Downstream Transcription Factors c-Jun Research Articles

Dehydration stress alters the mitogen-activated-protein kinase signaling and chaperone stress response in Xenopus laevis.

In arid conditions, the African Clawed frog Xenopus laevis enters a state of estivation dormancy as an adaptive survival strategy. Under estivation, X. laevis experience severe dehydration stress as 25-35% of total body water is lost. Dehydration in X. laevis can lead to periods of hypoxia due to elevated blood viscosity that impedes tissue perfusion. To understand how X. laevis survives under such stress, we studied the regulation pattern of key mitogen-activated protein kinases (MAPK) and their downstream transcription factors, along with several heat shock proteins in the oxygen sensitive brain and heart tissue of X. laevis under dehydration stress. Our study revealed that the activation phosphorylation residues of MAPK including JNK and MSK and their downstream transcription factors c-Jun and ATF2 are significantly decreased in the heart under dehydration. Given that JNK, c-Jun, and ATF2 are known positive regulators of apoptosis, this regulatory pattern suggest that a state of pro-survival signals may be established in the dehydrated heart. In support of this, protein levels of HSP60, a pro-apoptotic mitochondrial chaperone, was also downregulated in the heart in response to dehydration stress. In the brain tissue, most proteins remain unchanged with the exception of the apoptosis regulating p53 transcription factor, which showed a significant decrease in its activating phosphorylation residue under dehydration. Overall, our study revealed that in the Xenopus brain and heart, a specific suppression pattern of MAPK, transcription factors, and HSP takes place to potentially establish a state of pro-survival under dehydration stress.

Physciosporin suppresses the proliferation, motility and tumourigenesis of colorectal cancer cells

BackgroundLichens, which represent symbiotic associations of fungi and algae, are potential sources of numerous natural products. Physciosporin (PHY) is a potent secondary metabolite found in lichens and was recently reported to inhibit the motility of lung cancer cells via novel mechanisms. PurposeThe present study investigated the anticancer potential of PHY on colorectal cancer (CRC) cells. MethodsPHY was isolated from lichen extract by preparative TLC. The effect of PHY on cell viability, motility and tumourigenicity was elucidated by MTT assay, hoechst staining, flow cytometric analysis, transwell invasion and migration assay, soft agar colony formation assay, Western blotting, qRT-PCR and PCR array in vitro as well as tumorigenicity study in vivo. ResultsPHY decreased the viability of various CRC cell lines (Caco2, CT26, DLD1, HCT116 and SW620). Moreover, PHY elicited cytotoxic effects by inducing apoptosis at toxic concentrations. At non-toxic concentrations, PHY dose-dependently suppressed the invasion, migration and colony formation of CRC cells. PHY inhibited the motility of CRC cells by suppressing epithelial-mesenchymal transition and downregulating actin-based motility markers. In addition, PHY downregulated β-catenin and its downstream target genes cyclin-D1 and c-Myc. Moreover, PHY modulated KAI1 C-terminal-interacting tetraspanin and KAI1 expression, and downregulated the downstream transcription factors c-jun and c-fos. Finally, PHY administration showed considerable bioavailability and effectively decreased the growth of CRC xenografts in mice without causing toxicity. ConclusionPHY suppresses the growth and motility of CRC cells via novel mechanisms.

ERβ targets ZAK and attenuates cellular hypertrophy via SUMO-1 modification in H9c2 cells.

Aberrant expression of leucine zipper- and sterile ɑ motif-containing kinase (ZAK) observed in pathological human myocardial tissue is associated with the progression and elevation of hypertrophy. Our previous reports have correlated high levels of estrogen (E2) and abundant estrogen receptor (ER) α with a low incidence of pathological cardiac-hypertrophy and heart failure in the premenopause female population. However, the effect of elevated ERβ expression is not well known yet. Therefore, in this study, we have analyzed the cardioprotective effects and mechanisms of E2 and/or ERβ against ZAK overexpression-induced cellular hypertrophy. We have used transient transfection to overexpress ERβ into the ZAK tet-on H9c2 cells that harbor the doxycycline-inducible ZAK plasmid. The results show that ZAK overexpression in H9c2 cells resulted in hypertrophic effects, which was correlated with the upregulation of p-JNK and p-p38 MAPKs and their downstream transcription factors c-Jun and GATA-4. However, ERβ and E2 with ERβ overexpressions totally suppressed the effects of ZAK overexpression and inhibited the levels of p-JNK, p-p38, c-Jun, and GATA-4 effectively. Our results further reveal that ERβ directly binds with ZAK under normal conditions; however, ZAK overexpression reduced the association of ZAK-ERβ. Interestingly, increase in ERβ and E2 along with ERβ overexpression both enhanced the binding strengths of ERβ and ZAK and reduced the ZAK protein level. ERβ overexpression also suppressed the E3 ligase-casitas B-lineage lymphoma (CBL) and attenuated CBL-phosphoinositide 3-kinase (PI3K) protein association to prevent PI3K protein degradation. Moreover, ERβ and/or E2 blocked ZAK nuclear translocation via the inhibition of small ubiquitin-like modifier (SUMO)-1 modification. Taken together, our results further suggest that ERβ overexpression strongly suppresses ZAK-induced cellular hypertrophy and myocardial damage.

Transcriptional regulation of stress kinase JNK2 in pro-arrhythmic CaMKIIδ expression in the aged atrium.

c-jun N-terminal kinase (JNK) is a critical stress response kinase that activates in a wide range of physiological and pathological cellular processes. We recently discovered a pivotal role of JNK in the development of atrial arrhythmias in the aged heart, while cardiac CaMKIIδ, another pro-arrhythmic molecule, was also known to enhance atrial arrhythmogenicity. Here, we aimed to reveal a regulatory role of the stress kinase JNK2 isoform on CaMKIIδ expression. Activated JNK2 leads to increased CaMKIIδ protein expression in aged human and mouse atria, evidenced from the reversal of CaMKIIδ up-regulation in JNK2 inhibitor treated wild-type aged mice. This JNK2 action in CaMKIIδ expression was further confirmed in HL-1 myocytes co-infected with AdMKK7D-JNK2, but not when co-infected with AdMKK7D-JNK1. JNK2-specific inhibition (either by a JNK2 inhibitor or overexpression of inactivated dominant-negative JNK2 (JNK2dn) completely attenuated JNK activator anisomycin-induced CaMKIIδ up-regulation in HL-1 myocytes, whereas overexpression of JNK1dn did not. Moreover, up-regulated CaMKIIδ mRNA along with substantially increased phosphorylation of JNK downstream transcription factor c-jun [but not activating transcription factor2 (ATF2)] were exhibited in both aged atria (humans and mice) and transiently JNK activated HL-1 myocytes. Cross-linked chromatin-immunoprecipitation assays (XChIP) revealed that both c-jun and ATF2 were bound to the CaMKIIδ promoter, but significantly increased binding of c-jun only occurred in the presence of anisomycin and JNK inhibition alleviated this anisomycin-elevated c-jun binding. Mutated CaMKII consensus c-jun binding sites impaired its promoter activity. Enhanced transcriptional activity of CaMKIIδ by anisomycin was also completely reversed to the baseline by either JNK2 siRNA or c-jun siRNA knockdown. JNK2 activation up-regulates CaMKIIδ expression in the aged atrium. This JNK2 regulation in CaMKIIδ expression occurs at the transcription level through the JNK downstream transcription factor c-jun. The discovery of this novel molecular mechanism of JNK2-regulated CaMKII expression sheds new light on possible anti-arrhythmia drug development.

Long noncoding RNA LINP1: scaffolding non-homologous end joining.

Non-homologous end joining (NHEJ) is a principal pathway of DNA double-strand break (DSB) repair in mammalian cells,1 but is also involved in assembly of antigen receptor genes and telomere maintenance.2,3 Loss of NHEJ function can result in chromosome instability promoting malignant transformation, in particular when other cellular safeguards are compromised. Inherited NHEJ defects underlie radiosensitive severe combined immunodeficiency. NHEJ is initiated by binding of Ku70–Ku80 heterodimers to both ends of a DNA break, followed by recruitment of catalytic subunits of DNA-dependent protein kinase (DNA-PKcs) to form juxtaposed DNA-PK holoenzymes which join the ends in a synaptic complex. Cohesive blunt DNA ends can be joined directly by the XFL-XRCC4-DNA ligase IV complex, but in many cases DNA ends contain damaged bases or DNA backbone sugars that require processing before ligation. End processing may involve nucleases such as Artemis, DNA polymerases and polynucleotide kinase among other enzymes.4,5

The role of bioactive nanofibers in enamel regeneration mediated through integrin signals acting upon C/EBPα and c-Jun

Enamel formation involves highly orchestrated intracellular and extracellular events; following development, the tissue is unable to regenerate, making it a challenging target for tissue engineering. We previously demonstrated the ability to trigger enamel differentiation and regeneration in the embryonic mouse incisor using a self-assembling matrix that displayed the integrin-binding epitope RGDS (Arg-Gly-Asp-Ser). To further elucidate the intracellular signaling pathways responsible for this phenomenon, we explore here the coupling response of integrin receptors to the biomaterial and subsequent downstream gene expression profiles. We demonstrate that the artificial matrix activates focal adhesion kinase (FAK) to increase phosphorylation of both c-Jun N-terminal kinase (JNK) and its downstream transcription factor c-Jun (c-Jun). Inhibition of FAK blocked activation of the identified matrix-mediated pathways, while independent inhibition of JNK nearly abolished phosphorylated-c-Jun (p-c-Jun) and attenuated the pathways identified to promote enamel regeneration. Cognate binding sites in the amelogenin promoter were identified to be transcriptionally up-regulated in response to p-c-Jun. Furthermore, the artificial matrix induced gene expression as evidenced by an increased abundance of amelogenin, the main protein expressed during enamel formation, and the CCAAT enhancer binding protein alpha (C/EBPα), which is the known activator of amelogenin expression. Elucidating these cues not only provides guidelines for the design of synthetic regenerative strategies and opportunities to manipulate pathways to regulate enamel regeneration, but can provide insight into the molecular mechanisms involved in tissue formation.

JNK Regulates MCP-1 Expression in Adenovirus Type 19–Infected Human Corneal Fibroblasts

Previous studies indicate that adenovirus type 19 (Ad19) infection of human corneal fibroblasts (HCFs) induces the expression of several proinflammatory mediators, including IL-8 and monocyte chemoattractant protein-1 (MCP-1), and that the tyrosine kinase c-Src and its downstream target, the mitogen-activated protein kinase ERK1/2, mediate IL-8 expression. In this context, the authors sought to investigate the potential role of another mitogen-activated protein kinase, c-Jun N-terminal kinase (JNK), in adenoviral ocular pathogenesis. Ad19- and mock-infected HCFs were solubilized at various time points after infection, and cell lysates were subjected to SDS-PAGE followed by immunoblot analysis with a panel of antibodies against components of the MKK7/JNK/c-Jun pathway or immunoprecipitated for JNK assay. The induction of chemokine mRNA and protein was determined by real-time PCR and ELISA, respectively. Ad19 induced the phosphorylation of MKK7, JNK, and the downstream transcription factor c-Jun in HCFs at 15 and 30 minutes after infection. JNK activity was demonstrated at 30 minutes after infection using the GST-c-Jun fusion protein as a target substrate. SP600125, a specific pharmacologic inhibitor of JNK, blocked MCP-1 but not IL-8 mRNA and protein expression. Finally, PP2, a specific inhibitor of c-Src previously shown to inhibit the expression of both IL-8 and MCP-1 in Ad19-infected HCFs, also blocked JNK phosphorylation after infection. The MKK7/JNK/c-Jun cascade is rapidly activated and mediates MCP-1 expression in Ad19-infected HCFs. Furthermore, the activation of c-Src on Ad19 infection appears to regulate both the ERK and the JNK pathways.

Nitric Oxide-Donating Aspirin Inhibits Colon Cancer Cell Growth via Mitogen-Activated Protein Kinase Activation

Nitric oxide-donating aspirin (NO-aspirin), representing a new concept in the development of more efficacious nonsteroidal anti-inflammatory drugs, consists of traditional aspirin bearing -ONO(2), which releases NO. Conventional aspirin prevents human colon cancer, but its toxicity precludes its application as a chemopreventive agent. NO-aspirin seems safer and in cultured cancer cells it is >1000-fold more potent than aspirin. To determine the mechanism by which NO-aspirin inhibits cell growth, we studied its effect on mitogen-activated protein kinase (MAPK) signaling in HT-29 human colon cancer cells. NO-aspirin stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 and Akt only marginally. The greatest increases in phosphorylation were seen in cJun NH(2)-terminal kinase (JNK) and p38 MAP kinases, which were observed as early as 5 min and after 1 h of treatment, averaged more than 10-fold over control. The activation of JNK and p38 was accompanied by large increases in the phosphorylation of the downstream transcription factors cJun and activating transcription factor 2 (ATF-2). We used specific MAPK inhibitors, small interfering (siRNA) gene silencing methods, and dominant-negative cJun to determine the relevance of these phosphorylation events to the ability of NO-aspirin to inhibit colon cancer cell growth. Only the dual inhibitor of p38 and JNK and the use of combined siRNA silencing of p38 and cJun abrogated the ability of NO-aspirin to block cell growth. Our data indicate that NO-aspirin is dependent on both the p38 and the JNK MAP kinase pathways for its ability to inhibit the growth of colon cancer cells.

Acrolein induces activation of the epidermal growth factor receptor of human keratinocytes for cell death.

Acrolein, which is a highly reactive formaldehyde generated by lipid peroxidation, can affect skin and cause various disorders. The effect of exposure of human keratinocytes to acrolein on cell surface-oriented signal transduction into cells was examined. Incubation of human keratinocytes with a relatively low concentration (50 microM) of acrolein caused a prompt and selective induction of tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) as a 180-kDa molecule during the period from 5-30 min after the start of incubation. This early event was followed by an increase in the density and number of phosphotyrosine-containing proteins during the period from 60-120 min after the start of incubation. The catalytic activity of EGFR as measured by the levels of autophorphorylation and phosphorylation of an exogenously added substrate, casein, in in vitro kinase assay, greatly increased as early as 1 min after the start of incubation and then decreased gradually 30 min later. MAP family kinases, including ERK, JNK, and p38 kinase, and the potentially downstream transcription factor c-Jun were all promoted for phosphorylation/activation during a period of 5-30 min. Selective prompt phosphorylation/activation of EGFR followed by phosphorylation of MAP family kinases and c-Jun and their blockade by a specific EGFR inhibitor, AG1478, suggested that activation of EGFR is the major, and possibly single, cell surface element for intracellular signal transduction in acrolein-treated cells. Incubation of human keratinocytes with 50 microM of acrolein induced atypical apoptosis with morphologic apoptotic features with low-grade oligonucleoside-sized DNA fragmentation. Partial inhibition of such a cytopathic effect of acrolein on human keratinocytes by preincubation with AG1478 suggests the involvement of an EGFR-mediated signal pathway for atypical apoptosis. These results provide new information on acrolein-induced cell surface-oriented signal transduction to human keratinocytes, and this information may be useful for understanding the pathogenesis of a number of skin diseases in response to environmental acrolein and acrolein-generating ultraviolet irradiation.