR plays a key role in AR transactivation including androgen-independent activation of the AR in castration-resistant prostate cancer (CRPC) cells. We recently reported that amino acids (a.a.) 50-250 of the NTD are capable of modulating AR nucleocytoplasmic trafficking. To further explore the mechanism associated with a.a. 50-250, GFP pull-down assays were performed in C4-2 CRPC cells transfected with GFP tagged a.a. 50-250 of the AR. Mass spectrometry analysis of the pulled down proteins identified poly (A) binding protein cytoplasmic 1 (PABPC1) interaction with this region of the AR. In silico analysis of gene expression data revealed PABPC1 up-regulation in prostate cancer tissue specimens and this up-regulation correlates to increased disease recurrence. Co-immunoprecipitation assays confirmed the association of PABPC1 with a.a. 50-250 of the NTD of the AR. Knockdown of PABPC1 decreased nuclear AR protein levels and inhibited androgen activation of the AR target PSA in LNCaP and C4-2 cells. Additionally, knockdown of PABPC1 inhibited transactivation of the PSA promoter by NAR (AR lacking the LBD) and attenuated proliferation of AR-positive prostate cancer cells. These findings suggest that PABPC1 is a novel co-regulator of the AR and may be a potential target for blocking activation of the AR in CRPC.

No MeSH data available.


Related in: MedlinePlus

Knockdown of PABPC1 with siRNA decreases nuclear AR protein levels and PSA mRNA expression levels.(A) C4-2 cells were transfected with control siRNA (siC) (40 pmol/mL) or siRNA (40 pmol/mL) specific for PABPC1 (siPABPC1) for 48 hours followed by treatment with 0.1nM R1881 for 24 hours in the presence of 5% charcoal-stripped FBS RPMI. Nuclear and cytoplasmic extracts were analyzed by Western blot. GAPDH (Santa Cruz) and Lamin A/C (Genscript, Piscataway, NJ) were used as loading and cell compartment controls. PSA (B-C) and PABPC1 (D-E) mRNA expression levels were detected in LNCaP and C4-2 cells following siPABPC1 treatment as in (A). Expression was normalized to GAPDH. Experiments were reproduced twice. Significance was determined by Student’s t-test (**p<0.01, ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4503479&req=5

pone.0128495.g006: Knockdown of PABPC1 with siRNA decreases nuclear AR protein levels and PSA mRNA expression levels.(A) C4-2 cells were transfected with control siRNA (siC) (40 pmol/mL) or siRNA (40 pmol/mL) specific for PABPC1 (siPABPC1) for 48 hours followed by treatment with 0.1nM R1881 for 24 hours in the presence of 5% charcoal-stripped FBS RPMI. Nuclear and cytoplasmic extracts were analyzed by Western blot. GAPDH (Santa Cruz) and Lamin A/C (Genscript, Piscataway, NJ) were used as loading and cell compartment controls. PSA (B-C) and PABPC1 (D-E) mRNA expression levels were detected in LNCaP and C4-2 cells following siPABPC1 treatment as in (A). Expression was normalized to GAPDH. Experiments were reproduced twice. Significance was determined by Student’s t-test (**p<0.01, ***p<0.001).

Mentions: To determine the effect of inhibition of PABPC1 expression on AR nucleocytoplasmic trafficking, C4-2 cells were transfected with siPABPC1 for 48 hours followed by treatment with 0.1nM R1881 or vehicle for an additional 24 hours. Western blot analysis of nuclear and cytoplasmic fractions revealed that nuclear AR protein levels were reduced with the knockdown of PABPC1 in C4-2 cells (Fig 6A). Additionally, hormone induction of PSA mRNA levels in both LNCaP (Fig 6B) and C4-2 cells (Fig 6C) was significantly reduced by knockdown of PABPC1 (Fig 6D and 6E). Another androgen response gene, FKBP5, was tested for specificity of PABPC1 to androgen response gene expression. Inhibition of PABPC1 was shown to decrease the androgen up-regulation of FKBP5 by R1881 (S1 Fig). These results suggest that PABPC1 controls the amount of the AR in the nucleus and plays a role in the transactivation of AR target genes in these cell lines.


Poly (A) Binding Protein Cytoplasmic 1 Is a Novel Co-Regulator of the Androgen Receptor.

Eisermann K, Dar JA, Dong J, Wang D, Masoodi KZ, Wang Z - PLoS ONE (2015)

Knockdown of PABPC1 with siRNA decreases nuclear AR protein levels and PSA mRNA expression levels.(A) C4-2 cells were transfected with control siRNA (siC) (40 pmol/mL) or siRNA (40 pmol/mL) specific for PABPC1 (siPABPC1) for 48 hours followed by treatment with 0.1nM R1881 for 24 hours in the presence of 5% charcoal-stripped FBS RPMI. Nuclear and cytoplasmic extracts were analyzed by Western blot. GAPDH (Santa Cruz) and Lamin A/C (Genscript, Piscataway, NJ) were used as loading and cell compartment controls. PSA (B-C) and PABPC1 (D-E) mRNA expression levels were detected in LNCaP and C4-2 cells following siPABPC1 treatment as in (A). Expression was normalized to GAPDH. Experiments were reproduced twice. Significance was determined by Student’s t-test (**p<0.01, ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4503479&req=5

pone.0128495.g006: Knockdown of PABPC1 with siRNA decreases nuclear AR protein levels and PSA mRNA expression levels.(A) C4-2 cells were transfected with control siRNA (siC) (40 pmol/mL) or siRNA (40 pmol/mL) specific for PABPC1 (siPABPC1) for 48 hours followed by treatment with 0.1nM R1881 for 24 hours in the presence of 5% charcoal-stripped FBS RPMI. Nuclear and cytoplasmic extracts were analyzed by Western blot. GAPDH (Santa Cruz) and Lamin A/C (Genscript, Piscataway, NJ) were used as loading and cell compartment controls. PSA (B-C) and PABPC1 (D-E) mRNA expression levels were detected in LNCaP and C4-2 cells following siPABPC1 treatment as in (A). Expression was normalized to GAPDH. Experiments were reproduced twice. Significance was determined by Student’s t-test (**p<0.01, ***p<0.001).
Mentions: To determine the effect of inhibition of PABPC1 expression on AR nucleocytoplasmic trafficking, C4-2 cells were transfected with siPABPC1 for 48 hours followed by treatment with 0.1nM R1881 or vehicle for an additional 24 hours. Western blot analysis of nuclear and cytoplasmic fractions revealed that nuclear AR protein levels were reduced with the knockdown of PABPC1 in C4-2 cells (Fig 6A). Additionally, hormone induction of PSA mRNA levels in both LNCaP (Fig 6B) and C4-2 cells (Fig 6C) was significantly reduced by knockdown of PABPC1 (Fig 6D and 6E). Another androgen response gene, FKBP5, was tested for specificity of PABPC1 to androgen response gene expression. Inhibition of PABPC1 was shown to decrease the androgen up-regulation of FKBP5 by R1881 (S1 Fig). These results suggest that PABPC1 controls the amount of the AR in the nucleus and plays a role in the transactivation of AR target genes in these cell lines.

Bottom Line: Mass spectrometry analysis of the pulled down proteins identified poly (A) binding protein cytoplasmic 1 (PABPC1) interaction with this region of the AR.Knockdown of PABPC1 decreased nuclear AR protein levels and inhibited androgen activation of the AR target PSA in LNCaP and C4-2 cells.These findings suggest that PABPC1 is a novel co-regulator of the AR and may be a potential target for blocking activation of the AR in CRPC.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
The androgen receptor (AR) is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner. The NTD of the AR plays a key role in AR transactivation including androgen-independent activation of the AR in castration-resistant prostate cancer (CRPC) cells. We recently reported that amino acids (a.a.) 50-250 of the NTD are capable of modulating AR nucleocytoplasmic trafficking. To further explore the mechanism associated with a.a. 50-250, GFP pull-down assays were performed in C4-2 CRPC cells transfected with GFP tagged a.a. 50-250 of the AR. Mass spectrometry analysis of the pulled down proteins identified poly (A) binding protein cytoplasmic 1 (PABPC1) interaction with this region of the AR. In silico analysis of gene expression data revealed PABPC1 up-regulation in prostate cancer tissue specimens and this up-regulation correlates to increased disease recurrence. Co-immunoprecipitation assays confirmed the association of PABPC1 with a.a. 50-250 of the NTD of the AR. Knockdown of PABPC1 decreased nuclear AR protein levels and inhibited androgen activation of the AR target PSA in LNCaP and C4-2 cells. Additionally, knockdown of PABPC1 inhibited transactivation of the PSA promoter by NAR (AR lacking the LBD) and attenuated proliferation of AR-positive prostate cancer cells. These findings suggest that PABPC1 is a novel co-regulator of the AR and may be a potential target for blocking activation of the AR in CRPC.

No MeSH data available.


Related in: MedlinePlus