Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action

Abstract

In NIH3T3 cells, 0.001 nM of the synthetic androgen R1881 induces and stimulates association of androgen receptor (AR) with Src and phosphatidylinositol 3-kinase (Pl3-kinase), respectively, thereby triggering S-phase entry. 10 nM R1881 stimulates Rac activity and membrane ruffling in the absence of the receptor-Src-PI3-kinase complex assembly. The antiandrogen Casodex and specific inhibitors of Src and PI3-kinase prevent both hormonal effects, DNA synthesis and cytoskeletal changes. Neither low nor high R1881 concentration allows receptor nuclear translocation and receptor-dependent transcriptional activity in fibroblasts, although they harbor the classical murine AR. The very low amount of AR in NIH3T3 cells (7% of that present in LNCaP cells) activates the signaling pathways, but apparently is not sufficient to stimulate gene transcription. This view is supported by the appearance of receptor nuclear translocation as well as receptor-mediated transcriptional activity after overexpression of AR in fibroblasts. In addition, AR-negative Cos cells transiently transfected with a very low amount of hAR cDNA respond to low and high R1881 concentrations with signaling activation. Interestingly, they do not show significant transcriptional activation under the same experimental conditions. Fibroblasts are the first example of cells that respond to steroid hormones with activation of signaling pathways in the absence of endogenous receptor transcriptional activity. The data reported also show that hormone concentration can be crucial in determining the type of cell responsiveness.

Figure 1.

A low androgen concentration specifically stimulates the S-phase entry of NIH3T3 cells. (A) Quiescent fibroblasts on coverslips were left untreated (○) or treated (•) for the indicated times with 0.001 nM R1881. After in vivo labeling with 100 μM BrdU, the cells were fixed, permeabilized, and stained for BrdU incorporation. DNA synthesis was calculated by the formula: percentage of BrdU-positive cells = (number of BrdU-positive cells/number of total cells) × 100. (B) The cells were left untreated or treated with the indicated compounds, labeled with BrdU, and were fixed and stained after 18 h. DNA synthesis was calculated as in A. Different experiments were performed and multiple coverslips were analyzed. The means and SEM are shown. (C) Lysates from quiescent NIH3T3, LNCaP, MCF-7, and T47D cells were immunoblotted with the antibodies against the indicated receptors. Endogenous AR in NIH3T3 cells was revealed by two different antibodies (either C-19 or N-20). The optical density/mm² measured by densitometric analysis of blot with C-19 anti-AR antibody was 6.12 and 0.47 for AR expressed in LNCaP and NIH3T3 cells, respectively. In the lowest section of C, lysates from primary fibroblasts (mouse fibroblasts, MF, or mouse embryo fibroblasts, MEF, both at second passage) were immunoblotted with the C-19 anti-AR antibody.

Figure 2.

Requirement for the Src–ERK and PI3-kinase–Akt pathways in the androgen-stimulated S-phase entry is correlated to AR–Src–PI3-kinase association in NIH3T3 cells. Fibroblasts on coverslips were either untransfected or transfected with the plasmids expressing the indicated proteins. (A) Transfected cells were made quiescent, and then left unstimulated or stimulated with 0.001 nM R1881. BrdU was added and coverslips were analyzed for BrdU incorporation. In transfected cells, BrdU incorporation was calculated by the formula: percentage of BrdU-positive cells = (number of transfected BrdU-positive cells/number of transfected cells) × 100 and compared with BrdU incorporation of untransfected cells from the same coverslips. For each plasmid, data are derived from at least 200 scored cells. The results of more than two independent experiments have been averaged; means and SEM are shown. The statistical significance of these results was also evaluated by paired t test. P values were <0.001 for cells transfected with either Src K⁻, Δp85α, Akt K⁻, or A221–MEK-1. The difference in BrdU incorporation between the cells transfected with Src K⁻ and those transfected with Src wt was significant (P < 0.005). Also significant (P < 0.001) was the difference in BrdU incorporation between the cells transfected with Δp85α and those transfected with p85α wt. No significance was attributed to the difference in BrdU incorporation between the cells transfected with either Src wt or p85α wt and nontransfected cells stimulated with the androgen R1881. (B) Representative images of one of the experiments in A. Fluorescence in the left panels is from reactivity with either the anti-Src mAb (top) or the anti–MEK-1 Ab (bottom). Arrows and arrowheads mark the cells transfected with either Src K⁻ or A221–MEK-1 expressing plasmids. The central panels show staining with anti-BrdU antibody. Hoechst 33258 nuclear staining is presented in the right panels. Quiescent NIH3T3 cells were either left untreated or treated for 2 min with the indicated compounds. (C) Lysate proteins were immunoprecipitated with either control antibody (ctrl) or the 327 anti-Src monoclonal antibody (anti-Src mAb). (D) Lysate proteins were immunoprecipitated with either control antibody (ctrl) or rabbit polyclonal anti-p85 antibody (anti-p85 Ab). (C and D) Immunocomplexes were analyzed by immunoblot with antibodies against the indicated proteins. (D) By an NIH 1.61 image program, a 38% increase of AR/p85 association was detected on 0.001 nM R1881 stimulation of cells. This experiment was reproduced with similar findings. (E) Lysate proteins from NIH3T3 cells challenged for 2 min with the indicated compounds were immunoblotted with the C-19 anti-AR antibody.

Figure 2.

Requirement for the Src–ERK and PI3-kinase–Akt pathways in the androgen-stimulated S-phase entry is correlated to AR–Src–PI3-kinase association in NIH3T3 cells. Fibroblasts on coverslips were either untransfected or transfected with the plasmids expressing the indicated proteins. (A) Transfected cells were made quiescent, and then left unstimulated or stimulated with 0.001 nM R1881. BrdU was added and coverslips were analyzed for BrdU incorporation. In transfected cells, BrdU incorporation was calculated by the formula: percentage of BrdU-positive cells = (number of transfected BrdU-positive cells/number of transfected cells) × 100 and compared with BrdU incorporation of untransfected cells from the same coverslips. For each plasmid, data are derived from at least 200 scored cells. The results of more than two independent experiments have been averaged; means and SEM are shown. The statistical significance of these results was also evaluated by paired t test. P values were <0.001 for cells transfected with either Src K⁻, Δp85α, Akt K⁻, or A221–MEK-1. The difference in BrdU incorporation between the cells transfected with Src K⁻ and those transfected with Src wt was significant (P < 0.005). Also significant (P < 0.001) was the difference in BrdU incorporation between the cells transfected with Δp85α and those transfected with p85α wt. No significance was attributed to the difference in BrdU incorporation between the cells transfected with either Src wt or p85α wt and nontransfected cells stimulated with the androgen R1881. (B) Representative images of one of the experiments in A. Fluorescence in the left panels is from reactivity with either the anti-Src mAb (top) or the anti–MEK-1 Ab (bottom). Arrows and arrowheads mark the cells transfected with either Src K⁻ or A221–MEK-1 expressing plasmids. The central panels show staining with anti-BrdU antibody. Hoechst 33258 nuclear staining is presented in the right panels. Quiescent NIH3T3 cells were either left untreated or treated for 2 min with the indicated compounds. (C) Lysate proteins were immunoprecipitated with either control antibody (ctrl) or the 327 anti-Src monoclonal antibody (anti-Src mAb). (D) Lysate proteins were immunoprecipitated with either control antibody (ctrl) or rabbit polyclonal anti-p85 antibody (anti-p85 Ab). (C and D) Immunocomplexes were analyzed by immunoblot with antibodies against the indicated proteins. (D) By an NIH 1.61 image program, a 38% increase of AR/p85 association was detected on 0.001 nM R1881 stimulation of cells. This experiment was reproduced with similar findings. (E) Lysate proteins from NIH3T3 cells challenged for 2 min with the indicated compounds were immunoblotted with the C-19 anti-AR antibody.

Figure 3.

A high androgen concentration induces membrane ruffling mediated by PI3-kinase and Src. Serum-starved NIH3T3 cells on coverslips were left untreated or treated for 20 min with the indicated compounds. The chemical inhibitors PP2 and LY294002 (Qbiogene) at 5 and 10 μM, respectively, were added 10 min before the hormonal stimulation. A 1,000-fold excess of Casodex was added 5 min before the hormonal stimulation. Cells stained with Texas red–phalloidin were visualized by a fluorescent microscope. Micrographs are representative of three independent experiments.

Figure 4.

PI3-kinase and Src mediate Rac activation induced by high androgen concentration. (A) Serum-starved NIH3T3 cells were left untreated or treated for the indicated times with either 0.001 or 10 nM R1881. (B) The cells were left untreated or treated for 2 min with 10 nM R1881 in the absence or presence of PP2 or LY294002 (at 5 and 10 mM, respectively). The chemical inhibitors were added 10 min before the hormonal stimulation. Lysate proteins were submitted to a Rac pull-down assay, and the eluted proteins were immunoblotted with the anti-Rac antibody.

Figure 5.

Immunostaining of AR in NIH3T3 and LNCaP cells. Quiescent NIH3T3 and LNCaP cells on coverslips were untreated or treated for 30 min with 10 nM R1881. Cells were permeabilized as described in Materials and methods, and AR was visualized by immunofluorescence using the indicated antibodies. (A–B₁) Regardless of the antibody used (either C-19 or N-20), AR is localized in the extranuclear compartment of untreated (A and B) or androgen-treated NIH3T3 cells (A₁ and B₁). Exposure time in NIH3T3 cells was enhanced to better visualize cytoplasm AR. (C and C₁) AR is localized in both the nuclear and extranuclear compartment of untreated LNCaP cells (C). 10 nM R1881 induced nuclear import of AR in LNCaP cells (C₁). The cells that fall into the category of predominantly nuclear staining are marked with arrows. Immunostaining of AR from at least 200 scored LNCaP cells was also monitored, and the data were expressed as a percentage of cells that fall into the category of predominantly nuclear staining. In LNCaP cells, this value was ∼20% and shifted to ∼65% when they were stimulated for 30 min with 10 nM R1881. (D and D₁) Quiescent NIH3T3 cells on coverslips were fixed and permeabilized as described in Materials and methods. AR was visualized by immunofluorescence using the C-19 anti-AR antibody alone (D) or in the presence (D₁) of a 100-fold excess of a competing peptide (AR/C-19 P; Santa Cruz Biotechnology, Inc.).

Figure 5.

Immunostaining of AR in NIH3T3 and LNCaP cells. Quiescent NIH3T3 and LNCaP cells on coverslips were untreated or treated for 30 min with 10 nM R1881. Cells were permeabilized as described in Materials and methods, and AR was visualized by immunofluorescence using the indicated antibodies. (A–B₁) Regardless of the antibody used (either C-19 or N-20), AR is localized in the extranuclear compartment of untreated (A and B) or androgen-treated NIH3T3 cells (A₁ and B₁). Exposure time in NIH3T3 cells was enhanced to better visualize cytoplasm AR. (C and C₁) AR is localized in both the nuclear and extranuclear compartment of untreated LNCaP cells (C). 10 nM R1881 induced nuclear import of AR in LNCaP cells (C₁). The cells that fall into the category of predominantly nuclear staining are marked with arrows. Immunostaining of AR from at least 200 scored LNCaP cells was also monitored, and the data were expressed as a percentage of cells that fall into the category of predominantly nuclear staining. In LNCaP cells, this value was ∼20% and shifted to ∼65% when they were stimulated for 30 min with 10 nM R1881. (D and D₁) Quiescent NIH3T3 cells on coverslips were fixed and permeabilized as described in Materials and methods. AR was visualized by immunofluorescence using the C-19 anti-AR antibody alone (D) or in the presence (D₁) of a 100-fold excess of a competing peptide (AR/C-19 P; Santa Cruz Biotechnology, Inc.).

Figure 6.

Androgen-stimulated gene transcription in NIH3T3 cells before and after overexpression of hAR and hAR intracellular localization. NIH3T3 cells were transfected with either ARE 3424 (A) or 3416 (B) constructs with or without hAR-expressing plasmid. Cells were left unstimulated or stimulated for 18 h with the indicated concentrations of the androgen R1881. The luciferase activity was assayed, normalized using β-gal as an internal control, and expressed as fold induction. The same lysates were used for Western blot analysis with C-19 anti-AR Ab (C). (D and D₂) NIH3T3 cells were transfected with hAR-expressing plasmid, and then made quiescent. Cells were left unstimulated (D) or stimulated for 1 h with either 0.001 nM (D₁) or 10 nM R1881 (D₂). Fixed cells on coverslips were permeabilized as described in Materials and methods, and hAR was visualized by immunofluorescence using the rabbit polyclonal anti-AR (N-20) antibody.

Figure 7.

Molecular analysis of AR-RNA expressed in NIH3T3 and LNCaP cells. Total RNA and poly(A)⁺ RNA was obtained from growing LNCaP or NIH3T3 cells, respectively. cDNA was synthesized and amplified by PRC using specific primers for the NH₂ terminus (A), DNA binding (B), and LBD (C) of mouse AR. The PCR products were submitted to agarose gel. PCR products generated without reverse transcriptase from poly(A)⁺ RNA of NIH3T3 cells were submitted to the same analysis, as a control (ctrl).

Figure 8.

In Cos cells expressing very low amount of hAR, stimulation by 0.001 nM R1881 triggers the Src kinase activity but not gene transcription. Cos cells were transfected with the ARE-3416-Luc reporter gene in the absence or presence of the indicated amounts of hAR cDNA. (A) Cells were left unstimulated or stimulated for 18 h with the indicated concentrations of the androgen R1881. The luciferase activity was assayed, normalized using β-gal as an internal control, and expressed as fold induction. The same lysates were used for Western blot analysis with the C-19 anti-AR Ab (A, inset). (B) Quiescent cells were left unstimulated or stimulated for 2 min with the indicated concentrations of R1881. Lysates were either immunoblotted with the C-19 anti-AR Ab (top) or immunoprecipitated with anti-Src mAb. Immunoprecipitates were either blotted with anti-Src mAb (middle) or assayed for Src kinase activity using enolase as a substrate (bottom).