Development of a DNA transfer technique for human lung cells with synthetic peptide Tat-RGD and its application for regulatory and functional analysis of RELMB
The presented study is organized in two sections. In the first part a DNA-transfection procedure for human lung cells employing a peptide conjugate has been established and the corresponding transfection process characterized. In the second part this new transfection strategy has been applied for studying functional aspects of the human RELMBbeta gene.
Cell-lines and primary cells exhibit a varying degree of resistance to DNA
transfection strategies. In this study, we employed the synthetic peptide TatRGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into human pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded in significant transgene expression. When combining TRD with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by ~30-fold over TRD mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL mediated transfection efficiency was observed by a factor of ~ 2 and ~ 3 over that of DL. Laser scanning confocal microscopy for visualizing TR dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the TatRGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with standard cationic lipid based transfection reagent. The enhancement of DNA uptake by TatRGD is suggested to be mediated by caveoli dependent endocytosis.
RELMBbeta(resistin-like molecule) represents the most related human homologue of mouse RELMBbeta also known as hypoxic-induced-mitogenic-factor (HIMF) for which no human orthologue gene exist. In this part of the study, we isolated RELMBbeta cDNA from human lung tissue and performed regulatory and functional expression studies with the TatRGD procedure. RELMBbeta mRNA was upregulated in hypoxia in human lung A549 cell line as well as primary cultured adventitial fibroblasts (FB) and smooth muscle cells (SMC) of pulmonary artery. Upon transfection of a RELMBbeta encoding expression plasmid into these cells, we observed significant induction of proliferation particularly in SMC and A549 cells. The results suggest that human RELMBbeta may contribute to hypoxic induced pulmonary vascular remodeling processes or hypoxia related fibrotic lung disease.
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