Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. The activity of Nrf2 is regulated by the Cul3 adaptor Keap1, yet little is known regarding mechanisms of regulation of Keap1 itself. Here, we have used immunopurification of Keap1 and mass spectrometry, in addition to immunoblotting, to identify sequestosome 1 (SQSTM1) as a cellular binding partner of Keap1. SQSTM1 serves as a scaffold in various signaling pathways and shuttles polyubiquitinated proteins to the proteasomal and lysosomal degradation machineries. Ectopic expression of SQSTM1 led to a decrease in the basal protein level of Keap1 in a panel of cells. Furthermore, RNA interference (RNAi) depletion of SQSTM1 resulted in an increase in the protein level of Keap1 and a concomitant decrease in the protein level of Nrf2 in the absence of changes in Keap1 or Nrf2 mRNA levels. The increased protein level of Keap1 in cells depleted of SQSTM1 by RNAi was linked to a decrease in its rate of degradation; the half-life of Keap1 was almost doubled by RNAi depletion of SQSTM1. The decreased level of Nrf2 in cells depleted of SQSTM1 by RNAi was associated with decreases in the mRNA levels, protein levels, and function of several Nrf2-regulated cell defense genes. SQSTM1 was dispensable for the induction of the Keap1-Nrf2 pathway, as Nrf2 activation by tert-butylhydroquinone or iodoacetamide was not affected by RNAi depletion of SQSTM1. These findings demonstrate a physical and functional interaction between Keap1 and SQSTM1 and reveal an additional layer of regulation in the Keap1-Nrf2 pathway.

Nucleotide activation of P2 receptors is important in autocrine and paracrine regulation in many tissues. In the epidermis, nucleotides are involved in proliferation, differentiation, and apoptosis. In this study, we have used a combination of luciferin-luciferase luminometry, pharmacological inhibitors, and confocal microscopy to demonstrate that HaCaT keratinocytes release ATP into the culture medium, and that there are three mechanisms for nucleotide interconversion, resulting in ATP generation at the cell surface. Addition of ADP, GTP, or UTP to culture medium elevated the ATP concentration. ADP to ATP conversion was inhibited by diadenosine pentaphosphate, oligomycin, and UDP, suggesting the involvement of cell surface adenylate kinase, F 1 F 0 ATP synthase, and nucleoside diphosphokinase (NDPK), respectively, which was supported by immunohistochemistry. Simultaneous addition of ADP and GTP elevated ATP above that for each nucleotide alone indicating that GTP acts as a phosphate donor. However, the activity of NDPK, F 1 F 0 ATP synthase or the forward reaction of adenylate kinase could not fully account for the culture medium ATP content. We postulate that this discrepancy is due to the reverse reaction of adenylate kinase utilizing AMP. In normal human skin, F 1 F 0 ATP synthase and NDPK were differentially localized, with mitochondrial expression in the basal layer, and cell surface expression in the differentiated layers. We and others have previously demonstrated that keratinocytes express multiple P2 receptors. In this study we now identify the potential sources of extracellular ATP required to activate these receptors and provide better understanding of the role of nucleotides in normal epidermal homeostasis and wound healing.Extracellular nucleotides, such as ATP and ADP, are now recognized as important autocrine and paracrine factors involved in the regulation of many cellular processes in a wide range of tissues. They act via activation of the P2 family of receptors of which there are two subgroups: the P2X receptors (ligand-gated ion channels) and the P2Y receptors (G proteincoupled receptors) as determined by their molecular structure, transduction pathways, and pharmacological properties (1). There are currently seven members of the P2X subgroup (P2X 1-7 ) (2) and eight members of the P2Y subgroup (P2Y 1,2,4,6,11-14 ) identified in mammalian cell types (3).In the epidermis, P2 receptors are involved in the regulation of proliferation, differentiation, and apoptosis, and thus subtypes are expressed in different regions. Differential expression of multiple P2 receptor subtypes is species-and cell type-dependent (4 -7). Although P2Y 2 receptor expression is almost entirely confined to the proliferative basal layer (7, 8), P2X 5 receptors are expressed in keratinocytes in the early stages of differentiation, i.e. the spinous layer, and P2X 7 receptors are confined to the terminally differentiated cells of the cornified layer (8, 9). In vitro, normal human keratinocytes express mRNA for P2Y 1 , P2Y 2...

Allergic contact dermatitis is commonly associated with exposure to p-phenylenediamine. The aim of this study was to determine whether p-phenylenediamine (PPD) and/or Bandrowski's base (BB) stimulate T cells from allergic patients and volunteers, and to explore the relationship between T-cell immunogenicity and allergy. Lymphocytes from allergic patients proliferated with PPD and BB (n=8). Lymphocytes from 14/16 non-allergic individuals also proliferated following stimulation, but only with BB; cord blood lymphocytes failed to respond (n=6). Glutathione, which prevented BB formation, but not binding of PPD to cells and serum, did not prevent p-phenylenediamine-specific stimulation of patient lymphocytes. T-cell clones generated from allergic patients were stimulated separately with PPD and BB, while clones from volunteers proliferated with BB alone. Patient and volunteer clones secreted IL-4, IL-5, IL-13, TNF-alpha, MIP-1alpha, MIP-1beta, and RANTES. These data show that activation of T lymphocytes from allergic individuals alone with PPD represents an important discrimination between allergic and non-allergic groups. BB-specific T cells are found in both allergic patients and volunteers, but not in cord blood. Their presence seems to reflect an acquired immune response, which is not translated into an allergic reaction.

Juvenile neuronal ceroid lipofuscinosis, or Batten disease, is an autosomal recessive disorder characterized by progressive loss of motor and cognitive functions, loss of vision, progressively severe seizures, and death. The disease is associated with mutations in the gene CLN3, which encodes a novel 438 amino acid protein, the function of which is currently unknown. Protein secondary structure prediction programs suggest that the CLN3 protein has ¢ve to seven membrane-spanning domains (MSDs). To distinguish among a number of hypothetical models for the membrane topology of CLN3 we used in vitro translation of native, Flag epitope-labeled and glycosylation site-mutated CLN3 protein in the presence or absence of canine pancreatic microsomes. These were immunoprecipitated using antibodies speci¢c for Flag or peptide sequences within CLN3 or left untreated. The results indicate that CLN3 contains ¢ve MSDs, an extracellular/intraluminal amino-terminus, and a cytoplasmic carboxy-terminus.