Dual Mkk4 and Mkk7 Gene Deletion in Adult Mouse Causes an Impairment of Hippocampal Immature Granule Cells.

(1) Background: The c-Jun-NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase involved in regulating physiological processes in the central nervous system. However, the dual genetic deletion of Mkk4 and Mkk7 (upstream activators of JNK) in adult mice is not reported. The aim of this study was to induce the genetic deletion of Mkk4/Mkk7 in adult mice and analyze their effect in hippocampal neurogenesis. (2) Methods: To achieve this goal, Actin-CreERT2 (Cre+/-), Mkk4flox/flox, Mkk7flox/flox mice were created. The administration of tamoxifen in these 2-month-old mice induced the gene deletion (Actin-CreERT2 (Cre+/-), Mkk4∆/∆, Mkk7∆/∆ genotype), which was verified by PCR, Western blot, and immunohistochemistry techniques. (3) Results: The levels of MKK4/MKK7 at 7 and 14 days after tamoxifen administration were not eliminated totally in CNS, unlike what happens in the liver and heart. These data could be correlated with the high levels of these proteins in CNS. In the hippocampus, the deletion of Mkk4/Mkk7 induced a misalignment position of immature hippocampal neurons together with alterations in their dendritic architecture pattern and maturation process jointly to the diminution of JNK phosphorylation. (4) Conclusion: All these data supported that the MKK4/MKK7-JNK pathway has a role in adult neurogenic activity.

Regulation of the Golgi Apparatus by p38 and JNK Kinases during Cellular Stress Responses.

p38 and c-Jun N-terninal kinase (JNK) are activated in response to acute stress and inflammatory signals. Through modification of a plethora of substrates, these kinases profoundly re-shape cellular physiology for the optimal response to a harmful environment and/or an inflammatory state. Here, we utilized phospho-proteomics to identify several hundred substrates for both kinases. Our results indicate that the scale of signaling from p38 and JNK are of a similar magnitude. Among the many new targets, we highlight the regulation of the transcriptional regulators grb10-interacting GYF protein 1 and 2 (GIGYF1/2) by p38-dependent MAP kinase-activated protein kinase 2 (MK2) phosphorylation and 14-3-3 binding. We also show that the Golgi apparatus contains numerous substrates, and is a major target for regulation by p38 and JNK. When activated, these kinases mediate structural rearrangement of the Golgi apparatus, which positively affects protein flux through the secretory system. Our work expands on our knowledge about p38 and JNK signaling with important biological ramifications.

Exposure of the extracellular matrix and colonization of the ovary in metastasis of fallopian-tube-derived cancer.

High-grade serous ovarian cancer (HGSOC) can originate in the fallopian tube epithelium (FTE), but the role of the ovary in these tumors is unclear. Tumorigenic murine oviductal epithelial (MOE) cells allografted in the ovarian bursa resulted in aggressive tumors that spread throughout the peritoneum whereas intraperitoneal xenografting the same number of cells did not form tumors, indicating that colonization of the ovary may play a role in metastasis. Physical tearing of the ovarian surface to mimic rupture of the ovary during ovulation (independent of hormonal changes) resulted in more MOE and HGSOC cells adhering to the ovary compared with intact ovaries. More MOE cells also adhered to three-dimensional (3D) collagen and primary ovarian stromal cells than to ovarian surface epithelia, indicating that FTE cells adhered to the extracellular matrix exposed during ovulation. However, plating cells on 3D collagen reduced the viability of normal FTE but not cancer cells. Mutation of p53 (R273H or R248W) and activation of Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) (G12V) did not increase the viability of MOE cells on 3D collagen. In contrast, loss of phosphatase and tensin homolog (PTEN) allowed MOE cells to retain normal viability on 3D collagen. Loss of PTEN activated AKT and RAC1/c-jun N-terminal kinase signaling that each contributed to the increased viability, invasion and attachment in the collagen rich ovarian microenvironment. These results show that loss of PTEN activates multiple pathways that together enhance colonization of the ovary due to access to 3D collagen, which is a critical organ in the colonization of FTE-derived HGSOC.

Reciprocal signals between microglia and neurons regulate α-synuclein secretion by exophagy through a neuronal cJUN-N-terminal kinase-signaling axis.

BACKGROUND: Secretion of proteopathic α-synuclein (α-SNC) species from neurons is a suspected driving force in the propagation of Parkinson's disease (PD). We have previously implicated exophagy, the exocytosis of autophagosomes, as a dominant mechanism of α-SNC secretion in differentiated PC12 or SH-SY5Y nerve cells. Here we have examined the regulation of exophagy associated with different forms of nerve cell stress relevant to PD. RESULTS: We identify cJUN-N-terminal kinase (JNK) activity as pivotal in the secretory fate of autophagosomes containing α-SNC. Pharmacological inhibition or genetic (shRNA) knockdown of JNK2 or JNK3 decreases α-SNC secretion in differentiated PC12 and SH-SY5Y cells, respectively. Conversely, expression of constitutively active mitogen-activated protein kinase kinase 7 (MKK7)-JNK2 and -JNK3 constructs augment secretion. The transcriptional activity of cJUN was not required for the observed effects. We establish a causal relationship between increased α-SNC release by exophagy and JNK activation subsequent to lysosomal fusion deficiency (overexpression of Lewy body-localized protein p25α or bafilomycin A1). JNK activation following neuronal ER or oxidative stress was not correlated with exophagy, but of note, we demonstrate that reciprocal signaling between microglia and neurons modulates α-SNC secretion. NADPH oxidase activity of microglia cell lines was upregulated by direct co-culture with α-SNC-expressing PC12 neurons or by passive transfer of nerve cell-conditioned medium. Conversely, inflammatory factors secreted from activated microglia increased JNK activation and α-SNC secretion several-fold in PC12 cells. While we do not identify these factors, we extend our observations by showing that exposure of neurons in monoculture to TNFα, a classical pro-inflammatory mediator of activated microglia, is sufficient to increase α-SNC secretion in a mechanism dependent on JNK2 or JNK3. In continuation hereof, we show that also IFNß and TGFß increase the release of α-SNC from PC12 neurons. CONCLUSIONS: We implicate stress kinases of the JNK family in the regulation of exophagy and release of α-SNC following endogenous or exogenous stimulation. In a wider scope, our results imply that microglia not only inflict bystander damage to neurons in late phases of inflammatory brain disease but may also be active mediators of disease propagation.

Glycyrrhizic Acid Promotes M1 Macrophage Polarization in Murine Bone Marrow-Derived Macrophages Associated with the Activation of JNK and NF-κB.

The roots and rhizomes of Glycyrrhiza species (licorice) have been widely used as natural sweeteners and herbal medicines. The aim of this study is to investigate the effect of glycyrrhizic acid (GA) from licorice on macrophage polarization. Both phenotypic and functional activities of murine bone marrow-derived macrophages (BMDMs) treated by GA were assessed. Our results showed that GA obviously increased the cell surface expression of CD80, CD86, and MHCII molecules. Meanwhile, GA upregulated the expression of CCR7 and the production of TNF-α, IL-12, IL-6, and NO (the markers of classically activated (M1) macrophages), whereas it downregulated the expression of MR, Ym1, and Arg1 (the markers of alternatively activated (M2) macrophage). The functional tests showed that GA dramatically enhanced the uptake of FITC-dextran and E. coli K88 by BMDMs and decreased the intracellular survival of E. coli K88 and S. typhimurium. Moreover, we demonstrated that JNK and NF-κB activation are required for GA-induced NO and M1-related cytokines production, while ERK1/2 pathway exhibits a regulatory effect via induction of IL-10. Together, these findings indicated that GA promoted polarization of M1 macrophages and enhanced its phagocytosis and bactericidal capacity. The results expanded our knowledge about the role of GA in macrophage polarization.

Nitric oxide inhibits autophagy via suppression of JNK in meniscal cells.

OBJECTIVE: Autophagy is a potential protective mechanism that is involved in several degenerative diseases. Nitric oxide (NO) is associated with programmed cellular death in meniscal cells, but whether it can induce autophagy is still undetermined. This study aims to investigate the interaction between autophagy and NO in normal human meniscal cells. METHODS: Normal meniscal cells were harvested from female patients. NO donors and NO synthase inhibitors were used to regulate the level of NO. Changes in the incidence of autophagy and apoptosis were examined using flow cytometry, western blot and immunofluorescence methods. The effects of NO-mediated autophagy regulation of the expression of MMPs and aggrecanases (ADAMTS-4 and -5) were analysed by real-time PCR. RESULTS: NO donors inhibited autophagy as well as augmented apoptosis in human meniscal cells with serum deprivation. Conversely, treatment with NOS inhibitors resulted in up-regulation of the autophagy level while repressing apoptosis. NOS inhibitor treatment also resulted in down-regulation of MMPs and aggrecanase mRNA expression. This effect of NOS inhibitor was also blocked by autophagy inhibitors. Our results also showed that NOS inhibitor enhanced Jun-N-terminal kinase (JNK) activation. Furthermore, SP600125, a selective JNK inhibitor, blocked up-regulation of autophagy by NOS inhibitor. CONCLUSION: Our results demonstrated that NO augmented serum deprivation-induced apoptosis of meniscal cells via inhibition of autophagy through inactivation of JNK. Up-regulation of autophagy may be a potential approach in the treatment of meniscal tissue degeneration.

A role for the NF-κB pathway in cell protection from complement-dependent cytotoxicity.

Nucleated cells are equipped with several mechanisms that support their resistance to complement-dependent cytotoxicity (CDC). The role of the NF-κB pathway in cell protection from CDC was examined. Elevated sensitivity to CDC was demonstrated in cells lacking the p65 subunit of NF-κB or the IκB kinases IKKα or IKKß, and in cells treated with p65 small interfering RNA. Pretreatment with the IKK inhibitor PS-1145 also enhanced CDC of wild-type cells (WT) but not of p65(-/-) cells. Furthermore, reconstitution of p65 into p65(-/-) cells and overexpression of p65 in WT cells lowered their sensitivity to CDC. The postulated effect of p65 on the JNK-mediated death-signaling pathway activated by complement was examined. p65 small interfering RNA enhanced CDC in WT cells but not in cells lacking JNK. JNK phosphorylation induced by complement was more pronounced in p65(-/-) cells than in WT cells. The results indicate that the NF-κB pathway mediates cell resistance to CDC, possibly by suppressing JNK-dependent programmed necrotic cell death.

Involvement of α2-antiplasmin in dendritic growth of hippocampal neurons.

The α2-Antiplasmin (α2AP) protein is known as a principal physiological inhibitor of plasmin, but we previously demonstrated that it acts as a regulatory factor for cellular functions independent of plasmin. α2AP is highly expressed in the hippocampus, suggesting a potential role for α2AP in hippocampal neuronal functions. However, the role for α2AP was unclear. This study is the first to investigate the involvement of α2AP in the dendritic growth of hippocampal neurons. The expression of microtubule-associated protein 2, which contributes to neurite initiation and neuronal growth, was lower in the neurons from α2AP⁻/⁻ mice than in the neurons from α2AP⁺/⁺ mice. Exogenous treatment with α2AP enhanced the microtubule-associated protein 2 expression, dendritic growth and filopodia formation in the neurons. This study also elucidated the mechanism underlying the α2AP-induced dendritic growth. Aprotinin, another plasmin inhibitor, had little effect on the dendritic growth of neurons, and α2AP induced its expression in the neurons from plaminogen⁻/⁻ mice. The activation of p38 MAPK was involved in the α2AP-induced dendritic growth. Therefore, our findings suggest that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.

Using MKK4's metastasis suppressor function to identify and dissect cancer cell-microenvironment interactions during metastatic colonization.

Host tissue microenvironment plays key roles in cancer progression and colonization of secondary organs. One example is ovarian cancer, which colonizes the peritoneal cavity and especially the omentum. Our research indicates that the interaction of ovarian cancer cells with the omental microenvironment can activate a stress-kinase pathway involving the mitogen-activated protein kinase kinase 4 (MKK4). A combination of clinical correlative and functional data suggests that MKK4 activation suppresses growth of ovarian cancer cells lodged in omentum. These findings prompted us to turn our focus to the cellular composition of the omental microenvironment and its role in regulating cancer growth. In this review, in addition to providing an overview of MKK4 function, we highlight a use for metastasis suppressors as a molecular tool to study cancer cell interaction with its microenvironment. We review features of the omentum that makes it a favorable microenvironment for metastatic colonization. In conclusion, a broader, evolutionary biology perspective is presented which we believe needs to be considered when studying the evolution of cancer cells within a defined microenvironment. Taken together, this approach can direct new multi-dimensional lines of research aimed at a mechanistic understanding of host tissue microenvironment, which could be used to realize novel targets for future research.

Nod2-induced autocrine interleukin-1 alters signaling by ERK and p38 to differentially regulate secretion of inflammatory cytokines.

BACKGROUND & AIMS: Stimulation of nucleotide-binding oligomerization domain-containing (Nod)2 and other pattern recognition receptors (PRR) in human monocyte-derived macrophages induces interleukin (IL)-1, which increases mitogen-activated protein kinase (MAPK) activation and cytokine secretion. Activation of MAPK by PRR has varied effects on inflammatory cytokine secretion. We investigated whether different levels of autocrine IL-1 mediate these varied effects. METHODS: Macrophage responses to PRR ligands were analyzed by enzyme-linked immunosorbent assay and flow cytometry. We overexpressed or reduced MAPK levels (using small inhibitory RNA). RESULTS: Nod2 and other PRR activated signaling via extracellular signal-related kinase (ERK) and p38 that inhibited inflammatory cytokine production by human monocyte-derived macrophages; autocrine IL-1 production prevented this inhibition. ERK and p38 inhibited inflammatory cytokine production by human macrophages that produce low levels of IL-1 (such as M2, endotoxin-tolerant, and intestinal macrophages); adding exogenous IL-1 caused ERK and p38 to stimulate production of inflammatory cytokines in these cells. In mouse macrophages, which do not produce IL-1 in response to PRR stimulation alone, addition of exogenous IL-1 reversed the ERK-mediated inhibition of IL-12p40. Increasing activation of c-Jun N-terminal kinase in Nod2-stimulated human monocyte-derived macrophages, in the absence of autocrine IL-1 signaling, caused ERK and p38 to stimulate inflammatory cytokines secretion. Importantly, infection of human intestinal macrophages with pathogens that induce IL-1 production reversed the inhibition of inflammatory cytokine production by ERK and p38. CONCLUSIONS: In response to PRR stimulation of macrophages, the level of MAPK signaling is regulated by autocrine IL-1 and determines whether production of inflammatory cytokines is inhibited or stimulated. This mechanism could account for reported differences in MAPK regulation of inflammatory cytokines and propagate the inflammatory response to pathogens.

Pak1 as a novel therapeutic target for antihypertrophic treatment in the heart.

BACKGROUND: Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. Although many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy, the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study, we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator that antagonizes cardiac hypertrophy. METHODS AND RESULTS: Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs) or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an antihypertrophic role. To investigate the in vivo role of Pak1 in the heart, we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1(cko)). When subjected to 2 weeks of pressure overload, Pak1(cko) mice developed greater cardiac hypertrophy with attendant blunting of JNK activation compared with controls, and these knockout mice underwent the transition into heart failure when prolonged stress was applied. Chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1(cko) mice. Moreover, we discovered that the Pak1 activator FTY720, a sphingosine-like analog, was able to prevent pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. Meanwhile, FTY720 failed to exert such an effect on Pak1(cko) mice, suggesting that the antihypertrophic effect of FTY720 likely acts through Pak1 activation. CONCLUSIONS: These results, for the first time, establish Pak1 as a novel antihypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.

PTEN loss defines a TGF-ß-induced tubule phenotype of failed differentiation and JNK signaling during renal fibrosis.

We investigated the signaling basis for tubule pathology during fibrosis after renal injury. Numerous signaling pathways are activated physiologically to direct tubule regeneration after acute kidney injury (AKI) but several persist pathologically after repair. Among these, transforming growth factor (TGF)-ß is particularly important because it controls epithelial differentiation and profibrotic cytokine production. We found that increased TGF-ß signaling after AKI is accompanied by PTEN loss from proximal tubules (PT). With time, subpopulations of regenerating PT with persistent loss of PTEN (phosphate and tension homolog) failed to differentiate, became growth arrested, expressed vimentin, displayed profibrotic JNK activation, and produced PDGF-B. These tubules were surrounded by fibrosis. In contrast, PTEN recovery was associated with epithelial differentiation, normal tubule repair, and less fibrosis. This beneficial outcome was promoted by TGF-ß antagonism. Tubule-specific induction of TGF-ß led to PTEN loss, JNK activation, and fibrosis even without prior AKI. In PT culture, high TGF-ß depleted PTEN, inhibited differentiation, and activated JNK. Conversely, TGF-ß antagonism increased PTEN, promoted differentiation, and decreased JNK activity. Cre-Lox PTEN deletion suppressed differentiation, induced growth arrest, and activated JNK. The low-PTEN state with JNK signaling and fibrosis was ameliorated by contralateral nephrectomy done 2 wk after unilateral ischemia, suggesting reversibility of the low-PTEN dysfunctional tubule phenotype. Vimentin-expressing tubules with low-PTEN and JNK activation were associated with fibrosis also after tubule-selective AKI, and with human chronic kidney diseases of diverse etiology. By preventing tubule differentiation, the low-PTEN state may provide a platform for signals initiated physiologically to persist pathologically and cause fibrosis after injury.

Apoptosis signal-regulating kinase 1 inhibits hepatocarcinogenesis by controlling the tumor-suppressing function of stress-activated mitogen-activated protein kinase.

UNLABELLED: The stress-activated mitogen-activated protein kinases (MAPKs), c-Jun NH2-terminal kinase (JNK), and p38 have been implicated in hepatocarcinogenesis. Although the many interrelated functions of JNK and p38 are precisely regulated by upstream signaling molecules, little is known about upstream regulators. We investigated the role of apoptosis signal-regulating kinase 1 (ASK1), a major player in the regulation of JNK and p38 activities, in hepatocarcinogenesis using a mouse hepatocellular carcinoma (HCC) model. ASK1-deficient (ASK1(-/-) ) and wildtype (WT) mice were treated with diethylnitrosamine on postnatal day 14. Strikingly, after 7 months, approximately three times as many tumors developed in ASK1(-/-) mice as in WT mice. Although JNK and p38 activation were attenuated in ASK1(-/-) HCCs relative to WT HCCs, cell proliferation was comparable in HCCs from both types of mice. On the other hand, both cancer cell apoptosis and hyperphosphorylation of BimEL, a proapoptotic Bcl-2 family member, were suppressed in the ASK1(-/-) HCCs. ASK1(-/-) mice showed remarkable resistance to Fas-induced hepatocyte apoptosis in vivo, probably because of attenuated JNK-mediated BimEL phosphorylation and mitochondrial apoptotic pathway activation. The reintroduction of ASK1 to ASK1(-/-) mouse liver using an adenoviral vector restored Fas-induced hepatocyte death and phosphorylation of JNK and BimEL. Similar findings were obtained in tumor necrosis factor alpha-induced hepatocyte apoptosis. Furthermore, ASK1 was involved in DNA damage-induced p21 up-regulation through a p38 pathway. CONCLUSION: ASK1 is involved in death receptor-mediated apoptosis and DNA-damage response by way of stress-activated MAPK in the liver, and thus acts as a tumor suppressor in hepatocarcinogenesis. This study provides new insight into the regulation of stress- activated MAPK signaling in hepatocarcinogenesis.

ABCA1 and ABCG1 protect against oxidative stress-induced macrophage apoptosis during efferocytosis.

RATIONALE: Antiatherogenic effects of plasma high-density lipoprotein (HDL) include the ability to inhibit apoptosis of macrophage foam cells. The ATP-binding cassette transporters ABCA1 and ABCG1 have a major role in promoting cholesterol efflux from macrophages to apolipoprotein A-1 and HDL and are upregulated during the phagocytosis of apoptotic cells (efferocytosis). OBJECTIVE: The goal of this study was to determine the roles of ABCA1 and ABCG1 in preserving the viability of macrophages during efferocytosis. METHODS AND RESULTS: We show that despite similar clearance of apoptotic cells, peritoneal macrophages from Abca1(-/-)Abcg1(-/-), Abcg1(-/-), and, to a lesser extent, Abca1(-/-) mice are much more prone to apoptosis during efferocytosis compared to wild-type cells. Similar findings were observed following incubations with oxidized phospholipids, and the ability of HDL to protect against oxidized phospholipid-induced apoptosis was markedly reduced in Abca1(-/-)Abcg1(-/-) and Abcg1(-/-) cells. These effects were independent of any role of ABCA1 and ABCG1 in mediating oxidized phospholipid efflux but were reversed by cyclodextrin-mediated cholesterol efflux. The apoptotic response observed in Abca1(-/-)Abcg1(-/-) macrophages after oxidized phospholipid exposure or engulfment of apoptotic cells was dependent on an excessive oxidative burst secondary to enhanced assembly of NADPH oxidase (NOX)2 complexes, leading to sustained Jnk activation which turned on the apoptotic cell death program. Increased NOX2 assembly required Toll-like receptors 2/4 and MyD88 signaling, which are known to be enhanced in transporter deficient cells in a lipid raft-dependent fashion. CONCLUSIONS: We identified a new beneficial role of ABCA1, ABCG1 and HDL in dampening the oxidative burst and preserving viability of macrophages following exposure to oxidized phospholipids and/or apoptotic cells.

Activation and crosstalk between the endoplasmic reticulum road and JNK pathway in ischemia-reperfusion brain injury.

BACKGROUND: Recent studies suggest that endoplasmic reticulum stress (ERS) is the key process in ischemic brain injury. The JNK pathway is also involved in the process of ischemic brain injury. METHOD: We established a middle cerebral artery occlusion/reperfusion (MCAO/R) model in rats; detected the changes in c-Jun N-terminal kinase (JNK), GADD153 and caspase-12 at different reperfusion time points by immunohistochemistry, Western blot and double-label immunofluorescence; and observed the effect of JNK inhibitor SP600125 on the expression of JNK, GADD153 and caspase-12 to explore the relationship between the endoplasmic reticulum road and JNK pathway. RESULTS: The expression of the two hallmarks of ERS-GADD153 and caspase-12-significantly increased, and the activation of JNK also obviously increased. After interference by SP600125, the expression of p-JNk and caspase-12 obviously decreased, whereas the decrease of GADD153 occurred only after 24 h reperfusion. CONCLUSIONS: Both ERS and JNK pathways are involved in the pathological process of ischemic brain injury. The JNK pathway may be involved in the process of ERS, but perhaps has more effect on the caspase-12 pathway.

Nonvisual arrestins function as simple scaffolds assembling the MKK4-JNK3α2 signaling complex.

Arrestins make up a small family of proteins with four mammalian members that play key roles in the regulation of multiple G protein-coupled receptor-dependent and -independent signaling pathways. Although arrestins were reported to serve as scaffolds for MAP kinase cascades, promoting the activation of JNK3, ERK1/2, and p38, the molecular mechanisms involved were not elucidated, and even the direct binding of arrestins with MAP kinases was never demonstrated. Here, using purified proteins, we show that both nonvisual arrestins directly bind JNK3α2 and its upstream activator MKK4, and that the affinity of arrestin-3 for these kinases is higher than that of arrestin-2. Reconstitution of the MKK4-JNK3α2 signaling module from pure proteins in the presence of different arrestin-3 concentrations showed that arrestin-3 acts as a "true" scaffold, facilitating JNK3α2 phosphorylation by bringing the two kinases together. Both the level of JNK3α2 phosphorylation by MKK4 and JNK3α2 activity toward its substrate ATF2 increase at low and then decrease at high arrestin-3 levels, yielding a bell-shaped concentration dependence expected with true scaffolds that do not activate the upstream kinase or its substrate. Thus, direct binding of both kinases and true scaffolding is the molecular mechanism of action of arrestin-3 on the MKK4-JNK3α2 signaling module.

AlphaV-integrins mediate the mechanoprotective action of osteopontin in podocytes.

Increased mechanical load in podocytes due to glomerular hypertension is one of the important factors leading to podocyte damage and chronic kidney disease. In previous studies, we have shown that mechanical stretch increases osteopontin (OPN) expression in podocytes and that exogenous OPN is mechanoprotective via facilitating cytoskeletal reorganization of podocytes. In the present study, we asked whether the mechanoprotective effect of OPN in podocytes is mediated through specific integrins and whether endogenous OPN of podocytes is required for mechanoprotection. Conditionally immortalized mouse podocytes and primary podocytes (PP) from OPN-/- and OPN+/+ mice were used. Cyclic biaxial mechanical stretch (0.5 Hz, 7% linear strain) was applied for up to 3 days. Stretch-induced cell loss was ∼30% higher in OPN-/- PP compared with OPN+/+ PP. Increased cell loss of OPN-/- PP was rescued by OPN coating. Analysis of integrin expression by RT-PCR, application of RGD and SLAYGLR peptides and anti-integrin antibodies, small-interfering RNA knockdown of integrins, and application of kinase inhibitors identified αV-integrins (αVß1, αVß3, and αVß5) to mediate the mechano-protective effect of OPN in podocytes involving focal adhesion kinase, Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Our results demonstrate that endogenous OPN of podocytes plays a nonredundant role in podocyte adaptation to mechanical stretch, and that OPN signaling via α(V)-integrins may represent a relevant therapeutical target in podocytes.

Endonuclease G mediates endothelial cell death induced by carbamylated LDL.

End-stage kidney disease is a terminal stage of chronic kidney disease, which is associated with a high incidence of cardiovascular disease. Cardiovascular disease frequently results from endothelial injury caused by carbamylated LDL (cLDL), the product of LDL modification by urea-derived cyanate. Our previous data suggested that cLDL induces mitogen-activated protein kinase-dependent mitotic DNA fragmentation and cell death. However, the mechanism of this pathway is unknown. The current study demonstrated that cLDL-induced endothelial mitotic cell death is independent of caspase-3. The expression of endonuclease G (EndoG), the nuclease implicated in caspase-independent DNA fragmentation, was significantly increased in response to cLDL exposure to the cells. The inhibition of EndoG by RNAi protected cLDL-induced DNA fragmentation, whereas the overexpression of EndoG induced more DNA fragmentation in endothelial cells. Ex vivo experiments with primary endothelial cells isolated from wild-type (WT) and EndoG knockout (KO) mice demonstrated that EndoG KO cells are partially protected against cLDL toxicity compared with WT cells. To determine cLDL toxicity in vivo, we administered cLDL or native LDL (nLDL) intravenously to the WT and EndoG KO mice and then measured floating endothelial cells in blood using flow cytometry. The results showed an increased number of floating endothelial cells after cLDL versus nLDL injection in WT mice but not in EndoG KO mice. Finally, the inhibitors of MEK-ERK1/2 and JNK-c-jun pathways decreased cLDL-induced EndoG overexpression and DNA fragmentation. In summary, our data suggest that cLDL-induced endothelial toxicity is caspase independent and results from EndoG-dependent DNA fragmentation.

Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B leukemic cells.

BACKGROUND: Cucurbitacin-I (JSI-124) is potent inhibitor of JAK/STAT3 signaling pathway and has anti-tumor activity in a variety of cancer including B cell leukemia. However, other molecular targets of JSI-124 beyond the JAK/STAT3 pathway are not fully understood. METHODS: BJAB, I-83, NALM-6 and primary CLL cells were treated with JSI-124 as indicated. Apoptosis was measured using flow cytometry for accumulation of sub-G1 phase cells (indicator of apoptosis) and Annexin V/PI staining. Cell cycle was analyzed by FACS for DNA content of G1 and G2 phases. Changes in phosphorylation and protein expression of p38, Erk1/2, JNK, c-Jun, and XIAP were detected by Western blot analysis. STAT3 and c-Jun genes were knocked out using siRNA transfection. VEGF expression was determined by mRNA and protein levels by RT-PCR and western blotting. Streptavidin Pull-Down Assay was used to determine c-Jun binding to the AP-1 DNA binding site. RESULTS: Herein, we show that JSI-124 activates c-Jun N-terminal kinase (JNK) and increases both the expression and serine phosphorylation of c-Jun protein in the B leukemic cell lines BJAB, I-83 and NALM-6. JSI-124 also activated MAPK p38 and MAPK Erk1/2 albeit at lower levels than JNK activation. Inhibition of the JNK signaling pathway failed to effect cell cycle arrest or apoptosis induced by JSI-124 but repressed JSI-124 induced c-Jun expression in these leukemia cells. The JNK pathway activation c-Jun leads to transcriptional activation of many genes. Treatment of BJAB, I-83, and NALM-6 cells with JSI-124 lead to an increase of Vascular Endothelial Growth Factor (VEGF) at both the mRNA and protein level. Knockdown of c-Jun expression and inhibition of JNK activation significantly blocked JSI-124 induced VEGF expression. Pretreatment with recombinant VEGF reduced JSI-124 induced apoptosis. CONCLUSIONS: Taken together, our data demonstrates that JSI-124 activates the JNK signaling pathway independent of apoptosis and cell cycle arrest, leading to increased VEGF expression.

Interleukin 18 induces angiogenesis in vitro and in vivo via Src and Jnk kinases.

BACKGROUND: Interleukin 18 (IL-18) is a novel mediator of angiogenesis in rheumatoid arthritis (RA). OBJECTIVE: To examine the role of IL-18 in RA angiogenesis and the signalling mechanisms involved. METHODS: Human dermal microvascular endothelial cell (HMVEC) chemotaxis, capillary morphogenesis assays and Matrigel plug angiogenesis assays were performed in vivo using IL-18 with or without signalling inhibitors. A novel model of angiogenesis was devised using dye-tagged HMVECs to study their homing into RA and normal (NL) synovial tissues (STs) engrafted in severe combined immunodeficient (SCID) mice. RESULTS: IL-18-mediated angiogenesis depended on Src and Jnk, as the inhibitors of Src and Jnk blocked IL-18-induced HMVEC chemotaxis, tube formation and angiogenesis in Matrigel plugs. However, inhibitors of Janus kinase 2, p38, MEK, phosphatidylinositol-3-kinase and neutralising antibodies to vascular endothelial growth factor or stromal derived factor-1α did not alter IL-18-induced HMVEC migration. These results were confirmed with Jnk or Src sense or antisense oligodeoxynucleotides. Moreover, IL-18 induced phosphorylation of Src and Jnk in HMVECs. As proof of principle, IL-18 null mice had a significantly decreased angiogenesis compared with wild-type mice in Matrigel plug angiogenesis assays in vivo. IL-18 markedly enhanced mature HMVEC homing to human RA ST compared with NL ST in SCID mice, confirming the role of IL-18-induced angiogenesis in RA ST in vivo. CONCLUSION: Targeting IL-18 or its signalling intermediates may prove to be a potentially novel therapeutic strategy for angiogenesis-dependent diseases, such as RA.