TTF-1 action on the transcriptional regulation of cyclooxygenase-2 gene in the rat brain

Abstract

We have recently found that thyroid transcription factor-1 (TTF-1), a homeodomain-containing transcription factor, is postnatally expressed in discrete areas of the hypothalamus and closely involved in neuroendocrine functions. We now report that transcription of cyclooxygenase-2 (COX-2), the rate limiting enzyme in prostaglandin biosynthesis, was inhibited by TTF-1. Double immunohistochemistry demonstrated that TTF-1 was expressed in the astrocytes and endothelial cells of blood vessel in the hypothalamus. Promoter assays and electrophoretic mobility shift assays showed that TTF-1 inhibited COX-2 transcription by binding to specific binding domains in the COX-2 promoter. Furthermore, blocking TTF-1 synthesis by intracerebroventricular injection of an antisense oligomer induced an increase of COX-2 synthesis in non-neuronal cells of the rat hypothalamus, and resulted in animals' hyperthermia. These results suggest that TTF-1 is physiologically involved in the control of thermogenesis by regulating COX-2 transcription in the brain.

Figure 1. Expression of TTF-1 in non-neuronal cells of the POA.

Double IHC was performed on brain sections derived from 2-month-old male rats. TTF-1 protein (red, A, C, D and F) was detected with a monoclonal antibody. Endothelial cells of blood vessels (green, B and C) were identified using antibody against vWF, an endothelial cell-specific marker. Astroglial cells (green, E and F) were determined using GFAP antibody. A, red fluorescence signals representing TTF-1 immuno-positive cells in the POA. B, green signals revealing vWF-ir in the endothelial cells of blood vessel on the same section with A. C, merged image of A and B showing some cells co-expressing TTF-1 and vWF (indicated as arrows). Inset highlighting higher magnification image of TTF-1 colocalized with vWF. D and E, TTF-1 (red, D) and GFAP (green, E) immunoreactivities in cells of the POA. F, merged image of D and E revealing some cells with colocalization of TTF-1 and GFAP (arrow). Higher magnification image in inset highlighting colocalization of TTF-1 and GFAP. Scale bar = 50 µm.

Figure 2. Effect of TTF-1 synthesis blockade on the COX-2 expression in the hypothalamus.

The AS TTF-1 ODN or SCR ODN was injected into the lateral ventricle of 2-month-old male rats. One day after the injection, the hypothalamus was collected for western blot and real-time PCR analyses, or was examined by IHC. A, western blots showing a decrease in TTF-1 protein level caused by administration of the AS TTF-1 ODN (AS) compared with SCR ODN. B and C, AS TTF-1 ODN significantly increased both COX-2 mRNA (B) and protein (C) levels determined by real-time PCR analysis and western blotting. D, real-time PCR analysis showing increased POMC mRNA by the AS ODN (n = 6). **, p<0.01 versus SCR. E, the POA section showing a decrease of TTF-1-ir (red) and an increase of COX-2-ir (green) caused by the AS TTF-1 ODN. F, higher magnification images showing co-localization of TTF-1-ir (red) and COX-2-ir (green) in the AS ODN injected rat POA. Note that COX-2-ir appears in some cells expressing TTF-1-ir (merged, arrow). G, higher magnification images showing COX-2-ir (green) in the AS ODN injected rat POA. Notice that COX-2 is present only in cells with an absence of the NeuN-ir (red). Scale bar = 100 µm.

Figure 3. Regulation of COX-2 transcription by TTF-1.

Luciferase reporter constructs (pGL2) containing 5′-flanking region of the rat COX-2 gene were cotransfected into C6 glioma and B35 neuroblastoma cells with an expression vector carrying the rat TTF-1-coding region (TTF-1-pcDNA) at the final concentrations indicated. The cells were harvested for luciferase and β-galactosidase assays 24 h after transfection. A, dose-dependent inhibition of the COX-2 promoter activity by increasing concentrations of TTF-1 in C6 cells. Each value represents mean±SEM (n = 6). ***, p<0.001 versus 0 ng. B, absence of TTF-1 effect on COX-2 promoter in B35 cells. C and D, data from real-time PCR analysis showing effect of TTF-1 (TTF-1-pcDNA, 500 ng) on the endogenous COX-2 mRNA expression in the C6 cells (C) and B35 cells (D). E and F, data from western blotting revealing effect of TTF-1 on the COX-2 protein in the C6 cells (E) and B35 cells (F). G and H, PGE2 release from C6 cells (G) and B35 cells (H) by overexpression of TTF-1. Each value represents mean±SEM (n = 6). **, p<0.01 versus pcDNA.

Figure 4. EMSAs and ChIP assays.

EMSAs were performed with double-stranded oligomer probes containing the putative TTF-1 binding sites shown in Fig. S2 and table 1. A, relative binding activities calculated as a percentage of TTF-1 HD binding to the positive control probe C. Cβ, negative control probe carrying mutations in the TTF-1 binding domain (TBD). ND, not detectable. B, ChIP assays of rat COX-2 promoter DNA using TTF-1 Ab. DNA was immunoprecipitated from C6 cells with TTF-1 Ab (TTF-1) or IgG (as a negative control), and was PCR-amplified with primer sets, shown in Information S1, for COX-2 promoter fragments including TTF-1 binding sites indicated as numbers at TBD. Input represents the DNA extracted from the C6 cells before immunoprecipitation. C, hypothalamic nuclear extracts were incubated with oligonucleotide probes containing −2039 TTF-1 binding site, in the presence (+) or absence (-) of 5- or 20-fold excess of cold oligonecleotide C and Cβ and TTF-1 antibody (TTF-1 Ab) or preimmune serum (Pre IS). Incubation of nuclear proteins with a TTF-1 Ab prior to the protein-DNA binding reaction delays (arrow S, supershift) the migration of the protein-DNA complex (arrow B). F, free probe.

Figure 5. Effect of site-specific deletion of TTF-1 binding core motifs on the TTF-1-induced inhibition of COX-2 promoter activity.

TTF-1 expression vector (TTF-1-pcDNA, 500 ng) was cotransfected with nine single mutants of the COX-2 promoter (COX-2-P) deleted with core TTF-1 binding sites (showing relatively strong or moderate binding with TTF-1 in EMSAs) or with combined mutants deleted with −2039, −1657, and/or −223 sites. Positions of the deleted binding sites are indicated. The data are the means±SEM (n = 4). ###, p<0.001 versus COX-2-P + pcDNA; *, p<0.05; ***, p<0.001 versus COX-2-P + TTF-1-pcDNA.

Figure 6. Effect of TTF-1 synthesis blockade by AS TTF-1 ODN on the change of body temperature.

Body temperature was measured for 24 h after icv administration of AS TTF-1 ODN or SCR ODN. A, temperature began to increase 2 h after icv injection (arrow at 0 h) of the AS TTF-1 ODN (AS) and remained high until about 20 h after the injection compared with SCR ODN injected group (SCR). To determine involvement of prostaglandins in the AS-induced hyperthermia, rats were i.p.-injected with indomethacin (Indo) 30 min prior to the injection of the AS. Pretreatment of Indo significantly reduced the AS-induced increase of body temperature. The shaded area indicates dark period. The values represent means±SEM (n = 6). B, mean temperature after the injection indicating a significant difference between groups. ***, p<0.001 versus SCR; ###, p<0.001 versus AS.