Phorbol ester stimulates expression of the human tryptophanyl-tRNA synthetase gene

The effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on expression of the human interferon (IFN)-inducible tryptophanyl-tRNA synthetase (WRS) gene was studied. PMA caused an increase in the basal and IFN-induced WRS protein content in HeLa and HEK293 cultured cells. Besides, PMA upregulated WRS mRNA level in HeLa cells. Since PMA is known as a selective activator of protein kinase C (PKC) and is widely used to study the PKC-related pathways, these results show possible PKC involvement in regulation of the WRS gene expression. PKC inhibition by staurosporine (10 and 100 nM) had no effect on either basal or IFN-induced expression of WRS in either cell line. Consequently, PKC is not an indispensable element in WRS induction by IFN. Rather, PKC may activate WRS gene expression only by a distinct pathway.     

An Escherichia coli gene divergently transcribed from a promoter overlapping the metA promoter

The upstream region of the metA gene in Escherichia coli contains two promoters. We have identified by lacZ fusion an additional promoter in this region, and showed that it is transcribed in the opposite orientation from the metA gene. The putative translation product corresponds to a peptide of 147 amino acids – ORF19 by molecular mass. This peptide is probably not essential for growth, as an insertion mutant is viable.     

Functional role of the cis-acting elements in human monocyte chemotactic protein-1 gene in the regulation of its expression by phorbol ester in human glioblastoma cells

The monocyte chemotactic protein-1 (MCP-1) is a 76 amino acid protein that specifically attracts monocytes. The expression of MCP-1 gene can be induced by lipopolysaccharides (LPS), phorbol esters (TPA) and several cytokines. However, how they regulate MCP-1 gene expression is not known. We tested whether the two putative TPA-responsive elements (TREs) and one B enhancer-like region found in the MCP-1 promoter region, are involved in this regulation of MCP-1 gene expression. The 5 untranslated region of MCP-1 gene was linked to chloramphenicol acetyl transferase (CAT) reporter gene and transfected into human glioblastoma cells in which endogenous MCP gene expression was found to be stimulated by TPA and tumor necrosis factor- (TNF-). The 128 bp 5-flanking region containing one TRE was adequate for basal promoter activity but the presence of both TREs in the MCP-1 promoter region were needed to give TPA responsive enhancement (2.5 fold) of expression of the marker gene. Mutations in either of the TRE's could abolish the TPA induction of CAT expression. Replacement of the B enhancer-like element with a TRE-like sequence caused a 10-fold enhancement of CAT expression by TPA treatment. Random mutation of B enhancer-like element did not affect CAT expression or its TPA induction. None of the MCP promoter constructs showed significant increase in CAT expression by treatment with tumor necrosis factor- (TNF-). This result suggested that the TNF regulation of MCP-1 gene involves other parts of the gene besides the proximal 5 flanking region. (Mol Cell Biochem141: 121–128, 1994)     

Sp1 and Sp3 transcription factors upregulate the proximal promoter of the human prostate-specific antigen gene in prostate cancer cells

The serum level of prostate-specific antigen (PSA) is useful as a clinical marker for diagnosis and assessment of the progression of prostate cancer, and in evaluating the effectiveness of treatment. We characterized four Sp1/Sp3 binding sites in the proximal promoter of the PSA gene. In a luciferase assay, these sites contributed to the basal promoter activity in prostate cancer cells. In an electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we confirmed that Sp1 and Sp3 bind to these sites. Overexpression of wild-type Sp1 and Sp3 further upregulated the promoter activity, whereas overexpression of the Sp1 dominant-negative form or addition of mithramycin A significantly reduced the promoter activity and the endogenous mRNA level of PSA. Among the four binding sites, a GC box located at nucleotides -53 to -48 was especially critical for basal promoter activity. These results indicate that Sp1 and Sp3 are involved in the basal expression of PSA in prostate cancer cells.     

Phorbol ester potentiates the growth inhibitory effects of troglitazone via up-regulation of PPARgamma in A549 cells

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to induce growth arrest and differentiation of various cancer cells. In the current study, we investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of PPARgamma and proliferation of A549 cells. TPA elicited a dose- and time-dependent increase in PPARgamma mRNA and protein levels. PPARgamma expression in response to TPA was attenuated by pretreatment with bisindolylmaleimide I, N-acetyl-L-cysteine (NAC) and PD98059. TPA-induced protein kinase C (PKC) activation was linked to the generation of reactive oxygen species (ROS), both of which were indispensable for PPARgamma expression in A549 cells. Pretreatment with bisindolylmaleimide I or NAC blocked TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK), suggesting that ERK-mediated signaling is also involved in the induction of PPARgamma. Furthermore, the growth inhibitory effect of troglitazone was significantly potentiated by prolonged incubation with TPA and was attenuated in the presence of GW9662, a specific inhibitor of PPARgamma. These effects were associated with an induction of cell cycle arrest at G0/G1 phase, which was accompanied by the induction of p21Waf1/Cip1 expression and decreased cyclin D1 expression. Taken together, these observations indicate that TPA synergizes with PPARgamma ligand to inhibit cell growth through up-regulation of PPARgamma expression.     

Signal transduction and virulence regulation in human and animal pathogens

Abstract Pathogens have developed many strategies for survival in animals and humans which possess very effective defense mechanisms. Although there are many different ways, in which pathogenic bacteria solved the problem to overcome the host defense, some common features of virulence mechanisms can be detected even in phylogenetically very distant bacteria (Finlay and Falkow (1989) Microb. Rev. 6 1375–1383). One important feature is that the regulation of expression of virulence factors and the exact timing of their expression is very important for many of the pathogenic bacteria, as most of them have to encounter different growth situations during an infection cycle, which require a fast adaptation to the new situation by the expression of different factors. This review gives an overview about the mechanisms used by pathogenic bacteria to accomplish the difficult task of regulation of their virulence potential in response to environmental changes. In addition, the relationship of these virulence regulatory systems with other signal transduction mechanisms not involved in pathogenicity is discussed.