Mechanical stretch induces vascular permeability factor in human mesangial cells: Mechanisms of signal transduction

Hemodynamic abnormalities have been implicated in the pathogenesis of the increased glomerular permeability to protein of diabetic and other glomerulopathies. Vascular permeability factor (VPF) is one of the most powerful promoters of vascular permeability. We studied the effect of stretch on VPF production by human mesangial cells and the intracellular signaling pathways involved. The application of mechanical stretch (elongation 10%) for 6 h induced a 2.4-fold increase over control in the VPF mRNA level (P < 0.05). There was a corresponding 3-fold increase in VPF protein level by 12 h (P < 0.001), returning to the baseline by 24 h. Stretch-induced VPF secretion was partially prevented both by the protein kinase C (PKC) inhibitor H7 (50 μM: 72% inhibition, P < 0.05) and by pretreatment with phorbol ester (phorbol-12-myristate-13 acetate 10−7 M: 77% inhibition, P < 0.05). A variety of protein tyrosine kinase (PTK) inhibitors, genistein (20 μg/ml), herbimycin A (3.4 μM), and a specific pp60src peptide inhibitor (21 μM) also significantly reduced, but did not entirely prevent, stretch-induced VPF protein secretion (respectively 63%, 80%, and 75% inhibition; P < 0.05 for all). The combination of both PKC and PTK inhibition completely abolished the VPF response to mechanical stretch (100% inhibition, P < 0.05). Stretch induces VPF gene expression and protein secretion in human mesangial cells via PKC- and PTK-dependent mechanisms.

A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

An improved mammalian two-hybrid system designed for interaction trap screening is described in this paper. CV-1/EBNA-1 monkey kidney epithelial cells expressing Epstein–Barr virus nuclear antigen 1 (EBNA-1) were stably transfected with a reporter plasmid for GAL4-dependent expression of the green fluorescent protein (GFP). A resulting clone, GB133, expressed GFP strongly when transfected transiently with transcriptional activators fused to GAL4 DNA-binding domain with minimal background GFP expression. GB133 cells maintained plasmids containing the OriP Epstein–Barr virus replication origin that directs replication of plasmids in mammalian cells in the presence of the EBNA-1 protein. GB133 cells transfected stably with a model bait expressed GFP when further transfected transiently with an expression plasmid for a known positive prey. When the bait-expressing GB133 cells were transfected transiently with an OriP-containing expression plasmid for the positive prey together with excess amounts of empty vector, cells that received the positive prey were readily identified by green fluorescence in cell culture and eventually formed green fluorescent microcolonies, because the prey plasmid was maintained by the EBNA-1/Ori-P system. The green fluorescent microcolonies were harvested directly from the culture dishes under a fluorescence microscope, and total DNA was then prepared. Prey-encoding cDNA was recovered by PCR using primers annealing to the vector sequences flanking the insert-cloning site. This system should be useful in mammalian cells for efficient screening of cDNA libraries by two-hybrid interaction.