The mitogen-activated protein kinase kinase (MEK) kinase 1 (MEKK1) mediates activin B signals required for eyelid epithelium morphogenesis during mouse fetal development. The present study investigates the role of MEKK1 in epithelial wound healing, another activin-regulated biological process. In a skin wound model, injury markedly stimulates MEKK1 expression and activity, which are in turn required for the expression of genes involved in extracellular matrix (ECM) homeostasis. MEKK1 ablation or down-regulation by interfering RNA significantly delays skin wound closure and impairs activation of Jun NH 2 -terminal kinases, induction of plasminogen activator inhibitor (PAI)-1, and restoration of cell-cell junctions of the wounded epidermis. Conversely, expression of wild-type MEKK1 accelerates reepithelialization of full-thickness skin and corneal debridement wounds by mechanisms involving epithelial cell migration, a cell function that is partially abolished by neutralizing antibodies for PAI-1 and metalloproteinase III. Our data suggest that MEKK1 transmits wound signals, leading to the transcriptional activation of genes involved in ECM homeostasis, epithelial cell migration, and wound reepithelialization.

Arsenic is a widespread environmental toxic agent that has been shown to cause diverse tissue and cell damage and at the same time to be an effective anti-cancer therapeutic agent. The objective of this study is to explore the signaling mechanisms involved in arsenic toxicity. We show that the IB kinase ␤ (IKK␤) plays a crucial role in protecting cells from arsenic toxicity. Ikk␤ ؊/؊ mouse 3T3 fibroblasts have decreased expression of antioxidant genes, such as metallothionein 1 (Mt1). In contrast to wild type and IKK␤-reconstituted Ikk␤ ؊/؊ cells, IKK␤-null cells display a marked increase in arsenic-induced reactive oxygen species (ROS) accumulation, which leads to activation of the MKK4-c-Jun NH 2 -terminal kinase (JNK) pathway, c-Jun phosphorylation, and apoptosis. Pretreatment with the antioxidant N-acetylcysteine (NAC) and expression of MT1 in the Ikk␤ ؊/؊ cells prevented JNK activation; moreover, NAC pretreatment, MT1 expression, MKK4 ablation, and JNK inhibition all protected cells from death induced by arsenic. Our data show that two signaling pathways appear to be important for modulating arsenic toxicity. First, the IKK-NF-B pathway is crucial for maintaining cellular metallothionein-1 levels to counteract ROS accumulation, and second, when this pathway fails, excessive ROS leads to activation of the MKK4-JNK pathway, resulting in apoptosis.Arsenic is a highly toxic ubiquitous environmental contaminant. Chronic low level exposure may cause skin, lung, bladder, and kidney cancer, whereas higher doses of arsenic lead to tissue damage through the induction of cell death. Such dose and cell type-dependent effects are the basis for arsenic trioxide to be used in the treatment of acute promyelocytic leukemia. Initially it was thought that arsenic, like all-trans-retinoic acid, could target the promyelocytic leukemia gene product retinoic acid receptor (PML-RAR) fusion protein, causing its degradation; however, many PML-RAR-negative cell lines and cells from PML knock-out mice are also sensitive to arsenic, suggesting alternative mechanisms for toxicity (1-9).As a redox-active metalloid, arsenic in a dose-dependent manner elicits an immediate burst of intracellular ROS (10 -14). Cells normally respond to these changes by activating the defense mechanisms to re-establish redox homeostasis and protect from oxidative damage (15, 16). We and others have previously reported the transcriptional activation by arsenite of a battery of antioxidant genes, including hemeoxygenase-1, metallothionein 1 and 2, and thioredoxin reductase 1, which may result in parallel changes in protein levels and enzyme or binding activity (17,18).Antioxidant up-regulation, however, is insufficient to counteract the ROS 2 caused by high dose and prolonged arsenic exposure. Numerous studies have shown that chronic exposure to arsenic from drinking water in humans and experimental animals are often associated with increased oxidative stress, a state when cellular ROS production exceeds antioxidant capacity (19 -22). Depending on their level a...

Cytokine-activated inhibitor of B kinase ␤ (IKK␤) is a key mediator of immune and inflammatory responses, but recent studies suggest that IKK␤ is also required for tissue homeostasis in physiopathological processes. Here we report a novel role for IKK␤ in maintenance of constitutive levels of the redox scavenger GSH. Inactivation of IKK␤ by genetic or pharmacological means results in low cellular GSH content and marked reduction of redox potential. Similar to Ikk␤(Ϫ/Ϫ) cells, Tnfr1(Ϫ/Ϫ) and p65(Ϫ/Ϫ) cells are also GSH-deficient. As a consequence, cells deficient in IKK␤ signaling are extremely susceptible to toxicity caused by environmental and pharmacological agents, including oxidants, genotoxic agents, microtubule toxins, and arsenic. GSH biosynthesis depends on the activity of the rate-limiting enzyme glutamate-cysteine ligase (GCL), consisting of a catalytic subunit (GCLC) and a modifier subunit (GCLM). We found that loss of IKK␤ signaling significantly reduces basal NF-B activity and decreases binding of NF-B to the promoters of Gclc and Gclm, leading to reduction of GCLC and GCLM expression. Conversely, overexpression of GCLC and GCLM in IKK␤-null cells partially restores GSH content and prevents stress-induced cytotoxicity. We suggest that maintenance of GSH is a novel physiological role of the IKK␤-NF-B signaling cascade to prevent oxidative damage and preserve the functional integrity of the cells.