Regulation of Pituitary Adenylate Cyclase-activating Polypeptide Gene Transcription by TTF-1, a Homeodomain-containing Transcription Factor

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an important hypophysiotrophic factor as well as a regulator for immune, reproductive, and neural tissues. We recently found that TTF-1, a homeodomain-containing transcription factor essential for the development of the fetal diencephalon, is postnatally expressed in the hypothalamic area and plays a transcription regulatory role for certain neurohormones. Based on the similarity of synthesis sites between PACAP and TTF-1 and, moreover, on the presence of conserved core TTF-1 binding motifs in the 5-flanking region of the PACAP gene, we sought to uncover a regulatory role of TTF-1 in PACAP gene transcription. The TTF-1 homeodomain binds to six of the seven putative binding domains observed in the 5-flanking region of the PACAP gene. In the C6 glioma cell-line, TTF-1 activates the PACAP promoter in a dose-dependent manner. This transactivation of PACAP by TTF-1 was totally removed when the core TTF-1 binding motif at ؊369 was deleted. RNase protection assays showed that TTF-1 and PACAP mRNAs have daily fluctuations in the rat hypothalamus. They both were at low levels during the day and high levels during the night. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased the PACAP mRNA level as well as TTF-1 protein content in the rat hypothalamus, suggesting that TTF-1 also regulates PACAP transcription in vivo. Moreover, the TTF-1 promoter was inhibited by molecular oscillators of CLOCK and BMAL-1. Taken together, these data suggest that TTF-1 plays an important regulatory role in the gene transcription for PACAP, which may be important for the generation of a daily rhythm of hypothalamic PACAP gene expression.Pituitary adenylate cyclase-activating polypeptide (PACAP) 1 was originally isolated from the ovine hypothalamus (1, 2) and was classified as a member of the secretin-glucagon-vasoactive intestinal peptide family (1). PACAP plays the role of a hypophysiotropic factor and regulates the synthesis and secretion of pituitary hormones through the hypothalamo-hypophysial portal system (3). Although PACAP immunoreactivity is found in nearly all brain regions, its highest concentration is in the hypothalamus of both rat and human (3, 4). In the hypothalamus, PACAP immunoreactivity is primarily localized to the cells of the paraventricular and supraoptic nuclei (3, 4). PACAP is found both in neurons terminating on hypophysial blood vessels of the median eminence as well as in neurons projecting to the posterior pituitary. Therefore, PACAP may act not only as a hypophysiotropic factor regulating anterior pituitary hormone release but as a posterior pituitary hormone as well. It is also an important factor in the regulation of peripheral tissues such as ovary and testis and of the immune system.Recently, the 5Ј-flanking region of PACAP gene was characterized and shown to have several sequence motifs homologous to the cAMP response element (CRE), the TPA response element (TRE), and the growth hormone factor...

TTF-1, a Homeodomain-containing Transcription Factor, Participates in the Control of Body Fluid Homeostasis by Regulating Angiotensinogen Gene Transcription in the Rat Subfornical Organ

“…The AS TTF-1 ODN used to disrupt TTF-1 synthesis (5Ј-GAC TCA TCG ACA TGA TTC GGC GTC-3Ј) was directed against the sequence surrounding the first ATG codon of TTF-1 mRNA as previously reported (22). As a control, a scrambled (SCR) sequence of identical base composition was used (5Ј-AGT CCT ACT CGG TAC GTA TGC AGC-3Ј).…”

“…Oligodeoxynucleotides C and C␤ were used as a positive and negative control, respectively (34). EMSA was performed as previously described (22,33,34). Electrophoretically separated signals corresponding to the protein-bound and free DNA were quantitated with Multi-analyst software.…”

“…C6 cells were grown in Dulbecco's modified Eagle's medium/F-12 containing 10% fetal bovine serum. Twenty-four h after seeding the cells in six-well plates, they were transiently transfected with the AoGen promoter-luciferase reporter construct (AoGen-P) using LipofectAMINE (Invitrogen) along with different concentrations (100 -500 ng/well) of the expression vector, pcDNA 3.1-zeo (Invitrogen), containing the TTF-1 coding region (TTF-1-pcDNA) (20,22). Transfection efficiency was normalized by cotransfecting the ␤-galactosidase reporter plasmid (pCMV-␤-gal; Clontech, Palo Alto, CA) at 20 ng/ml.…”

“…As a control, a scrambled (SCR) sequence of identical base composition was used (5Ј-AGT CCT ACT CGG TAC GTA TGC AGC-3Ј). For the intracerebral injection, the ODNs were diluted to a final concentration of 100 ng/ l artificial cerebrospinal fluid (22). Under pentobarbital (7.5 mg/kg body weight) and ketamine hydrochloride (25 mg/kg body weight) anesthesia, a polyethylene cannula (outer diameter, 1.05 mm; inner diameter, 0.35 mm) was stereotaxically implanted into the brain with its opening protruding onto the top of the SFO (coordinates: AP ϭ 1.3 mm caudal to the bregma; V ϭ 4.3 mm from the dura mater; L ϭ 0.0 mm from the midsagittal line) as recommended (37).…”

“…In the hypothalamus, TTF-1 is present in sets of neurons containing luteinizing hormone-releasing hormone, enkephalin, and pituitary adenylate cyclase-activating polypeptide (20,22), in addition to ependymoglial cells lining the third ventricle and astrocytic tanycytes of the median eminence, where it is coexpressed with the epidermal growth factor-related receptor ErbB2 (20). We also showed that TTF-1 binds to specific recognition motifs present in each one of these promoters to regulate transcriptional activity (20,22).…”