MAP3K4 promotes fetal and placental growth by controlling the receptor tyrosine kinases IGF1R/IR and Akt signaling pathway.

Disruption of fetal growth results in severe consequences to human health, including increased fetal and neonatal morbidity and mortality, as well as potential lifelong health problems. Molecular mechanisms promoting fetal growth represent potential therapeutic strategies to treat and/or prevent fetal growth restriction (FGR). Here, we identify a previously unknown role for the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) in promoting fetal and placental growth. We demonstrate that inactivation of MAP3K4 kinase activity causes FGR due in part to placental insufficiency. Significantly, MAP3K4 kinase-inactive mice display highly penetrant lethality prior to weaning and persistent growth reduction of surviving adults. Additionally, we elucidate molecular mechanisms by which MAP3K4 promotes growth through control of the insulin-like growth factor 1 receptor (IGF1R), insulin receptor (IR), and Akt signaling pathway. Specifically, MAP3K4 kinase inactivation in trophoblast stem (TS) cells results in reduced IGF1R and IR expression and decreased Akt activation. We observe these changes in TS cells also occur in differentiated trophoblasts created through in vitro differentiation of cultured TS cells and in vivo in placental tissues formed by TS cells. Furthermore, we show that MAP3K4 controls this pathway by promoting Igf1r transcript expression in TS cells through activation of CREB-binding protein (CBP). In the MAP3K4 kinase-inactive TS cells, Igf1r transcripts are repressed because of reduced CBP activity and increased histone deacetylase 6 expression and activity. Together, these data demonstrate a critical role for MAP3K4 in promoting fetal and placental growth by controlling the activity of the IGF1R/IR and Akt signaling pathway.

A non-translational role of threonyl-tRNA synthetase in regulating JNK signaling during myogenic differentiation.

Aminoacyl-tRNA synthetases (aaRSs) are house-keeping enzymes that are essential for protein synthesis. However, it has become increasingly evident that some aaRSs also have non-translational functions. Here we report the identification of a non-translational function of threonyl-tRNA synthetase (ThrRS) in myogenic differentiation. We find that ThrRS negatively regulates myoblast differentiation in vitro and injury-induced skeletal muscle regeneration in vivo. This function is independent of amino acid binding or aminoacylation activity of ThrRS, and knockdown of ThrRS leads to enhanced differentiation without affecting the global protein synthesis rate. Furthermore, we show that the non-catalytic new domains (UNE-T and TGS) of ThrRS are both necessary and sufficient for the myogenic function. In searching for a molecular mechanism of this new function, we find the kinase JNK to be a downstream target of ThrRS. Our data further reveal MEKK4 and MKK4 as upstream regulators of JNK in myogenesis and the MEKK4-MKK4-JNK pathway to be a mediator of the myogenic function of ThrRS. Finally, we show that ThrRS physically interacts with Axin1, disrupts Axin1-MEKK4 interaction and consequently inhibits JNK signaling. In conclusion, we uncover a non-translational function for ThrRS in the maintenance of homeostasis of skeletal myogenesis and identify the Axin1-MEKK4-MKK4-JNK signaling axis to be an immediate target of ThrRS action.

LncRNA-MAP3K4 regulates vascular inflammation through the p38 MAPK signaling pathway and cis-modulation of MAP3K4.

Chronic vascular inflammation plays a key role in the pathogenesis of atherosclerosis. Long non-coding RNAs (lncRNAs) have emerged as essential inflammation regulators. We identify a novel lncRNA termed lncRNA-MAP3K4 that is enriched in the vessel wall and regulates vascular inflammation. In the aortic intima, lncRNA-MAP3K4 expression was reduced by 50% during the progression of atherosclerosis (chronic inflammation) and 70% during endotoxemia (acute inflammation). lncRNA-MAP3K4 knockdown reduced the expression of key inflammatory factors (eg, ICAM-1, E-selectin, MCP-1) in endothelial cells or vascular smooth muscle cells and decreased monocytes adhesion to endothelium, as well as reducing TNF-α, IL-1ß, COX2 expression in macrophages. Mechanistically, lncRNA-MAP3K4 regulates inflammation through the p38 MAPK signaling pathway. lncRNA-MAP3K4 shares a bidirectional promoter with MAP3K4, an upstream regulator of the MAPK signaling pathway, and regulates its transcription in cis. lncRNA-MAP3K4 and MAP3K4 show coordinated expression in response to inflammation in vivo and in vitro. Similar to lncRNA-MAP3K4, MAP3K4 knockdown reduced the expression of inflammatory factors in several different vascular cells. Furthermore, lncRNA-MAP3K4 and MAP3K4 knockdown showed cooperativity in reducing inflammation in endothelial cells. Collectively, these findings unveil the role of a novel lncRNA in vascular inflammation by cis-regulating MAP3K4 via a p38 MAPK pathway.

MAP3K4 kinase activity dependent control of mouse gonadal sex determination†.

Sex determination requires the commitment of bipotential gonads to either a testis or an ovarian fate. Gene deletion of the kinase Map3k4 results in gonadal sex reversal in XY mice, and transgenic re-expression of Map3k4 rescues the sex reversal phenotype. Map3k4 encodes a large, multi-functional protein possessing a kinase domain and several, additional protein-protein interaction domains. Although MAP3K4 plays a critical role in male gonadal sex determination, it is unknown if the kinase activity of MAP3K4 is required. Here, we use mice expressing full-length, kinase-inactive MAP3K4 from the endogenous Map3k4 locus to examine the requirement of MAP3K4 kinase activity in sex determination. Although homozygous kinase-inactivation of MAP3K4 (Map3k4KI/KI) is lethal, a small fraction survive to adulthood. We show Map3k4KI/KI adults exhibit a 4:1 female-biased sex ratio. Many adult Map3k4KI/KI phenotypic females have a Y chromosome. XY Map3k4KI/KI adults with sex reversal display female mating behavior, but do not give rise to offspring. Reproductive organs are overtly female, but there is a broad spectrum of ovarian phenotypes, including ovarian absence, primitive ovaries, reduced ovarian size, and ovaries having follicles in all stages of development. Further, XY Map3k4KI/KI adults are smaller than either male or female Map3k4WT/WT mice. Examination of the critical stage of gonadal sex determination at E11.5 shows that loss of MAP3K4 kinase activity results in the loss of Sry expression in XY Map3k4KI/KI embryos, indicating embryonic male gonadal sex reversal. Together, these findings demonstrate the essential role for kinase activity of MAP3K4 in male gonadal sex determination.

Activation of JNK signaling in osteoblasts is inversely correlated with collagen synthesis in age-related osteoporosis.

The age-related reduction in the function of osteoblasts plays a central role in the pathogenesis of bone loss and osteoporosis. Collagen synthesis is a primary function of differentiated osteoblasts, however, the mechanisms for age-related changes in collagen synthesis in human osteoblasts remain elusive. We use Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) analysis to exploit the transcriptional profiles of osteoblasts from young and old donors. A panel of collagen members was downregulated in aged osteoblasts, including COL12A1, COL5A1, COL5A3, COL8A1 and COL8A2. Co-expression analysis followed by GO analysis revealed that oxidoreductase activity and kinase activity were inversely correlated with collagen synthesis in osteoblasts. GESA analysis further showed that JNK signaling was upregulated in aged osteoblasts. Consistently, MAP3K4 and MAP4K2, upstream of JNK, were also increased in aged osteoblasts. Moreover, expression levels of MAP3K4 were significantly inversely correlated with levels of the collagen genes. Those transcriptomic results were further verified by examining clinical specimens of osteoporosis by immunohistochemistry. These results provide transcriptomic evidence that deregulated JNK signaling may impair collagen synthesis in osteoblasts and imply a therapeutic value of JNK inhibitors for treating osteoporosis and preventing skeletal aging by counteracting the age-related reduction in the function of osteoblasts.

MEKK4 Signaling Regulates Sensory Cell Development and Function in the Mouse Inner Ear.

Mechanosensory hair cells (HCs) residing in the inner ear are critical for hearing and balance. Precise coordination of proliferation, sensory specification, and differentiation during development is essential to ensure the correct patterning of HCs in the cochlear and vestibular epithelium. Recent studies have revealed that FGF20 signaling is vital for proper HC differentiation. However, the mechanisms by which FGF20 signaling promotes HC differentiation remain unknown. Here, we show that mitogen-activated protein 3 kinase 4 (MEKK4) expression is highly regulated during inner ear development and is critical to normal cytoarchitecture and function. Mice homozygous for a kinase-inactive MEKK4 mutation exhibit significant hearing loss. Lack of MEKK4 activity in vivo also leads to a significant reduction in the number of cochlear and vestibular HCs, suggesting that MEKK4 activity is essential for overall development of HCs within the inner ear. Furthermore, we show that loss of FGF20 signaling in vivo inhibits MEKK4 activity, whereas gain of Fgf20 function stimulates MEKK4 expression, suggesting that Fgf20 modulates MEKK4 activity to regulate cellular differentiation. Finally, we demonstrate, for the first time, that MEKK4 acts as a critical node to integrate FGF20-FGFR1 signaling responses to specifically influence HC development and that FGFR1 signaling through activation of MEKK4 is necessary for outer hair cell differentiation. Collectively, this study provides compelling evidence of an essential role for MEKK4 in inner ear morphogenesis and identifies the requirement of MEKK4 expression in regulating the specific response of FGFR1 during HC development and FGF20/FGFR1 signaling activated MEKK4 for normal sensory cell differentiation. SIGNIFICANCE STATEMENT: Sensory hair cells (HCs) are the mechanoreceptors within the inner ear responsible for our sense of hearing. HCs are formed before birth, and mammals lack the ability to restore the sensory deficits associated with their loss. In this study, we show, for the first time, that MEKK4 signaling is essential for the development of normal cytoarchitecture and hearing function as MEKK4 signaling-deficient mice exhibit a significant reduction of HCs and a hearing loss. We also identify MEKK4 as a critical hub kinase for FGF20-FGFR1 signaling to induce HC differentiation in the mammalian cochlea. These results reveal a new paradigm in the regulation of HC differentiation and provide significant new insights into the mechanism of Fgf signaling governing HC formation.

Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4.

Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level.

Molecular cloning, characterization and functional analysis of a putative mitogen-activated protein kinase kinase kinase 4 (MEKK4) from blood clam Tegillarca granosa.

The mitogen-activated protein kinase (MAPK) cascades stand for one of the most important signaling mechanisms in response to environmental stimuli. In the present study, we cloned and identified for the first time the full-length cDNA of MAPK kinase kinase 4 (TgMEKK4) from Blood clam Tegillarca granosa using rapid amplification of cDNA ends method. The full-length cDNA of TgMEKK4 was of 1605 bp in length, encoding a polypeptide of 364 amino acids with a predicted molecular mass of 41.22 kDa and theoretical isoelectric point of 6.29. The conserved MEKK4-domain was identified in TgMEKK4 by SMART program analysis. Homology analysis of the deduced amino acid sequence of TgMEKK4 with other known sequences revealed that TgMEKK4 shared 58%-80% identity to MEKK4s from other species. TgMEKK4 mRNA transcripts could be detected in all tissues examined with the highest expression level in the gill by qRT-PCR. The mRNA expression of TgMEKK4 was up-regulated significantly in hemocytes after Vibrio parahaemolyticus, Vibrio alginolyticus and Lipopolysaccharide (LPS) challenges. Overexpression of TgMEKK4 in HEK 293T cells resulted in the activation of JNK and ERK, but not p38. Consistently, In vivo study indicated that LPS stimulation enhanced JNK, ERK and p38 phosphorylation in blood clams. These results suggest that TgMEKK4 is a powerful factor in the regulation of genes that may be involved in innate immune response of blood clam.

Transgenic expression of Map3k4 rescues T-associated sex reversal (Tas) in mice.

Disorders of sex development in the human population range in severity from mild genital defects to gonadal sex reversal. XY female development has been associated with heterozygous mutations in several genes, including SOX9, WT1 and MAP3K1. In contrast, XY sex reversal in mice usually requires complete absence of testis-determining gene products. One exception to this involves T-associated sex reversal (Tas), a phenomenon characterized by the formation of ovotestes or ovaries in XY mice hemizygous for the hairpin-tail (T(hp)) or T-Orleans (T(Orl)) deletions on proximal mouse chromosome 17. We recently reported that mice heterozygous for a null allele of Map3k4, which resides in the T(hp) deletion, exhibit XY ovotestis development and occasional gonadal sex reversal on the sensitized C57BL/6J-Y(AKR) (B6-Y(AKR)) genetic background, reminiscent of the Tas phenotype. However, these experiments did not exclude the possibility that loss of other loci in the T(hp) deletion, or other effects of the deletion itself, might contribute to Tas. Here, we show that disruption to Sry expression underlies XY gonadal defects in B6-Y(AKR) embryos harbouring the T(hp) deletion and that a functional Map3k4 bacterial artificial chromosome rescues these abnormalities by re-establishing a normal Sry expression profile. These data demonstrate that Map3k4 haploinsufficiency is the cause of T-associated sex reversal and that levels of this signalling molecule are a major determinant of the expression profile of Sry.

Identification of novel HIV-1 dependency factors in primary CCR4(+)CCR6(+)Th17 cells via a genome-wide transcriptional approach.

BACKGROUND: The HIV-1 infection is characterized by profound CD4(+) T cell destruction and a marked Th17 dysfunction at the mucosal level. Viral suppressive antiretroviral therapy restores Th1 but not Th17 cells. Although several key HIV dependency factors (HDF) were identified in the past years via genome-wide siRNA screens in cell lines, molecular determinants of HIV permissiveness in primary Th17 cells remain to be elucidated. RESULTS: In an effort to orient Th17-targeted reconstitution strategies, we investigated molecular mechanisms of HIV permissiveness in Th17 cells. Genome-wide transcriptional profiling in memory CD4(+) T-cell subsets enriched in cells exhibiting Th17 (CCR4(+)CCR6(+)), Th1 (CXCR3(+)CCR6(-)), Th2 (CCR4(+)CCR6(-)), and Th1Th17 (CXCR3(+)CCR6(+)) features revealed remarkable transcriptional differences between Th17 and Th1 subsets. The HIV-DNA integration was superior in Th17 versus Th1 upon exposure to both wild-type and VSV-G-pseudotyped HIV; this indicates that post-entry mechanisms contribute to viral replication in Th17. Transcripts significantly enriched in Th17 versus Th1 were previously associated with the regulation of TCR signaling (ZAP-70, Lck, and CD96) and Th17 polarization (RORγt, ARNTL, PTPN13, and RUNX1). A meta-analysis using the NCBI HIV Interaction Database revealed a set of Th17-specific HIV dependency factors (HDFs): PARG, PAK2, KLF2, ITGB7, PTEN, ATG16L1, Alix/AIP1/PDCD6IP, LGALS3, JAK1, TRIM8, MALT1, FOXO3, ARNTL/BMAL1, ABCB1/MDR1, TNFSF13B/BAFF, and CDKN1B. Functional studies demonstrated an increased ability of Th17 versus Th1 cells to respond to TCR triggering in terms of NF-κB nuclear translocation/DNA-binding activity and proliferation. Finally, RNA interference studies identified MAP3K4 and PTPN13 as two novel Th17-specific HDFs. CONCLUSIONS: The transcriptional program of Th17 cells includes molecules regulating HIV replication at multiple post-entry steps that may represent potential targets for novel therapies aimed at protecting Th17 cells from infection and subsequent depletion in HIV-infected subjects.

Mitogen-activated protein kinase kinase kinase (MAPKKK) 4 from rapeseed (Brassica napus L.) is a novel member inducing ROS accumulation and cell death.

MAPKKK is the largest family of MAPK cascade, which is known to play important roles in plant growth, development and immune responses. So far, only a few have been functionally characterized even in the model plant, Arabidopsis due to the potential functional redundancy of MAPKKK. We previously identified and cloned a few MAPKKK family genes from rapeseed. In this study, BnaMAPKKK4 was characterized as a member in eliciting accumulation of reactive oxygen species (ROS) and hypersensitive response (HR)-like cell death. This is accompanied with accumulation of malondialdehyde (MDA), anthocyanin as well as nuclear DNA fragmentation. The transcript abundance of a series of ROS accumulation, cell death, and defense response related genes were up-regulated by the expression of MAPKKK4. Further investigation identified BnaMAPKKK4 elicited ROS through the downstream MPK3. These results indicate that BnaMAPKKK4 and its downstream components function in the ROS-induced cell death.

A dual role for receptor-interacting protein kinase 2 (RIP2) kinase activity in nucleotide-binding oligomerization domain 2 (NOD2)-dependent autophagy.

Autophagy is triggered by the intracellular bacterial sensor NOD2 (nucleotide-binding, oligomerization domain 2) as an anti-bacterial response. Defects in autophagy have been implicated in Crohn's disease susceptibility. The molecular mechanisms of activation and regulation of this process by NOD2 are not well understood, with recent studies reporting conflicting requirements for RIP2 (receptor-interacting protein kinase 2) in autophagy induction. We examined the requirement of NOD2 signaling mediated by RIP2 for anti-bacterial autophagy induction and clearance of Salmonella typhimurium in the intestinal epithelial cell line HCT116. Our data demonstrate that NOD2 stimulates autophagy in a process dependent on RIP2 tyrosine kinase activity. Autophagy induction requires the activity of the mitogen-activated protein kinases MEKK4 and p38 but is independent of NFκB signaling. Activation of autophagy was inhibited by a PP2A phosphatase complex, which interacts with both NOD2 and RIP2. PP2A phosphatase activity inhibited NOD2-dependent autophagy but not activation of NFκB or p38. Upon stimulation of NOD2, the phosphatase activity of the PP2A complex is inhibited through tyrosine phosphorylation of the catalytic subunit in a process dependent on RIP2 activity. These findings demonstrate that RIP2 tyrosine kinase activity is not only required for NOD2-dependent autophagy but plays a dual role in this process. RIP2 both sends a positive autophagy signal through activation of p38 MAPK and relieves repression of autophagy mediated by the phosphatase PP2A.

High yield purification of JNK1ß1 and activation by in vitro reconstitution of the MEKK1→MKK4→JNK MAPK phosphorylation cascade.

The c-Jun N-terminal kinase (JNK) pathway forms part of the mitogen-activated protein kinase (MAPK) signaling pathways comprising a sequential three-tiered kinase cascade. Here, an upstream MAP3K (MEKK1) phosphorylates and activates a MAP2K (MKK4 and MKK7), which in turn phosphorylates and activates the MAPK, JNK. The C-terminal kinase domain of MEKK1 (MEKK-C) is constitutively active, while MKK4/7 and JNK are both activated by dual phosphorylation of S/Y, and T/Y residues within their activation loops, respectively. While improvements in the purification of large quantities of active JNKs have recently been made, inadequacies in their yield, purity, and the efficiency of their phosphorylation still exist. We describe a novel and robust method that further improves upon the purification of large yields of highly pure, phosphorylated JNK1ß1, which is most suitable for biochemical and biophysical characterization. Codon harmonization of the JNK1ß1 gene was used as a precautionary measure toward increasing the soluble overexpression of the kinase. While JNK1ß1 and its substrate ATF2 were both purified to >99% purity as GST fusion proteins using GSH-agarose affinity chromatography and each cleaved from GST using thrombin, constitutively-active MEKK-C and inactive MKK4 were separately expressed in E. coli as thioredoxin-His(6)-tagged proteins and purified using urea refolding and Ni(2+)-IMAC, respectively. Activation of JNK1ß1 was then achieved by successfully reconstituting the JNK MAPK activation cascade in vitro; MEKK-C was used to activate MKK4, which in turn was used to efficiently phosphorylate and activate large quantities of JNK1ß1. Activated JNK1ß1 was thereafter able to phosphorylate ATF2 with high catalytic efficiency.

GADD45beta enhances Col10a1 transcription via the MTK1/MKK3/6/p38 axis and activation of C/EBPbeta-TAD4 in terminally differentiating chondrocytes.

GADD45beta (growth arrest- and DNA damage-inducible) interacts with upstream regulators of the JNK and p38 stress response kinases. Previously, we reported that the hypertrophic zone of the Gadd45beta(-/-) mouse embryonic growth plate is compressed, and expression of type X collagen (Col10a1) and matrix metalloproteinase 13 (Mmp13) genes is decreased. Herein, we report that GADD45beta enhances activity of the proximal Col10a1 promoter, which contains evolutionarily conserved AP-1, cAMP-response element, and C/EBP half-sites, in synergism with C/EBP family members, whereas the MMP13 promoter responds to GADD45beta together with AP-1, ATF, or C/EBP family members. C/EBPbeta expression also predominantly co-localizes with GADD45beta in the embryonic growth plate. Moreover, GADD45beta enhances C/EBPbeta activation via MTK1, MKK3, and MKK6, and dominant-negative p38alphaapf, but not JNKapf, disrupts the combined trans-activating effect of GADD45beta and C/EBPbeta on the Col10a1 promoter. Importantly, GADD45beta knockdown prevents p38 phosphorylation while decreasing Col10a1 mRNA levels but does not affect C/EBPbeta binding to the Col10a1 promoter in vivo, indicating that GADD45beta influences the transactivation function of DNA-bound C/EBPbeta. In support of this conclusion, we show that the evolutionarily conserved TAD4 domain of C/EBPbeta is the target of the GADD45beta-dependent signaling. Collectively, we have uncovered a novel molecular mechanism linking GADD45beta via the MTK1/MKK3/6/p38 axis to C/EBPbeta-TAD4 activation of Col10a1 transcription in terminally differentiating chondrocytes.

MEKK4 sequesters RIP2 to dictate NOD2 signal specificity.

The Crohn's-disease-susceptibility protein, NOD2, coordinates signaling responses upon intracellular exposure to bacteria. Although NOD2 is known to activate NFkappaB, little is known about the molecular mechanisms by which NOD2 coordinates functionally separate signaling pathways such as NFkappaB, JNK, and p38 to regulate cytokine responses. Given that one of the characteristics of Crohn's disease is an altered cytokine response to normal bacterial flora, the coupling of signaling pathways could be important for Crohn's-disease pathophysiology. We find that a MAP3K, MEKK4, binds to RIP2 to sequester RIP2 from the NOD2 signaling pathway. This MEKK4:RIP2 complex dissociates upon exposure to the NOD2 agonist, MDP, allowing NOD2 to bind to RIP2 and activate NFkappaB. MEKK4 thus sequesters RIP2 to inhibit the NOD2:RIP2 complex from activating NFkappaB signaling pathways, and Crohn's-disease-associated NOD2 polymorphisms cannot compete with MEKK4 for RIP2 binding. Lastly, we find that MEKK4 helps dictate signal specificity downstream of NOD2 activation as knockdown of MEKK4 in macrophages exposed to MDP causes increased NFkappaB activity, absent p38 activity, and hyporesponsiveness to TLR2 and TLR4 agonists. These biochemical findings suggest that basal inhibition of the NOD2-driven NFkappaB pathway by MEKK4 could be important in the pathogenesis of Crohn's disease.

Regulation of JNK activity in the apoptotic response of intestinal epithelial cells.

We have studied apoptosis of gastrointestinal epithelial cells by examining the receptor-mediated and DNA damage-induced pathways using TNF-α and camptothecin (CPT), respectively. TNF-α requires inhibition of antiapoptotic protein synthesis by cycloheximide (CHX). CHX also results in high levels of active JNK, which are necessary for TNF-induced apoptosis. While CPT induces apoptosis, the increase in JNK activity was not proportional to the degree of apoptosis. Thus the mechanism of activation of JNK and its role in apoptosis are unclear. We examined the course of JNK activation in response to a combination of TNF-α and CPT (TNF + CPT), which resulted in a three- to fourfold increase in apoptosis compared with CPT alone, indicating an amplification of apoptotic signaling pathways. TNF + CPT caused apoptosis by activating JNK, p38, and caspases-8, -9, and -3. TNF-α stimulated a transient phosphorylation of JNK1/2 and ERK1/2 at 15 min, which returned to basal by 60 min and remained low for 4 h. CPT increased JNK1/2 activity between 3 and 4 h. TNF + CPT caused a sustained and robust JNK1/2 and ERK1/2 phosphorylation by 2 h, which remained high at 4 h, suggesting involvement of MEKK4/7 and MEK1, respectively. When administered with TNF + CPT, SP-600125, a specific inhibitor of MEKK4/7, completely inhibited JNK1/2 and decreased apoptosis. However, administration of SP-600125 at 1 h after TNF + CPT failed to prevent JNK1/2 phosphorylation, and the protective effect of SP-600125 on apoptosis was abolished. These results indicate that the persistent activation of JNK might be due to inhibition of JNK-specific MAPK phosphatase 1 (MKP1). Small interfering RNA-mediated knockdown of MKP1 enhanced TNF + CPT-induced activity of JNK1/2 and caspases-9 and -3. Taken together, these results suggest that MKP1 activity determines the duration of JNK1/2 and p38 activation and, thereby, apoptosis in response to TNF + CPT.

Butin (7,3',4'-trihydroxydihydroflavone) reduces oxidative stress-induced cell death via inhibition of the mitochondria-dependent apoptotic pathway.

Recently, we demonstrated that butin (7,3',4'-trihydroxydihydroflavone) protected cells against hydrogen peroxide (H(2)O(2))-induced apoptosis by: (1) scavenging reactive oxygen species (ROS), activating antioxidant enzymes such superoxide dismutase and catalase; (2) decreasing oxidative stress-induced 8-hydroxy-2'-deoxyguanosine levels via activation of oxoguanine glycosylase 1, and (3), reducing oxidative stress-induced mitochondrial dysfunction. The objective of this study was to determine the cytoprotective effects of butin on oxidative stress-induced mitochondria-dependent apoptosis, and possible mechanisms involved. Butin significantly reduced H(2)O(2)-induced loss of mitochondrial membrane potential as determined by confocal image analysis and flow cytometry, alterations in Bcl-2 family proteins such as decrease in Bcl-2 expression and increase in Bax and phospho Bcl-2 expression, release of cytochrome c from mitochondria into the cytosol and activation of caspases 9 and 3. Furthermore, the anti-apoptotic effect of butin was exerted via inhibition of mitogen-activated protein kinase kinase-4, c-Jun NH(2)-terminal kinase (JNK) and activator protein-1 cascades induced by H(2)O(2) treatment. Finally, butin exhibited protective effects against H(2)O(2)-induced apoptosis, as demonstrated by decreased apoptotic bodies, sub-G(1) hypodiploid cells and DNA fragmentation. Taken together, the protective effects of butin against H(2)O(2)-induced apoptosis were exerted via blockade of membrane potential depolarization, inhibition of the JNK pathway and mitochondria-involved caspase-dependent apoptotic pathway.

Expression of chondroitin sulfate proteoglycan 4 (CSPG4) in melanoma cells is downregulated upon inhibition of BRAF.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growth­factor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAF­mutant CSPG4­positive or ­negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patient­derived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4­positive cells as compared to pre­therapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drug­resistance in melanoma cells.

Activation of MTK1/MEKK4 induces cardiomyocyte death and heart failure.

MTK1 (MEKK4) is a mitogen-activated protein kinase kinase kinase that regulates the activity of its downstream mitogen-activated kinases, p38, and c-Jun N-terminal kinase (JNK). However, the physiological function of MTK1 in the heart remains to be determined. Here, we attempted to elucidate the function of MTK1 in the heart using in vitro and in vivo models. MTK1 was activated in the hearts of mice subjected to pressure overload-induced heart failure. Overexpression of a constitutively active mutant of MTK1 (MTK1DeltaN) induced apoptosis in isolated neonatal rat cardiomyocytes, whereas a kinase domain-deleted form of MTK1 attenuated H(2)O(2)-induced apoptosis. Specific inhibitors of p38 or JNK effectively protected cardiomyocytes from MTK1DeltaN-induced cell death. In mice, cardiac-specific overexpression of MTK1DeltaN resulted in early mortality compared with the lifespan of littermate controls. Echocardiographic analysis revealed increases in end-diastolic and end-systolic left ventricular internal dimensions and a decrease in fractional shortening in MTK1DeltaN transgenic mice. In addition, the mice showed characteristic phenotypes of heart failure such as an increase in lung weight. The number of TUNEL-positive myocytes and the level of cleaved caspase 3 protein were both increased in MTK1DeltaN transgenic mice. Thus, MTK1 plays an important role in the regulation of cell death and is also involved in the pathogenesis of heart failure.

Trouble making the first move: interpreting arrested neuronal migration in the cerebral cortex.

Postmitotic cortical neurons that fail to initiate migration can remain near their site of origin and form persistent periventricular nodular heterotopia (PH). In human telencephalon, this malformation is most commonly associated with Filamin-A (FLNa) mutations. The lack of genetic animal models that reliably produce PH has delayed our understanding of the underlying molecular mechanisms. This review examines PH pathogenesis using a new mouse model. Although PH have not been observed in Flna-deficient mice generated thus far, the loss of MEKK4, a regulator of Flna, produces striking PH in mice and offers insight into the mechanisms involved in neuronal migration initiation. Elucidating the basic functions of FLNa and associated molecules is crucial for understanding the causes of PH and for developing prevention for at-risk patients.