Ovarian steroid action on tryptophan hydroxylase protein and serotonin compared to localization of ovarian steroid receptors in midbrain of guinea pigs.

The effect of estrogen (E) and progesterone (P) on the protein expression of the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase (TPH), and the level of serotonin in the hypothalamic terminal field was examined in guinea pigs. In addition, we questioned whether serotonin neurons of guinea pigs contain ovarian steroid receptors (estrogen receptoralpha[ERalpha], estrogen receptor beta[ERbeta], progestin receptors [PRs]) that could directly mediate the actions of E or P. Western blot and densitometric analysis for TPH were used on raphe extracts from untreated-ovariectomized (OVX), OVX-E-treated (28 d), and OVX-E+P-treated (14 d E+14 d E+P) guinea pigs. The medial basal hypothalami from the same animals were extracted and subjected to high-performance liquid chromatography analysis for serotonin, dopamine, 5-hydroxyindole acetic acid, and homovanillic acid. The brains from other animals treated in an identical manner were perfusion fixed and examined for the colocalization of ERalpha plus serotonin and PR plus serotonin with double immunohistochemistry or for expression of ERbeta mRNA with in situ hybridization. E and E+P treatment significantly increased TPH protein levels compared to the untreated control group (p < 0.05), but TPH levels were similar in the E and E+P-treated groups. By contrast, serotonin (nanogram/milligram of protein) in the hypothalamus was significantly increased by E+P treatment, but not by E alone. Neither ERalpha nor PR proteins were detected within serotonin neurons of the guinea pig raphe nucleus. However, ERbeta mRNA was expressed in the dorsal raphe. In summary, E alone increased TPH protein expression and the addition of P had no further effect, whereas E+P increased hypothalamic serotonin and E alone had no effect. The localization of ERbeta, but not ERalpha or PR, in the dorsal raphe nucleus suggests that E acting via ERbeta within serotonin neurons increases expression of TPH, but that P acting via other neurons and transsynaptic stimulation may effect changes in TPH enzymatic activity, which in turn, would lead to an increase in serotonin synthesis.

PU.1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells.

Cytokines, important for lineage commitment and differentiation during hematopoiesis, exert their influence by binding specific receptors. Receptor expression is tightly regulated and examining the factors that govern their expression will allow better understanding of the events that determine lineage commitment. The granulocyte colony-stimulating factor (G-CSF) receptor is expressed exclusively in myeloid cells and the placenta. We show here that the G-CSF receptor transcription start site is identical in each of these tissues. A 1,391-bp fragment of the G-CSF receptor promoter is both active in myeloid cell lines and tissue specific. We have also found two regions that are important for G-CSF receptor promoter activity. One region, located at bp -49, contains a GCAAT site that specifically binds the C/EBP alpha transcription factor in myeloid nuclear extracts. Mutation of this site prevents C/EBP alpha binding and reduces promoter activity by 60%. The other functionally important region of the G-CSF receptor promoter is in the 5' untranslated region, at bp +36 and +43, where there are two sites for the ets family member PU.1. Mutation of these sites prevents PU.1 binding and reduces promoter activity by 75%. These results reinforce the importance of both PU.1 and C/EBP alpha in the expression of myeloid-specific genes and neutrophil development.

Sp1 is a critical factor for the monocytic specific expression of human CD14.

CD14 is a membrane glycoprotein expressed specifically on monocytes and macrophages, and its expression is markedly increased during the process of monocyte differentiation. In order to study CD14 gene regulation, the human CD14 gene was cloned from a partial EcoRI digested chromosome 5 library. A 5.5-kilobase genomic clone contained the full-length CD14 coding sequence and 4.2 kilobases of 5'-upstream sequence. One major and one minor transcription start site were identified 101 and 130 base pairs (bp) upstream, respectively, from the protein translation start ATG. A DNA fragment containing 128 bp of upstream sequence had strong, monocyte-specific promoter activity in the CD14 positive monocytic cell line Mono Mac 6 as compared to the nonmonocytic cell lines HeLa and REX. Four regions in this DNA fragment interact with nuclear proteins isolated from monocytic cells. The Sp1 transcription factor bound to three different regions in the CD14 promoter. Mutation of the major Sp1 binding site (-110 bp) decreased tissue-specific promoter activity, and these results, together with transactivation experiments, demonstrate that Sp1 plays a critical role in the tissue-specific expression of CD14 in monocytic cells. CD14 Sp1 site oligonucleotides bound preferentially to a 105-kDa Sp1 species, which is present in higher relative levels in monocytic than non-monocytic cells, suggesting that modification of Sp1, such as phosphorylation, may explain how the Sp1 site mediates monocytic specific promoter activity.