Analysis of genetic control elements in eukaryotes: transcriptional activity or nuclear hitchhiking?

A common way to analyse basal and stimulated activity of eukaryotic genetic control elements, such as promoters and enhancers, is to introduce them into cells via DNA vectors containing an easily assayable reporter gene. Activity is then studied by measurement of transiently produced mRNA or reporter protein. In such assays, it is assumed that the variable measured is proportional to the transcriptional activity of the control element under investigation. Here we question the validity of this generally accepted assumption. Specifically, recent observations indicate that control elements, in addition to modulating transgene transcription, can facilitate the nuclear uptake of their carrier plasmids. This process is mediated by transcription factors or other nuclear proteins harbouring nuclear localisation signals, which bind to the control elements in the cytoplasm and transport the DNA into the nucleus through the protein nuclear import machinery. As the number of mRNA transcripts produced for an epi-chromosomally expressed transgene is directly related to its copy number inside the nucleus, such transport activity may lead to substantial overestimation of the transcriptional potency of the control element(s) studied.

Surfactant protein D-coated Klebsiella pneumoniae stimulates cytokine production in mononuclear phagocytes.

Encapsulated Klebsiella pneumoniae strains K21a, K10, and K50, all of which contain dimannose sequences in their capsular polysaccharides that are recognized by the mannose receptor of macrophages, stimulated interleukin secretion and cytokine mRNA expression by human monocyte-derived macrophages. By contrast, the corresponding unencapsulated phase variants and the K2 strain, which lack the dimannose sequence, did not. Coating of unencapsulated phase variants of Klebsiella strains with surfactant protein (SP)-D resulted in marked stimulation of cytokine mRNA accumulation. The induction of cytokine mRNA via the mannose receptor occurred only in monocyte-derived macrophages, whereas that caused by SP-D-coated Klebsiella strains occurred in both macrophages and peripheral-blood monocytes. The results suggested that innate immunity against pulmonary pathogens might be mediated by SP-D, which acts as an opsonin to enhance the interaction of macrophages with unencapsulated phase variants originating from the upper respiratory tract, and by macrophage mannose receptors, which recognize encapsulated variants expressing capsular dimannose residues.

A regulated, NFkappaB-assisted import of plasmid DNA into mammalian cell nuclei.

The success of synthetic DNA delivery systems in human gene therapy will be enhanced by increasing transfection efficiencies and by providing tighter control over targeting of the DNA into the nucleus. Here, we used DNA vectors that contain repetitive binding sites for the inducible transcription factor NFkappaB, which is transported into the nucleus by the nuclear import machinery. Nuclear entry of the modified vectors was augmented 12-fold and was associated with corresponding increase in gene expression. Depending on their position, the binding sites could also function as transcriptional enhancers, increasing gene expression levels up to an additional 19-fold. Notably, nuclear targeting of the DNA and transgene transcription could both be regulated by exogenous stimulators which modulate the intracellular distribution of NFkappaB. The approach provides a framework for the controlled targeting of constitutive or transcriptionally regulated synthetic vectors into mammalian cell nuclei.

Surfactant protein D enhances phagocytosis and killing of unencapsulated phase variants of Klebsiella pneumoniae.

Pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin (collectin) that is secreted into the alveoli and distal airways of the lung. We have studied the interactions of SP-D and alveolar macrophages with Klebsiella pneumoniae, a common cause of nosocomial pneumonia. SP-D does not agglutinate encapsulated K. pneumoniae but selectively agglutinates spontaneous, unencapsulated phase variants, such as Klebsiella strain K50-3OF, through interactions with their lipopolysaccharides (LPS). These effects are calcium dependent and inhibited with maltose but not lactose, consistent with involvement of the SP-D carbohydrate recognition domain. Precoating of K50-3OF with SP-D enhances the phagocytosis and killing of these organisms by rat alveolar macrophages in cell culture and stimulates the production of nitric oxide by the NR-8383 rat alveolar macrophage cell line. SP-D similarly enhances the NO response to K50-3OF LPS adsorbed to Latex beads under conditions where soluble LPS or SP-D, or soluble complexes of SP-D and LPS, do not stimulate NO production. Our studies demonstrate that interactions of SP-D with exposed arrays of Klebsiella LPS on a particulate surface can enhance the host defense activities of alveolar macrophages and suggest that activation of macrophages by SP-D requires binding to microorganisms or other particulate ligands. Because unencapsulated phase variants are likely to be responsible for the initial stages of tissue invasion and infection, we speculate that SP-D-mediated agglutination and/or opsonization of K. pneumoniae is an important defense mechanism for this respiratory pathogen in otherwise healthy individuals.