Mechanical stretch induces platelet-activating factor receptor gene expression through the NF-kappaB transcription factor.
In this study, we used the platelet-activating factor (PAF) receptor gene as a model of a mechano-sensitive gene to investigate how mechanical stimuli regulate gene expression and cell function. We utilized a culture system of pulmonary artery smooth muscle cells and a well-defined in vitro mechanical 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 levels after 1 h exposure of the cells to a 5% strain at a frequency of 1 Hz. To investigate the mechanism of activation of this gene by stretch, we performed transfection experiments with a luciferase reporter gene linked to segments 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 was enhanced in mechanically stretched cells indicating a direct effect on transcriptional activity. When truncated to leave the nucleotides between -610 to +27, the promoter-reporter construct lost stretch inducibility suggesting that the region between -1099 and -610 was required for stretch responsiveness. This region contains four copies of NF-kappaB binding sites. These elements are in close proximity to one another and can form a complex with nuclear proteins derived from stretched cells as demonstrated by gel mobility shift assay. Moreover, in experiments using cycloheximide, we found that de novo protein synthesis was not necessary for the induction of the PAF receptor gene expression by mechanical stretch. Conversely, preincubation of the cells with protein kinase C inhibitors suppressed mechanical stretch-induced PAF receptor gene expression at the mRNA levels and abrogated upstream events of NF-kappaB activation in the cytoplasm. These data strongly suggest that stretch-induced PAF receptor gene expression is mediated by NF-kappaB binding to the PAF receptor gene promoter and that protein kinase C activation is among the molecular features of NF-kappaB activation and translocation into the nucleus in mechanically stretched cells.
University of Pennsylvania, Department of Histology, 416 Levy Research Building, Philadelphia, PA 19104, USA. chaqour@biochem.dental.upenn.edu
B
B
Chaqour
P S
PS
Howard
C F
CF
Richards
E J
EJ
Macarak
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D009281:Naphthalenes / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010812:Physical Stimulation; D010980:Platelet Membrane Glycoproteins / Q000235:genetics*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D019311:Staurosporine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009281:Naphthalenes; D019384:Nucleic Acid Synthesis Inhibitors; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D003513:Cycloheximide; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C