Cell interactions of osteoarthritic subchondral bone osteoblasts and articular chondrocytes aggravate MMP-2 and MMP-9 production through the mediation of ERK1/2 and JNK phosphorylation

Matrix Metalloproteinases (MMP) play a key role in osteoarthritis (OA) development. The aim of the present study was to investigate whether, the cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of MMPs, and also to test the potential involvement of mitogen activated protein kinase (MAPK) signalling pathway during this process.

Alkyl chain substituted 1,9-pyrazoloanthrones exhibit prominent inhibitory effect on c-Jun N-terminal kinase (JNK)

N-Alkyl substituted pyrazoloanthrone derivatives were synthesized, characterized and tested for their in vitro inhibitory activity over c-Jun N-terminal kinase (JNK). Among the tested molecules, a few derivatives showed significant inhibitory activity against JNK with minimal off-target effect on other mitogen-activated protein kinase (MAP kinase) family members such as MEK1/2 and MKK3,6. These results suggested that N-alkyl (propyl and butyl) bearing pyrazoloanthrone scaffolds provide promising therapeutic inhibitors for JNK in regulating inflammation associated disorders.

Anthrapyrazolone analogues intercept inflammatory JNK signals to moderate endotoxin induced septic shock

Severe sepsis or septic shock is one of the rising causes for mortality worldwide representing nearly 10% of intensive care unit admissions. Susceptibility to sepsis is identified to be mediated by innate pattern recognition receptors and responsive signaling pathways of the host. The c-Jun N-terminal Kinase (JNK)-mediated signaling events play critical role in bacterial infection triggered multi-organ failure, cardiac dysfunction and mortality. In the context of kinase specificities, an extensive library of anthrapyrazolone analogues has been investigated for the selective inhibition of c-JNK and thereby to gain control over the inflammation associated risks. In our comprehensive biochemical characterization, it is observed that alkyl and halogen substitution on the periphery of anthrapyrazolone increases the binding potency of the inhibitors specifically towards JNK. Further, it is demonstrated that hydrophobic and hydrophilic interactions generated by these small molecules effectively block endotoxin-induced inflammatory genes expression in in vitro and septic shock in vivo, in a mouse model, with remarkable efficacies. Altogether, the obtained results rationalize the significance of the diversity oriented synthesis of small molecules for selective inhibition of JNK and their potential in the treatment of severe sepsis.

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 mu M) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading Delta psi m dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.

Organization of mammary epithelial cells into 3D acinar structures requires glucocorticoid and JNK signaling

Mammary epithelial cells cultured on a concentrated laminin-rich extracellular matrix formed 3D acinar structures that matured to polarized monolayers surrounding a lumen. In the absence of glucocorticoids mature acinus formation failed and the expression of an acinus-associated, activator protein 1 (AP1) and nuclear factor kappaB transcription factor DNA-binding profile was lost. Treatment with the JNK inhibitor, SP600125, caused similar effects, whereas normal organization of the mammary epithelial cells as acini caused JNK activation in a glucocorticoid-dependent manner. The forming acini expressed BRCA1, GADD45beta, MEKK4, and the JNK activating complex GADD 45beta-MEKK4 in a glucocorticoid-dependent fashion. JNK catalyzed phosphorylation of c-Jun was also detected in the acini. In addition, expression of beta4 integrin and in situ occupation of its promoter by AP1 components, c-Jun and Fos, was glucocorticoid dependent. These results suggest that glucocortocoid signaling regulates acinar integrity through a pathway involving JNK regulation of AP1 transcription factors and beta4 integrin expression.

Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1

The breast cancer susceptibility gene BRCA1 encodes a protein implicated in the cellular response to DNA damage, with postulated roles in homologous recombination as well as transcriptional regulation. To identify downstream target genes, we established cell lines with tightly regulated inducible expression of BRCA1. High-density oligonucleotide arrays were used to analyze gene expression profiles at various times following BRCA1 induction. A major BRCA1 target is the DNA damage-responsive gene GADD45. Induction of BRCA1 triggers apoptosis through activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), a signaling pathway potentially linked to GADD45 gene family members. The p53-independent induction of GADD45 by BRCA1 and its activation of JNK/SAPK suggest a pathway for BRCA1-induced apoptosis.

Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.

IB1/JIP-1 controls JNK activation and increased during prostatic LNCaP cells neuroendocrine differentiation.

The scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1) is a modulator of the c-Jun N-terminal kinase (JNK) activity, which has been implicated in pleiotrophic cellular functions including cell differentiation, division, and death. In this study, we described the presence of IB1/JIP-1 in epithelium of the rat prostate as well as in the human prostatic LNCaP cells. We investigated the functional role of IB1/JIP-1 in LNCaP cells exposed to the proapoptotic agent N-(4-hydroxyphenyl)retinamide (4-HPR) which induced a reduction of IB1/JIP-1 content and a concomittant increase in JNK activity. Conversely, IB1/JIP-1 overexpression using a viral gene transfer prevented the JNK activation and the 4-HPR-induced apoptosis was blunted. In prostatic adenocarcinoma cells, the neuroendocrine (NE) phenotype acquisition is associated with tumor progression and androgen independence. During NE transdifferentiation of LNCaP cells, IB1/JIP-1 levels were increased. This regulated expression of IB1/JIP-1 is secondary to a loss of the neuronal transcriptional repressor neuron restrictive silencing factor (NRSF/REST) function which is known to repress IB1/JIP-1. Together, these results indicated that IB1/JIP-1 participates to the neuronal phenotype of the human LNCaP cells and is a regulator of JNK signaling pathway.

Febuxostat, an Inhibitor of Xanthine Oxidase, Suppresses Lipopolysaccharide-Induced MCP-1 Production via MAPK Phosphatase-1-Mediated Inactivation of JNK.

Excess reactive oxygen species (ROS) formation can trigger various pathological conditions such as inflammation, in which xanthine oxidase (XO) is one major enzymatic source of ROS. Although XO has been reported to play essential roles in inflammatory conditions, the molecular mechanisms underlying the involvement of XO in inflammatory pathways remain unclear. Febuxostat, a selective and potent inhibitor of XO, effectively inhibits not only the generation of uric acid but also the formation of ROS. In this study, therefore, we examined the effects of febuxostat on lipopolysaccharide (LPS)-mediated inflammatory responses. Here we show that febuxostat suppresses LPS-induced MCP-1 production and mRNA expression via activating MAPK phosphatase-1 (MKP-1) which, in turn, leads to dephosphorylation and inactivation of JNK in macrophages. Moreover, these effects of febuxostat are mediated by inhibiting XO-mediated intracellular ROS production. Taken together, our data suggest that XO mediates LPS-induced phosphorylation of JNK through ROS production and MKP-1 inactivation, leading to MCP-1 production in macrophages. These studies may bring new insights into the novel role of XO in regulating inflammatory process through MAPK phosphatase, and demonstrate the potential use of XO inhibitor in modulating the inflammatory processes.

A human ribonuclease induces apoptosis associated with p21WAF1/CIP1 induction and JNK inactivation

Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase. Methods: Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.n Results: We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP. Conclusions: We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase

Involvement of ERK, Akt and JNK signalling in H2O2-induced cell injury and protection by hydroxytyrosol and its metabolite homovanillic alcohol

The olive oil polyphenol, hydroxytyrosol (HT), is believed to be capable of exerting protection against oxidative kidney injury. In this study we have investigated the ability of HT and its O-methylated metabolite, homovanillic alcohol (HVA) to protect renal cells against oxidative damage induced by hydrogen peroxide. We show that both compounds were capable of inhibiting hydrogen peroxide-induced kidney cell injury via an ability to interact with both MAP kinase and PI3 kinase signalling pathways, albeit at different concentrations. HT strongly inhibited death and prevented peroxide-induced increases in ERK1/2 and JNK1/2/3 phosphorylation at 0.3 microM, whilst HVA was effective at 10 microM. At similar concentrations, both compounds also prevented peroxide-induced reductions in Akt phosphorylation. We suggest that one potential protective effect exerted by olive oil polyphenols against oxidative kidney cell injury may be attributed to the interactions of HT and HVA with these important intracellular signalling pathways.