Abstract
Induction of drug-metabolizing microsomal cytochromes p450 (p450s) results in a striking proliferation of the smooth endoplasmic reticulum (ER). Overexpression of P450s in yeast and cultured cells produces a similar response. The signals mediating this process are not known but probably involve signal transduction pathways involved in the unfolded protein response (UPR) or the ER overload response (EOR). We have examined the temporal response of specific genes in these pathways and genes globally to overexpression of p450 in cultured cells. Activity of NFkappaB, an EOR component, was substantially increased by overexpression of full-length p450 2C2 or a chimera with the 28-amino acid signal anchor sequence of p450 2C2 in HepG2 cells, and the activation correlated temporally with the accumulation of p450 in the cells. In the UPR pathway, activation of the transcription factor XBP1 by IRE1 also correlated with the accumulation of p450 in the cells, and in contrast, maximum activation of the BiP/grp78 promoter preceded the accumulation. Differential effects of expression of p450 on apoptosis were observed in nonhepatic COS1 and hepatic HepG2 cells. In COS1 cells, apoptosis was induced, and this correlated with sustained activation of the pro-apoptotic JNK pathway, induction of CHOP, and an absence of the increased NFkappaB activity. In HepG2 cells, JNK was only transiently activated, and CHOP expression was not induced. As assessed by DNA microarray analysis, up-regulation of signaling genes was predominant including those involved in anti-apoptosis and ER stress. These results suggest that both the EOR and UPR pathways are involved in the cellular response to induction of p450 expression and that in hepatic cells genes are also induced to block apoptosis, which may be a physiologically relevant response to prevent cell death during xenobiotic induced expression of p450 in the liver.
Highlights
The endoplasmic reticulum (ER)1 represents the central organelle of the cell in which the crucial steps of folding and
As assessed by DNA microarray analysis, up-regulation of signaling genes was predominant including those involved in anti-apoptosis and ER stress. These results suggest that both the endoplasmic reticulum overload response (EOR) and unfolded protein response (UPR) pathways are involved in the cellular response to induction of P450 expression and that in hepatic cells genes are induced to block apoptosis, which may be a physiologically relevant response to prevent cell death during xenobiotic induced expression of P450 in the liver
ER stress-activated signal transduction is mediated by three ER transmembrane proteins as follows: 1) transcriptional activator ATF6, which upon ER stress is proteolytically released from the membrane and up-regulates ER stress-response element (ERSE)-containing promoters; 2) translation initiation factor 2 kinase, PERK, which mainly attenuates translation, slowing down the accumulation of ER proteins and allowing the ER to accommodate its cargo load; and 3) the transmembrane kinase/ endoribonuclease IRE1 (4 – 8)
Summary
Materials—Tissue culture materials were purchased from Invitrogen. The enhanced GFP vector was from Clontech. The phRK-TK reporter vector and dual luciferase reporter activity assay kit were from Promega. RT-PCR—RNA was prepared from transfected cells cultured on 6-well plates with the Qiagen RNeasy kit and used in RT-PCR performed with the Omniscript RT kit (Qiagen). Cells were transfected on 24-well plates with the amounts of DNA indicated in the figures legends. Fluorescent Protein Concentration Assay—To measure the concentration of C2/GFP chimera in transfected cells, cells cultured in 6-well plates were washed with PBS, scraped, and collected by brief centrifugation. CDNA Microarray Analysis—Total RNA from transfected HepG2 cells was isolated using the Qiagen RNeasy kit. Double-stranded cDNA was synthesized with 5– 8 g of total RNA using the T7-oligo(dT) primer (Genset Oligos, Boulder, CO) and the SuperScript doublestranded cDNA synthesis kit (Invitrogen) following the manufacturers’ protocols. A total of 22,867 putative promoter sequences were searched for XBP1 and NFB-binding sites using a pattern matching program written in Perl.
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