Ability of Egr1 to activate tyrosine hydroxylase transcription in PC12 cells. Cross-talk with AP-1 factors.

We have recently identified an Egr1 motif that overlaps with the Sp1 element in the tyrosine hydroxylase (TH) promoter. Here we examine whether this motif has a functional role in the regulation of TH transcription in PC12 cells. In nuclear extracts from control PC12 cells, an oligonucleotide containing the TH Sp1/Egr1 motif binds Sp1-containing complexes. Treatment of PC12 cells with phorbol ester (2 micrometer 12-O-tetradecanoylphorbol-13-acetate (TPA)) gives rise to a new Egr1-containing complex. TPA treatment reduces the steady-state levels of the Sp1 protein and leads to the appearance of immunoreactive Egr1 protein within 30-60 min. Expression of the Egr1 protein in PC12 cells stimulates the chloramphenicol acetyltransferase reporter gene placed under the control of the first 272 nucleotides of the rat TH promoter. Site-directed mutagenesis of either the Sp1/Egr1 motif or of an upstream AP-1 motif or both abolishes the Egr1-mediated induction of chloramphenicol acetyltransferase activity. An oligonucleotide encompassing the AP-1/E-box sequence of the rat TH promoter competes in electrophoretic mobility shift assays for binding of nuclear extracts from control and TPA-treated cells to an oligonucleotide containing the Sp1/Egr1 element, indicating that these two enhancers may interact. The results show that Egr1 can activate TH transcription and reveals cross-talk between Sp1/Egr1 and AP-1 factors.

Sp1/Egr1 motif: a new candidate in the regulation of rat tyrosine hydroxylase gene transcription by immobilization stress.

The rat tyrosine hydroxylase (TH) promoter contains a GC box (Sp1 motif) whose function is currently unclear. In this study, we examined the effect of immobilization (IMO) stress on binding of adrenomedullary transcription factors to that site. DNase I footprinting analysis reveals the binding of proteins to the Sp1 motif. Extracts from the adrenal medulla of IMO-treated rats generate a footprint on the Sp1 motif that is smaller than that generated by control extracts. Electrophoretic mobility shift assays using an oligonucleotide containing the Sp1 region show that two Sp1 protein-containing complexes (I and III) form with control extracts. In contrast, extracts from the adrenal medulla of IMO-treated rats form a novel complex (II) that contains the Egr1 protein. This is the first report to reveal changes in the binding pattern on the TH Sp1 motif in response to stress and to identify an overlapping Egr1 site.

Cellular changes and induction of apoptosis in human promyelocytic HL-60 cells infected with the agent of human granulocytic ehrlichiosis (HGE).

Human granulocytic ehrlichiosis (HGE) is an emerging and occasionally fatal human infectious disease whose pathogenesis is largely unknown. Goodman et al. (1) recently described the successful cultivation of the HGE infectious agent in human promyelocytic HL-60 leukemic cells. It was reported in the same study that infectivity invariably led to host cell death, although the mechanism by which HGE infection triggers cellular self-destruction is as yet undetermined. In this communication, we show that in vitro passage of HGE pathogen-infected blood elicits a significantly dysfunctional G1-to-S transition. Moreover, we provide evidence that the cytopathic properties of the HGE pathogen are attributed to its ability to induce apoptosis in host HL-60 cells. Determination of specific protein expression changes by Western blot analysis showed that HGE infection resulted in reduced expression of PCNA and pRB, both of which play a role in cell cycling. Moreover, the steady state level of bcl-2, which protects eukaryotic cells against apoptosis, is suppressed by exposure to the HGE agent. These results suggest that this pathogen HGE induces apoptosis in HL-60 cells by a mechanism involving the shut-off of multiple cell cycle and apoptosis regulatory events.