Abstract
Previous studies have shown that the promoter of the type II TGF-beta receptor gene (TbetaR-II) is strongly stimulated by Elf3, a member of the Ets transcription factor family. The TbetaR-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 TbetaR-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 TbetaR-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 TbetaR-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 TbetaR-II promoter in these cells. In contrast, PEA3 stimulates the TbetaR-II promoter in F9-differentiated cells, but it inhibits this promoter in F9 cells. Thus, the effects of PEA3 on the TbetaR-II promoter are cell context-dependent. We also show that the effects of Elf3 are cell context-dependent. Elf3 strongly stimulates the TbetaR-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 TbetaR-II promoter. Together, our findings indicate that Elf3 is not the only Ets protein capable of stimulating the TbetaR-II promoter. Importantly, our findings also indicate that each of the five Ets proteins influences the TbetaR-II promoter in a unique manner because of important differences in their biochemical properties or their patterns of cellular expression.
Highlights
TGF-1 consists of three isoforms that exert potent, pleiotropic effects during growth and differentiation [1, 2]
Elf3 Synergistically Increases the Activity of the TR-II Promoter via Two Overlapping Ets Sites— Ets proteins bind to an essential core sequence of 5Ј-GGA(A/T)-3Ј [63], their binding to DNA is affected by flanking sequences
The findings described in this study demonstrate that the TR-II promoter is strongly stimulated by Elf3, it is highly responsive to Ets1 and PEA3 in F9-differentiated cells
Summary
Materials—Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Invitrogen. Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT). In addition to 15 g of TR-IIϪ108/ϩ56 promoter/ reporter gene constructs, the cells were transfected with 1 g of the internal control plasmid, pCMV--gal, to normalize for any differences in transfection efficiency or general effects on transcription In this regard, Ets, Elf, and PU., but not PEA3, Ets, Elf3⌬N233, or Elf3⌬N270, appeared to decrease the activity of pCMV--gal more than 3-fold when their expression vectors were used at concentrations greater than 3 g. Transfection and Expression of GFP and YFP Fusion Proteins—F9 EC cells or F9-differentiated cells were transiently transfected with expression vectors for Ets-fluorescent fusion proteins using LipofectAMINE in conjunction with the LipofectAMINE PLUS reagent (Invitrogen) as described previously [62] This transfection protocol was used, because previous studies determined that only small differences in transfection efficiency are observed when these reagents are used [62]. The sequence, TAGC, was added to the 5Ј end of each oligonucleotide for use in radiolabeling
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