Selective inhibition of prolactin gene transcription by the ETS-2 repressor factor.

Regulation of prolactin gene transcription requires cooperative interactions between the pituitary-specific POU domain protein Pit-1 and members of the ETS transcription factor family. We demonstrate here that the ETS-2 repressor factor (ERF) is expressed in pituitary tumor cells and that overexpression of recombinant ERF inhibits prolactin promoter activity, but not the closely related growth hormone promoter. In non-pituitary cell lines, coexpression of ERF disrupts the cooperative interactions between Pit-1 and ETS-1 and blocks the induction of Pit-1-dependent prolactin promoter activity by cAMP. The potential role of ERF in the inhibitory response of the prolactin promoter to dopamine was examined using pituitary tumor cells stably expressing dopamine D2 receptors. The inhibitory responses of the prolactin promoter to ERF and dopamine are additive, suggesting that ERF has a complementary role in this hormonal response. A single Pit-1 DNA-binding element from the prolactin promoter is sufficient to reconstitute the inhibitory response to ERF. DNA binding analysis using either a composite Pit-1/ETS protein-binding site or a Pit-1 element with no known affinity for ETS proteins revealed that ERF interferes with Pit-1 binding. Together, these results demonstrate that ERF is a specific inhibitor of basal and hormone-regulated transcription of the prolactin gene and suggest a new level of complexity for the interaction of ETS factors with Pit-1 target genes.

Cell type-specific expression of the pituitary transcription activator pit-1 in the human pituitary and pituitary adenomas.

Pit-1 is a transcription factor that has been shown to be critical for pituitary-specific activation of the GH and PRL genes. In rodents and humans, differentiation and/or maintenance of somatotroph, lactotroph, and thyrotroph phenotypes are dependent on expression of a functional pit-1 gene. In rodents, Pit-1 protein is detectable in only these three cell types; however, pit-1 mRNA transcripts appear to be present at comparable levels in all adenohypophysial cell types, suggesting that translational controls may dictate the pattern of Pit-1 expression. We examined the distribution of pit-1 transcripts in the human pituitary and pituitary adenomas. All tumors were characterized by immunocytochemistry, electron microscopy, and tissue culture for accurate classification. Northern blot analysis demonstrated abundant levels of pit-1 mRNA in somatotroph, mammosomatotroph, and lactotroph adenomas. Two clinically silent adenomas that expressed TSH as well as gonadotropins contained detectable levels of pit-1 mRNA. No pit-1 expression was otherwise detected in corticotroph, gonadotroph, null cell, or oncocytic adenomas. In situ hybridization localized pit-1 mRNA transcripts in adenomas that contained GH, PRL, or TSH, but not in adenomas composed of other cell types. Pit-1 mRNA was also localized to selected subpopulations of the human nontumorous adenohypophysis that contained immunoreactivity for GH, PRL, and/or TSH. Pit-1 protein immunoreactivity was detected in the nuclei of adenomas that expressed pit-1 mRNA, but not in those that were negative for pit-1 mRNA; it was also localized only in cells containing GH, PRL, or TSH beta in the nontumorous adenohypophysis. These data demonstrate selective expression of the human pit-1 gene in adenohypophysial cell types responsible for GH, PRL, and/or TSH synthesis and are consistent with a predominantly pretranslational regulatory mechanism for Pit-1 expression in the human.

Inhibitory control of prolactin and Pit-1 gene promoters by dopamine. Dual signaling pathways required for D2 receptor-regulated expression of the prolactin gene.

Transcription of the prolactin gene is suppressed by dopaminergic activation of D2 receptors in pituitary lactotrophs. The mechanism of signal transduction at the nuclear level and the cell surface was examined in the dopamine-responsive GH4ZR7 cell line. Dopamine treatment caused a 40-50% decrease in endogenous prolactin mRNA that was specifically blocked by addition of (-)-sulpiride. To define dopamine-responsive elements, plasmids containing 5'-regulatory regions of the prolactin gene fused to the coding sequences for luciferase were transiently or stably transfected into GH4ZR7 cells. Chimeric transcripts initiated at the authentic transcription start site were regulated in a promoter-selective manner; dopamine or the agonist bromocryptine inhibited prolactin promoter (position -422) activity by 70%, but had no regulatory effects on other cellular or viral promoters. A shorter prolactin promoter (position -78) or a prolactin TATAA box linked to heterologous binding sites for transcription factor Pit-1 was sufficient to confer dopamine inhibition (40%). In addition to the prolactin promoter, we found that dopamine inhibited transcriptional activity of the Pit-1 promoter (positions -258 to +8) by 60%. Surprisingly, deletion of two cAMP response elements in the Pit-1 promoter only partially eliminated dopamine responsiveness. These data suggest that sequences in the Pit-1 promoter between positions -92 and +8, which include an autoregulatory Pit-1-binding site and the TATAA box, are sufficient for negative regulation. In this study, we also examined the signal transduction pathways that link D2 receptor activation and the inhibition of prolactin gene transcription. We found, as suggested in earlier studies, that a dopamine-dependent decrease in cAMP may be important for mediating negative regulation of transcription. However, high extracellular K+ concentrations that prevent dopamine effects on membrane potential and [Ca2+]i, but not cAMP levels, completely blocked dopamine regulation of the prolactin promoter. This suggests that two distinct signaling pathways initiated at D2 receptors may be required for transcriptional regulation of the prolactin gene.

Immunoreactive somatostatin and beta-endorphin content in the brain of mature rats after neonatal exposure to propylthiouracil.

The contents of immunoreactive somatostatin (IR-SRIF) and beta-endorphin (IR-beta-EP) in 12 brain regions were examined in rats exposed neonatally to propylthiouracil (PTU) through the mother's milk. Since the dose of PTU used in this study is lower than the usual dose employed to induce hypothyroidism, a milder form of neonatal hypothyroidism resulted. This conclusion is supported by the only mild subnormal growth of rats to adulthood and serum T4 and T3 concentrations in the normal range. Adult rats treated with PTU neonatally had significantly higher IR-SRIF contents in several brain regions compared to controls, whereas IR-beta-EP levels were not significantly different (significant increase only in the thalamus) in most regions. The results indicate that even mild hypothyroidism during early postnatal development causes permanent impairment of brain function, which manifests itself in part by an altered brain content of IR-SRIF.

Isolation and identification of a new potent inhibitor of creatine kinase from human serum.

Human serum contains a very potent inhibitor of creatine kinase in addition to urate which was characterized as an inhibitor of this enzyme by Warren (Warren, W.A. (1975) Clin. Biochem. 8, 247). This new inhibitor was isolated from human serum by dialysis, negative DEAE-cellulose adsorption and a series of gel filtration chromatographies. The inhibitor so isolated was an organic compound. Kinetic investigation showed noncompetitive inhibition versus creatine, as well as MgATP. Infrared spectroscopy revealed amino and carboxyl groups. The isolated material was ninhydrin-positive. Thin-layer chromatography of the dansyl derivative showed several fluorescent spots. Quantitative amino acid analysis revealed the presence of several amino acids. Of these only cystine and cysteine were inhibitory in the standard inhibitor assay, using rabbit MM creatine kinase. Testing with human MM creatine kinase only cystine was found inhibitory. Cystine has a specific inhibitor activity of 600 anti-Units per mg, using rabbit or human MM creatine kinase, respectively. Urate by comparison has a specific inhibitor activity of 45 anti-Units per mg using human MM creatine kinase.