Hinokitiol inhibits RANKL-induced osteoclastogenesis in vitro and prevents ovariectomy-induced bone loss in vivo.

Osteoporosis is a metabolic bone-loss disease characterized by abnormally excessive osteoclast formation and bone resorption. Identification of natural medicines that can inhibit osteoclastogenesis, bone resorption, and receptor activator of nuclear factor-κB ligand (RANKL)-induced signaling is necessary for improved treatment of osteoporosis. In this study, hinokitiol, a tropolone-related compound extracted from the heart wood of several cupressaceous plants, was found to inhibit RANKL-induced osteoclast formation and bone resorption in vitro. Hinokitiol inhibited early activation of the ERK, p38, and JNK-MAPK pathways, thereby suppressing the activity and expression of downstream factors (c-Jun, c-Fos, and NFATC1). Consistent with the above in vitro findings, hinokitiol treatment protected against ovariectomy-induced bone loss in vivo. Collectively, our results imply that hinokitiol can potentially serve as an effective agent for treating osteoclast-induced osteoporosis.

Selective antagonism of cJun for cancer therapy.

The activator protein-1 (AP-1) family of transcription factors modulate a diverse range of cellular signalling pathways into outputs which can be oncogenic or anti-oncogenic. The transcription of relevant genes is controlled by the cellular context, and in particular by the dimeric composition of AP-1. Here, we describe the evidence linking cJun in particular to a range of cancers. This includes correlative studies of protein levels in patient tumour samples and mechanistic understanding of the role of cJun in cancer cell models. This develops an understanding of cJun as a focal point of cancer-altered signalling which has the potential for therapeutic antagonism. Significant work has produced a range of small molecules and peptides which have been summarised here and categorised according to the binding surface they target within the cJun-DNA complex. We highlight the importance of selectively targeting a single AP-1 family member to antagonise known oncogenic function and avoid antagonism of anti-oncogenic function.

Discovery of phenanthridine analogues as novel chemical probes disrupting the binding of DNA to ΔFosB homodimers and ΔFosB/JunD heterodimers.

The transcription factor ΔFosB accumulates in response to chronic insults such as drugs of abuse, L-3,4-dihydroxyphenylalanine (l-DOPA) or stress in specific regions of the brain, triggering long lasting neural and behavioral changes that underlie aspects of drug addiction, dyskinesia, and depression. Thus, small molecule chemical probes are urgently needed to investigate biological functions of ΔFosB. Herein we describe the identification of a novel phenanthridine analogue ZL0220 (27) as an active and promising ΔFosB chemical probe with micromolar inhibitory activities against ΔFosB homodimers and ΔFosB/JunD heterodimers.

Coupling Computational and Intracellular Screening and Selection Toward Co-compatible cJun and cFos Antagonists.

Basic leucine-zipper (bZIP) proteins represent difficult, yet compelling, oncogenic targets since numerous cell-signaling cascades converge upon them, where they function to modulate the transcription of specific gene targets. bZIPs are widely recognized as important regulators of cellular processes that include cell proliferation, apoptosis, and differentiation. Once such validated transcriptional regulator, activator protein-1, is typically composed of heterodimers of Fos and Jun family members, with cFos-cJun being the best described. It has been shown to be key in the progression and development of a number of different diseases. As a proof-of-principle for our approach, we describe the first use of a novel combined in silico/in cellulo peptide-library screening platform that facilitates the derivation of a sequence that displays high selectivity for cJun relative to cFos, while also avoiding homodimerization. In particular, >60 million peptides were computationally screened and all potential on/off targets ranked according to predicted stability, leading to a reduced size library that was further refined by intracellular selection. The derived sequence is predicted to have limited cross-talk with a second previously derived peptide antagonist that is selective for cFos in the presence of cJun. The study provides new insight into the use of multistate screening with the ability to combine computational and intracellular approaches in evolving multiple cocompatible peptides that are capable of satisfying conflicting design requirements.

The MAP2K4/JNK/c-Jun Signaling Pathway Plays A Key Role In Dexmedetomidine Protection Against Acetaminophen-Induced Liver Toxicity.

PURPOSE: Dexmedetomidine [DEX; (S)-4-[1-(2,3-dimethylphenyl)ethyl]-3H-imidazole] is a selective α2-adrenergic receptor (α2-AR) agonist that attenuates the liver damage associated with local or systemic inflammation. However, it remains unclear whether DEX has protective effects against acetaminophen (Paracetamol, PARA)-induced liver toxicity (PILT). METHODS: PILT mice were established by intraperitoneal administration of a hepatotoxic dose of acetaminophen (300 mg/kg). Thirty minutes later, the mice were treated with DEX at a concentration of 0, 5, 25, or 50 µg/kg. Blood and liver samples were obtained for further analysis. RESULTS: DEX treatment significantly attenuated PILT in mice, with the strongest beneficial effects at a dose of 25 µg/kg. The levels of hepatic cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), in addition to myeloperoxidase (MPO) activity, were significantly decreased following DEX treatment. Moreover, DEX treatment reduced macrophage recruitment around the area of hepatotoxicity and the expression levels of hepatic phosphorylated mitogen-activated protein kinase kinase 4 (MAP2K4), c-jun N-terminal kinase (JNK), and c-Jun expression induced by acetaminophen overdose. CONCLUSION: The data suggest that DEX likely downregulates the JNK signaling pathway and its downstream effectors to promote its hepatoprotective effect, providing a clinical application of DEX for the attenuation of PILT.

Ambroxol alleviates ventilator-induced lung injury by inhibiting c-Jun expression.

OBJECTIVE: Ventilator-induced lung injury (VILI) remains a challenge. This study was designed to investigate the effects of ambroxol on VILI and the underlying mechanisms in a rodent model. MATERIALS AND METHODS: Male Wistar rats weighing 310-380 g were divided into four groups (n=8 per group): 1) saline only, 2) ventilation plus saline, 3) ventilation plus ambroxol (2 mg/kg), and 4) ventilation plus ambroxol (50 mg/kg). Rats in groups 1 and 2 were treated (i.p.) with 2.5 ml of saline once a day for six days and last injected 1 h prior to tracheotomy. Rats in groups 3 and 4 received ambroxol on the same schedule. Rats were ventilated for 90 minutes at a tidal volume (VT) of 30 ml/kg. The expression levels of c-Jun, a component of activator protein-1 (AP-1), and gamma-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme in the synthesis of glutathione (gamma-glutamyl-cysteinyl-glycine, GSH), an endogenous antioxidant, were measured with immunohistochemical staining and in situ hybridization. Both AP-1 and GSH are involved in VILI. RESULTS: Ambroxol at 50 mg/kg inhibited ventilation-induced lung inflammation, significantly elevated the ventilation-induced down-regulation of γ-GCS mRNA and protein, and significantly decreased the ventilation-induced up-regulation of c-Jun mRNA and protein. It has been reported that reactive oxygen species (ROS) can activate AP-1, leading to the production of pro-inflammatory cytokines and lung inflammation. CONCLUSIONS: Ambroxol increases γ-GCS to promote GSH production, which in turn, inhibits ROS-dependent AP-1 activation and inflammation.

Inhibition of the TIRAP-c-Jun interaction as a therapeutic strategy for AP1-mediated inflammatory responses.

Bacterial endotoxin-induced sepsis causes 30-40% of the deaths in the intensive care unit (ICU) globally, for which there is no pharmacotherapy. Lipopolysaccharide (LPS), a bacterial endotoxin, stimulates the Toll-like receptor (TLR)-4 signalling pathways to upregulate the expression of various inflammatory mediators. Here, we show that the TIRAP and c-Jun protein signalling complex forms in macrophages in response to LPS stimulation, which increases the AP1 transcriptional activity, thereby amplifying the expression of inflammatory mediators. Using a computer-aided molecular docking platform, we identified gefitinib as a putative inhibitor of the TIRAP-c-Jun signalling complex. Further, we demonstrated the ability of gefitinib to inhibit the interaction of TIRAP-c-Jun with in vitro experiments and with a mouse model of sepsis. Importantly, pre-treatment with gefitinib increased the survival of the mice that received a lethal dose of LPS compared to that of the controls. These findings verify the ability of gefitinib to directly disrupt the interaction of TIRAP and c-Jun, thereby inhibiting a major inflammatory response that is often observed in patients experiencing sepsis.

Olive oil inhibits ageing signs induced by chronic stress in ex vivo human skin via inhibition of extracellular-signal-related kinase 1/2 and c-JUN pathways.

OBJECTIVE: Chronic stress-induced oxidative damage and protease synthesis cause a loss of extracellular matrix components promoting human skin ageing. The administration of antioxidant compounds, such as those observed in olive oil, may attenuate stress-induced ageing signs in human skin. Thus, the aim of this study was to investigate the effect of olive oil administration in ex vivo stressed human skin. METHODS: Explants of human skin were treated with high levels of epinephrine (as observed in stressed patients) and olive oil in medium for 13 days. Cultures treated with medium alone were used as controls. RESULTS: Olive oil reversed the high epinephrine level-induced reduction in epidermis and dermis thickness and collagen fibre content in ex vivo human skin. The increase in the production of reactive oxygen species (ROS) and malondialdehyde levels (an index of lipid peroxidation) promoted by high levels of epinephrine were also attenuated by olive oil in ex vivo human skin. Moreover, olive oil was able to reverse the high epinephrine level-induced increase in extracellular signal-related kinase 1/2 (ERK 1/2) and c-JUN (a major component of transcription factor AP-1) phosphorylation and protein matrix metalloproteinase-2 (MMP-2) expression in ex vivo human skin. CONCLUSION: Olive oil attenuates stress-induced ageing signs (thinner dermis and collagen fibre loss) in ex vivo human skin by reducing MMP-2 expression, ROS production, and ERK 1/2 and c-JUN phosphorylation.

OBJECTIF: Le dommage oxydatif chronique induit par le stress et la synthèse de protéases entraînent une dégradation des composants de la matrice extracellulaire favorisant le vieillissement de la peau humaine. L'administration de composés antioxydants, tels que ceux observés dans l'huile d'olive, peut atténuer les signes de vieillissement induits par le stress sur la peau humaine. L'objectif de cette étude était donc d'étudier l'effet de l'administration d'huile d'olive sur une peau humaine stressée ex vivo. MÉTHODES: Des explants de peau humaine ont été traités avec des niveaux élevés d'épinéphrine (comme observé chez les patients stressés) et d'huile d'olive dans un milieu pendant 13 jours. Les cultures traitées avec le milieu seul ont été utilisées comme témoins. RÉSULTATS: L'huile d'olive a renversé la réduction de l'épaisseur de l'épiderme et du derme et des fibres de collagène, induite par le niveau élevé d'épinéphrine dans la peau humaine ex vivo. L'augmentation de la production d'espèces réactives de l'oxygène (ROS) et de malondialdéhyde (indice de péroxydation lipidique) favorisée par des taux élevés d'épinéphrine a également été atténuée par l'huile d'olive dans la peau humaine ex vivo. En outre, l'huile d'olive a pu inverser l'augmentation, induite par le niveau élevé d'épinéphrine, de la phosphorylation de la kinase liée au signal extracellulaire 1/2 (ERK 1/2) et c-JUN (un composant majeur du facteur de transcription AP-1) et de la expression de la métalloprotéinase matricielle protéique-2 (MMP-2) dans la peau humaine ex vivo. CONCLUSION: L'huile d'olive atténue les signes du vieillissement induit par le stress (mincissement du derme et perte de fibres de collagène) dans la peau humaine ex vivo en réduisant l'expression de MMP-2, la production de ROS et la phosphorylation de ERK 1/2 et de cJUN.

Hyaluronan-CD44 interaction promotes HPV 16 E6 oncogene-mediated oropharyngeal cell carcinoma survival and chemoresistance.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy that often involves the oral cavity, pharynx, larynx, or paranasal sinuses. There is a compelling evidence of the human papilloma virus including HPV16 E6 oncogene drives cell transformation and oncogenic processes of HPV positive (HVP+) HNSCC [in particular, Oropharyngeal Squamous Cell Carcinoma (OPSCC)]. In this study, we determined that human OPSCC-derived, HPV16 E6+ cells (UMSCC-104 and UMSCC-47 cell lines) express CD44 and a regulatory transcription factor, c-Jun. Importantly, interaction between matrix hyaluronan (HA) and CD44 (an HA receptor) promotes c-Jun phosphorylation followed by phospho-c-Jun nuclear translocation and co-localization with HPV16 E6 in the nucleus of both UMSCC-104 and UMSCC-47 cells. Further analyses revealed that HPV16 E6 expression is regulated by an upstream promoter containing AP1/c-Jun binding site(s), and chromatin immunoprecipitation (ChIP) assays demonstrated that stimulation of HPV16 E6 expression by HA-CD44 interaction is phospho-c-Jun dependent in these HPV16+ UMSCC-104 and UMSCC-47 cells. This process results in an upregulation of survival proteins, inhibitors of the apoptosis family of proteins (IAPs) and chemoresistance in these HPV16+ cells. Treatment of UMSCC-104 or UMSCC-47 cells with c-Jun-specific or HPV16 E6-specific small interfering RNAs effectively blocks HA/CD44-mediated c-Jun signaling and abrogates HPV16 E6 expression as well as causes downregulation of survival proteins (cIAP-1 and cIAP-2) expression and enhancement of chemosensitivity. Together, these findings suggest that the HA/CD44-induced c-Jun signaling plays a pivotal role in HPV16 E6 upregulation leading to survival protein (cIAP-1/cIAP-2) production and chemoresistance in HPV16+ UMSCC-104 and UMSCC-47 cells. Most importantly, using a mouse xenograft model, we have observed that Cisplatin chemotherapy combined with the suppression of CD44, c-Jun and HPV16 E6 (by treating both UMSCC-104 cells and UMSCC-47 cells with CD44shRNA or c-Jun shRNA or HPV16 E6 shRNA) appears to be more effective in tumor size reduction than chemotherapy alone. Thus, these newly-discovered HA/CD44-c-Jun/HPV16E6 signaling pathways may provide new drug targets for overcoming cisplatin chemoresistance in HPV16E6-positive OPSCC cells.

Computational Competitive and Negative Design To Derive a Specific cJun Antagonist.

Basic leucine zipper (bZIP) proteins reside at the end of cell-signaling cascades and function to modulate transcription of specific gene targets. bZIPs are recognized as important regulators of cellular processes such as cell growth, apoptosis, and cell differentiation. One such validated transcriptional regulator, activator protein-1, is typically comprised of heterodimers of Jun and Fos family members and is key in the progression and development of a number of different diseases. The best described component, cJun, is upregulated in a variety of diseases such as cancer, osteoporosis, and psoriasis. Toward our goal of inhibiting bZIP proteins implicated in disease pathways, we here describe the first use of a novel in silico peptide library screening platform that facilitates the derivation of sequences exhibiting a high affinity for cJun while disfavoring homodimer formation or formation of heterodimers with other closely related Fos sequences. In particular, using Fos as a template, we have computationally screened a peptide library of more than 60 million members and ranked hypothetical on/off target complexes according to predicted stability. This resulted in the identification of a sequence that bound cJun but displayed little homomeric stability or preference for cFos. The computationally selected sequence maintains an interaction stability similar to that of a previous experimentally derived cJun antagonist while providing much improved specificity. Our study provides new insight into the use of tandem in silico screening/ in vitro validation and the ability to create a peptide that is capable of satisfying conflicting design requirements.

Transcription factors SF1 and cJUN cooperate to activate the Fdx1 promoter in MA-10 Leydig cells.

The Ferredoxin 1 (FDX1) protein supports steroid biosynthesis in steroidogenic cells through electron transfer to the rate-limiting steroidogenic enzyme, CYP11A1. The latter catalyzes the conversion of cholesterol to pregnenolone through side chain cleavage inside the mitochondria. Thus far, only several transcription factors have been implicated in the regulation of mouse Fdx1 promoter activity in Leydig cells. These include the nuclear receptor SF1 and SP1. Since two conserved regulatory elements for AP1 transcription factors have been located at -764 and -617bp of the Fdx1 promoter, we hypothesized that cJUN may cooperate with other partners to regulate Fdx1 in Leydig cells. Indeed, we report that SF1 and cJUN interact and cooperate to activate the Fdx1 promoter in MA-10 and TM3 Leydig cells. Furthermore, we found that such activation requires different regulatory elements located between -124 and -306bp of the Fdx1 promoter and involves recruitment of SF1 to this region. Using RNA interference, the importance of SF1 in transcriptional regulation of Fdx1 was confirmed, whereas cJUN was dispensable even though it cooperated with SF1 to upregulate Fdx1 expression in MA-10 cells. Thus, our data provides new insights in the molecular mechanisms that control mouse Fdx1 transcription, possibly leading to regulation of CYP11A1 enzyme activation, in Leydig cells.

XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage-independent growth of bladder cancer cell.

BACKGROUND: The X-linked inhibitor of apoptosis protein (XIAP) is a well-known potent apoptosis suppressor and also participates in cancer cell biological behaviors, therefore attracting great attentions as a potential antineoplastic therapeutic target for past years. Anti-IAP therapy is reported to be closely related to epidermal growth factor receptor (EGFR) expression level. However, whether and how XIAP modulates EGFR expression remains largely unknown. METHODS: Human XIAP was knockdown with short-hairpin RNA in two different bladder cancer cell lines, T24T and UMUC3. Two XIAP mutants, XIAP ∆BIR (deletion of N-terminal three BIR domains) and XIAP ∆RING (deletion of C-terminal RING domain and keeping the function of BIR domains), were generated to determine which domain is involved in regulating EGFR. RESULTS: We found here that lacking of XIAP expression resulted in a remarkable suppression of EGFR expression, consequently leading to the deficiency of anchorage-independent cell growth. Further study demonstrated that BIR domain of XIAP was crucial for regulating the EGFR translation by suppressing the transcription and expression of miR-200a. Mechanistic studies indicated that BIR domain activated the protein phosphatase 2 (PP2A) activity by decreasing the phosphorylation of PP2A at Tyr307 in its catalytic subunit, PP2A-C. Such activated PP2A prevented the deviant phosphorylation and activation of MAPK kinases/MAPKs, their downstream effector c-Jun, and in turn inhibiting transcription of c-Jun-regulated the miR-200a. CONCLUSIONS: Our study uncovered a novel function of BIR domain of XIAP in regulating the EGFR translation, providing significant insight into the understanding of the XIAP overexpression in the cancer development and progression, further offering a new theoretical support for using XIAP BIR domain and EGFR as targets for cancer therapy.

JunD/AP-1 Antagonizes the Induction of DAPK1 To Promote the Survival of v-Src-Transformed Cells.

The increase in AP-1 activity is a hallmark of cell transformation by tyrosine kinases. Previously, we reported that blocking AP-1 using the c-Jun dominant negative mutant TAM67 induced senescence, adipogenesis, or apoptosis in v-Src-transformed chicken embryo fibroblasts (CEFs) whereas inhibition of JunD by short hairpin RNA (shRNA) specifically induced apoptosis. To investigate the role of AP-1 in Src-mediated transformation, we undertook a gene profiling study to characterize the transcriptomes of v-Src-transformed CEFs expressing either TAM67 or the JunD shRNA. Our study revealed a cluster of 18 probe sets upregulated exclusively in response to AP-1/JunD impairment and v-Src transformation. Four of these probe sets correspond to genes involved in the interferon pathway. One gene in particular, death-associated protein kinase 1 (DAPK1), is a C/EBPß-regulated mediator of apoptosis in gamma interferon (IFN-γ)-induced cell death. Here, we show that inhibition of DAPK1 abrogates cell death in v-Src-transformed cells expressing the JunD shRNA. Chromatin immunoprecipitation data indicated that C/EBPß was recruited to the DAPK1 promoter while the expression of a dominant negative mutant of C/EBPß abrogated the induction of DAPK1 in response to the inhibition of AP-1. In contrast, as determined by chromatin immunoprecipitation (ChIP) assays, JunD was not detected on the DAPK1 promoter under any conditions, suggesting that JunD promotes survival by indirectly antagonizing the expression of DAPK1 in v-Src transformed cells. IMPORTANCE: Transformation by the v-Src oncoprotein causes extensive changes in gene expression in primary cells such as chicken embryo fibroblasts. These changes, determining the properties of transformed cells, are controlled in part at the transcriptional level. Much attention has been devoted to transcription factors such as AP-1 and NF-κB and the control of genes associated with a more aggressive phenotype. In this report, we describe a novel mechanism of action determined by the JunD component of AP-1, a factor enhancing cell survival in v-Src-transformed cells. We show that the loss of JunD results in the aberrant activation of a genetic program leading to cell death. This program requires the activation of the tumor suppressor death-associated protein kinase 1 (DAPK1). Since DAPK1 is phosphorylated and inhibited by v-Src, these results highlight the importance of this kinase and the multiple mechanisms controlled by v-Src to antagonize the tumor suppressor function of DAPK1.

JUN is a key transcriptional regulator of the unfolded protein response in acute myeloid leukemia.

The transcription factor JUN is frequently overexpressed in multiple genetic subtypes of acute myeloid leukemia (AML); however, the functional role of JUN in AML is not well defined. Here we report that short hairpin RNA (shRNA)-mediated inhibition of JUN decreases AML cell survival and propagation in vivo. By performing RNA sequencing analysis, we discovered that JUN inhibition reduces the transcriptional output of the unfolded protein response (UPR), an intracellular signaling transduction network activated by endoplasmic reticulum (ER) stress. Specifically, we found that JUN is activated by MEK signaling in response to ER stress, and that JUN binds to the promoters of several key UPR effectors, such as XBP1 and ATF4, to activate their transcription and allow AML cells to properly negotiate ER stress. In addition, we observed that shRNA-mediated inhibition of XBP1 or ATF4 induces AML cell apoptosis and significantly extends disease latency in vivo tying the reduced survival mediated by JUN inhibition to the loss of pro-survival UPR signaling. These data uncover a previously unrecognized role of JUN as a regulator of the UPR as well as provide key new insights into the how ER stress responses contribute to AML and identify JUN and the UPR as promising therapeutic targets in this disease.

Neuroprotective effects of curcumin on endothelin-1 mediated cell death in hippocampal neurons.

OBJECTIVES: Alzheimer's disease is a progressive neurodegenerative disease characterized by loss of hippocampal neurons leading to memory deficits and cognitive decline. Studies suggest that levels of the vasoactive peptide endothelin-1 (ET-1) are increased in the brain tissue of Alzheimer's patients. Curcumin, the main ingredient of the spice turmeric, has been shown to have anti-inflammatory, anti-cancer, and neuroprotective effects. However, the mechanisms underlying some of these beneficial effects are not completely understood. The objective of this study was to determine if curcumin could protect hippocampal neurons from ET-1 mediated cell death and examine the involvement of c-Jun in this pathway. METHODS: Primary hippocampal neurons from rat pups were isolated using a previously published protocol. Viability of the cells was measured by the live/dead assay. Immunoblot and immunohistochemical analyses were performed to analyze c-Jun levels in hippocampal neurons treated with either ET-1 or a combination of ET-1 and curcumin. Apoptotic changes were evaluated by immunoblot detection of cleaved caspase-3, cleaved fodrin, and a caspase 3/7 activation assay. RESULTS: ET-1 treatment produced a 2-fold increase in the levels of c-Jun as determined by an immunoblot analysis in hippocampal neurons. Co-treatment with curcumin significantly attenuated the ET-1 mediated increase in c-Jun levels. ET-1 caused increased neuronal cell death of hippocampal neurons indicated by elevation of cleaved caspase-3, cleaved fodrin and an increased activity of caspases 3 and 7 which was attenuated by co-treatment with curcumin. Blockade of JNK, an upstream effector of c-Jun by specific inhibitor SP600125 did not fully protect from ET-1 mediated activation of pro-apoptotic enzymes in primary hippocampal cells. DISCUSSION: Our data suggests that one mechanism by which curcumin protects against ET-1-mediated cell death is through blocking an increase in c-Jun levels. Other possible mechanisms include decreasing pro-apoptotic signaling activated by ET-1 in primary hippocampal neurons.

Pentraxin-2 suppresses c-Jun/AP-1 signaling to inhibit progressive fibrotic disease.

Pentraxin-2 (PTX-2), also known as serum amyloid P component (SAP/APCS), is a constitutive, antiinflammatory, innate immune plasma protein whose circulating level is decreased in chronic human fibrotic diseases. Here we show that recombinant human PTX-2 (rhPTX-2) retards progression of chronic kidney disease in Col4a3 mutant mice with Alport syndrome, reducing blood markers of kidney failure, enhancing lifespan by 20%, and improving histological signs of disease. Exogenously delivered rhPTX-2 was detected in macrophages but also in tubular epithelial cells, where it counteracted macrophage activation and was cytoprotective for the epithelium. Computational analysis of genes regulated by rhPTX-2 identified the transcriptional regulator c-Jun along with its activator protein-1 (AP-1) binding partners as a central target for the function of rhPTX-2. Accordingly, PTX-2 attenuates c-Jun and AP-1 activity, and reduces expression of AP-1-dependent inflammatory genes in both monocytes and epithelium. Our studies therefore identify rhPTX-2 as a potential therapy for chronic fibrotic disease of the kidney and an important inhibitor of pathological c-Jun signaling in this setting.

Intermittent hypoxia confers pro-metastatic gene expression selectively through NF-κB in inflammatory breast cancer cells.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Treatment options are limited and the mechanisms underlying its aggressiveness are poorly understood. Intermittent hypoxia (IH) causes oxidative stress and is emerging as important regulator of tumor metastasis. Vessels in IBC tumors have been shown to be immature, which is a primary cause of IH. We therefore investigated the relevance of IH for the modulation of gene expression in IBC cells in order to assess IH as potential regulator of IBC aggressiveness. Gene array analysis of IBC cells following chronic IH (45-60 days) demonstrated increased expression of pro-metastatic genes of the extracellular matrix, such as tenascin-C (TNC; an essential factor of the metastatic niche) and matrix metalloproteinase 9 (MMP9), and of pro-inflammatory processes, such as cyclooxygenase-2 (COX-2). Investigating the oxidative stress-dependent regulation of TNC, we found a gradual sensitivity on mRNA and protein levels. Oxidative stress activated NF-E2-related factor 2 (Nrf2), c-Jun N-terminal kinase (JNK), c-Jun and nuclear factor κB (NF-κB), but TNC upregulation was only dependent on NF-κB activation. Pharmacological inhibition of inhibitor of NF-κB α (IκBα) phosphorylation as well as overexpression of IκBα prevented TNC, MMP9 and COX-2 induction, whereas the pro-inflammatory cytokine interleukin-1ß (IL-1ß) increased their expression levels. Analysis of the gene array data showed NF-κB binding sites for 64% of all upregulated genes, linking NF-κB with IH-dependent regulation of pro-metastatic gene expression in IBC cells. Our results provide a first link between intermittent hypoxia and pro-metastatic gene expression in IBC cells, revealing a putative novel mechanism for the high metastatic potential of IBC.

SPANXA suppresses EMT by inhibiting c-JUN/SNAI2 signaling in lung adenocarcinoma.

SPANXA (Sperm Protein Associated with the Nucleus on the X-chromosome, family members A1/A2) acts as a cancer-testis antigen expressed in normal testes, but dysregulated in various tumors. We found that SPANXA is highly expressed in low-invasive CL1-0 cells compared with isogenous high-invasive CL1-5 cells. SPANXA was preferably expressed in tumor tissues and associated with the prolonged survival of lung adenocarcinomas. SPANXA suppressed the invasion and metastasis of lung cancer cells in vitro and in vivo. By the expression microarray and pathway analysis, we found that the SPANXA-altered genes were enriched in the epithelial-mesenchymal transition (EMT) pathway. SPANXA reduced SNAI2 expression resulted in up-regulating E-cadherin. c-JUN acts as the positive-regulator of EMT. Silencing SPANXA increased c-JUN mRNA expression and blockage of c-JUN led to SNAI2 down-regulation. Our results clearly characterized SPANXA as an EMT inhibitor by suppressing c-JUN-SNAI2 axis in lung adenocarcinoma.

Synergistic effects of c-Jun and SP1 in the promotion of TGFß1-mediated diabetic nephropathy progression.

Diabetic nephropathy (DN) is a major complication of diabetes mellitus. Transforming growth factor beta 1 (TGFß1) is a well-distinguished mediator of progressive renal fibrosis in DN. However, the molecular mechanisms contributing to enhanced TGFß1 expression in the progression of DN are not fully understood. Herein, we reported that c-Jun and specificity protein 1 (SP1) were critical upstream regulators of TGFß1 expression in DN. The increase in c-Jun and SP1 expressions was positively correlated with TGFß1 in both high glucose-treated human renal mesangial cells (HRMCs) and diabetic kidneys. Furthermore, c-Jun dose-dependently promoted SP1-mediated TGFß1 transcription and vice versa. The synergistic effects of c-Jun and SP1 were attributed to their auto-regulation and cross-activation. Moreover, enhanced phosphorylation levels of c-Jun and SP1 were accompanied with increased TGFß1 expression in diabetic kidneys. Accordingly, dephosphorylation of c-Jun and SP1 by the specific c-Jun N-terminal kinase (JNK) inhibitor SP600125 prevented the increase in TGFß1 expression. These results suggested that c-Jun and SP1 synergistically activated profibrotic TGFß1 expression in the development of DN by auto-regulation, cross-activation and phospho-modification.

Inhibitors of c-Jun phosphorylation impede ovine primordial follicle activation.

STUDY HYPOTHESIS: Is the c-Jun-N-terminal kinase (JNK) pathway implicated in primordial follicle activation? STUDY FINDING: Culture of ovine ovarian cortex in the presence of two different c-Jun phosphorylation inhibitors impeded pre-antral follicle activation. WHAT IS KNOWN ALREADY: Despite its importance for fertility preservation therapies, the mechanisms of primordial follicle activation are poorly understood. Amongst different signalling pathways potentially involved, the JNK pathway has been previously shown to be essential for cell cycle progression and pre-antral follicle development in mice. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Ovine ovarian cortex pieces were cultured with varying concentrations of SP600125, JNK inhibitor VIII or anti-Mullerian hormone (AMH) in the presence of FSH for 9 days. Follicular morphometry and immunohistochemistry for proliferating cell nuclear antigen (PCNA), apoptosis and follicle activation (Foxo3a) were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Inhibition of primordial follicle activation occurred in the presence of SP600125, JNK inhibitor VIII and AMH when compared with controls (all P < 0.05) after 2 days of culture. However, only in the highest concentrations used was the inhibition of activation associated with induction of follicular apoptosis (P < 0.05). In growing follicles, PCNA antigen expression was reduced when the JNK inhibitors or AMH were used (P < 0.05 versus control), indicating reduced proliferation of the somatic compartment. LIMITATIONS, REASONS FOR CAUTION: Although we evaluated the effects of inhibition of c-Jun phosphorylation on primordial follicle development, we did not determine the cellular targets and mechanism of action of the inhibitors. WIDER IMPLICATIONS OF THE FINDINGS: These results are the first to implicate the JNK pathway in primordial follicle activation and could have significant consequences for the successful development of fertility preservation strategies and our understanding of primordial follicle activation. LARGE SCALE DATA: n/a. STUDY FUNDING AND COMPETING INTERESTS: Dr Michael J. Bertoldo and the laboratories involved in the present study were supported by a grant from 'Région Centre' (CRYOVAIRE, Grant number #320000268). There are no conflicts of interest to declare.