A role for MEK kinase 1 in TGF-beta/activin-induced epithelium movement and embryonic eyelid closure

MEKK1-deficient mice show an eye open at birth phenotype caused by impairment in embryonic eyelid closure. MEK kinase 1 (MEKK1) is highly expressed in the growing tip of the eyelid epithelium, which displays loose cell-cell contacts and prominent F-actin fibers in wild-type mice, but compact cell contacts, lack of polymerized actin and a concomitant impairment in c-Jun N-terminal phosphorylation in MEKK1-deficient mice. In cultured keratinocytes, MEKK1 is essential for JNK activation by TGF-beta and activin, but not by TGF-alpha. MEKK1-driven JNK activation is required for actin stress fiber formation, c-Jun phosphorylation and cell migration. However, MEKK1 ablation does not impair other TGF-beta/activin functions, such as nuclear translocation of Smad4. These results establish a specific role for the MEKK1-JNK cascade in transmission of TGF-beta and activin signals that control epithelial cell movement, providing the mechanistic basis for the regulation of eyelid closure by MEKK1. This study also suggests that the signaling mechanisms that control eyelid closure in mammals and dorsal closure in Drosophila are evolutionarily conserved.     

Nuclear factor-kappa B activation by the CXC chemokine melanoma growth-stimulatory activity/growth-regulated protein involves the MEKK1/p38 mitogen-activated protein kinase pathway

Melanoma growth stimulatory activity/growth-regulated protein (MGSA/GRO), a CXC chemokine, plays an important role in inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. Constitutive expression of MGSA/GROalpha in melanoma tumors is associated with constitutive nuclear factor (NF)-kappaB activity. We show here that either exogenous addition or continuous expression of MGSA/GROalpha in immortalized melanocytes enhances NF-kappaB activation, as well as mitogen-activated protein (MAP) kinase kinase kinase (MEKK) 1, MAP kinase kinase (MEK) 3/6, and p38 MAP kinase activation. Expression of dominant negative M-Ras (S27N), dominant negative MEKK1 (K432M), or specific chemical inhibitors for p38 MAP kinase (SB202190 and SB203580) block MGSA/GROalpha-induced NF-kappaB transactivation, demonstrating that Ras, MEKK1, and p38 are involved in the signal pathways of MGSA/GROalpha activation of NF-kappaB. Expression of dominant active Ras or dominant active MEKK1 alone can also stimulate NF-kappaB activation. The expression of dominant negative MEKK1 inhibits the Ras-induced NF-kappaB activation, suggesting that MEKK1 is a downstream target of Ras. Moreover, MGSA/GROalpha induction of NF-kappaB is independent of the MEK1/ERK cascade, because MGSA/GROalpha failed to increase ERK and ELK activation, and specific chemical inhibitors for MEK1 (PD98059) had no effect on MGSA/GROalpha-enhanced NF-kappaB activation. These data demonstrate that NF-kappaB activation is required for MGSA/GROalpha-induced melanocyte transformation through a Ras/MEKK1/p38 cascade in melanocytes.     

MEK kinase 1 (MEKK1) transduces c-Jun NH2-terminal kinase activation in response to changes in the microtubule cytoskeleton

Cell shape change and the restructuring of the cytoskeleton are important regulatory responses that influence the growth, differentiation, and commitment to apoptosis of different cell types. MEK kinase 1 (MEKK1) activates the c-Jun NH2-terminal kinase (JNK) pathway in response to exposure of cells to microtubule toxins, including taxol. MEKK1 expression is elevated 3-fold in mitosis and microtubule toxin-treated cells accumulated at G2/M of the cell cycle. Targeted disruption of MEKK1 expression in embryonic stem cells resulted in the loss of JNK activation and increased apoptosis in response to taxol. Targeted disruption of the MEK kinase 2 gene had no effect on activation of the JNK pathway in response to microtubule toxins demonstrating a specific role of MEKK1 in this response. Cytochalasin D-mediated disruption of actin fibers activates JNK and stimulates apoptosis similarly in MEKK1(-/-) and wild type cells. The results show that MEKK1 is required for JNK activation in response to microtubule but not actin fiber toxins in embryonic stem cells. MEKK1 activation can protect cells from apoptosis in response to change in the integrity of the microtubule cytoskeleton.     

Inflammatory levels of nitric oxide inhibit airway epithelial cell migration by inhibition of the kinase ERK1/2 and activation of hypoxia-inducible factor-1 alpha

Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1alpha and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1alpha stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1alpha activation. Activation of HIF-1alpha by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1alpha mutant construct inhibited epithelial wound repair, implicating HIF-1alpha in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1alpha. Finally, NO-mediated inhibition of cell migration was associated with HIF-1alpha-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1alpha and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.     

Matrix metalloproteinase (MMP) and TGF-β1-stimulated cell migration in skin and cornea wound healing

Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGF-β) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGF-β-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins, and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.     

MEK kinase 2 and the adaptor protein Lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src

Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex.     

Cyclic Mechanical Stretch Decreases Cell Migration by Inhibiting Phosphatidylinositol 3-Kinase- and Focal Adhesion Kinase-mediated JNK1 Activation

Epithelial cell migration during wound healing requires coordinated signaling pathways that direct polarization of the leading and trailing ends of the cells, cytoskeletal organization, and remodeling of focal adhesions. These inherently mechanical processes are disrupted by cyclic stretch (CS), but the specific signaling molecules involved in this disruption are not well understood. In this study, we demonstrate that inhibition of phosphatidylinositol 3-kinase (PI3K) or expression of a dominant-negative form of PI3K caused inhibition of airway epithelial cell wound closure. CS caused a sustained decrease in activation of PI3K and inhibited wound healing. Expression of constitutively active PI3K stimulated translocation of Tiam1 to the membrane, increased Rac1 activity, and increased wound healing of airway epithelial cells. Increased Rac1 activity resulted in increased phosphorylation of JNK1. PI3K activation was not regulated by association with focal adhesion kinase. Restoration of efficient cell migration during CS required coexpression of constitutively active PI3K, focal adhesion kinase, and JIP3.     

Axin forms a complex with MEKK1 and activates c-Jun NH(2)-terminal kinase/stress-activated protein kinase through domains distinct from Wnt signaling

Axin negatively regulates the Wnt pathway during axis formation and plays a central role in cell growth control and tumorigenesis. We found that Axin also serves as a scaffold protein for mitogen-activated protein kinase activation and further determined the structural requirement for this activation. Overexpression of Axin in 293T cells leads to differential activation of mitogen-activated protein kinases, with robust induction for c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase, moderate induction for p38, and negligible induction for extracellular signal-regulated kinase. Axin forms a complex with MEKK1 through a novel domain that we term MEKK1-interacting domain. MKK4 and MKK7, which act downstream of MEKK1, are also involved in Axin-mediated JNK activation. Domains essential in Wnt signaling, i. e. binding sites for adenomatous polyposis coli, glycogen synthase kinase-3beta, and beta-catenin, are not required for JNK activation, suggesting distinct domain utilization between the Wnt pathway and JNK signal transduction. Dimerization/oligomerization of Axin through its C terminus is required for JNK activation, although MEKK1 is capable of binding C terminus-deleted monomeric Axin. Furthermore, Axin without the MEKK1-interacting domain has a dominant-negative effect on JNK activation by wild-type Axin. Our results suggest that Axin, in addition to its function in the Wnt pathway, may play a dual role in cells through its activation of JNK/stress-activated protein kinase signaling cascade.     

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration.     

Glycogen synthase kinase 3 beta is a natural activator of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1)

Glycogen synthase kinase 3beta (GSK3 beta) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and insulin response. GSK3 beta has now been shown to induce activation of the mitogen-activated protein kinase kinase kinase MEKK1 and thereby to promote signaling by the stress-activated protein kinase pathway. GSK3 beta-binding protein blocked the activation of MEKK1 by GSK3 beta in human embryonic kidney 293 (HEK293) cells. Furthermore, co-immunoprecipitation analysis revealed a physical association between endogenous GSK3 beta and MEKK1 in HEK293 cells. Overexpression of axin1, a GSK3 beta-regulated scaffolding protein, did not affect the physical interaction between GSK3 beta and MEKK1 in transfected HEK293 cells. Exposure of cells to insulin inhibited the activation of MEKK1 by GSK3 beta, and this inhibitory effect of insulin was abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Furthermore, MEKK1 activity under either basal or UV- or tumor necrosis factor alpha-stimulated conditions was reduced in embryonic fibroblasts derived from GSK3 beta knockout mice compared with that in such cells from wild-type mice. Ectopic expression of GSK3 beta increased both basal and tumor necrosis factor alpha-stimulated activities of MEKK1 in GSK3 beta(-/-) cells. Together, these observations suggest that GSK3 beta functions as a natural activator of MEKK1.     

Smad4 disruption accelerates keratinocyte reepithelialization in murine cutaneous wound repair

Keratinocyte reepithelialization is a rate-limiting event in cutaneous wound repair, which involves the migration and proliferation of keratinocytes to cover the denuded dermal surface. Transforming growth factor-β1 (TGF-β1) has the ability to induce epithelial cell migration while inhibiting proliferation, and controversial results have been generated regarding the effect of TGF-β signaling on reepithelialization. In this study, full-thickness skin wounds were made in keratinocyte-specific Smad4 knockout and the control mice. The wound closure, reepithelialization, keratinocyte proliferation, myofibroblast numbers and collagen deposition of were assessed. The results showed that the proliferation of keratinocytes increased, which accelerated the reepithelialization, and led to faster wound repair in the epidermis of Smad4 mutant mice. Upregulation of keratin 17, 14-3-3 sigma and phosphorylated AKT in the hyperproliferative epidermis may be correlated with the accelerated reepithelialization. We conclude that Smad4 plays an inhibitory role in the keratinocyte-mediated reepithelialization of wound healing.     

Bmx tyrosine kinase transgene induces skin hyperplasia, inflammatory angiogenesis, and accelerated wound healing

The Bmx gene, a member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in arterial endothelium and in certain hematopoietic and epithelial cells. Previous in vitro studies have implicated Bmx signaling in cell migration and survival and suggested that it contributes to the progression of prostate carcinomas. However, the function of Bmx in normal tissues in vivo is unknown. We show here that Bmx expression is induced in skin keratinocytes during wound healing. To analyze the role of Bmx in epidermal keratinocytes in vivo, we generated transgenic mice overexpressing Bmx in the skin. We show that Bmx overexpression accelerates keratinocyte proliferation and wound reepithelialization. Bmx expression also induces chronic inflammation and angiogenesis in the skin, and gene expression profiling suggests that this occurs via cytokine-mediated recruitment of inflammatory cells. Our studies provide the first data on Bmx function in vivo and form the basis of evaluation of its role in epithelial neoplasia.     

MEKK1 is required for inducible urokinase-type plasminogen activator expression

Urokinase-type plasminogen activator (uPA) regulates the remodeling of extracellular matrix and controls reparative processes such as wound healing and liver regeneration. Here we show inducible uPA expression is controlled by MEKK1, a MAPK kinase kinase that regulates the ERK1/2 and JNK pathways. MEKK1 is activated in response to growth factors and cytoskeletal changes. We have found MEKK1 to be necessary for uPA up-regulation in response to treatment with phorbol 12-myristate 13-acetate or basic fibroblast growth factor. We demonstrate that growth factor-treated MEKK1-deficient fibroblasts display greatly reduced uPA expression and activity compared with control fibroblasts. Further, we show that growth factor-induced uPA expression requires MEKK1-dependent MKK1 and JNK activity and that transfection of MEKK1 into knockout cells restores inducible uPA expression and activity. Importantly, disrupted expression of MEKK2, a related MAPK kinase kinase, had no effect on uPA activity. Therefore, we conclude that MEKK1 expression is required for PMA- or FGF-2-induced signals to control uPA expression and function.     

Rapid protein kinase D1 signaling promotes migration of intestinal epithelial cells

We have examined the role of protein kinase D1 (PKD1) signaling in intestinal epithelial cell migration. Wounding monolayer cultures of intestinal epithelial cell line IEC-18 or IEC-6 induced rapid PKD1 activation in the cells immediately adjacent to the wound edge, as judged by immunofluorescence microscopy with an antibody that detects the phosphorylated state of PKD1 at Ser(916), an autophosphorylation site. An increase in PKD1 phosphorylation at Ser(916) was evident as early as 45 s after wounding, reached a maximum after 3 min, and persisted for ≥15 min. PKD1 autophosphorylation at Ser(916) was prevented by the PKD family inhibitors kb NB 142-70 and CRT0066101. A kb NB 142-70-sensitive increase in PKD autophosphorylation was also elicited by wounding IEC-6 cells. Using in vitro kinase assays after PKD1 immunoprecipitation, we corroborated that wounding IEC-18 cells induced rapid PKD1 catalytic activation. Further results indicate that PKD1 signaling is required to promote migration of intestinal epithelial cells into the denuded area of the wound. Specifically, treatment with kb NB 142-70 or small interfering RNAs targeting PKD1 markedly reduced wound-induced migration in IEC-18 cells. To test whether PKD1 promotes migration of intestinal epithelial cells in vivo, we used transgenic mice that express elevated PKD1 protein in the small intestinal epithelium. Enterocyte migration was markedly increased in the PKD1 transgenic mice. These results demonstrate that PKD1 activation is one of the early events initiated by wounding a monolayer of intestinal epithelial cells and indicate that PKD1 signaling promotes the migration of these cells in vitro and in vivo.     

MEKK1 regulates calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of migrating fibroblasts

Herein, we define how MEKK1, a MAPK kinase kinase, regulates cell migration. MEKK1 is associated with actin fibers and focal adhesions, localizing MEKK1 to sites critical in the control of cell adhesion and migration. EGF-induced ERK1/2 activation and chemotaxis are inhibited in MEKK1-/- fibroblasts. MEKK1 deficiency causes loss of vinculin in focal adhesions of migrating cells, increased cell adhesion and impeded rear-end detachment. MEKK1 is required for activation of the cysteine protease calpain and cleavage of spectrin and talin, proteins linking focal adhesions to the cytoskeleton. Inhibition of ERK1/2 or calpain, but not of JNK, mimics MEKK1 deficiency. Therefore, MEKK1 regulates calpain-mediated substratum release of migrating fibroblasts.