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(Received for publication, April 30, 1997, and in revised form, May 14, 1997)
From the Activation of stress response genes can impart
cellular tolerance to environmental stress. Iodoacetamide (IDAM) is an
alkylating toxicant that up-regulates expression of hsp70
(Liu, H., Lightfoot, D. L., and Stevens, J. L. (1996)
J. Biol. Chem. 271, 4805-4812) and grp78
in LLC-PK1 renal epithelial cells. Therefore, we used IDAM to determine
the role of these genes in tolerance to toxic chemicals. Prior heat
shock did not protect cells from IDAM but pretreatment with
trans-4,5-dihydroxy-1,2-dithiane (DTTox), thapsigargin, or
tunicamycin enhanced expression of the endoplasmic reticulum (ER)
chaperones GRP78 and GRP94 and rendered cells tolerant to IDAM. Cells
expressing a 524-base pair antisense grp78 fragment (pkASgrp78) had a diminished capacity to up-regulate grp78
and grp94 expression after ER stress. Protection against
IDAM due to prior ER stress was also attenuated in pkASgrp78 cells
suggesting that ER chaperones of the GRP family are critical for
tolerance. Covalent binding of IDAM to cellular macromolecules and
depletion of cellular thiols was similar in tolerant and naïve
cells. However, DTTox pretreatment blocked the increases in cellular
Ca2+ and lipid peroxidation observed after IDAM treatment.
Overexpressing the ER Ca2+-binding protein calreticulin
prevented IDAM-induced cell death, the rise in cytosolic
Ca2+, and oxidative stress. Although activation of the ER
stress response did not prevent toxicity due to Ca2+
influx, EGTA-AM and ruthenium red both blocked cell death suggesting that redistribution of intracellular Ca2+ to the
mitochondria may be important in toxicity. The data support a model in
which induction of ER stress proteins prevents disturbances of
intracellular Ca2+ homeostasis, thus uncoupling toxicant
exposure from oxidative stress and cell death. Multiple ER stress
proteins are likely to be involved in this tolerance response.
Volume 272, Number 35,
Issue of August 29, 1997
pp. 21751-21759
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
,§
,
W. Alton Jones Cell Science Center,
Lake Placid, New York 12946, the ¶ Department of
Chemistry, Clarkson University, Potsdam, New York 13676, and
§ Division of Toxicology, Leiden Amsterdam Center for
Drug Research, Leiden University, Leiden, The Netherlands
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