The proto-oncoprotein Brx activates estrogen receptor beta by a p38 mitogen-activated protein kinase pathway

Abstract

The estrogen receptors (ERs) are ligand-inducible transcription factors that play key roles in the control of growth and differentiation in reproductive tissues. We showed that the novel Dbl family proto-oncoprotein Brx enhances ligand-dependent activity of ERalpha via a Cdc42-dependent pathway. Brx also significantly enhances ligand-dependent activity of ERbeta. This enhancement is not affected by inhibition of p44/42 mitogen-activated protein kinase (MAPK) activation by PD98059. However, addition of the p38 MAPK inhibitor SB202190 abrogates the enhancement of ERbeta activity by Brx, showing that p38 MAPK activity is required for the enhancement of ERbeta function by Brx. In COS-7 cells, transfection of Brx leads to activation of endogenous p38 MAPK activity. Co-expression of the beta2 isoform of human p38 MAPK and a constitutively active form of the p38 MAPK kinase MKK6 (MKK6-EE) synergistically augments ligand-dependent activity of ERbeta. Our findings suggest that p38 MAPKs may be important regulators of ERbeta activity.

Fig. 1

A, Brx co-expression augments ligand-dependent activation of a G4E1b-luciferase reporter plasmid by GAL4-ERβ. Augmentation of ERβ-mediated activation of G4E1b-luciferase by Brx co-expression is inhibited by the addition of 4-hydroxy tamoxifen. Ishikawa cells were transfected with 1 μg of G4E1b-luciferase and expression vectors for GAL4-ERβ3 (500 ng) and Brx (1 μg) or the respective amounts of empty vectors as control. Estradiol (10 nm, solid bars) or vehicle control (gray bars), and vehicle control or 4-hydroxytamoxifen (40 nm) was added to the cells as indicated. Cells were harvested after 20 h. Luciferase values represent -fold activation (mean) over control (RSV-0 with G4E1b-luciferase) in the absence of ligand, under each condition from three experiments performed in triplicate. Error bars represent standard deviations. B, Brx did not affect receptor expression levels. COS-7 cells were transfected with expression vectors for Brx (lane 1), GAL4-ERβ (lane 2), or both (lane 3). Brx and GAL4-ERβ were detected in lysates of transfected cells by Western blotting. Plasmid amounts were held constant by addition of empty expression vectors. C, Brx does not augment the activity of a chimeric GAL4-VP16 transcription factor. Ishikawa cells were transfected with 1 μg of G4E1b-luciferase reporter plasmid and expression vectors for GAL4-VP16 (0, 400, 800, or 1200 ng, as indicated) and Brx (1 μg, gray bars) or the same amounts of empty expression vectors (solid bars). Cells were harvested and assayed for luciferase activity after 20 h. Luciferase values represent means of-fold activation (with activation given by 400 ng of GAL4-VP16 arbitrarily assigned a value of 1.0) from two experiments performed in duplicate. Error bars represent standard deviations.

Fig. 2

A, augmentation of ERβ activity by Brx is not affected by the inhibition of mitogen-activated protein kinase (MAPK) activity. The two panels show activation of (ERE)₂-tk-Luc reporter plasmid by ERβ with co-expression of Brx (left panel) and without (right panel). Addition of PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, did not attenuate the activation of ERβ with or without Brx co-expression. Ishikawa cells were transfected with 1 μg of (ERE)₂-tk-Luc and expression vectors for ERβ (100 ng) and Brx (1 μg) or the respective amounts of empty vectors as control. Estradiol (10 nm, solid bars) or vehicle control (gray bars) and PD98059 (10 μm) (+) or an equal amount of vehicle (−) was added to the cells as indicated. Cells were harvested after 20 h. Luciferase values represent-fold activation (mean) over control (RSV-0 with (ERE)₂-tk Luc in the absence of ligand) from three experiments performed in triplicate. Error bars represent standard deviations. B, PD98059 inhibits MAPK in Ishikawa cells. Activation of a serum response element reporter in Ishikawa cells is attenuated by the addition of PD98059. Ishikawa cells were serum-starved overnight and transfected with 1.0 μg of a c-fos serum response element SRE-tk-luciferase reporter. The two columns show activation of the SRE-tk-Luc reporter by co-transfection of an expression vector encoding MEKE, a constitutively active mutant form of MEK. PD98059 (10 μm) (+) or an equal amount of vehicle (−) was added to the cells. Cells were harvested after 20 h. Luciferase values represent -fold activation compared with reporter activity after transfection of empty expression vector (not shown).

Fig. 3. Activation of ERβ by Brx is dependent on p38 MAPK activity

A, activation of ERβ by Brx is inhibited by the p38 MAPK inhibitor SB202190 (2 μm). Ishikawa cells were transfected with 1 μg of a G4E1b-luciferase reporter plasmid and expression vectors for GAL4-ERβ (100 ng) and Brx (1 μg) or the same amounts of empty expression vectors. Estradiol (10 nm, solid bars) or vehicle control (EtOH, gray bars) and SB202190 or control (SB202474) were added as indicated. Cells were harvested after 20 h. Luciferase values represent -fold activation (mean) over control (RSV0 with G4E1b-luciferase in the absence of ligand) from two experiments performed in duplicate. Error bars represent standard deviations. B, the p38 MAPK inhibitor SB202190 does not inhibit the activity of GAL4-VP16. Ishikawa cells were transfected with 1 μg of a G4E1b-luciferase reporter plasmid and 400 ng of an expression vector for GAL4-VP16. SB202190 (3 μm) was added as indicated. Luciferase values represent -fold activation (with activation given by 400 ng of GAL4-VP16 arbitrarily assigned a value of 1.0) from two experiments performed in duplicate. Error bars represent standard deviations.

Fig. 4

A and B, MKK6-EE and p38β2 synergistically activate ERβ (A) and Elk-1 (B) in Ishikawa. Cells were transfected with 1 μg of G4E1b-luciferase reporter and 500 ng of expression vectors for GAL4-Elk-1 or GAL4-ERβ, MKK6-EE, and p38β2 or the same amounts of empty expression vectors, as indicated. Cells were harvested for assay after 20 h. Values represent -fold activation (mean) over control (G4E1b-luciferase with empty vectors) from two experiments performed in duplicate. C, Brx activates endogenous p38 MAPK in COS-7 cells. Cells were transfected with expression vectors for Brx or MKK6-EE or with empty vector (RSV-0). Transfected cells were serum-starved overnight, and active p38 MAPK was detected in lysates of transfected cells by Western blotting. Positive and negative control lanes contained C-6 glioma cell extracts prepared with or without anisomycin treatment, respectively. D, ERβ is efficiently phosphorylated by p38β2. Active recombinant human p38β2 MAPK was incubated in vitro with [³²P]ATP and substrate proteins for 15 min at 30 °C. Labeled proteins were resolved by electrophoresis and detected by autoradiography. Phosphorylation substrates were no protein (lane 1), recombinant GST-ATF-2 (lane 2), BSA (lane 3), and recombinant ERβ (lane 4).

Fig. 5

Brx augments ligand-dependent activity of ERβ via a p38 MAPK pathway.