FHL2, a novel tissue-specific coactivator of the androgen receptor

Abstract

The control of target gene expression by nuclear receptors requires the recruitment of multiple cofactors. However, the exact mechanisms by which nuclear receptor-cofactor interactions result in tissue-specific gene regulation are unclear. Here we characterize a novel tissue-specific coactivator for the androgen receptor (AR), which is identical to a previously reported protein FHL2/DRAL with unknown function. In the adult, FHL2 is expressed in the myocardium of the heart and in the epithelial cells of the prostate, where it colocalizes with the AR in the nucleus. FHL2 contains a strong, autonomous transactivation function and binds specifically to the AR in vitro and in vivo. In an agonist- and AF-2-dependent manner FHL2 selectively increases the transcriptional activity of the AR, but not that of any other nuclear receptor. In addition, the transcription of the prostate-specific AR target gene probasin is coactivated by FHL2. Taken together, our data demonstrate that FHL2 is the first LIM-only coactivator of the AR with a unique tissue-specific expression pattern.

Fig. 1. FHL2 interacts with the AR in yeast in an agonist-dependent manner. (A) Schematic representation of the AGA bait protein and the FHL2 protein. (B) β-galactosidase activity in yeast expressing AGA and Gal4AD–FHL2 in the presence or absence of the agonist R1881 or the antagonist CPA (bar 2). Bars 1 and 3 show the negative and bar 4 shows the positive controls, respectively.

Fig. 2. Analysis of FHL2 expression. (A) FHL2 is specifically expressed in the heart. Northern blots of human tissues (fetal, adult and adult muscle) and adult mouse tissues were probed with FHL2. β–actin was used as an internal control. (B) FHL2 is mainly expressed in the heart ventricles. Northern blot analysis of mRNA of left and right ventricles (LV, RV) and left and right atria (LA, RA) of an explanted human heart. GAPDH was used as a control. (C) FHL2 and AR protein expression in human heart. Extract (200 μg) from a left ventricle was analysed in a Western blot. Controls are shown in lane 2 (10 μg of 293 cell extract transfected with human AR) and lane 3 (100 ng of purified His-tagged FHL2). The upper panel was decorated with an α–AR antibody, the lower panel with an α–FHL2-specific antibody. (D) Immunohistochemical staining of FHL2 in cardiac muscle cells (a and b) or FHL2 and AR in prostate epithelium (c–f). Controls are shown in b, d and f. In the heart, FHL2 antibodies stained specifically myocardial fibres (my), but not vascular smooth muscle cells (sm) (a). Two hematoxilin–eosin stained arteriolae (at) shown in (a) entirely lack FHL2 immunoreactivity. Both cytoplasmic and nuclear FHL2 immunoreactivity was detected in the secretory epithelium of the prostate (c). AR immunoreactivity was confined to the nucleus of the secretory epithelium (e). Abbreviations: at, arteriola; br, brain; co, colon; ep, epithelial cells; ht, heart; kd, kidney; li, liver; lm, lumen; lu, lung; my, myofibres; pan, pancreas; pl, placenta; pr, prostate; skm, skeletal muscle; si, small intestine; sm, smooth muscle; sp, spleen; st, stomach; str, stroma; te, testis; ut, uterus.

Fig. 2. Analysis of FHL2 expression. (A) FHL2 is specifically expressed in the heart. Northern blots of human tissues (fetal, adult and adult muscle) and adult mouse tissues were probed with FHL2. β–actin was used as an internal control. (B) FHL2 is mainly expressed in the heart ventricles. Northern blot analysis of mRNA of left and right ventricles (LV, RV) and left and right atria (LA, RA) of an explanted human heart. GAPDH was used as a control. (C) FHL2 and AR protein expression in human heart. Extract (200 μg) from a left ventricle was analysed in a Western blot. Controls are shown in lane 2 (10 μg of 293 cell extract transfected with human AR) and lane 3 (100 ng of purified His-tagged FHL2). The upper panel was decorated with an α–AR antibody, the lower panel with an α–FHL2-specific antibody. (D) Immunohistochemical staining of FHL2 in cardiac muscle cells (a and b) or FHL2 and AR in prostate epithelium (c–f). Controls are shown in b, d and f. In the heart, FHL2 antibodies stained specifically myocardial fibres (my), but not vascular smooth muscle cells (sm) (a). Two hematoxilin–eosin stained arteriolae (at) shown in (a) entirely lack FHL2 immunoreactivity. Both cytoplasmic and nuclear FHL2 immunoreactivity was detected in the secretory epithelium of the prostate (c). AR immunoreactivity was confined to the nucleus of the secretory epithelium (e). Abbreviations: at, arteriola; br, brain; co, colon; ep, epithelial cells; ht, heart; kd, kidney; li, liver; lm, lumen; lu, lung; my, myofibres; pan, pancreas; pl, placenta; pr, prostate; skm, skeletal muscle; si, small intestine; sm, smooth muscle; sp, spleen; st, stomach; str, stroma; te, testis; ut, uterus.

Fig. 3. FHL2 interacts with the AR in vitro and in vivo. (A) Selective interaction of FHL2 with the AR. GST pulldown assays were performed with in vitro translated, labelled AR, PR, GR or MR in the presence of their cognate ligands and GST–FHL2 fusion protein. GST protein was used as a control. (B) AR coimmunoprecipitates with FHL2 in the presence of the natural agonist DHT. Nuclear extracts of 293 cells transfected with AR and Flag-FHL2 were immunoprecipitated with α–Flag antibody. Ten percent of the extract used for immunoprecipitation was loaded as input in lanes 1 and 3. The immunoprecipitate (IP) is loaded in lanes 2 and 4. Western blots were either decorated with an α–Flag- or an α–AR-specific antibody.

Fig. 4. AR associates with FHL2 in an AF-2-dependent manner. (A) GST pulldown assays were performed using GST–FHL2 fusion protein and in vitro translated, labelled AR or AR mutants in the presence of the agonist R1881. (B) Interaction between the AR and either GST, GST–FHL2, GST–FHL2(1–162) or GST–FHL2(163–279) fusion proteins in the presence of the agonist R1881. Numbers above the scheme indicate the amino acid position.

Fig. 4. AR associates with FHL2 in an AF-2-dependent manner. (A) GST pulldown assays were performed using GST–FHL2 fusion protein and in vitro translated, labelled AR or AR mutants in the presence of the agonist R1881. (B) Interaction between the AR and either GST, GST–FHL2, GST–FHL2(1–162) or GST–FHL2(163–279) fusion proteins in the presence of the agonist R1881. Numbers above the scheme indicate the amino acid position.

Fig. 5. FHL2 contains an autonomous transactivation function. The Gal–FHL2 fusion protein transactivates the G5E1b-LUC reporter gene in different cell lines (CV-1, 293 and HL-1). The coactivators Gal–TIF2.1 and Gal–SRC1a were used as controls.

Fig. 6. FHL2 is a specific coactivator of the AR. (A) FHL2 coactivates the AR in an agonist- and AF-2-dependent manner. Expression plasmids coding for AR, ARΔH12, PR, GR or MR were cotransfected with the MMTV-LUC reporter with or without FHL2 in 293 cells. (B) FHL2, TIF2, SRC1e or SRC1a coactivate the AR to a similar extent in 293 cells. (C) FHL2 functions as a coactivator of the AR-specific probasin-LUC reporter (PB-LUC). AR and FHL2 were cotransfected as indicated in the presence of two different AR agonists (R1881, DHT) or the antagonist CPA in CV-1 cells.

Fig. 6. FHL2 is a specific coactivator of the AR. (A) FHL2 coactivates the AR in an agonist- and AF-2-dependent manner. Expression plasmids coding for AR, ARΔH12, PR, GR or MR were cotransfected with the MMTV-LUC reporter with or without FHL2 in 293 cells. (B) FHL2, TIF2, SRC1e or SRC1a coactivate the AR to a similar extent in 293 cells. (C) FHL2 functions as a coactivator of the AR-specific probasin-LUC reporter (PB-LUC). AR and FHL2 were cotransfected as indicated in the presence of two different AR agonists (R1881, DHT) or the antagonist CPA in CV-1 cells.

Fig. 6. FHL2 is a specific coactivator of the AR. (A) FHL2 coactivates the AR in an agonist- and AF-2-dependent manner. Expression plasmids coding for AR, ARΔH12, PR, GR or MR were cotransfected with the MMTV-LUC reporter with or without FHL2 in 293 cells. (B) FHL2, TIF2, SRC1e or SRC1a coactivate the AR to a similar extent in 293 cells. (C) FHL2 functions as a coactivator of the AR-specific probasin-LUC reporter (PB-LUC). AR and FHL2 were cotransfected as indicated in the presence of two different AR agonists (R1881, DHT) or the antagonist CPA in CV-1 cells.