Coordinated gene regulation within the vascular endothelium is required for normal cardiovascular patterning during development and for vascular homeostasis during adulthood, yet little is known about the mechanisms that regulate endothelial transcriptional events. Vascular endothelial zinc finger 1 (Vezf1)/DB1 is a recently identified zinc finger-containing protein that is expressed specifically within endothelial cells during development. In this report, we demonstrate that Vezf1/ DB1 is a nuclear localizing protein that potently and specifically activates transcription mediated by the human endothelin-1 promoter, in a Tax-independent manner, in transient transfection assays. Using a combination of deletion mutagenesis and electrophoretic mobility shift assays, a novel Vezf1/DB1-responsive element was localized to a 6-base pair (bp) motif, ACCCCC, located 47 bp upstream of the endothelin-1 transcription start site. Recombinant Vezf1/DB1 also bound to this sequence, and a 2-bp mutation in this element abolished Vezf1/DB1 responsiveness by the endothelin-1 promoter. Vezf1/DB1 could be identified with a specific antibody in nuclear complexes from endothelial cells that bound to this element. Regulation of endothelin-1 promoter activity by Vezf1/DB1 provides a mechanism for endothelin-1 expression in the vascular endothelium during development and to maintain vascular tone; Vezf1/DB1 itself is a candidate transcription factor for modifying endothelial cell phenotypes in order to appropriately assemble and maintain the cardiovascular system.

Transcription of the transforming growth factor-␤2 (TGF-␤2) gene is dependent on a cAMP-response element/activating transcription factor (CRE/ATF) site that is bound by CREB and ATF-1 as well as an E-box motif that is bound by upstream stimulatory factors 1 and 2 (USF1 and USF2). To identify additional factors involved in the expression of the TGF-␤2 gene, we employed F9 embryonal carcinoma (EC) cells, which express TGF-␤2 only after the cells differentiate. We show that overexpression of the transcription factors, CREB, ATF-1, USF1, and USF2 dramatically increases TGF-␤2 promoter activity in F9-differentiated cells. We further show that the coactivators p300 and CBP up-regulate the TGF-␤2 promoter when CREB and ATF-1 are expressed in conjunction with protein kinases that phosphorylate CREB on serine 133 and ATF-1 on serine 63. Importantly, we identify the presence of serine 133-phosphorylated CREB in the nucleus of F9-differentiated cells but not in the nucleus of F9 EC cells. This phosphorylated form is present in whole cell extracts of both the parental and differentiated cells, suggesting that nuclear accumulation of serine 133-phosphorylated CREB is regulated during differentiation of F9 EC cells and is likely to play an important role in the activation of the TGF-␤2 gene.

Transforming growth factor-␤2 (TGF-␤2) is an important regulator of cell proliferation and differentiation; however, its transcriptional regulation is not well understood. Here we report characterization of an essential E-box motif, positioned at ؊50/؊45 between a previously described functional cAMP response element/ activating transcription factor site and the TATA box of the human TGF-␤2 promoter. By site-directed mutagenesis, we demonstrate that this E-box motif is necessary for the promoter activity, not only in differentiated cells derived from embryonal carcinoma cells, but also in choriocarcinoma cells and in MCF-7 breast carcinoma cells. We also demonstrate that the transcription factors USF1 and USF2 bind to this E-box motif in vitro when nuclear extracts from each of these cell lines are examined by gel retardation assays. Moreover, using a dominant-negative USF2 protein, we show that USF proteins are critical for TGF-␤2 promoter activity in vivo. The importance of the E-box motif described in this study is supported by the presence of an E-box motif in the same position in the chicken TGF-␤2 gene promoter.Transforming growth factor-␤2 (TGF-␤2), 1 like other growth factors in the TGF-␤ family, is involved in the regulation of many different cellular functions, including cell proliferation and differentiation as well as production and maintenance of extracellular matrices (reviewed in Refs. 1 and 2). Through its multifaceted effects, TGF-␤2 plays important regulatory roles in a host of biological events, from embryogenesis through repair processes, to regulation of the immune system. Hence, the regulation of TGF-␤2 gene in various systems warrants detailed investigation. The studies presented here focus on the transcriptional regulation of the TGF-␤2 gene in embryonal carcinoma (EC) cells and their differentiated counterparts, which represent a model system of early embryonic development (reviewed in Ref. 3). Given the importance of TGF-␤2 production in implantation and in tumor malignancy (4 -6), we extended our studies to two choriocarcinoma cell lines and a breast carcinoma cell line. Previous work demonstrated the presence of a critical positive regulatory region in the TGF-␤2 gene promoter, localized between Ϫ77 and ϩ63, where ϩ1 is the transcription start site (7)(8)(9). This region contains a functional CRE/ATF motif, which is indispensable for the positive effect of this promoter region in different cell types and capable of binding activating transcription factor 1 (ATF-1) in vitro.Recently, computerized sequence analysis demonstrated that the human TGF-␤2 promoter also contains a CACGTG motif between Ϫ50 and Ϫ45, which conforms to the consensus sequence of an E-box motif, CANNTG (10). Interestingly, this E-box motif appears to be evolutionarily conserved, since the same CACGTG sequence is present in the chicken TGF-␤2 promoter, and it is located in the same position relative to the similarly conserved CRE/ATF site and the TATA box of the chicken promoter (11). Thus, the putative E-box motif l...

The endothelium is required for maintenance of vascular integrity and homeostasis during vascular development and in adulthood. However, little is known about the coordinated interplay between transcription factors and signaling molecules that regulate endothelial cell-dependent transcriptional events. Vascular endothelial zinc finger-1 (Vezf1) is a zinc finger-containing transcription factor that is specifically expressed within the endothelium during vascular development. We have previously shown that Vezf1 potently activates transcription of the endothelin-1 promoter. We now report the identification of p68RacGAP, a novel Vezf1-interacting 68-kDa RhoGAP domain-containing protein. p68RacGAP mRNA is highly expressed in vascular endothelial cells by Northern blot analysis, and immunohistochemical staining of adult mouse tissues identified p68RacGAP in endothelial cells, vascular smooth muscle cells, and epithelial cells in vivo. Rac1 and Vezf1 both bind avidly to p68RacGAP, suggesting that p68RacGAP is not only a GTPase-activating protein for Rac1 but that p68RacGAP may also be part of the protein complex that binds to and modulates Vezf1 transcriptional activity. Functionally p68RacGAP specifically activates the GTPase activity of Rac1 in vivo but not Cdc42 or RhoA. In addition, p68RacGAP potently inhibits Vezf1/DB1-mediated transcriptional activation of the human endothelin-1 promoter and modulates endothelial cell capillary tube formation. Taken together, these data suggest that p68RacGAP is a multifunctional regulatory protein that has a Rac1-specific GTPase-activating activity, regulates transcriptional activity of the endothelin-1 promoter, and is involved in the signal transduction pathway that regulates endothelial cell capillary tube formation during angiogenesis.

To study the role of glucocorticoid receptor (GR) at different stages of mammary gland development, mammary anlage were rescued from GR-/- mice by transplantation into the cleared fat pad of wild-type mice. In virgin mice, GR-/- outgrowths displayed abnormal ductal morphogenesis characterized by distended lumena, multiple layers of luminal epithelial cells in some regions along the ducts, and increased periductal stroma. In contrast, the loss of GR did not result in overt phenotypic changes in mammary gland development during pregnancy, lactation, and involution. Surprisingly, despite the known synergism between glucocorticoids and prolactin in the regulation of milk protein gene expression, whey acidic protein and beta-casein mRNA levels were unaffected in GR-/- transplants as compared with wild-type transplants. That mineralocorticoid receptor (MR) might compensate for the loss of GR was suggested by the detection of MR in the mammary gland at d 1 of lactation. This hypothesis was tested using explant cultures derived from the GR-/- transplants in which the mineralocorticoid fludrocortisone was able to synergistically induce beta-casein gene expression in the presence of prolactin and insulin. These studies suggest that MR may compensate for the absence of GR at some, but not at all stages of mammary gland development.