Yusen Liu

Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury.

The mitogen-activated protein kinase (MAPK) family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. In this study we investigated the factors controlling MAPK activation by HO and explored the impact of altering the pathways to kinase activation on cell survival following HO exposure. Potent activation (10-20-fold) of extracellular signal-regulated protein kinase (ERK2) occurred within 10 min of HO treatment, whereupon rapid inactivation ensued. HO activated ERK2 in several cell types and also moderately activated (3-5-fold) both c-Jun N-terminal kinase and p38/RK/CSBP. Additionally, HO increased the mRNA expression of MAPK-dependent genes c-jun, c-fos, and MAPK phosphatase-1. Suramin pretreatment completely inhibited HO stimulation of ERK2, highlighting a role for growth factor receptors in this activation. Further, ERK2 activation by HO was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol, indicating that metal-catalyzed free radical formation mediates the initiation of signal transduction by HO. HO-stimulated activation of ERK2 was abolished in PC12 cells by inducible or constitutive expression of the dominant negative Ras-N-17 allele. Interestingly, PC12/Ras-N-17 cells were more sensitive than wild-type PC12 cells to HO toxicity. Moreover, NIH 3T3 cells expressing constitutively active MAPK kinase (MEK, the immediate upstream regulator of ERK) were more resistant to HO toxicity, while those expressing kinase-defective MEK were more sensitive, than cells expressing wild-type MEK. Taken together, these studies provide insight into mechanisms of MAPK regulation by HO and suggest that ERK plays a critical role in cell survival following oxidant injury.

Restraint of Proinflammatory Cytokine Biosynthesis by Mitogen-Activated Protein Kinase Phosphatase-1 in Lipopolysaccharide-Stimulated Macrophages

Exposure of macrophages to LPS elicits the production of proinflammatory cytokines, such as TNF-α, through complex signaling mechanisms. Mitogen-activated protein (MAP) kinases play a critical role in this process. In the present study, we have addressed the role of MAP kinase phosphatase-1 (MKP-1) in regulating proinflammatory cytokine production using RAW264.7 macrophages. Analysis of MAP kinase activity revealed a transient activation of c-Jun N-terminal kinase (JNK) and p38 after LPS stimulation. Interestingly, MKP-1 was induced concurrently with the inactivation of JNK and p38, whereas blocking MKP-1 induction by triptolide prevented this inactivation. Ectopic expression of MKP-1 accelerated JNK and p38 inactivation and substantially inhibited the production of TNF-α and IL-6. Induction of MKP-1 by LPS was found to be extracellular signal-regulated kinase dependent and involved enhanced gene expression and increased protein stability. Finally, MKP-1 expression was also induced by glucocorticoids as well as cholera toxin B subunit, an agent capable of preventing autoimmune diseases in animal models. These findings highlight MKP-1 as a critical negative regulator of the macrophage inflammatory response, underscoring its premise as a potential target for developing novel anti-inflammatory drugs.

Acute hypertension activates mitogen-activated protein kinases in arterial wall.

Mitogen-activated protein (MAP) kinases are rapidly activated in cells stimulated with various extracellular signals by dual phosphorylation of tyrosine and threonine residues. They are thought to play a pivotal role in transmitting transmembrane signals required for cell growth and differentiation. Herein we provide evidence that two distinct classes of MAP kinases, the extracellular signal-regulated kinases (ERK) and the c-Jun NH2-terminal kinases (JNK), are transiently activated in rat arteries (aorta, carotid and femoral arteries) in response to an acute elevation in blood pressure induced by either restraint or administration of hypertensive agents (i.e., phenylephrine and angiotensin II). Kinase activation is followed by an increase in c-fos and c-jun gene expression and enhanced activating protein 1 (AP-1) DNA-binding activity. Activation of ERK and JNK could contribute to smooth muscle cell hypertrophy/hyperplasia during arterial remodeling due to frequent and/or persistent elevations in blood pressure.

Differential activation of ERK, JNK/SAPK and P3/CSBP/RK map kinase family members during the cellular response to arsenite

Exposure of cells to either proliferative or stressful stimuli elicits a complex response involving one or more distinct phosphorylation cascades culminating in the activation of multiple members of the mitogen-activated protein kinase (MAPK) family, including extracellular signal regulated kinase (ERK), stress-activated c-Jun N-terminal kinase (JNK/SAPK), and p38/RK/CSBP protein kinase. While the pathways transducing mitogenic stimuli to these kinases are relatively well established, the early signalling events leading to their activation in response to stress are poorly understood. In the present study, we examined ERK, JNK/SAPK, and p38 activation in cells treated with the sulfhydryl-reactive agent sodium arsenite. Arsenite treatment potently activated both JNK/SAPK and p38, but only moderately activated ERK. Activation of all three kinases was prevented by the free radical scavenger N-Acetyl-L-cysteine, suggesting that an oxidative signal initiates the responses. Suramin, a growth factor receptor poison, significantly inhibited ERK activation by arsenite, but had little effect on either JNK/SAPK or p38 activity. In contrast, suramin inhibited the activation of all three kinases by short wavelength ultraviolet light (UVC) irradiation. In addition, comparative studies with wild-type PC12 cells and PC12 cells expressing a dominant negative Ras mutant allele indicated that arsenite activates ERK primarily through a Ras-dependent pathway(s), while activation of both JNK/SAPK and p38 occurs through a mechanism relatively independent of Ras. These results suggest that JNK/SAPK and p38 may share common upstream regulators distinct from those involved in ERK activation.

Transcriptional induction of MKP-1 in response to stress is associated with histone H3 phosphorylation-acetylation.

ABSTRACT Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) has been shown to play a critical role in mediating the feedback control of MAP kinase cascades in a variety of cellular processes, including proliferation and stress responsiveness. Although MKP-1expression is induced by a broad array of extracellular stimuli, the mechanisms mediating its induction remain poorly understood. Here we show that MKP-1 mRNA was potently induced by arsenite and ultraviolet light and modestly increased by heat shock and hydrogen peroxide. Interestingly, arsenite also dramatically induces phosphorylation-acetylation of histone H3 at a global level which precedes the induction of MKP-1 mRNA. The transcriptional induction of MKP-1, histone H3 modification, and elevation in MKP-1 mRNA in response to arsenite are all partially prevented by the p38 MAP kinase inhibitor SB203580, suggesting that the p38 pathway is involved in these processes. Finally, analysis of the DNA brought down by chromatin immunoprecipitation (ChIP) reveals that arsenite induces phosphorylation-acetylation of histone H3 associated with the MKP-1 gene and enhances binding of RNA polymerase II to MKP-1 chromatin. ChIP assays following exposure to other stress agents reveal various degrees of histone H3 modification at the MKP-1 chromatin. The differential contribution of p38 and ERK MAP kinases in mediating MKP-1 induction by different stress agents further illustrates the complexity and versatility of stress-induced MKP-1 expression. Our results strongly suggest that chromatin remodeling after stress contributes to the transcriptional induction of MKP-1.

Tumor promoter arsenite activates extracellular signal-regulated kinase through a signaling pathway mediated by epidermal growth factor receptor and Shc.

ABSTRACT Although arsenite is an established carcinogen, the mechanisms underlying its tumor-promoting properties are poorly understood. Previously, we reported that arsenite treatment leads to the activation of the extracellular signal-regulated kinase (ERK) in rat PC12 cells through a Ras-dependent pathway. To identify potential mediators of the upstream signaling cascade, we examined the tyrosine phosphorylation profile in cells exposed to arsenite. Arsenite treatment rapidly stimulated tyrosine phosphorylation of several proteins in a Ras-independent manner, with a pattern similar to that seen in response to epidermal growth factor (EGF) treatment. Among these phosphorylated proteins were three isoforms of the proto-oncoprotein Shc as well as the EGF receptor (EGFR). Tyrosine phosphorylation of Shc allowed for enhanced interactions between Shc and Grb2 as identified by coimmunoprecipitation experiments. The arsenite-induced tyrosine phosphorylation of Shc, enhancement of Shc and Grb2 interactions, and activation of ERK were all drastically reduced by treatment of cells with either the general growth factor receptor poison suramin or the EGFR-selective inhibitor tyrphostin AG1478. Down-regulation of EGFR expression through pretreatment of cells with EGF also attenuated ERK activation and Shc tyrosine phosphorylation in response to arsenite treatment. These results demonstrate that the EGFR and Shc are critical mediators in the activation of the Ras/ERK signaling cascade by arsenite and suggest that arsenite acts as a tumor promoter largely by usurping this growth factor signaling pathway.

COUP-TF acts as a competitive repressor for estrogen receptor-mediated activation of the mouse lactoferrin gene.

We previously demonstrated that the estrogen response module (mERM) of the mouse lactoferrin gene, which contains an overlapping chicken ovalbumin upstream promoter transcription factor (COUP-TF)- and estrogen receptor-binding element, is responsible for estrogen induction. In this report we show that COUP-TF represses the mERM response to estrogen stimulation. Mutation and deletion of the COUP-TF-binding element or reduction of the endogenous COUP-TF increases mERM estrogen responsiveness. Likewise, overexpression of the COUP-TF expression vector blocked the estrogen-stimulated response of mERM in transfected cells. The molecular mechanism of this repression is due to the competition between COUP-TF and the estrogen receptor for binding at identical contact sites in the overlapping region of the mERM. Our results indicate that two members of the steroid-thyroid receptor superfamily work in concert to modulate lactoferrin gene expression.

Arsenic Trioxide Promotes Histone H3 Phosphoacetylation at the Chromatin of CASPASE-10 in Acute Promyelocytic Leukemia Cells

Arsenic trioxide (As2O3) is highly effective for the treatment of acute promyelocytic leukemia, even in patients who are unresponsive to all-trans-retinoic acid therapy. As2O3 is believed to function primarily by promoting apoptosis, but the underlying molecular mechanisms remain largely unknown. In this report, using cDNA arrays, we have examined the changes in gene expression profiles triggered by clinically achievable doses of As2O3 in acute promyelocytic leukemia NB4 cells. CASPASE-10 expression was found to be potently induced by As2O3. Accordingly, caspase-10 activity also substantially increased in response to As2O3 treatment. A selective inhibitor of caspase-10, Z-AEVD-FMK, effectively blocked caspase-3 activation and significantly attenuated As2O3-triggered apoptosis. Interestingly, the treatment of NB4 cells with As2O3 markedly increased histone H3 phosphorylation at serine 10, an event that is associated with acetylation of the lysine 14 residue. Chromatin immunoprecipitation assays revealed that As2O3 potently enhances histone H3 phosphoacetylation at the CASPASE-10 locus. These results suggest that the effect of As2O3 on histone H3 phosphoacetylation at the CASPASE-10 gene may play an important role in the induction of apoptosis and thus contribute to its therapeutic effects on acute promyelocytic leukemia.

Induction of Mitogen-Activated Protein Kinase Phosphatase-1 During Acute Hypertension

Recently, we demonstrated that elevated blood pressure activates mitogen-activated protein (MAP) kinases in rat aorta. Here we provide evidence that the vascular response to acute hypertension also includes induction of MAP kinase phosphatase-1 (MKP-1), which has been shown to function in the dephosphorylation and inactivation of MAP kinases. Restraint or immobilization stress, which leads to a rapid rise in blood pressure, resulted in a rapid and transient induction of MKP-1 mRNA followed by elevated MKP-1 protein expression in rat aorta. That the induction of MKP-1 by restraint was due to the rise in blood pressure was supported by the finding that several different hypertensive agents (phenylephrine, vasopressin, and angiotensin II) were likewise capable of eliciting the response, and sodium nitroprusside, a nonspecific vasodilator agent that prevented the acute rise in blood pressure in response to the hypertensive agents, abrogated MKP-1 mRNA induction. The in vivo effects could not be mimicked by treatment of cultured aortic smooth muscle cells with similar doses of the hypertensive agents. These findings support a role for MKP-1 in the in vivo regulation of MAP kinase activity during hemodynamic stress.

The carboxyl-terminal domains of MKP-1 and MKP-2 have inhibitory effects on their phosphatase activity.

Both the mitogen-activated protein kinase (MAPK) phosphatases MKP-1 and MKP-2 exert important feedback control of MAPK-mediated signaling events. The function of MKP-1 and MKP-2 is regulated via complex mechanisms, ranging from increased transcription of the MKP-1 and MKP-2 genes to post-translational catalytic activation of MKP-1 and MKP-2 proteins upon binding to their substrate MAPKs. In addition, MKP-1 stability increases upon ERK-dependent phosphorylation of two serine residues in its C-terminus. The C-terminal regions of MKP-1 and MKP-2, but not those of other MKPs, are homologous. To investigate the role of this domain, we have deleted the C-terminal tails from MKP-1 and MKP-2 and examined the effect of these deletions on their enzymatic activity. C-terminally truncated MKP-1 and MKP-2 exhibited, both in vivo and in vitro, substantially greater phosphatase activity towards their substrate MAPKs than did the full-length counterparts. However, C-terminal truncations did not significantly change either their substrate affinity, or their substrate-mediated catalytic activation. Basal phosphatase activity of the truncated proteins was also significantly higher than that of the wild-type counterparts. Collectively, these results suggest that the C-terminal domain may potentially play a role in the regulation of MKP-1 and MKP-2.