Abstract
Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide (LPS) or interleukin-1beta. We have studied the mechanism of induction, and found that transcriptional activation of CYP2E1 occurred within 3 h, and CYP2E1 dependent catalytic activity was induced more than 4-fold within 5 h. The induction was sensitive to several tyrosine kinase inhibitors, and was further modulated by inhibitors of p38 MAP kinase. MAP kinase kinase-3 (MKK3) was phosphorylated in response to LPS, and expression of constitutively active MKK3, but not the MAP kinase kinases MEKK1 or MKK1, activated CYP2E1. Transcriptional activation was mediated through a C/EBPbeta and -delta binding element situated at -486/-474, and appeared to involve activation of prebound factors as well as recruitment of newly synthesized C/EBPbeta and -delta. It is thus suggested that LPS induces MKK3 activation in astrocytes, which in turn stimulates a C/EBPbeta and -delta binding element to mediate transcriptional activation of CYP2E1.
Highlights
Cytochrome P450 2E1 (CYP2E1) is a member of the cytochrome P450 (CYP) gene superfamily and is active in the oxidation of fatty acids and ketones
We have studied the mechanism of induction, and found that transcriptional activation of CYP2E1 occurred within 3 h, and CYP2E1 dependent catalytic activity was induced more than 4-fold within 5 h
The 6-hydroxylation of CZN was inhibited by CYP2E1 antisera, but not preimmune sera, to ϳ85%, as we have described in previous reports (11, 18, 19)
Summary
Primary Cortical Glial Cultures—Rat primary cortical glial cultures were established from cortices of newborn rats (6 –12 h) as previously described (18). Cell cultures were analyzed with microscopy for the composition of cells, and the expression of CYP2E1 and luciferase protein. Preparation of Microsomal Fractions—After harvest of the rat primary cortical glial cultures, the cells were washed twice in phosphatebuffered saline, pH 7.4, and subsequently sonicated in 5 volumes of ice-cold 10 mM Tris-HCl buffer, pH 7.4, with 20% glycerol, 1.14% (w/v) KCl, 0.2 mM EDTA, 0.1 mM dithiothreitol, and, freshly dissolved, 0.1 mM phenylmethylsulfonyl fluoride and 20 M butylated hydroxytoluene. One mg of microsomal protein was incubated with 500 M CZN and 0.5 mM NADPH in 50 mM potassium phosphate buffer, for 120 min. Large plates (ဧ 139 mm) with cortical glial cells 85% confluent were transfected with 4 –17 g of pFC-MEKK1, pFC-MKK3, or empty plasmid DNA, using DMRIE-C (35 l) and 35 ml of Opti-MEM medium. Antibodies to C/EBP and NFB proteins were from Santa Cruz Biotechnology
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