GSTp: Guardian of Stress Kinases and Regulator of the Cellular Stress Response
By Shoichi Takahashi, PhD, and Ze’ev Ronai, PhD
Glutathione s-transferases (gst) are a group of multifunctional enzymes that catalyze the nucleophilic addition of glutathione to a widely heterogeneous group of compounds. These compounds are involved in the conjugation of glutathione to numerous xenobiotics and are believed to be responsible for the detoxification of a variety substances.1,2 On the basis of primary sequence similarity, mammalian cytosolic GST can be divided into at least five subclasses: alpha, mu, zeta, omega, and pi.3 There has been considerable interest in the properties of GST pi (GSTp) because its altered expression has been linked to carcinogenesis and tumor progression.4 GSTp overexpression is common in a variety of human tumors, including lung, colon, ovary, testis, and bladder, as well as in hepatocellular carcinoma.5-10 Moreover, increased GSTp concentration and drug resistance are correlated.11 These findings suggest that GSTp’s biological action may contribute to tumor cell survival and the multidrug-resistant phenotype.
Introduction
One of the most important facts of GSTp biology is its relationship to reactive oxygen species (ROS). ROS play an important role in stress kinase and transcription factor activation and are a key determinant in the cellular stress response.12,13 The reduction-oxidation (redox) state of the cell is a result of the balance between the levels of oxidizing and reducing equivalents. Elevation of ROS in excess of the buffer capacity results in potentially cytotoxic "oxidative stress." Altered ROS balance directly affects cellular proliferation, apoptosis, and senescence.14-16 Almost every gene that has been implicated in the stress response also has been affected by altered redox potential or increased ROS levels.
Altered ROS in response to a wide range of exogenous stimuli, including x-rays, ultraviolet (UV) irradiation, cytokines, and chemotherapeutic drugs, induce cellular oxidative stress and cause the activation of multiple stress kinases, including MAP kinase/ERK kinase kinase 1 (MEKK1), apoptosis signal-regulating kinase 1 (ASK1), MAP kinase kinase (MAPKK), and MAP kinase (MAPK).17,18 Both MEKK1 and ASK1 have been implicated in the activation of JNK and p38, which result in the phosphorylation of various transcription factors, including c-Jun, ATF2, and p53.19,20 GSTp’s ability to elicit protection against ROS-generating agents has been associated with a coordinated increase in its own expression that is attributed to AP1 and ARE elements within the GSTp promoter.21
Recent studies identified the association of GSTp with JNK, which contributes to low basal JNK activity in non-stressed cells.18 Whereas GSTp inhibits JNK activity in normal growing cells, it is not associated with and does not inhibit JNK activity in cells exposed to DNA damage or reactive oxygen radicals. JNK, in its non-active form, targets the ubiquitination and degradation of JNK-associated proteins, including its substrates c-Jun, ATF2, and p53. Therefore, maintaining a low basal level JNK activity in non-stressed cells shortens the half-life of JNK substrates, which is among the mechanisms that play an important role in maintaining controlled cell growth.
GSTp in JNK Inhibition Activity
Important to our understanding of GSTp’s ability to elicit JNK inhibitory activity is the finding that such inhibition is mediated primarily by the low molecular weight form of GSTp. When first identified, the Jun-JNK-associated protein exhibited a molecular weight of 23 kDa. Only the low molecular weight form of GSTp could elicit JNK inhibitory activity. UV irradiation or H2O2 treatment reduces the GSTp-JNK association, probably as a result of the GST-GST multimers formation. Because disulfide bonds induce steric constraints, multimers can no longer accommodate the Jun-JNK complex. The switch from a low molecular weight to a multimer form is thought to provide the underlying mechanism for GSTp’s ability to transmit changes in redox potential at the JNK signaling level.18
Important confirmation for the finding that GSTp is a regulator of JNK activity comes from the study of GSTp1/p2(-/-)-null mice cells.18 GSTp1/p2(-/-)-derived mouse embryo fibroblasts revealed a higher basal level of JNK activity. GSTp transfection into MEF GST-p1/p2(-/-) cells caused decreased JNK phosphorylation, kinase activity, and Jun-mediated transactivation, further supporting GSTp’s role as an inhibitor of JNK signaling.
GSTp overexpression has been associated with malignancy and acquired resistance to electrophilic anticancer drugs. The finding that GSTp is a modulator of JNK inhibition and the relationship between expression of this protein and JNK inhibition suggest that cancer cells prone to GSTp overexpression also may exhibit high intrinsic JNK inhibitory activity. In this model, GSTp effects on JNK may confer the ability to resist apoptosis on tumor cells.
Effect of GSTp Expression on Other Stress Kinases
To further assess GSTp’s role in regulating stress kinases, we established cultures of mouse fibroblasts that express GSTp under a tetracyclin-off inducible promoter.22 This system allows us to determine the effect of GSTp expression not only on JNK but also on other stress kinases under normal and ROS-generating conditions. In addition, the system allows us to determine whether the changes in stress kinases contribute to the well-established role of GSTp as a protector of cells from ROS-mediated death. Systematic analysis of major stress kinase pathways revealed that increased GSTp expression was sufficient to elevate the activity of ERK, p38, and IKK, which are among the major stress-activated signaling cascades, while it maintained inhibition of JNK. GSTp’s ability to increase activity of these kinases also was observed in cells exposed to H2O2 treatment. While H2O2 elicits efficient JNK activation, the degree of JNK activation is attenuated in the presence of GSTp. The findings that GSTp can increase ERK, IKK, and p38, while decreasing JNK activity, position GSTp as a coordinator of stress kinases.
GSTp’s interaction with stress kinases is expected to affect tumor cells, in which GSTp often is found to exhibit deregulated expression. Many tumors were found to exhibit a high level of GSTp expression, coinciding with their drug resistance and reduced potential to undergo apoptosis. An interesting angle on GSTp activities is given by tumors in which GSTp is underexpressed, as often happens in prostate tumors. Intriguingly, JNK was reported to exhibit high basal levels of activity in prostate tumor-derived cell lines, and antisense JNK, to efficiently reduce the tumorigenicity of such prostate cancer cells in nude mice.23
Summary
The mechanism(s) underlying GSTp’s ability to serve as coordinator of stress kinases are yet to be determined. Several hypotheses currently are being tested. GSTp may affect the upstream stress signaling molecule(s) implicated in the regulation of multiple stress kinases. It is equally possible that GSTp exerts effects on scaffold proteins that are a part of stress signaling regulatory cascades. The GSTp promoter consists of several response elements, including c-Jun, which has been shown to play an important part in the regulation of GST expression. Thus, it is possible to envision the existence of an autoregulatory loop in which new GSTp, synthesized in response to activation of c-Jun, may resume JNK inhibition shortly after stress.
A guardian of stress kinases activities in normally growing and stressed cells, GSTp may serve as a sensor of intracellular changes in redox potential and a regulator of the cellular stress response. (Dr. Takahashi is a Postdoctoral Fellow and Dr. Ronai is a Professor at the Derald H. Ruttenburg Cancer Center, Mt. Sinai School of Medicine, New York, NY.)
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