We have shown that osteogenic protein-1 (OP-1) (bone morphogenetic protein-7) is responsible for the induction of nephrogenic mesenchyme during embryonic kidney development. Gene knock-out studies showed that OP-1 null mutant mice die of renal failure within the first day of postnatal life. In the present study, we evaluated the effect of recombinant human OP-1 for the treatment of acute renal failure after 60 min bilateral renal artery occlusion in rats. Bioavailability studies in normal rats indicate that ‫ف‬ 1.4 g OP-1/ml is available in the circulation 1 min after intravenous administration of 250 g/kg, which then declines steadily with a half life of 30 min. About 0.5% of the administered OP-1 dose/g tissue is targeted for OP-1 receptors in the kidney. We show that OP-1 preserves kidney function, as determined by reduced blood urea nitrogen and serum creatinine, and increased survival rate when administered 10 min before or 1 or 16 h after ischemia, and then at 24-h intervals up to 72 h after reperfusion.

We used a cell-free HeLa cell transcription system to identify and characterize transcription factors and the promoter elements that they recognize in RNA polymerase II-transcribed genes. Deletion of the region (-71 to -83) containing the GTCAAA direct repeat resulted in a marked decrease of specific transcription of the ovalbumin gene; transcription could be competed with DNA fragments containing this sequence. Furthermore, DNase I footprinting identified a protein-binding site including this direct repeat with crude extracts and one of the partially purified protein fractions required for transcription. We propose that a soluble factor activates transcription through binding to the direct repeat of GTCAAA sequence upstream from the ovalbumin gene.Recombinant DNA methodology and the techniques of gene transfer have facilitated the characterization of promoters for RNA polymerase II-transcribed genes. The efficiency of mutated promoters in directing accurate initiation has been tested in vivo and in vitro (1,2,10,32). These experiments suggested the existence of distinct promoter elements upstream from the initiation site of transcription. The TATA box, a highly conserved homology among mRNA coding genes and located at -24 to -32 base pairs from the initiation site, is essential for efficient and accurate initiation of most genes both in vivo and in vitro (2, 32). In addition, sequences upstream from the TATA box have been shown to be required in vivo (32) and in vitro (20,22,24,27,29,40,41) for efficient transcription of many eucaryotic genes. This is clearly the case for the ovalbumin gene in vivo (28). We previously showed that accurate initiation of the ovalbumin gene transcription in vitro requires the TATA homology (39, 42). We demonstrate here that sequences upstream from the TATA box are important for quantitative transcription of the ovalbumin gene in crude extracts from HeLa cells. This upstream promoter element is located approximately 80 base pairs from the cap site and includes a direct repeat of GTCAAA sequence. Since this region was originally defined by Benoist et al. (1) and Efstratiadis et al. (11) as the CAAT-box region, we tentatively designate the direct repeat sequence as the CAAT box for reference purposes.The requirement for a distal promoter element was initially indicated by 5' deletion mapping. To test whether a transcription factor interacts with this promoter element, we performed transcription competition assays and found that crucial sequences for the binding of a transcription factor include the CAAT box. We have shown previously by exonuclease footprinting that a protein which binds to this region of the ovalbumin gene is present in HeLa cell fractions (13). We now confirm and extend this footprinting analysis using DNase I with either crude nuclear extracts or the partially purified fractions (38) required for transcription in reconstituted systems. These results suggest that the upstream element delineated by the functional transcription assays is the specific binding site fo...

The expression of the human presenilin-1 cellular gene is suppressed by the p53 protooncogene. The rapid kinetic of the down-regulation has suggested that it may result from a primary mechanism. We show here that p53 also suppresses the transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene in transient infection assays in neuroblastoma (SK-N-SH) and hepatoma (HepG2) cell lines. Only a minimum promoter including sequences from ؊35 to ؉ 6 from the transcription initiation is sufficient to confer down-regulation. We have previously defined a crucial DNA element controlling 90% of the expression of the gene within the same short area, and the identification of the transcription factors involved should also provide insights into the regulation of PS1 by p53. This region contains an Ets transcription factor binding motif, and a 2-base pair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by more than 90%. We now show that Ets1 and Ets2 indeed transactivate a PS1 promoter-chloramphenicol acetyltransferase reporter including the (؊35 to ؉6) fragment. Furthermore, in vitro translated Ets2 binds specifically to the ؊10 Ets motif in electrophoretic mobility shift assays. Therefore, Ets1/2 factors bind specifically to the ؊10 Ets element and activate PS1 transcription. We also show that the coactivator p300 enhances the activation by Ets1 and Ets2 as well as the repression by p53. p300 is known to interact with p53 as well as with Ets1 and Ets2. We show that p53 does not bind directly to the PS1 promoter. Hence the repression of PS1 transcription by p53 is likely to be mediated through protein-protein interactions.Presenilin genes (PS1 and PS2) encode highly homologous integral membrane proteins (1, 2). A majority of early onset or familial Alzheimer's disease (FAD) 1 cases results from mutations in PS1, PS2, or amyloid precursor protein (APP) with a majority of cases in PS1 (3, 4). The pathogenesis of FAD includes as an early invariant the development of amyloid plaques containing specifically A␤42/43 polypeptide (3, 5). A␤42 is produced by sequential proteolytic cleavage of ␤ APP (6). PS1 appears to play a crucial role in the normal metabolism of APP as well as in the pathological increase of A␤42 (7). The exact function of PS1 in the processing of APP is still unclear. PS1 appears to be tightly associated within a multiprotein complex with ␥ secretase, the second of two proteases that cleave APP and that has not yet been identified (8). Recent evidence has suggested that PS1 itself may contain ␥ secretase catalytic activity (9 -11). Furthermore, evidence that an endoprotease activity crucial for normal biological function is contained in PS1 or requires PS1 has also been derived from examining the function of PS1 in Notch receptor cleavage and activation. The PS1 homologue in Caenorhabditis elegans, SEL-12, is required for Notch receptor signaling and cell fate determination (12). Similar to APP, Notch undergoes intramembrane p...

Deletion mapping of the human presenilin-1 (PS1) promoter delineated the most active fragment from ؊118 to ؉178 in relation to the transcription start site mapped in this study, in both human neuroblastoma SK-N-SH and hepatoma HepG2 cells. 5 deletions revealed that a crucial element controlling over 90% of the promoter activity in these cell lines is located between ؊22 and ؊6. A mutation altering only two nucleotides of the ETS consensus sequence present at ؊12 (GGAA to TTAA) has a similar effect. Electrophoretic mobility shift assays showed that a set of specific complexes between nuclear factors and the PS1 promoter are eliminated by this point mutation, as well as by competition with an ETS consensus oligonucleotide. Competition experiments in DNase I footprinting correlated with electrophoretic mobility shift assays and showed that only one of several footprints over the PS1 promoter is eliminated by competition with an ETS consensus oligonucleotide. It extends from ؊14 to ؊6 and surrounds the ETS motif present at ؊12. Thus, a crucial ETS element is present at ؊12 and binds a protein(s) recognizing specifically the ETS consensus motif. At least one such complex is eliminated by preincubating the nuclear extract with an antibody with broad cross-reactivity with Ets-1 and Ets-2 proteins, thus confirming that an ETS transcription factor(s) recognizes the ؊12 motif. Several Sp1 binding motifs at positions ؊70, ؊55, and ؉20 surround this ETS element. Competition DNase I footprinting showed that Sp1-like nuclear factors recognize specifically these sites in both cell lines. Furthermore, a combination of 5 and 3 deletions indicated the presence of positive promoter elements between ؊96 and ؊35 as well as between ؉6 and ؉42. Thus, transfection and footprinting assays correlate to suggest that Sp1 transcription factor(s) bind at several sites upstream and downstream from the initiation site and activate the transcription of the PS1 promoter. Sequences downstream from the transcription initiation site also contain major control elements. 3 deletions from ؉178 to ؉107 decreased promoter activity by 80%. However, further deletion to ؉42 increased promoter activity by 3-4-fold. Collectively, these data indicate that sequences upstream and downstream from the transcription start site each control over 80% of the promoter activity. Hence, this suggests that protein-protein interactions between factors recognizing downstream and upstream sequences are involved.

We have identified DNA sequences required for the expression of the presenilin 1 (PS1) gene. A promoter region has been mapped in SK‐N‐SH cells and includes sequences between −118 and +178 flanking the major initiation site (+1). The PS1 gene is also efficiently transcribed in the SH‐SY5Y subclone of SK‐N‐SH cells. However the promoter appears to be utilized in alternative ways in both cell types. Sequences both upstream as well as downstream from the initiation site mapped in SK‐N‐SH cells were shown by 5′‐ and 3′‐deletion analysis to play a crucial role in both cell lines. However, in SH‐SY5Y cells either upstream or downstream sequences are sufficient to direct transcription, whereas in SK‐N‐SH cells 5′‐deletions past the +1 site eliminate over0 95% of transcription. Several Ets motifs (GGAA) as well as Sp1 motifs [(G/T)GGCGGRRY] are juxtaposed both upstream and downstream from +1. To understand how the promoter may be utilized alternatively in different cell types we have examined the effect of point mutations in these elements. Altering an Ets motif at −10 eliminates 80% of transcription in SK‐N‐SH cells whereas the same mutation has only a minor effect in SH‐SY5Y cells. Conversely, mutation of the Ets element at +90, which eliminates 70% of transcription in SH‐SY5Y cells, has a lesser effect in SK‐N‐SH cells. In both cell types a promoter including mutations at both −10 and +90 sites loses over 90% transcription activity indicating the crucial importance of these two Ets motifs. The effect of Sp1 mutations appears to be similar in both cell types. Hence the differential expression in each cell type may be at least partially determined by Ets factors and the −10/+90 sites. We have identified several Ets factors that recognize specifically the −10 Ets motif by the yeast one‐hybrid selection including avian erythroblastosis virus E26 oncogene homologue 2, Ets‐like gene 1, Ets translocation variant 1 and Ets related molecule (ERM). We show here that ERM specifically recognizes Ets motifs on the PS1 promoter located at −10 as well as downstream at +90, +129 and +165 and activates PS1 transcription with promoter fragments containing or not the −10 Ets site.