Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues.

Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.

TIP30 directly binds p53 tumor suppressor protein in vitro.

TIP30 (30 kDa HIV-1 TAT-interacting protein), also called HTATIP2 or CC3, is a tumor suppressor protein that acts as an angiogenesis inhibitor. TIP30 blocks nuclear import of the mRNA-binding protein HuR, and thereby promotes the cytoplasmic accumulation of HuR by binding to importin-ß, which is known to facilitate the cytoplasm-tonuclear transport of HuR. Accumulation of HuR in the cytoplasm, in turn, enhances the expression of the transcription factor p53, a tumor suppressor that plays an essential role in preserving genome stability and inhibiting cancer growth. In addition to such a post-transcriptional mechanism via which TIP30 increases the p53 level, it has been proposed that TIP30 may regulate p53 protein at the protein level by directly binding to it. In order to investigate the possibility of direct interaction between p53 and TIP30, we have used on three functional regions in p53 and examined their interactions with TIP30 using GST pull-down assay and surface plasmon resonance technique. The results show that that TIP30 binds to the DNA-binding domain and the C-terminal domain of p53.

Cytosolic malate dehydrogenase regulates senescence in human fibroblasts.

Carbohydrate metabolism changes during cellular senescence. Cytosolic malate dehydrogenase (MDH1) catalyzes the reversible reduction of oxaloacetate to malate at the expense of reduced nicotinamide adenine dinucleotide (NADH). Here, we show that MDH1 plays a critical role in the cellular senescence of human fibroblasts. We observed that the activity of MDH1 was reduced in old human dermal fibroblasts (HDFs) [population doublings (PD) 56], suggesting a link between decreased MDH1 protein levels and aging. Knockdown of MDH1 in young HDFs (PD 20) and the IMR90 human fibroblast cell line resulted in the appearance of significant cellular senescence features, including senescence-associated ß-galactosidase staining, flattened and enlarged morphology, increased population doubling time, and elevated p16(INK4A) and p21(CIP1) protein levels. Cytosolic NAD/NADH ratios were decreased in old HDFs to the same extent as in MDH1 knockdown HDFs, suggesting that cytosolic NAD depletion is related to cellular senescence. We found that AMP-activated protein kinase, a sensor of cellular energy, was activated in MDH1 knockdown cells. We also found that sirtuin 1 (SIRT1) deacetylase, a controller of cellular senescence, was decreased in MDH1 knockdown cells. These results indicate that the decrease in MDH1 and subsequent reduction in NAD/NADH ratio, which causes SIRT1 inhibition, is a likely carbohydrate metabolism-controlled cellular senescence mechanism.

Processed short neuropeptide F peptides regulate growth through the ERK-insulin pathway in Drosophila melanogaster.

The Drosophila sNPF gene regulates growth through the ERK-insulin pathway. sNPF encodes a precursor protein that is processed and produces biologically active sNPF peptides. However, the functions of these peptides are not known. In Drosophila neuronal cells in culture and in flies in vivo, sNPF1 and sNPF2 activated the ERK-insulin pathway and regulated body growth. In addition, the sNPF precursor and the processed sNPF peptide were co-localized in the neurons of the central nervous system. These results indicate that sNPF1 and sNPF2 peptides processed from the sNPF precursor are sufficient for regulating body growth through the ERK-insulin pathway in Drosophila.

Clusterin mRNA expression in apoptotic and activated rat thymocytes.

Clusterin is a 75-80 kDa heterodimeric glycoprotein, that is produced in most tissues but which exact biological role is still not clear. Particularly, its role in protection or promotion of apoptosis is heavily disputed, since data supporting both views have been reported in several independent studies. To clarify this issue, and also to determine whether clusterin expression itself might be affected by apoptosis, in the present study, rat thymocytes were treated with dexamethasone, -a synthetic glucocorticoid that elicits apoptosis in thymocytes-, and clusterin mRNA expression was analyzed by semi-quantitative RT-PCR before and after induction of apoptosis. Interestingly, neither the treatment with dexamethasone in vitro nor triggering of apoptosis in vivo up- regulated clusterin expression, opposing the view that clusterin is involved in apoptotic processes. On the other hand, a new clusterin mRNA isoform was detected and isolated, whose expression was restricted to freshly isolated thymocytes. This novel isoform lacks the post-translational proteolytic cleavage site and is therefore predicted to encode a monomeric protein. The biological function under normal circumstances, however, will need further investigations for clarification. While apoptosis could not modulate clusterin expression, activation of thymocytes with concanavalin A and interleukin-2 resulted in up-regulation of clusterin mRNA level, indicating that clusterin expression is rather under the control of cell activation-mediated rather than apoptosis-induced signals.

Leptin receptor isoform expression in rat osteoblasts and their functional analysis.

The genetic defect in producing the adipose hormone leptin results among others in a drastic increase of bone mass. The current understanding is that under normal circumstances, osteoblast activity is indirectly suppressed by a hypothalamic relay induced by leptin-signalling in the brain. To investigate whether leptin might also regulate osteoblast activity in a direct manner, expression of leptin receptors in rat osteoblasts was determined and their functionality was analyzed upon recombinant leptin treatment. Reverse transcription-PCR confirmed the expression of four among the six currently described receptor isoforms, which were also able to transduce cell signalling as shown by STAT3 phosphorylation after activation.