Glucocorticoids induce human glycoprotein hormone alpha-subunit gene expression in the gonadotrope

Abstract

The human glycoprotein hormone alpha-subunit (alphaGSU) gene is transcriptionally regulated by glucocorticoids in a cell type-specific fashion. In direct contrast to repression of alphaGSU by glucocorticoids in placenta, glucocorticoid receptor (GR) modulation in the pituitary is little understood. We show that glucocorticoids stimulate the alphaGSU promoter in immortalized pituitary gonadotrope-derived LbetaT2 cells, whereas estrogens, androgens, and progestins have no significant effect. Moreover, GR acts in a dose-dependent manner at physiological concentrations of glucocorticoids. Transient transfection of GR with dexamethasone (Dex) treatment further stimulates the alphaGSU promoter, but this induction is severely diminished using a receptor mutated in the DNA-binding domain. Truncation and cis mutations demonstrate that glucocorticoid response element 2 (GRE2) and cAMP-response element 2 (CRE2) within -168 bp of the human alphaGSU promoter are critical for induction. Moreover, dominant-negative CRE-binding protein markedly inhibits basal but also Dex induction of alphaGSU promoter activity. Additionally, GR specifically binds to GRE2 in the human alphaGSU promoter in vitro and to the 5' region of the endogenous mouse alphaGSU gene in vivo. Furthermore, overexpression of the homeobox factor, Distal-less 3 that regulates this gene in placental cells through a site partially overlapping GRE2, blocks Dex induction of alphaGSU in gonadotrope cells, indicating that placenta-specific expression of Dlx3 may interfere with GR, resulting in repression in placental cells vs. induction in gonadotrope cells. These results demonstrate the stimulatory role played by glucocorticoids in alphaGSU gene expression in the pituitary gonadotrope, in contrast to repression in placental cells, and highlight the tissue-specific nature of steroid hormone action.

Figure 1

A, Glucocorticoids specifically induce human αGSU gene expression in LβT2 gonadotrope cells. LβT2 cells were transiently cotransfected with the 1.8-kb αGSU-luc reporter gene and with 200 ng of the respective receptor expression vectors indicated on the graph. The cells were serum starved overnight and then treated with 100 nm R1881 (synthetic androgen), R5020 (synthetic progesterone), Dex (synthetic glucocorticoid), or 17β-estradiol for 24 h. Luciferase activity was assayed and normalized to β-galactosidase activity and shown relative to the empty reporter vector. B, The 1.8-kb αGSUluc reporter gene was transiently transfected into LβT2 cells without (endogenous GR) or with (exogenous GR) the GR expression vector. The cells were serum starved overnight and then treated for 24 h with 100 nm corticosterone, a natural glucocorticoid, or Dex, a synthetic glucocorticoid. C, The 1.8-kb αGSUluc reporter gene was transiently transfected into LβT2 cells without (endogenous GR) or with (exogenous GR) the GR expression vector. The cells were serum starved overnight and then treated for 24 h with the indicated Dex concentrations (100 pm to 1 μm). Data represent the mean ± sem of at least three experiments performed in triplicate and are presented as fold induction relative to the vehicle control. *, Dex induction is significantly different from the vehicle-treated control, Student’s t test, P < 0.05.

Figure 2

A, GR binds to the αGSU promoter in vivo. ChIP was performed using the cross-linked protein/chromatin from LβT2 cells (treated with vehicle or 100 nm Dex) using antibodies directed against GR or nonspecific IgG as a negative control: upper panel, PCR primers (Table 1) encompassing the proximal promoter of αGSU (−246 to −54) were used to detect precipitation of genomic DNA; lower panel, PCR primers encompassing the downstream αGSU-coding region (+932 to +1169) were used as a control for specificity. PCR amplification was performed on 0.2% of chromatin input. B, DNA binding by GR is required to facilitate human αGSU gene expression. The 1.8-kb αGSUluc reporter gene was transiently cotransfected into LβT2 cells with the wild-type GR, GR-DBD mutant expression vector, or GRdim4 mutant expression vector as indicated. The cells were serum starved overnight and then treated with 100 nm Dex for 24 h. Data represent the mean ± sem of at least three experiments performed in triplicate and are presented as fold induction relative to the vehicle control. The GR-DBD mutant response was significantly different from the GR response. Levels of induction not connected by the same letter are significantly different, using one-way ANOVA followed by Tukey’s post hoc test, P < 0.05.

Figure 3

Mapping the regions involved in induction of human αGSU by GR. The 1.8-kb αGSUluc, −845αGSUluc, −668αGSUluc, −391αGSUluc, −224αGSUluc, −168αGSUluc, −116αGSUluc, or −90αGSUluc reporter genes were transiently transfected into LβT2 cells along with the GR expression vector. The cells were serum starved overnight and then treated for 24 h with 100 nm Dex. Data represent the mean ± sem of at least three experiments performed in triplicate and are presented as fold induction relative to the control. The response with −1.8 kb and with the truncations −846, −668, −391, −224, and −168 bp are significantly different from the response with −116 and −90 truncations. Levels not connected by the same letter are significantly different, using one-way ANOVA followed by Tukey’s post hoc test, P < 0.05.

Figure 4

The cis elements involved in glucocorticoid regulation of human αGSU. A, Schematic representation of the 5′ proximal promoter of the human αGSU gene. The 220 bp of 5′ flanking sequences of the human αGSU promoter are shown encompassing the steroidogenic factor-1 (SF-1) binding site, the CAAT element, the CREs (CRE1 and CRE2), the JRE that is occupied in placental cells, the CAAT box, the pituitary homeobox (Ptx) element, and the TATA element. Overlapping with some of these elements are the putative GREs (GREs 1, 2, and 3). B, The sequences of three GREs and a GRE consensus are shown. Boxes indicate conservation of the key G and C residues with the consensus sequence. C, GRE2 and CRE2 have a significant role in the induction of human αGSU by GR. The wild-type 1.8-kb αGSUluc reporter gene or one of the three GRE/CRE cis mutants was transiently transfected into LβT2 along with GR expression vector. The cells were serum starved overnight and then treated for 24 h with 100 nm Dex. Data represent the mean ± sem of at least three experiments performed in triplicate and are presented as fold induction relative to the vehicle control. Induction with the GRE2 mutant, CRE2 mutant, and/or double GRE2/CRE2 mutant are significantly different from induction with wild-type GR. Levels not connected by the same letter are significantly different, using one-way ANOVA followed by Tukey’s post hoc test, P < 0.05.

Figure 5

DN-CREB decreases GR-induced expression of the human αGSU promoter. DN-CREB was overexpressed in LβT2 cells that had been transiently transfected with 1.8-kb αGSUluc and the GR expression vector. The cells were serum starved overnight and then treated for 24 h with 100 nm Dex. Data represent the mean ± sem of at least three experiments performed in triplicate. A, Bar graph depicting luc values normalized to β-galactosidase values; B, bar graph presented as fold induction relative to the control for empty vector vs. DN-CREB to show decrease in fold induction by Dex.

Figure 6

GR binds to the −111 GRE2 of the human αGSU promoter. Whole-cell extracts containing overexpressed GR from baculovirus-infected insect cells were incubated with ³²P-labeled oligonucleotides representing the three GREs as indicated (see Table 1) and compared with a positive control probe from the mouse FSHβ gene that has been previously shown to bind GR. A, The N499 GR rabbit polyclonal antibody was used to supershift GR, and nonspecific rabbit IgG was used as a negative control for binding; B, 1000-fold excess of the relevant oligonucleotide probes, either wild-type or mutant (Table 1), was used for competition.

Figure 7

Overexpression of Dlx3 inhibits GR-induced human αGSU promoter in LβT2 cells. A, Sequence overlap of the JRE and the GRE2. Sequence is shown from −124 to −97 of the human αGSU gene. The rounded box indicates the sequence of CRE2, squared box indicates the JRE sequence, and the oval indicates the full GRE2 with both 6-bp half-sites and the 3-bp spacer. B, Either pCI empty vector or Dlx3 expression vector (100 ng) was overexpressed in LβT2 cells that had been transiently transfected with 1.8-kb αGSUluc and the GR expression vector. The cells were serum starved overnight and then treated for 24 h with 100 nm Dex. Data represent the mean ± sem of at least three experiments performed in triplicate and are presented as fold induction relative to the vehicle control.

Figure 8

GnRH and activin modulate Dex-induced human αGSU gene expression synergistically. The 1.8-kb αGSUluc reporter gene was transiently transfected into LβT2 cells with or without overexpression of GR (200 ng). Cells were serum starved overnight and then treated for 24 h with vehicle, 100 nm Dex, 10 nm GnRH, 10 ng/ml activin, 100 nm Dex with 10 nm GnRH, or 100 nm Dex with 10 ng/ml activin. Data represent the mean ± sem of at least three experiments performed in triplicate and are presented as fold induction relative to the control. Daggers (†) represent synergy between hormone treatments by two-way ANOVA, and asterisks (*) represent significant induction by hormone by one-way ANOVA, P < 0.05.