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

Abstract

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 M 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 peroxidetreated 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.

DNA sequence determinants of nuclear protein binding to the c-Ha-ras antioxidant/electrophile response element in vascular smooth muscle cells: identification of Nrf2 and heat shock protein 90β as heterocomplex components

“…The size of this protein is consistent with preparative EMSA and UV cross-linking studies showing the presence of a predominant 80-kDa protein in ARE/EpRE protein complexes (Miller et al 2000;Holderman et al 2002). The PEI-purified protein fraction containing the 80-kDa protein was also found to contain proteins that bound specifically to the c-Ha-ras ARE/EpRE (Holderman et al 2002).…”

“…To identify c-Ha-ras ARE/EpRE binding proteins, a predominant 80-kDa band was resolved from nuclear ex- tracts of vSMCs after PEI purification (Holderman et al 2002). The size of this protein is consistent with preparative EMSA and UV cross-linking studies showing the presence of a predominant 80-kDa protein in ARE/EpRE protein complexes (Miller et al 2000;Holderman et al 2002).…”

“…It is likely that detection of Hsp90␤ by the PEI isolation method reflects its function as a chaperone with ARE/EpRE binding proteins pulled down with the PEI resin. As such, we previously have isolated and sequenced the zinc finger transcription factor Phi AP3 as a putative ARE/EpRE-binding protein (Holderman et al 2002). Because zinc finger proteins are known to interact with Hsp chaperones, this offers another possibility of the role of Hsp90␤ in our system.…”

“…The PEI purification used has been described previously (Holderman et al 2002). Slices were washed for 30 minutes in 1 mL of 30% methanol and then washed for 60 minutes in 500 L of 100 mM ammonium bicarbonate.…”

“…Several proteins have been identified to interact with ARE/EpREs (Li and Jaiswal 1994;Vasiliou et al 1995;Venugopal and Jaiswal 1996;Itoh et al 1997;Montano et al 1998Montano et al , 2000Moinova and Mulcahy 1999;Chen and Ramos 2000;Nguyen et al 2000;Zipper and Mulcahy 2000;Holderman et al 2002;Sankaranarayanan and Jaiswal 2004). Among these, the transcription factor Nrf2 has received much attention as a critical mediator of the cellular response to stress in multiple cells types, including HepG2 and neuronal cell lines.…”