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1.
Figure 3

Figure 3. In C4-2 cells, PXN regulates AR nuclear localization and androgen-induced mRNA expression.. From: Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation.

(A) PXN is required for AR nuclear localization. Immunofluorescence of AR (red), PXN (green), or PS-PXN (green) in cells treated with Nsp or PXN siRNA, followed by treatment with medium or DHT (25 nM; 30 minutes). PXN-knockdown cells were transfected with plasmids expressing WT PXN or PXN(S→A). Original magnification, ×40. (B) PXN regulates androgen-induced gene expression. Relative expression of DHT-induced FKBP5 and NKX3-1 mRNAs by qPCR in Nsp or siRNA-mediated PXN-knockdown in C4-2 cells. Data are represented as mean ± SEM (n = 3). *P ≤ 0.001; **P ≤ 0.05 relative to Nsp siRNA.

Aritro Sen, et al. J Clin Invest. 2012 July 2;122(7):2469-2481.
2.
Figure 8

Figure 8. PXN mediates extranuclear and nuclear AR and ERK signaling.. From: Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation.

Androgen binds ARs at or near the plasma membrane, which transactivates the EGFR via MMP-mediated release of EGFR ligands. EGFR can also be activated directly by EGF. This activates Src, which phosphorylates PXN on tyrosine residues, enabling Raf to activate MEK/ERK. ERK then mediates phosphorylation of PXN at serine residues (though not necessarily directly), and PS-PXN enters the nucleus. Meanwhile, androgen-bound AR also translocates to the nucleus. In the nucleus, PS-PXN interacts with androgen-bound AR to retain it in the nucleus. PS-PXN then associates with or near the AR on the PSA/NKX3-1 promoter to help promote AR-driven transcription (left). Activated ERK also enters the nucleus, where PS-PXN, p-ERK, and ELK1 form a complex and ERK phosphorylates ELK1, which induces c-FOS expression. The latter then activates cyclin D1 promoter activity/cyclin D1 expression (right), thereby promoting cell proliferation.

Aritro Sen, et al. J Clin Invest. 2012 July 2;122(7):2469-2481.
3.
Figure 5

Figure 5. PXN modulates ERK-mediated transcription independent of androgens.. From: Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation.

(A) PXN is required for EGF-induced cyclin D1 promoter activity. Nsp/PXN-specific siRNA-treated PC3 cells were transiently transfected with cyclin D1 promoter-luciferase plus cytomegalovirus–β-gal plasmid, followed by EGF stimulation (20 ng/ml; 24 hours). PXN-knockdown cells were transfected with plasmids expressing WT PXN or PXN lacking the MAPK-targeted serines (S→A). Cyclin D1 promoter-luciferase activity was normalized to β-gal expression and data represented as fold increase with respect to medium (mean ± SEM, n = 3). *P ≤ 0.001. (B) Nuclear localization of ERK is independent of PS-PXN. Immunofluorescence of ERK (red), PXN (green), p-ERK (green), or PS-PXN (green) in PC3 cells treated with Nsp siRNA or PXN siRNA, followed by medium or EGF (20 ng/ml; 30 minutes) stimulation. PXN-knockdown cells were transfected with plasmids expressing WT PXN or PXN lacking the MAPK-targeted serine (S→A). Adjacent Hoechst staining represents the nucleus (n = 5 experiments, all with identical results). Original magnification, ×40.

Aritro Sen, et al. J Clin Invest. 2012 July 2;122(7):2469-2481.
4.
Figure 1

Figure 1. In LNCaP cells, phosphorylated PXN is a nuclear protein that regulates AR-mediated transcription.. From: Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation.

(A) PS-PXN localizes to the nucleus. Immunofluorescence of PXN and PS-PXN with medium or DHT (25 nM) treatment (30 minutes). For all immunofluorescence, adjacent Hoechst staining represents the nucleus; experiments were repeated more than 3 times with identical results. Original magnification, ×40. (B) DHT stimulation of the PSA promoter requires PXN. Nsp or PXN siRNA–treated cells were transfected with PSA-luciferase and cytomegalovirus–β-gal plasmid; this was followed by DHT treatment (25 nM; 24 hours). PXN-knockdown cells were transfected with plasmids encoding WT PXN or PXN(S→A). Luciferase activity was normalized to β-gal and represented as fold increase over medium (n = 4). (C) PXN regulates expression of multiple androgen targets. Relative expression of DHT-induced PSA, FKBP5, NKX3-1, and c-FOS mRNAs in Nsp or siRNA-mediated PXN knockdown LNCaP cells. Data are represented as mean ± SEM (n = 3). *P ≤ 0.001; **P ≤ 0.05 relative to Nsp siRNA.

Aritro Sen, et al. J Clin Invest. 2012 July 2;122(7):2469-2481.
5.
Figure 2

Figure 2. In LNCaP cells, phosphorylated PXN maintains DHT-triggered AR nuclear localization and binds to promoter DNA.. From: Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation.

(A) PXN is required for AR nuclear localization. Immunofluorescence of AR (red), PXN (green), or PS-PXN (green) in cells treated with Nsp or PXN siRNA, followed by medium or DHT (25 nM; 30 minutes). PXN-knockdown cells were transfected with plasmids expressing WT PXN or PXN(S→A). (B) PXN retains AR within the nucleus. Cells were treated with PXN siRNA, then leptomycin B (15 ng/ml), followed by medium or DHT (25 nM; 30 minutes) stimulation. Original magnification, ×40. (C) PXN and AR form a complex in the nucleus. AR or PXN was precipitated from nuclear extracts of DHT-treated LNCaP cells (IP), followed by immunoblotting (IB) (n = 3 with identical results). IgG represents precipitation with Nsp antibody. (D) AR and PXN bind to the PSA and NKX3-1 promoters. Cells were starved overnight; this was followed by medium or DHT (25 nM; 45 minutes) treatment and chromatin IP using antibodies against the indicated proteins (IP). IgG represents Nsp antibody. Values are represented as percentage input (mean ± SEM, n = 3). *P ≤ 0.001; **P ≤ 0.05 relative to medium.

Aritro Sen, et al. J Clin Invest. 2012 July 2;122(7):2469-2481.
6.
Figure 6

Figure 6. PS-PXN regulates c-FOS mRNA expression in the nucleus by complexing with ERK and ELK1 to allow ERK-mediated activation of ELK1. . From: Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation.

(A and B) PS-PXN is essential for EGF-induced phosphorylation of ELK1 and c-FOS mRNA expression. Immunoblot (A) and qPCR (B) analysis in PC3 cells (treated with Nsp or PXN-specific siRNA) showing EGF-induced (20 ng/ml; 30 minutes) (A) phosphorylation of ELK1 and (B) c-FOS mRNA expression. PXN-knockdown cells were also transfected with plasmids expressing WT PXN or PXN lacking the MAPK-targeted serines (S→A). Data are represented as mean ± SEM (n = 3). *P ≤ 0.005 relative to medium. (C) PS-PXN forms a complex with ERK and ELK1 in the nucleus. IP assays were performed in nuclear extracts of LNCaP cells treated with EGF (20 ng/ml, 24 hours) (n = 3 experiments with identical results). Either PXN, PS-PXN, ERK, p-ERK, or p-ELK1 were precipitated, followed by immunoblotting as indicated. (D) ERK and ELK1, but not PXN, bind to the c-FOS promoter in EGF-stimulated PC3 cells. PC3 cells were serum starved overnight, followed by treatment with medium or EGF (20 ng/ml) for 24 hours, before chromatin IP. The occupancy of ERK, p-ELK1, and PXN on c-FOS promoter was examined. Values are presented as percentage input (mean ± SEM, n = 3). *P ≤ 0.005 with respect to medium. Representative immunoblot shows equal amounts of ERK, p-ELK1, and PXN were IP from medium (M) and EGF-treated samples.

Aritro Sen, et al. J Clin Invest. 2012 July 2;122(7):2469-2481.
7.
Figure 4

Figure 4. PXN specifically regulates AR nuclear localization in primary GCs.. From: Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation.

Primary GCs from C57BL/6J mouse ovaries were treated with DHT or estradiol (25 nM) for 30 minutes. (A) Immunofluorescence studies (n = 3 experiments with identical results) showed that, under basal conditions (medium), PXN is predominantly cytoplasmic. Under DHT or estradiol (E2) stimulation, PS-PXN is primarily nuclear. Adjacent Hoechst staining represents the nucleus. (B and C) Immunofluorescence studies (n = 3 experiments with identical results) of primary GCs treated with Nsp or PXN-specific siRNA. PXN ablation prevents DHT-induced nuclear translocation of AR (B), but has no effect on nuclear localization of ERα in medium or estradiol-treated cells (C). (D) Immunofluorescence studies (n = 3 experiments with identical results) in MCF7 breast cancer cells showing siRNA-mediated knockdown of PXN has no effect on ERα nuclear localization in medium or estradiol-treated cells. Original magnification, ×40. (E) PXN is not required for ERE-mediated transcription. Nsp or PXN-specific siRNA treated MCF7 cells were transiently transfected with ERE reporter luciferase construct plus cytomegalovirus–β-gal plasmid. ERE-luciferase activity was normalized to β-gal expression and data represented as fold increase with respect to medium treatment (mean ± SEM, n = 3). *P ≤ 0.001 relative to medium. The blot on the right shows PXN knockdown.

Aritro Sen, et al. J Clin Invest. 2012 July 2;122(7):2469-2481.
8.
Figure 7

Figure 7. PXN knockdown inhibits prostate cancer growth and tumor development, and PXN expression is upregulated in prostate cancer patient samples.. From: Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation.

(AD) PXN knockdown in PC3 cells suppresses tumor formation in xenografts. Nude mice (n = 15/group) were injected subcutaneously with 0.2 ml (1.5 × 106) PC3 cells infected with PXN shRNA or GFP shRNA-negative control. (A) The growth curve of tumors bearing PC3 cell xenografts. (B) The tumor weights of prostate cancer cell xenografts. *P ≤ 0.005 comparing PXN shRNA and GFP shRNA. (C and D) ELK1 phosphorylation and cyclin D1 expression are lower in xenograft tumors lacking PXN. Protein levels of PXN, p-ELK1, and t-ELK1 (C) as well as mRNA levels of cyclin D1 (D) in excised tumors that were treated with GFP or PXN shRNA. (E and F) PXN levels and downstream effectors are upregulated in prostate cancer TMAs. TMA sections were subjected to immunohistochemistry and PXN, AR, cyclin D1, and Ki67 expression measured by scoring intensity and cell number. (E) Data represented as percentage of maximum score for AR, PXN, and cyclin D1 and as percentage of positive cells for Ki67. *P ≤ 0.0001; **P ≤ 0.001 relative to control or BPH samples. All data are represented as mean ± SEM (n = 15). (F) Representative TMA sections showing PXN, cyclin D1, and AR expression in normal/BPH and cancer samples and PS-PXN expression in cancer samples (note that PS-PXN expression was not statistically different in benign versus cancerous prostate).

Aritro Sen, et al. J Clin Invest. 2012 July 2;122(7):2469-2481.

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