Vol. 61, Issue 5, 1174-1183, May 2002

Identification of Albumin Precursor Protein, Phi AP3, and -Smooth Muscle Actin as Novel Components of Redox Sensing Machinery in Vascular Smooth Muscle Cells

M. T. Holderman, K. P. Miller, L. J. Dangott, and K. S. Ramos

Departments of Physiology and Pharmacology (M.T.H., K.P.M., K.S.R.) and Biochemistry and Biophysics (L.J.D.), and The Center for Environmental and Rural Health, Texas A&M University, College Station, Texas (L.J.D., K.S.R.)

Aerobic organisms are continually subjected to environmental stressors that compromise redox homeostasis and induce cellular injury. In vascular smooth muscle cells (vSMCs), the activation/repression of redox-regulated genes after environmental stress often involves protein binding to cis-acting antioxidant response elements (AREs). The present study was conducted to identify proteins that participate in redox-regulated protein binding to human c-Ha-ras and mouse glutathione S-transferase A1 AREs in vSMCs after oxidant injury. Challenge of vSMCs with 0.3 or 3 µM hydrogen peroxide, 3-methylcholanthrene, benzo[a]pyrene-7,8-diol, 3-hydroxy benzo[a]pyrene, and benzo[a]pyrene-3,6-quinone induced concentration-related increases in ARE protein binding. The profiles of ARE complex assembly were comparable, but exhibited chemical specificity. Pretreatment with 0.5 mM N-acetylcysteine inhibited activation of ARE protein binding in hydrogen peroxide-treated cells. Preparative electrophoretic mobility shift assays coupled to Western analysis identified NF-E2-related proteins 1 and 2 and JunD in complexes assembled on AREs. Polyethylenimine affinity and sequence-specific serial immobilized DNA affinity chromatography followed by N-terminal sequencing identified albumin precursor protein, phi AP3, and -smooth muscle actin as members of the ARE signaling pathway. Sequence analysis of albumin protein revealed homology to the redox-regulated transcription factors Bach1 and 2, as well as cytoskeletal and molecular motor proteins. These results implicate albumin precursor protein, phi AP3, and -smooth muscle actin as participants in redox sensing in vSMCs, and suggest that protein complex assembly involves interactions between leucine zipper and zinc finger transcription factors with cytoskeletal proteins.