Genetic Control of MAP3K1 in Eye Development and Sex Differentiation

The MAP3K1 is responsible for transmitting signals to activate specific MAP2K-MAPK cascades. Following the initial biochemical characterization, genetic mouse models have taken center stage to elucidate how MAP3K1 regulates biological functions. To that end, mice were generated with the ablation of the entire Map3k1 gene, the kinase domain coding sequences, or ubiquitin ligase domain mutations. Analyses of the mutants identify diverse roles that MAP3K1 plays in embryonic survival, maturation of T/B cells, and development of sensory organs, including eye and ear. Specifically in eye development, Map3k1 loss-of-function was found to be autosomal recessive for congenital eye abnormalities, but became autosomal dominant in combination with Jnk and RhoA mutations. Additionally, Map3k1 mutation increased eye defects with an exposure to environmental agents such as dioxin. Data from eye developmental models reveal the nexus role of MAP3K1 in integrating genetic and environmental signals to control developmental activities. Here, we focus the discussions on recent advances in understanding the signaling mechanisms of MAP3K1 in eye development in mice and in sex differentiation from human genomics findings. The research works featured here lead to a deeper understanding of the in vivo signaling network, the mechanisms of gene–environment interactions, and the relevance of this multifaceted protein kinase in disease etiology and pathogenesis

Mitogen-activated Protein Kinase Kinase Kinase 1 Protects against Nickel-induced Acute Lung Injury

Nickel compounds are environmental and occupational hazards that pose serious health problems and are causative factors of acute lung injury. The c-jun N-terminal kinases (JNKs) are regulated through a mitogen-activated protein (MAP) 3 kinase-MAP2 kinase cascade and have been implicated in nickel toxicity. In this study, we used genetically modified cells and mice to investigate the involvement of two upstream MAP3Ks, MAP3K1 and 2, in nickel-induced JNK activation and acute lung injury. In mouse embryonic fibroblasts, levels of JNK activation and cytotoxicity induced by nickel were similar in the Map3k2-null and wild-type cells but were much lower in the Map3k1/Map3k2 double-null cells. Conversely, the levels of JNK activation and cytotoxicity were unexpectedly much higher in the Map3k1-null cells. In adult mouse tissue, MAP3K1 was widely distributed but was abundantly expressed in the bronchiole epithelium of the lung. Accordingly, MAP3K1 ablation in mice resulted in severe nickel-induced acute lung injury and reduced survival. Based on these findings, we propose a role for MAP3K1 in reducing JNK activation and protecting the mice from nickel-induced acute lung injury

MEKK1 Transduces Activin Signals in Keratinocytes To Induce Actin Stress Fiber Formation and Migration

Activins and other members of the transforming growth factor β family play a critical role in morphological changes of the epidermis that require epithelial cell movement. We investigated the molecular pathways in the transmission of activin signals that lead to actin reorganization and epithelial cell migration. We found that activins cause the activation of RhoA but not of Rac and CDC42, leading to MEKK1-dependent phosphorylation of JNK and transcription factor c-Jun. Through a RhoA-independent mechanism, the activins also induce p38 activity in keratinocytes from wild-type but not from MEKK1-deficient mice. Although neither pathway is dependent on Smad activation, the MEKK1-mediated JNK and p38 activities are both essential for activin-stimulated and transcription-dependent keratinocyte migration. Only JNK is involved in transcription-independent actin stress fiber formation, which needs also the activity of ROCK. Because ROCK is required for JNK activation by RhoA and its overexpression leads to MEKK1 activation, we propose a RhoA-ROCK-MEKK1-JNK pathway and a MEKK1-p38 pathway as Smad-independent mechanisms in the transmission of activin signals. Together, these pathways lead to the control of actin cytoskeleton reorganization and epithelial cell migration, contributing to the physiologic and pathological effects of activins on epithelial morphogenesis

Loss of IκB kinase β promotes myofibroblast transformation and senescence through activation of the ROS-TGFβ autocrine loop

ABSTRACT Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence

Corneal Wound Healing Requires IKB kinase β Signaling in Keratocytes

<div><p>IkB kinase β (IKKβ) is a key signaling kinase for inflammatory responses, but it also plays diverse cell type-specific roles that are not yet fully understood. Here we investigated the role of IKKβ in the cornea using <i>Ikkβ</i>^<i>ΔCS</i> mice in which the <i>Ikkβ</i> gene was specifically deleted in the corneal stromal keratocytes. The <i>Ikkβ</i>^<i>ΔCS</i> corneas had normal morphology, transparency and thickness; however, they did not heal well from mild alkali burn injury. In contrast to the <i>Ikkβ</i>^<i>F/F</i> corneas that restored transparency in 2 weeks after injury, over 50% of the <i>Ikkβ</i>^<i>ΔCS</i> corneas failed to fully recover. They instead developed recurrent haze with increased stromal thickness, severe inflammation and apoptosis. This pathogenesis correlated with sustained myofibroblast transformation with increased α smooth muscle actin (α-SMA) expression, higher levels of senescence β-Gal activity and scar tissue formation at the late stage of wound healing. In addition, the <i>Ikkβ</i>^<i>ΔCS</i> corneas displayed elevated expression of hemo-oxygenase-1 (HO-1), a marker of oxidative stress, and activation of stress signaling pathways with increased JNK, c-Jun and SMAD2/3 phosphorylation. These data suggest that IKKβ in keratocytes is required to repress oxidative stress and attenuate fibrogenesis and senescence in corneal wound healing.</p></div

Corneal wound healing in <i>Ikkβ</i>^<i>F</i> and <i>Ikkβ</i> ^<i>ΔCS</i> mice.

<p>The <i>Ikkβ</i>^<i>F</i> and <i>Ikkβ</i> ^<i>ΔCS</i> mice were subjected to mild alkali burn corneal injury, and the eyes were examined under a stereoscope and slit lamp. (A) Representative photos of the eyes at different days after injury, and (B) the average opacity score are presented as mean±SEM of at least 8 eyes examined under each genotype/experimental conditions. Significant differences between groups were calculated using 2-way repeated measures ANOVA followed by post hoc multiple comparisons of means (Tukey method), and *<i>p</i> < 0.05 is considered statistically significant between the genotypes on the given days of injury. (C) The wounded eyes were harvested at different days after injury and examined by H&E. ST: corneal stroma, EP, corneal epithelium, labeled with arrows.</p

Oxidative stress and stress signaling in the injured cornea.

<p>The <i>Ikkβ</i>^<i>F</i> and <i>Ikkβ</i> ^<i>ΔCS</i> eyes were harvested at 28 days after alkali burn injury. The tissues were processed and used for immunohistochemistry using anti-HO-1, a marker of oxidative stress, anti-pJNK and p-c-Jun, markers for the stress-activated JNK pathway, and anti-p-SMAD, a marker for active TGFβ signaling. Blue: DAPI (nuclei), Red: leukocytes. ST: corneal stroma, EP, corneal epithelium, labeled with arrows. (B) The number of positive cells was quantified and **p<0.01 and ***p<0.001 was considered significantly different between <i>Ikkβ</i>^<i>F</i> and <i>Ikkβ</i> ^<i>ΔCS</i> eyes. Results represent at least 5 slides/eye and 3 injured eyes examined.</p

Role of IKKβ in corneal development and maintenance.

<p>(A) The eyes of <i>Ikkβ</i>^<i>F</i> and <i>Ikkβ</i>^<i>ΔCS</i> mice were examined under a stereoscope and photographed. (B) The <i>Ikkβ</i>^<i>F</i> and <i>Ikkβ</i>^<i>ΔCS</i> eyes were analyzed by H&E staining, TUNEL assay and immunostaining using anti-PCNA. Blue: Hoechst for nuclei. Red: TUNEL and PCNA positive signals, which were absent in the cornea of adult mice. ST: corneal stroma, EP, corneal epithelium, labeled with arrows. Pictures represent results from at least 3 mouse corneas of each genotype.</p

Myofibroblast transformation, senescence and apoptosis of the injured cornea.

<p>(A) The <i>Ikkβ</i>^<i>F</i> and <i>Ikkβ</i> ^<i>ΔCS</i> eyes were harvested at 28 days after alkali burn injury. The tissues were processed and used for immunohistochemistry using anti-α-SMA, a marker for myofibroblast and TUNEL assays for the detection of apoptotic cells. Blue: DAPI (nuclei), Red: leukocytes. (B) The <i>Ikkβ</i>^<i>F</i> and <i>Ikkβ</i> ^<i>ΔCS</i> eyes were harvested at 4 days and 28 days after alkali burn injury. The tissue sections were examined by SA-β-Gal staining. The SA-β-Gal positive cells are stained with blue color. ST: corneal stroma, EP, corneal epithelium, labeled with arrows. (B and D) The number of staining positive cells was quantified and **p<0.01 and ***p<0.001 was considered significantly different between <i>Ikkβ</i>^<i>F</i> and <i>Ikkβ</i> ^<i>ΔCS</i> eyes. Data represent at least at least 5 slides/eye and 3 injured eyes examined.</p