Mechanical stretch induces platelet-activating factor receptor gene expression through the NF-kappa B transcription factor

In this study, we used the platelet-activating fatter (PAF) receptor gene a s a model of a mechano-sensitive gene to investigate how mechanical stimuli regulate gene expression and cell function, We utilized a culture system o f pulmonary artery smooth muscle cells and a well-defined ii? vitro mechani cal device that imparts an equibiaxial strain repeatedly to cells attached to an elastomeric membrane. Northern blot and immunohistochemical analyses revealed increased PAF receptor expression at both the mRNA and protein lev els after Ih exposure of the cells to a 5% strain at a frequency of 1Hz. To investigate the mechanism of activation of this gene by stretch, we perfor med transfection experiments with a luciferase reporter gene linked to segm ents of the 5' flanking region of the receptor gene promoter. Expression of the transfected reporter gene bearing a 1.1-kb fragment of the promoter wa s enhanced in mechanically stretched cells indicating a direct effect on tr anscriptional activity. When truncated to leave the nucleotides between -61 0 to +27, the promoter-reporter construct lost stretch inducibility suggest ing that the region between -1099 and -610 was required for stretch respons iveness, This region contains four copies of NF-kappa B binding sites. Thes e elements are in close proximity to one another and can form a complex wit h nuclear proteins derived From stretched cells as demonstrated by gel mobi lity shift assay. Moreover, in experiments using cycloheximide, we found th at de nova protein synthesis was not necessary for the induction of the PAF receptor gene expression by mechanical stretch. Conversely preincubation o f the cells with protein kinase C inhibitors suppressed mechanical stretch- induced PAF receptor gene expression at the mRNA levels and abrogated upstr eam events of NF-kappa B activation in the cytoplasm. These dal-a strongly suggest that stretch-induced PAF receptor gene expression is mediated by NF -kappa B binding to the PAF receptor gene promoter and that protein kinase C activation is among the molecular features of NF-kappa B activation and t ranslocation into the nucleus in mechanically stretched cells. (C) 1999 Aca demic Press.