NF-B and Matrix-Dependent Regulation of Osteopontin Promoter Activity in Allylamine-Activated Vascular Smooth Muscle Cells
Figure 5
Profiles of osteopontin promoter activity in control and allylamine-activated vascular smooth muscle cells. Panel (a) shows a schematic representation of the rat osteopontin promoter, cis-acting elements within the full-length promoter, and several deletions thereof. Panel (b) shows a comparison of osteopontin promoter activity in control and allylamine cells. Control and allylamine cells were seeded at equivalent densities and allowed to recover for 8 hours. Cultures were then transfected with the rat osteopontin promoter in pGL2-basic, a luciferase vector, or a deletion thereof (1157 rOPNpr, 672 rOPNpr, 329 rOPNpr, and 129 rOPNpr). Also included as a control was β-galactosidase, to allow normalization of luciferase signal. □: control, ■: allylamine. indicates significant difference from 2094 rOPNpr in control, + indicates significant difference from 1157 rOPNpr in control, # indicates significant difference from 657 rOPNpr in control (). Panel (c) identifies mutants generated site-directed mutagenesis of the rat osteopontin promoter on activity profiles in control and allylamine cells. Several sites were identified through computerized examination and mutated using Stratagene’s QuikChange Site-Directed Mutagenesis Kit. Alterations in each element are denoted by a capital letter. Panel (d) shows osteopontin promoter activity in control and allylamine cells after site-directed mutagenesis. Control and allylamine cells were seeded at equivalent densities and allowed to recover for 8 hours. Cultures were transfected with the rat osteopontin promoter (rOPNpr) in a luciferase vector, or a vector in which a cis-acting element has been mutated through site-directed mutagenesis. These mutants are denoted VDREx rOPNPr, AP-1x rOPNpr, NF-κBx rOPNpr, USF1x rOPNpr, USF2x rOPNpr, and USF3x rOPNpr. □: control, ■: allylamine. indicates significant difference from 2094 rOPNpr in control, + indicates significant difference from VDREx rOPNpr in control, # indicates significant difference from NF-κBx rOPNpr in control (). Similar results were seen in two separate experiments.
