Mixed lineage kinase 3 connects reactive oxygen species to c-Jun NH2-terminal kinase-induced mitochondrial apoptosis in genipin-treated PC3 human prostate cancer cells

It has been reported that genipin, the aglycone of geniposide, induces apoptotic cell death in human hepatoma cells via a NADPH oxidase-reactive oxygen species (ROS)-c-Jun NH(2)-terminal kinase (JNK)-dependent activation of mitochondrial pathway. This continuing work aimed to define that mixed lineage kinase 3 (MLK3) is a key mediator, which connect between ROS and JNK in genipin-induced cell death signaling. In PC3 human prostate cancer cells, genipin stimulated MLK3 activity in concentration- and time-dependent manner. The PC3 cells stably transfected with dominant-negative form of MLK3 was less susceptible to population of the sub-G1 apoptotic cells, activation of caspase, collapse of mitochondrial membrane potential, and release of cytochrome c triggered by genipin, suggesting a crucial role of MLK3 in genipin signaling to apoptotic cell death. Diphenyleneiodonium (DPI), a specific inhibitor of NADPH oxidase, markedly inhibited ROS generation and MLK3 phosphorylation in the genipin-treated cells. Pretreatment with SP0600125, a specific inhibitor of JNK but neither U0126, a specific inhibitor of MEK1/2 nor PD169316, a specific inhibitor of p38 suppressed genipin-induced apoptotic cell death. Notably, both the phosphorylation of JNK and induction of c-Jun induced by genipin were markedly inhibited in PC3-EGFP-MLK3 (K144R) cells expressing a dominant-negative MLK3 mutant. Taken together, our observations suggest genipin signaling to apoptosis of PC3 cells is mediated via activation of ROS-dependent MLK3, which leads to downstream activation of JNK.     

The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3.

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.     

Cdc2 and Cdk2 kinase activated by transforming growth factor-beta1 trigger apoptosis through the phosphorylation of retinoblastoma protein in FaO hepatoma cells.

The signaling pathway leading to TGF-beta1-induced apoptosis was investigated using a TGF-beta1-sensitive hepatoma cell line, FaO. Cell cycle analysis demonstrated that the accumulation of apoptotic cells was preceded by a progressive decrease of the cell population in the G(1) phase concomitant with a slight increase of the cell population in the G(2)/M phase in response to TGF-beta1. TGF-beta1 induced a transient increase in the expression of Cdc2, cyclin A, cyclin B, and cyclin D1 at an early phase of apoptosis. During TGF-beta1-induced apoptosis, the transient increase in cyclin-dependent kinase (Cdk) activities coincides with a dramatic increase in the hyperphosphorylated forms of RB. Treatment with roscovitine or olomoucine, inhibitors of Cdc2 and Cdk2, blocked TGF-beta1-induced apoptosis by inhibiting RB phosphorylation. Overexpression of Bcl-2 or adenovirus E1B 19K suppressed TGF-beta1-induced apoptosis by blocking the induction of Cdc2 mRNA and the subsequent activation of Cdc2 kinase, whereas activation of Cdk2 was not affected, suggesting that Cdc2 plays a more critical role in TGF-beta1-induced apoptosis. In conclusion, we present the evidence that Cdc2 and Cdk2 kinase activity transiently induced by TGF-beta1 phosphorylates RB as a physiological target in FaO cells and that RB hyperphosphorylation may trigger abrupt cell cycle progression, leading to irreversible cell death.     

Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC(50) values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.     

Prevention of anti-IgM-induced apoptosis accompanying G1 arrest in B lymphoma cells overexpressing dominant-negative mutant form of c-Jun N-terminal kinase 1

A family of mitogen-activated protein (MAP) kinases comprising the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAP kinases are involved in proliferation and apoptosis. However, there are some arguments concerning the role of these kinases in Ag-induced B cell apoptosis. Two of the B lymphoma cell lines (CH31 and WEHI-231) susceptible to anti-IgM-induced apoptosis were used as a model. To address these issues, we examined the kinetics of anti-IgM-induced activation of MAP kinases and established cell lines overexpressing a dominant-negative (dn) mutant form of JNK1 (dnJNK1). Anti-IgM induced a sustained JNK1 activation with a peak at 8 h, with a marginal activation of ERK1/ERK2 in CH31 cells. The sustained JNK1 activation was not a secondary event through a caspase activation. The peak point of the JNK1 activation was just before the onset of a decline in mitochondrial membrane potential, which preceded anti-IgM-induced cell death. Following anti-IgM stimulation, dnJNK1 prevented a decline in mitochondrial membrane potential at 24 h, with a prolonged inhibition up to 72 h in WEHI-231, although it did so only partially during a later time period in CH31. The dnJNK1 cells also demonstrated diminished procaspase-3 activation and a decreased rate of apoptosis upon anti-IgM stimulation, with a concomitant increased arrest in G(1) phase, which could be explained by enhanced levels of cyclin-dependent kinase inhibitor p27(Kip1) protein. Thus, anti-IgM-induced JNK activation might be implicated in cell cycle progression as well as in apoptosis regulation, probably involving p27(Kip1) protein.     

Transforming growth factor-beta1 (TGF-beta)-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGF-beta-activated kinase 1 and mitogen-activated protein kinase kinase 3

The inhibitory Smad7, a direct target gene for transforming growth factor-beta (TGF-beta), mediates TGF-beta1-induced apoptosis in several cell types. Herein, we report that apoptosis of human prostate cancer PC-3U cells induced by TGF-beta1 or Smad7 overexpression is caused by a specific activation of the p38 mitogen-activated protein kinase pathway in a TGF-beta-activated kinase 1 (TAK1)- and mitogen-activated protein kinase kinase 3 (MKK3)-dependent manner. Expression of dominant negative p38, dominant negative MKK3, or incubation with the p38 selective inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], prevented TGF-beta1-induced apoptosis. The expression of Smad7 was required for TGF-beta-induced activation of MKK3 and p38 kinases, and endogenous Smad7 was found to interact with phosphorylated p38 in a ligand-dependent manner. Ectopic expression of wild-type TAK1 promoted TGF-beta1-induced phosphorylation of p38 and apoptosis, whereas dominant negative TAK1 reduced TGF-beta1-induced phosphorylation of p38 and apoptosis. Endogenous Smad7 was found to interact with TAK1, and TAK1, MKK3, and p38 were coimmunoprecipitated with Smad7 in transiently transfected COS1 cells. Moreover, ectopically expressed Smad7 enhanced the coimmunoprecipitation of HA-MKK3 and Flag-p38, supporting the notion that Smad7 may act as a scaffolding protein and facilitate TAK1- and MKK3-mediated activation of p38.     

Transforming growth factor-beta1 potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling

Tubular atrophy resulting from epithelial cell loss is one of the characteristic features in the development of chronic renal interstitial fibrosis. Although the trigger(s) and mechanism for tubular cell loss remain undefined, the hyperactive transforming growth factor (TGF)-beta1 signaling has long been suspected to play an active role. Here we demonstrate that although TGF-beta1 did not induce cell death per se, it dramatically potentiated renal tubular cell apoptosis initiated by other death cues in vitro. Pre-incubation of human kidney epithelial cells (HKC) with TGF-beta1 markedly promoted staurosporine-induced cell death in a time- and dose-dependent manner. TGF-beta1 dramatically accelerated the cleavage and activation of pro-caspase-9, but not pro-caspase-8, in HKC cells. This event was followed by an accelerated activation of pro-caspase-3. To elucidate the mechanism underlying TGF-beta1 promotion of tubular cell death, we investigated the signaling pathways activated by TGF-beta1. Both Smad-2 and p38 mitogen-activated protein (MAP) kinase were rapidly activated by TGF-beta1, as demonstrated by the early induction of phosphorylated Smad-2 and p38 MAP kinase, respectively. We found that overexpression of inhibitory Smad-7 completely abolished Smad-2 phosphorylation and activation induced by TGF-beta1 but did not inhibit TGF-beta1-induced apoptosis. However, suppression of p38 MAP kinase with chemical inhibitor SC68376 not only abolished p38 MAP kinase phosphorylation but also obliterated apoptosis induced by TGF-beta1. These results suggest that hyperactive TGF-beta1 signaling potentiates renal tubular epithelial cell apoptosis by a Smad-independent, p38 MAP kinase-dependent mechanism.     

The MLK Family Mediates c-Jun N-Terminal Kinase Activation in Neuronal Apoptosis

Neuronal apoptotic death induced by nerve growth factor (NGF) deprivation is reported to be in part mediated through a pathway that includes Rac1 and Cdc42, mitogen-activated protein kinase kinases 4 and 7 (MKK4 and -7), c-Jun N-terminal kinases (JNKs), and c-Jun. However, additional components of the pathway remain to be defined. We show here that members of the mixed-lineage kinase (MLK) family (including MLK1, MLK2, MLK3, and dual leucine zipper kinase [DLK]) are expressed in neuronal cells and are likely to act between Rac1/Cdc42 and MKK4 and -7 in death signaling. Overexpression of MLKs effectively induces apoptotic death of cultured neuronal PC12 cells and sympathetic neurons, while expression of dominant-negative forms of MLKs suppresses death evoked by NGF deprivation or expression of activated forms of Rac1 and Cdc42. CEP-1347 (KT7515), which blocks neuronal death caused by NGF deprivation and a variety of additional apoptotic stimuli and which selectively inhibits the activities of MLKs, effectively protects neuronal PC12 cells from death induced by overexpression of MLK family members. In addition, NGF deprivation or UV irradiation leads to an increase in both level and phosphorylation of endogenous DLK. These observations support a role for MLKs in the neuronal death mechanism. With respect to ordering the death pathway, dominant-negative forms of MKK4 and -7 and c-Jun are protective against death induced by MLK overexpression, placing MLKs upstream of these kinases. Additional findings place the MLKs upstream of mitochondrial cytochrome c release and caspase activation.     

Transforming growth factor-beta stimulates parathyroid hormone-related protein and osteolytic metastases via Smad and mitogen-activated protein kinase signaling pathways

Transforming growth factor (TGF)-beta promotes breast cancer metastasis to bone. To determine whether the osteolytic factor parathyroid hormone-related protein (PTHrP) is the primary mediator of the tumor response to TGF-beta, mice were inoculated with MDA-MB-231 breast cancer cells expressing a constitutively active TGF-beta type I receptor. Treatment of the mice with a PTHrP-neutralizing antibody greatly decreased osteolytic bone metastases. There were fewer osteoclasts and significantly decreased tumor area in the antibody-treated mice. TGF-beta can signal through both Smad and mitogen-activated protein (MAP) kinase pathways. Stable transfection of wild-type Smad2, Smad3, or Smad4 increased TGF-beta-stimulated PTHrP secretion, whereas dominant-negative Smad2, Smad3, or Smad4 only partially reduced TGF-beta-stimulated PTHrP secretion. When the cells were treated with a variety of protein kinases inhibitors, only specific inhibitors of the p38 MAP kinase pathway significantly reduced both basal and TGF-beta-stimulated PTHrP production. The combination of Smad dominant-negative blockade and p38 MAP kinase inhibition resulted in complete inhibition of TGF-beta-stimulated PTHrP production. Furthermore, TGF-beta treatment of MDA-MB-231 cells resulted in a rapid phosphorylation of p38 MAP kinase. Thus, the p38 MAP kinase pathway appears to be a major component of Smad-independent signaling by TGF-beta and may provide a new molecular target for anti-osteolytic therapy.     

High glucose-mediated effects on endothelial cell proliferation occur via p38 MAP kinase

The mitogen-activated protein (MAP) kinases contribute to altered cell growth and function in a variety of disease states. However, their role in the endothelial complications of diabetes mellitus remains unclear. Human endothelial cells were exposed for 72 h to 5 mM (control) or 25 mM (high) glucose or 5 mM glucose plus 20 mM mannitol (osmotic control). The roles of p38 and p42/44 MAP kinases in the high glucose-induced growth effects were determined by assessment of phosphorylated MAP kinases and their downstream activators by Western blot and by pharmacological inhibition of these MAP kinases. Results were expressed as a percentage (means +/- SE) of control. High glucose increased the activity of total and phosphorylated p38 MAP kinase (P < 0.001) and p42/44 MAP kinase (P < 0.001). Coexposure of p38 MAP kinase blocker with high glucose reversed the antiproliferative but not the hypertrophic effects associated with high-glucose conditions. Transforming growth factor (TGF)-beta1 increased the levels of phosphorylated p38 MAP kinase, and p38 MAP kinase blockade reversed the antiproliferative effects of this cytokine. The high glucose-induced increase in phosphorylated p38 MAP kinase was reversed in the presence of TGF-beta1 neutralizing antibody. Although hyperosmolarity also induced antiproliferation (P < 0.0001) and cell hypertrophy (P < 0.05), there was no change in p38 activity, and therefore inhibition of p38 MAP kinase had no influence on these growth responses. Blockade of p42/44 MAP kinase had no effect on the changes in endothelial cell growth induced by either high glucose or hyperosmolarity. High glucose increased p42/44 and p38 MAP kinase activity in human endothelial cells, but only p38 MAP kinase mediated the antiproliferative growth response through the effects of autocrine TGF-beta1. High glucose-induced endothelial cell hypertrophy was independent of activation of the MAP kinases studied. In addition, these effects were independent of any increase in osmolarity associated with high-glucose exposure.     

Redox signaling and the MAP kinase pathways

The mitogen-activated protein (MAP) kinases are a large family of proline-directed, serine/threonine kinases that require tyrosine and threonine phosphorylation of a TxY motif in the activation loop for activation through a phosphorylation cascade involving a MAPKKK, MAPKK and MAPK, often referred to as the MAP kinase module. Three separate such modules have been identified, based on the TxY motif of the MAP kinase and the dual-specificity kinases that strictly phosphorylate their specific TxY sequence. They are the extracellular signal regulated kinases (ERKs), c-jun N-terminal kinases (JNKs) and p38 MAPKs. The ERKs are mainly associated with proliferation and differentiation while the JNKs and p38MAP kinases regulate responses to cellular stresses. Redox homeostasis is critical for proper cellular function. While reactive oxygen species (ROS) and oxidative stress have been implicated in injury, a rapidly growing literature suggests that a transient increase in ROS levels is an important mediator of proliferation and results in activation of various signaling molecules and pathways, among which the MAP kinases. This review will summarize the role of ROS in MAP kinase activation in various systems, including in macrophages, cells of myeloid origin that play an essential role in inflammation and express a multi-component NADPH oxidase that catalyzes the receptor-regulated production of ROS.     

A Novel Role for Mixed Lineage Kinase 3 (MLK3) in B-Raf Activation and Cell proliferation

The extracellular signal-regulated kinase (ERK) group of MAPKs is essential for cell proliferation, including that stimulated by mitogens, oncogenic ras and raf. The Raf kinases (especially B-Raf) are ERK-specific, mitogen-activated MAP3Ks. Mixed lineage kinase-3 (MLK3) is a MAP3K previously thought to be a selective regulator of the JNK group of MAPKs. Surprisingly, we found that silencing of mlk3 by RNAi suppresses mitogen and cytokine activation not only of JNK but of ERK and p38 as well. Silencing mlk3 also blocks mitogen-stimulated phosphorylation of B-Raf at Thr598 and Ser601—a step required for B-Raf activation. Finally, silencing mlk3 prevents serum-stimulated cell proliferation and the proliferation of tumor cells bearing either oncogenic Ki-Ras or loss of function neurofibromatosis-1 (NF1) or NF2 mutations. The proliferation of tumor cells with activating mutations in B-raf or raf-1 are unaffected by silencing mlk3. These results define a new role for MLK3 in B-Raf activation, ERK signaling and cell proliferation. Accordingly, targeting MLK3 could be beneficial to the treatment of tumors with activated receptor tyrosine kinase or ras mutations, and to the treatment of NF1 or NF2 tumors.     

Hsp90/p50cdc37 is required for mixed-lineage kinase (MLK) 3 signaling

Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase (MAPK) kinase kinase that activates MAPK pathways, including the c-Jun NH(2)-terminal kinase (JNK) and p38 pathways. MLK3 and its family members have been implicated in JNK-mediated apoptosis. A survey of human cell lines revealed high levels of MLK3 in breast cancer cells. To learn more about MLK3 regulation and its signaling pathways in breast cancer cells, we engineered the estrogen-responsive human breast cancer cell line, MCF-7, to stably, inducibly express FLAG epitope-tagged MLK3. FLAG.MLK3 complexes were isolated by affinity purification, and associated proteins were identified by in-gel trypsin digestion followed by liquid chromatography/tandem mass spectrometry. Among the proteins identified were heat shock protein 90alpha,beta (Hsp90) and its kinase-specific co-chaperone p50(cdc37). We show that endogenous MLK3 complexes with Hsp90 and p50(cdc37). Further experiments demonstrate that MLK3 associates with Hsp90/p50(cdc37) through its catalytic domain in an activity-independent manner. Upon treatment of MCF-7 cells with geldanamycin, an ansamycin antibiotic that inhibits Hsp90 function, MLK3 levels decrease dramatically. Furthermore, tumor necrosis factor alpha-induced activation of MLK3 and JNK in MCF-7 cells is blocked by geldanamycin treatment. Our finding that geldanamycin treatment does not affect the cellular levels of the downstream signaling components, MAPK kinase 4, MAPK kinase 7, and JNK, suggests that Hsp90/p50(cdc37) regulates JNK signaling at the MAPK kinase kinase level. Previously identified Hsp90/p50(cdc37) clients include oncoprotein kinases and protein kinases that promote cellular proliferation and survival. Our findings reveal that Hsp90/p50(cdc37) also regulates protein kinases involved in apoptotic signaling.     

Inhibition of mixed-lineage kinase (MLK) activity during G2-phase disrupts microtubule formation and mitotic progression in HeLa cells

The mixed-lineage kinases (MLK) are serine/threonine protein kinases that regulate mitogen-activated protein (MAP) kinase signaling pathways in response to extracellular signals. Recent studies indicate that MLK activity may promote neuronal cell death through activation of the c-Jun NH2-terminal kinase (JNK) family of MAP kinases. Thus, inhibitors of MLK activity may be clinically useful for delaying the progression of neurodegenerative diseases, such as Parkinson's. In proliferating non-neuronal cells, MLK may have the opposite effect of promoting cell proliferation. In the current studies we examined the requirement for MLK proteins in regulating cell proliferation by examining MLK function during G2 and M-phase of the cell cycle. The MLK inhibitor CEP-11004 prevented HeLa cell proliferation by delaying mitotic progression. Closer examination revealed that HeLa cells treated with CEP-11004 during G2-phase entered mitosis similar to untreated G2-phase cells. However, CEP-11004 treated cells failed to properly exit mitosis and arrested in a pro-metaphase state. Partial reversal of the CEP-11004 induced mitotic arrest could be achieved by overexpression of exogenous MLK3. The effects of CEP-11004 treatment on mitotic events included the inhibition of histone H3 phosphorylation during prophase and prior to nuclear envelope breakdown and the formation of aberrant mitotic spindles. These data indicate that MLK3 might be a unique target to selectively inhibit transformed cell proliferation by disrupting mitotic spindle formation resulting in mitotic arrest.     

TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling

Stress-activated mitogen-activated protein (MAP) kinase p38 mediates stress signaling in mammalian cells via threonine and tyrosine phosphorylation in its conserved TGY motif by upstream MAP kinase kinases (MKKs). In addition, p38 MAP kinase can also be activated by an MKK-independent mechanism involving TAB-1 (TAK-1-binding protein)-mediated autophosphorylation. Although TAB-1-mediated p38 activation has been implicated in ischemic heart, the biological consequences and downstream signaling of TAB-1-mediated p38 activation in cardiomyocytes is largely unknown. We show here that TAB-1 expression leads to a significant induction of p38 autophosphorylation and consequent kinase activation in cultured neonatal cardiomyocytes. In contrast to MKK3-induced p38 kinase downstream effects, TAB-1-induced p38 kinase activation does not induce expression of pro-inflammatory genes, cardiac marker gene expression, or changes in cellular morphology. Rather, TAB-1 binds to p38 and prevents p38 nuclear localization. Furthermore, TAB-1 disrupts p38 interaction with MKK3 and redirects p38 localization in the cytosol. Consequently, TAB-1 expression antagonizes the downstream activity of p38 kinase induced by MKK3 and attenuates interleukin-1beta-induced inflammatory gene induction in cardiomyocytes. These data suggest that TAB-1 can mediate MKK-independent p38 kinase activation while negatively modulating MKK-dependent p38 function. Our study not only redefines the functional role of TAB-1 in p38 kinase-mediated signaling pathways but also provides the first evidence that intracellular localization of p38 kinase and complex interaction dictates its downstream effects. These results suggest a previously unknown mechanism for stress-MAP kinase regulation in mammalian cells.     

MKK3 and -6-dependent activation of p38alpha MAP kinase is required for cytoskeletal changes in pulmonary microvascular endothelial cells induced by ICAM-1 ligation

Previous studies demonstrated that neutrophil adherence induces ICAM-1-dependent cytoskeletal changes in TNF-alpha-treated pulmonary microvascular endothelial cells that are prevented by a pharmacological inhibitor of p38 MAP kinase. This study determined whether neutrophil adherence induces activation of p38 MAP kinase in endothelial cells, the subcellular localization of phosphorylated p38, which MAP kinase kinases lead to p38 activation, which p38 isoform is activated, and what the downstream targets may be. Confocal microscopy showed that neutrophil adhesion for 2 or 6 min induced an increase in phosphorylated p38 in endothelial cells that was punctate and concentrated in the central region of the endothelial cells. Studies using small interfering RNA (siRNA) to inhibit the protein expression of MAP kinase kinase 3 and 6, either singly or in combination, showed that both MAP kinase kinases were required for p38 phosphorylation. Studies using an antisense oligonucleotide to p38alpha demonstrated that inhibition of the protein expression of p38alpha 1) inhibited activation of p38 MAP kinase without affecting the protein expression of p38beta; 2) prevented phosphorylation of heat shock protein 27, an actin binding protein that may induce actin polymerization upon phosphorylation; 3) attenuated cytoskeletal changes; and 4) attenuated neutrophil migration to the EC borders. Thus MAP kinase kinase3- and 6-dependent activation of the alpha-isoform of p38 MAP kinase is required for the cytoskeletal changes induced by neutrophil adherence and influences subsequent neutrophil migration toward endothelial cell junctions.     

Glycogen synthase kinase-3beta induces neuronal cell death via direct phosphorylation of mixed lineage kinase 3

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3beta has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3beta during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3beta, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3beta-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3beta activation, indicating that induction of GSK-3beta lies upstream of MLK3. In GSK-3beta-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3beta. The activation of MLK3 by GSK-3beta occurred via phosphorylation of MLK3 on two amino acid residues, Ser(789) and Ser(793), that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3beta was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3beta. Taken together, our data provide a direct link between GSK-3beta and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3beta. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.