Role of High Glucose–Induced Nuclear Factor– B Activation in Monocyte Chemoattractant Protein-1 Expression by Mesangial Cells

Although high glucose (HG) has been shown to induce nuclear factor– B (NFB) activation in vascular cells, the upstream regulation and the biologic significance of NFB activation in diabetic renal injury are not clear. It was, therefore, examined if HG-induced generation of reactive oxygen species (ROS) and protein kinase C (PKC) activation are involved in NFB activation in mesangial cells (MC), and the role of NFB activation in HG-induced monocyte chemoattractant protein-1 (MCP-1) expression by MC was further investigated. Recent observations suggest that MCP-1 may play a role in the development and progression of diabetic nephropathy. HG rapidly induced NFB activation in MC as estimated by electrophoretic mobility shift assay. Supershift assay suggests that most of the binding activity arose from p50/p50 and p50/p65 dimers. Antioxidants, pyrrolidine dithiocarbamate, N-acetyl-L-cystein, and trolox effectively inhibited HG-induced NFB activation in MC. HG rapidly generated dichlorofluorescin-sensitive intracellular ROS in MC as measured by laser-scanning confocal microscopy. HG also activated PKC rapidly in MC. Inhibition of PKC effectively blocked HG-induced intracellular ROS generation and NFB activation in MC. HG increased MCP-1 mRNA expression by 1.9-fold and protein secretion by 1.6-fold that of control glucose in MC transfected with control vector but not in MC transfected with dominant negative mutant inhibitor of NFB (I B M). Inhibition of either PKC or ROS effectively blocked HG-induced, but not basal, MCP-1 protein secretion by MC transfected with control vector. Thus this study demonstrates that HG rapidly activates NFB in MC through PKC and ROS and suggests that HG-induced NFB activation in MC may play a role in diabetic renal injury through upregulation of MCP-1 mRNA and protein expression. Nuclear factor– B (NFB) is a transcription factor widely distributed in most cell types, including glomerular mesangial cells (MC) (1,2). It mainly consists of dimers of the two subunits p50 and p65 (Rel A) and exists in inactive form in the cytoplasm associated with an inhibitory protein called I B (3). Activation of NFB results from the release of the inhibitory I B subunit from the heterotrimeric complex followed by translocation of dimer to the nucleus, where it initiates transcription (3). Activation of NFB can be induced by various molecules, such as cytokines, lipopolysaccharide (LPS), phorbol ester, and reactive oxygen species (ROS), and controls the transcription of several genes involved in immune and inflammatory responses, cell growth, and adhesion (3,4). Increasing evidences suggest that NFB may play an important role in MC activation leading to renal injury (1,2). High glucose (HG) is the main determinant of the development and progression of diabetic nephropathy in both type 1 (5) and type 2 diabetes (6). As in other diabetic vascular complications (7,8), we and others have proposed that oxidative stress generated by hyperglycemia is one of the major mediators of diabetic nephropathy (9–12). Recent studies suggest that the activation of NFB is one possible mechanism for oxidative stress-induced vascular complications in diabetes. NFB activation in isolated peripheral blood mononuclear cells of patients with poorly controlled type 1 diabetes was significantly prevented by an antioxidant thioctic acid (13). Cardiac tissues of diabetic rats also show increased NFB activation associated with oxidative stress (14). In addition, HG can induce NFB activation in endothelial (15–17) and vascular smooth muscle cells (18). A recent report (19) demonstrating higher renal expression of NFB system in experimental diabetic rats than in control animals suggests possible involvement of NFB activation in diabetic renal injury as well. However, it is not known if HG activates NFB in MC or if HG-induced NFB activation plays a role in diabetic renal injury. Thus, the following were the objectives of our present study: (1) to determine if HG can activate NFB in MC; (2) to examine if HG-induced ROS and protein kinase C (PKC) are involved in NFB activation to understand the upstream regulation of HG-induced NFB activation in MC; and (3) to evaluate the role of NFB Received March 7, 2001. Accepted October 3, 2001. Correspondence to: Dr. Hi Bahl Lee, Hyonam Kidney Laboratory, Soon Chun Hyang University, 657 Hannam Dong, Yongsan Ku, Seoul 140-743, Korea. Phone: 82-2-709-9171, 82-2-792-6657; Fax: 82-2-792-5812; E-mail: