Previous studies demonstrated that differentiation of mouse embryonal carcinoma cells leads to transcriptional up-regulation of the mouse type II transforming growth factor- receptor (mTR-II) gene. To elucidate the molecular mechanisms regulating transcription of this gene, we isolated the 5-flanking region of the mTR-II gene and characterized its expression in F9-differentiated cells. Analysis of mTR-II promoter/reporter gene constructs demonstrates that two conserved Ets-binding sites play an important role in the activity of the mTR-II promoter. Importantly, we present evidence that mElf-3, a member of the Ets family, plays a key role in the activation of the mTR-II promoter. Northern blot analysis reveals that the steadystate levels of mTR-II mRNA increase in parallel with those of mElf-3 mRNA during the differentiation of F9 embryonal carcinoma cells. We also demonstrate that mElf-3 contains one or more domains that influence its binding to DNA. Finally, we report that a single amino acid substitution in the transactivation domain of mElf-3 reduces its ability to transactivate and elevates its steady-state levels of expression. In conclusion, our data argue that mElf-3 plays a key role in the regulation of the mTR-II gene, and Elf-3 itself is regulated at multiple levels.
Low voltage-activated (T-type) calcium channels are responsible for burst firing and transmitter release in neurons and are important for exocytosis and hormone secretion in pituitary cells. T-type channels contain an α1 subunit, of which there are three subtypes, Cav3.1, 3.2 and 3.3, and each subtype has distinct kinetic characteristics. Although 17β-estradiol modulates T-type calcium channel expression and function, little is known about the molecular mechanisms involved. Presently, we used real-time PCR quantification of RNA extracted from hypothalamic nuclei and pituitary in vehicle and E2-treated C57BL/6 mice to elucidate E2-mediated regulation of Cav3.1, 3.2 and 3.3 subunits. The three subunits were expressed in both the hypothalamus and the pituitary. E2 treatment increased the mRNA expression of Cav3.1 and 3.2, but not Cav3.3, in the medial preoptic area and the arcuate nucleus. In the pituitary, Cav3.1 was increased with E2-treatment and Cav3.2 and 3.3 were decreased. In order to examine whether the classical estrogen receptors (ERs) were involved in the regulation, we used ERα-and ERβ-deficient C57BL/6 mice and explored the effects of E2 on T-type channel subtypes. Indeed, we found that the E2-induced increase in Cav3.1 in the hypothalamus was dependent on ERα, whereas the E2 effect on Cav3.2 was dependent on both ERα and ERβ. However, the E2-induced effects in the pituitary were dependent on only the expression of ERα. The robust E2-regulation of the T-type calcium channels could be an important mechanism by which E2 increases the excitability of hypothalamic neurons and modulates pituitary secretion.
Previous studies have shown that the promoter of the type II TGF- receptor gene (TR-II) is strongly stimulated by Elf3, a member of the Ets transcription factor family. The TR-II gene behaves as a tumor suppressor and it is expressed in nearly all cell types, whereas Elf3 is expressed primarily in epithelial cells. Hence, the TR-II gene is likely to be regulated by other Ets proteins in nonepithelial cells. In this study, we examined the effects of four other Ets family members (Ets1, Ets2, PEA3, and PU.1) on TR-II promoter/reporter constructs that contain the two essential ets sites of this gene. These studies employed F9 embryonal carcinoma cells and their differentiated cells, because transcription of the TR-II gene increases after F9 cells differentiate. Here we demonstrate that Ets2, which is expressed in F9-differentiated cells along with Elf3, does not stimulate or bind to the TR-II promoter in these cells. In contrast, PEA3 stimulates the TR-II promoter in F9-differentiated cells, but it inhibits this promoter in F9 cells. Thus, the effects of PEA3 on the TR-II promoter are cell context-dependent. We also show that the effects of Elf3 are cell context-dependent. Elf3 strongly stimulates the TR-II promoter in F9-differentiated cells, but not in F9 cells. In contrast to Elf3 and PEA3, Ets1 strongly stimulates this promoter in both F9 cells and F9-differentiated cells. Finally, we show that PU.1 exerts little or no effect on the activity of the TR-II promoter. Together, our findings indicate that Elf3 is not the only Ets protein capable of stimulating the TR-II promoter. Importantly, our findings also indicate that each of the five Ets proteins influences the TR-II promoter in a unique manner because of important differences in their biochemical properties or their patterns of cellular expression.
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