Signalling the molecular stress response to nephrotoxic and mutagenic cysteine conjugates: Differential roles for protein synthesis and calcium in the induction of c‐fos and c‐myc mRNA in LLC‐PK1 cells

Abstract

Nephrotoxic and mutagenic cysteine conjugates (NCC) are activated by the enzyme cysteine conjugate, beta-lyase, to reactive acylating species which bind covalently to cellular macromolecules. We now show that an early event after treatment of LLC-PK1 cells with NCC is the induction of mRNA for both c-fos and c-myc. Treatment with S-(1,2-dichlorovinyl)-L-cysteine (DCVC) induced c-fos (53-fold) and c-myc mRNA (20-fold) and increased transcription about 3-fold for both genes. Covalent binding was required for induction of both mRNAs. Dithiothreitol partially prevented induction of both c-fos and c-myc RNA. Buffering the DCVC-induced increase in cytosolic free calcium had no effect on c-fos mRNA, but partially blocked c-myc mRNA induction. Cycloheximide blocked the induction of c-myc mRNA in the absence of an effect on c-fos induction. The data suggest that the increase in c-fos mRNA is a primary response to DCVC toxicity and occurs without a requirement for protein synthesis or an increase in intracellular free calcium. In contrast, c-myc induction requires protein synthesis, suggesting that the presence of another primary response factor may regulate induction either transcriptionally or posttranscriptionally. The data suggest that different signalling pathways regulate induction of c-fos and c-myc mRNA in response to stress caused by reactive acylating species.

A Role for c-myc in Chemically Induced Renal-Cell Death

“…However, there are remarkably few studies that critically address the role of endogenous c-myc expression in apoptosis initiated by toxicants. However, the fact that c-myc mRNA is induced in vivo and in cells in culture by a variety of environmental stresses including cytokines, ionizing or UV irradiation, heavy metals, oxidative stress, and chemicals (13,14,43,59,61,71) suggests that this may be the case. This connection is strengthened by the observations that expression of bcl-2 or, in some but not all cases, mutant p53 blocks apoptosis induced by c-myc overexpression and by a variety of toxicants, cytokines, and other stressful insults (30,40,41,47,57,62,66).…”

“…c-myc mRNA levels increase in the kidney in vivo and in kidney epithelial cells in vitro after exposure to nephrotoxicants (13,45,61,63,71). Like myeloid and mesenchymal cells, enforced c-myc expression induces apoptosis in rat kidney epithelial cells deprived of serum (55), indicating that activation of c-myc might play a role in cell death in the renal epithelium.…”

“…The nephrotoxicity of DCVC has been well characterized; cells die after DCVC exposure either by apoptosis or necrosis via pathways involving covalent binding of a reactive metabolite, loss of glutathione, increased levels of cellular Ca 2ϩ and reactive oxygen species, disruption of the actin cytoskeleton, and peroxidation of membrane lipids (11,64,65). DCVC induces c-myc mRNA in a manner that is linked, at least in part, to an increase in cellular Ca 2ϩ content and oxidative stress (71). Thus, DCVC is a good model for investigating the role of c-myc induction in toxicantinduced cell death.…”

Proteomic approaches to characterize protein modifications: new tools to study the effects of environmental exposures.

“…Modification of keap1 causes dissociation from NRF2, thus permitting NRF2 entry into the nucleus, complexation with other proteins, and activation of the electrophile response element (57). Studies by Stevens and colleagues have documented the ability of the prototypical alkylating agent iodoacetamide to induce the synthesis of endoplasmic reticulum (ER)-associated stress proteins, including Grp78, Grp94, and calreticulin, and the cytoplasmic stress protein Hsp70 (58)(59)(60). Other studies have provided evidence that ER proteins are prominent targets for reactive electrophiles produced from P450 enzymes, although no adducts have been mapped to specific proteins (20).…”

Reduction of trans-4,5-Dihydroxy-1,2-dithiane by Cellular Oxidoreductases Activates gadd153/chop andgrp78 Transcription and Induces Cellular Tolerance in Kidney Epithelial Cells

“…For example, depletion of cellular GSH causes protein thiol oxidation and activation of hsp70 transcription (1,2). Likewise, perturbing cellular Ca 2ϩ with ionophores or toxicants that cause oxidative stress activates c-fos, c-myc, and the growth arrest and DNA damage-inducible gene, gadd153 (3)(4)(5)(6)(7). In some cases, oxidants may directly regulate transcription factors, such as oxyR, by protein thiol oxidation (8).…”