Inhibition Of c-Jun Research Articles

Protective Effects of SP600125 on Renal Ischemia-Reperfusion Injury in Rats

Ischemia/reperfusion injury (IRI) has a negative effect on renal allograft survival. Using a rat model of kidney IRI in this study, we investigated the overall effect of selective c-Jun N-terminal kinase (JNK) inhibitor SP600125 on renal IRI events. All 45 Fisher rats were anesthetized and renal IRI model was established by 45 min clamp of bilateral renal pedicles and 24 h reperfusion. Vehicle solution or SP600125 solution was intraperitoneally injected 45 min before ischemia, respectively. Analysis of renal histology, function, reactive oxygen species (ROS) expression, JNK phosphorylation status, as well as intra-renal pro-inflammatory cytokines expression was evaluated in this study. After IRI, the levels of blood urea nitrogen, creatinine, tissue malondialdehyde, TNF-α, IL-1β, IL-6 were all elevated significantly, while superoxide dismutase, catalase activity were decreased. Histologic findings showed severe devastating lesions and increased rodent cell apoptosis; SP600125 effectively improved morphologic features, reversed above-mentioned parameters, and significantly attenuated c-Jun phosphorylation, as well as intra-renal pro-inflammatory cytokines expression compared with vehicle-treated group. These data demonstrate that inhibition of c-Jun with SP600125 is capable of attenuating renal IRI, which might be a novel therapy target.

Enteral arginine modulates inhibition of AP-1/c-Jun by SP600125 in the postischemic gut

We previously demonstrated that enteral arginine increased c-Jun/activator protein-1 (AP-1) DNA-binding activity and iNOS expression in a rodent model of mesenteric ischemia/reperfusion (I/R). The objective of this study was to specifically investigate the role of AP-1 in arginine's deleterious effect on the postischemic gut. We hypothesized that AP-1 inhibition would mitigate the effects of arginine. Using a rodent model of mesenteric I/R we demonstrated that gut neutrophil infiltration, activity of c-Jun/AP-1, as well as iNOS expression were increased by I/R and further increased by arginine while lessened by inhibition of c-Jun using the pharmacologic c-Jun N-terminal kinase inhibitor, SP600125. Similar results were demonstrated using a cell culture model of oxidant stress in IEC-6 cells. Importantly, effects of SP600125 were comparable to those of c-Jun silencing. Lastly, the specific iNOS inhibitor, 1400W, had no effect on either AP-1 or c-Jun. In conclusion, SP600125 attenuated the activity of c-Jun/AP-1, iNOS expression, and neutrophil infiltration induced by arginine following mesenteric I/R. Our data suggest that AP-1 inhibition mitigates the injurious inflammatory effects of arginine in the postischemic gut. Further investigation into the pathologic role of enteral arginine in the postischemic gut is warranted.

The AP-1 repressor protein, JDP2, potentiates hepatocellular carcinoma in mice

BackgroundThe AP-1 transcription factor plays a major role in cell proliferation, apoptosis, differentiation and developmental processes. AP-1 proteins are primarily considered to be oncogenic. Gene disruption studies placed c-Jun as an oncogene at the early stage of a mouse model of hepatocellular carcinoma. Mice lacking c-Jun display reduced number and size of hepatic tumors attributed to elevated p53 expression and increased apoptosis. This suggests that c-Jun inhibition may serve as a therapeutic target for liver cancer. The c-Jun dimerization protein 2, JDP2 is an AP-1 repressor protein that potently inhibits AP-1 transcription. On the other hand, the JDP2 locus was found at a recurring viral integration site in T-cell lymphoma. We sought to examine the potential of JDP2 to inhibit c-Jun/AP-1 oncogenic activity in mice. Towards this end, we generated a tetracycline inducible transgenic mouse expressing JDP2 specifically in the liver. We used diethylnitrosamine (DEN) injection to initiate liver cancer in mice and assessed the extent of liver cancer in JDP2-transgenic and wild type control mice by biochemical and molecular biology techniques.ResultsJDP2-transgenic mice display normal liver function. JDP2-transgenic mice displayed potentiation of liver cancer, higher mortality and increased number and size of tumors. The expression of JDP2 at the promotion stage was found to be the most critical for enhancing liver cancer severity.ConclusionsThis study suggests that JDP2 expression may play a critical role in liver cancer development by potentiating the compensatory proliferative response and increased inflammation in the DEN liver cancer model.

C-Jun Regulates Phosphoinositide-dependent Kinase 1 Transcription: IMPLICATION FOR Akt AND PROTEIN KINASE C ACTIVITIES AND MELANOMA TUMORIGENESIS

Mutations in N-RAS and B-RAF, which commonly occur in melanomas, result in constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) signaling. Active ERK increases expression and activity of the c-Jun transcription factor, linking ERK and Jun N-terminal kinase (JNK) cascades. Here, we show that c-Jun regulates transcription of phosphoinositide-dependent kinase 1 (PDK1) with a concomitant impact on Akt and protein kinase C (PKC) activity and related substrates. Inhibition of c-Jun reduces PDK1 expression and attenuates Akt and PKC activity, which can be restored by exogenous PDK1. c-Jun regulation of PDK1 in melanoma contributes to growth rate and the ability to form tumors in mice. Correspondingly, increased levels of c-Jun in melanoma cell lines coincide with up-regulation of PDK1 and phosphorylation of PKC and Akt. The identification of c-Jun as a transcriptional regulator of PDK1 expression highlights key mechanisms underlying c-Jun oncogenic activity, and provides new insight into the nature of up-regulated Akt and PKC in melanoma.

Direct and Indirect Roles of Cyclin-dependent Kinase 5 as an Upstream Regulator in the c-Jun NH2-Terminal Kinase Cascade: Relevance to Neurotoxic Insults in Alzheimer's Disease

Significant increase in JNK, c-Jun, and Cdk5 activities are reported in Alzheimer's disease (AD). Inhibition of c-Jun prevents neuronal cell death in in vivo AD models, highlighting it as a major JNK effector. Both JNK and Cdk5 promote neurodegeneration upon deregulation; however, Cdk5 has not been mechanistically linked to JNK or c-Jun. This study presents the first mechanism showing Cdk5 as a major regulator of the JNK cascade. Deregulated Cdk5 induces biphasic activation of JNK pathway. The first phase revealed c-Jun as a direct substrate of Cdk5, whose activation is independent of reactive oxygen species (ROS) and JNK. In the second phase, Cdk5 activates c-Jun via ROS-mediated activation of JNK. Rapid c-Jun activation is supported by in vivo data showing c-Jun phosphorylation in cerebral cortex upon p25 induction in transgenic mice. Cdk5-mediated biphasic activation of c-Jun highlights c-Jun, rather than JNK, as an important therapeutic target, which was confirmed in neuronal cells. Finally, Cdk5 inhibition endows superior protection against neurotoxicity, suggesting that Cdk5 is a preferable therapeutic target for AD relative to JNK and c-Jun.

The role of the transcription factor AP-1 in colitis-associated and β-catenin-dependent intestinal tumorigenesis in mice

Chronic inflammation is an important cancer risk factor but the molecular pathways linking inflammation and cancer are incompletely understood. The transcription factor c-Jun/AP-1 (activator protein 1) is involved in inflammatory responses and tumorigenesis and has been proposed as an essential mediator of oncogenic beta-catenin signaling in the intestine. Here, we examined the functions of c-Jun in two distinct mouse models of conditional and intestine-specific activation of beta-catenin. c-Jun is strongly expressed in the small intestine of mutant mice. However, beta-catenin-dependent cell proliferation is surprisingly not affected in mice lacking c-jun in intestinal epithelium, suggesting that c-Jun is not an essential immediate target of beta-catenin signaling in the small intestine. To examine the functions of Jun and Fos proteins during inflammation and cancer in the colon, colitis-associated tumors were induced chemically in the respective knockout mice. Tumors were characterized by activated beta-catenin and strongly expressed c-Jun and JunB. However, tumorigenesis was not affected by inactivation of c-Jun in either intestinal epithelium or myeloid cells. Moreover, tumorigenesis was not altered in mice lacking junB, junD, c-fos, fra-1 or fra-2, suggesting that inhibition of c-Jun or other single AP-1 proteins is not a determining factor in colitis-associated cancer in mice.

Benzo(a)pyrene-caused increased G 1–S transition requires the activation of c-Jun through p53-dependent PI-3K/Akt/ERK pathway in human embryo lung fibroblasts

Benzo(a)pyrene (B(a)P) is a potent lung carcinogen mainly derived from tobacco smoking and environmental contamination, however, the molecular mechanisms by which it accelerates the cell cycle progression and induces the abnormal cell proliferation are still far away from understood. Our current analysis of human embryo lung fibroblasts (HELF) showed that B(a)P exposure was able to promote cell cycle G 1–S phase transition. This effect was correlated with c-Jun activation because inhibition of c-Jun by its dominant negative mutant (TAM67) reversed B(a)P action on cell cycle with the down-regulation of expression of cyclin D1, pRb and E2F1. Further study found that overexpression of dominant negative mutants of, PI-3K or Akt, dramatically reduced B(a)P-induced the activation of c-Jun and extracellular signaling regulated kinase (ERK), but not c-Jun NH2 terminal kinase (JNK). Inhibition of p53 by either its inhibitor pifithrin-α or p53 siRNA markedly increased B(a)P-induced the activation of c-Jun, Akt and ERK in this context. Take together, our results indicate that c-Jun activation by p53-dependent PI-3K/Akt/ERK pathway is responsible for B(a)P-induced cell cycle alternations in human embryo lung fibroblasts.

Endoplasmic Reticulum Stress Increases the Expression of Methylenetetrahydrofolate Reductase through the IRE1 Transducer

Methylenetetrahydrofolate reductase (MTHFR), an enzyme in folate and homocysteine metabolism, influences many cellular processes including methionine and nucleotide synthesis, methylation reactions, and maintenance of homocysteine at nontoxic levels. Mild deficiency of MTHFR is common in many populations and modifies risk for several complex traits including vascular disease, birth defects, and cancer. We recently demonstrated that MTHFR can be up-regulated by NF-kappaB, an important mediator of cell survival that is activated by endoplasmic reticulum (ER) stress. This observation, coupled with the reports that homocysteine can induce ER stress, prompted us to examine the possible regulation of MTHFR by ER stress. We found that several well characterized stress inducers (tunicamycin, thapsigargin, and A23187) as well as homocysteine could increase Mthfr mRNA and protein in Neuro-2a cells. The induction of MTHFR was also observed after overexpression of inositol-requiring enzyme-1 (IRE1) and was inhibited by a dominant-negative mutant of IRE1. Because IRE1 triggers c-Jun signaling, we examined the possible involvement of c-Jun in up-regulation of MTHFR. Transfection of c-Jun and two activators of c-Jun (LiCl and sodium valproate) increased MTHFR expression, whereas a reported inhibitor of c-Jun (SP600125) and a dominant-negative derivative of c-Jun N-terminal kinase-1 reduced MTHFR activation. We conclude that ER stress increases MTHFR expression and that IRE1 and c-Jun mediate this activation. These findings provide a novel mechanism by which the ER can regulate homeostasis and allude to an important role for MTHFR in cell survival.

A peptide inhibitor of c‐Jun promotes wound healing in a mouse full‐thickness burn model

Significant damage to tissue surrounding burn injuries occurs after the removal of the thermal source. This damage is caused by a combination of both necrotic and apoptotic cell death in the zone of stasis. Preserving the zone of stasis can reduce the wound size and thereby improve wound healing. We tested whether a peptide previously identified to inhibit necrotic and apoptotic cell death in neurons through c-Jun inhibition could enhance wound healing. We first tested the effects of this peptide on a keratinocyte and fibroblast cell line in culture. The peptide promoted proliferation of keratinocytes but had no effect on fibroblast proliferation, while the peptide also inhibited ultraviolet-induced apoptosis of keratinocytes. We finally tested the peptide in vivo, using a mouse model of burn injury. Wounds that were treated with the peptide reepithelialized faster than controls, while cell death surrounding the wound site was markedly reduced 24 hours postinjury, suggesting that the prevention of apoptosis as well as the proliferative effects of this peptide contribute to the wound healing process. Our data implicate c-Jun in multiple processes during wound repair and demonstrate that treatment of burn injuries using inhibitors of c-Jun dimerization at the time of injury can promote wound healing.

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

Activation of c-jun N-terminal kinase (JNK) has been described in islet isolation and engraftment, making JNK a key target in islet transplantation. The objective of this study was to investigate if JNK inhibition with a cell-permeable TAT peptide inhibitor (L-JNKI) protects functional beta cell mass in human islets and affects AKT and its substrates in islet cells. The effect of L-JNKI (10 micromol/l) on islet count, mitochondrial membrane potential, glucose-stimulated insulin release and phosphorylation of both AKT and its substrates, as well as on reversal of diabetes in immunodeficient diabetic Nu/Nu mice was studied. In vitro, L-JNKI reduced the islet loss in culture and protected from cell death caused by acute cytokine exposure. In vivo, treatment of freshly isolated human islets and diabetic Nu/Nu mice recipients of such islets resulted in improved functional beta cell mass. We showed that L-JNKI activates AKT and downregulates glycogen synthase kinase-3 beta (GSK-3B) in human islets exposed to cytokines, while other AKT substrates were unaffected, suggesting that a specific AKT/GSK-3B regulation by L-JNKI may represent one of its mechanisms of cytoprotection. In conclusion, we have demonstrated that targeting JNK in human pancreatic islets results in improved functional beta cell mass and in the regulation of AKT/GSK3B activity.

Reactive oxygen species mediated apoptosis of esophageal cancer cells induced by marine triprenyl toluquinones and toluhydroquinones

Marine invertebrates, algae, and microorganisms are prolific producers of novel secondary metabolites. Some of these secondary metabolites have the potential to be developed as chemotherapeutic agents for the treatment of a wide variety of diseases, including cancer. We describe here the mechanism leading to apoptosis of esophageal cancer cell lines in the presence of triprenylated toluquinones and toluhydroquinones originally isolated from the Arminacean nudibranch Leminda millecra. Triprenylated toluquinone-induced and toluhydroquinone-induced cell death is mediated via apoptosis after a cell cycle block. Molecular events include production of reactive oxygen species (ROS), followed by induction and activation of c-Jun (AP1) via c-Jun-NH2-kinase-mediated and extracellular signal-regulated kinase-mediated pathways. Partial resistance to these compounds could be conferred by the ROS scavengers Trolox and butylated hydroxyanisol, a c-Jun-NH2-kinase inhibitor, and inhibition of c-Jun with a dominant negative mutant (TAM67). Interestingly, the levels of ROS produced varied between compounds, but was proportional to the ability of each compound to kill cells. Because cancer cells are often more susceptible to ROS, these compounds present a plausible lead for new antiesophageal cancer treatments and show the potential of the South African marine environment to provide new chemical entities with potential clinical significance.

C-Jun N-terminal kinase signaling regulates events associated with both health and degeneration in motoneurons

The c-Jun N-terminal kinases (JNKs) are activated by various stimuli and are critical for neuronal development as well as for death following a stressful stimulus. Here, we have evaluated JNK activity in both healthy and dying motoneurons from developing chick embryos and found no apparent difference in overall JNK activity between the conditions, suggesting that this pathway maybe critical in both circumstances. Pharmacological inhibition of JNK in healthy motoneurons supplied with trophic support resulted in decreased mitochondrial membrane potential, neurite outgrowth, and phosphorylation of microtubule-associated protein 1B. On the other hand, in motoneurons deprived of trophic support, inhibition of JNK attenuated caspase activation, and nuclear condensation. We also examined the role of JNK’s downstream substrate c-Jun in mediating these events. While c-Jun expression and phosphorylation were greater in cells supplied with trophic support as compared with those deprived, inhibition of c-Jun had no effect on nuclear condensation in dying cells or neurite outgrowth in healthy cells, suggesting that JNK’s role in these events is independent of c-Jun. Together, our data underscore the dualistic nature of JNK signaling that is critical for both survival and degenerative changes in motoneurons.

Interferon-γ Suppresses Cyclooxygenase-2 Promoter Activity by Inhibiting C-Jun and C/EBPβ Binding

Cyclooxygenase-2 (COX-2) and interferon gamma (IFNgamma) are overexpressed in vascular inflammatory and atherosclerotic lesions. We postulated that IFNgamma suppresses COX-2 expression at the transcriptional level. The effect of IFNgamma on COX-2 expression was evaluated in several types of human cells stimulated with phorbol 12-myristate 13-acetate (PMA), interleukin (IL)-1beta, or tumor necrosis factor (TNF) alpha. IFNgamma concentration-dependently inhibited COX-2 proteins and promoter activities induced by PMA or cytokines in human fibroblasts and monocytic and endothelial cells. PMA and cytokines stimulate binding of C-Jun, C-Fos, CCAAT/enhancer binding protein beta (C/EBPbeta), or NF-kappaB to their respective regulatory elements on COX-2 promoter. IFNgamma blocked C-Jun and C/EBPbeta but not C-Fos or p50 NF-kappaB binding as determined by in vitro binding assays and chromatin immunoprecipitation assay. p300 binding to COX-2 promoter was inhibited by IFNgamma in a manner comparable to C-Jun and C/EBPbeta binding. IFNgamma suppresses proinflammatory mediator-induced COX-2 transcription by selective inhibition of C-Jun and C/EBPbeta DNA binding activity and p300 recruitment in human cells. Because IFNgamma is coexpressed with COX-2 in vascular lesions, it may play a role in controlling COX-2-mediated inflammatory changes.

Turning back the clock: regression of abdominal aortic aneurysms via pharmacotherapy

Abdominal aortic aneurysm (AAA) is a common disease that causes progressive expansion and rupture of the aorta with high mortality. There is a large and unmet need for nonsurgical treatment for AAA. Research has shown that an intricate network of inflammatory cells and interstitial cells contributes to the formation of AAA by producing pro-inflammatory mediators that activate enzymes to degrade the extracellular matrix (ECM) and impair ECM biosynthesis. Pharmacological agents such as statins and angiotensin-converting enzyme inhibitors may promote tissue stabilization in AAA by diminishing pro-inflammatory signaling and normalizing metabolism of the ECM. Our recent experiments in animal models demonstrate that inhibition of c-Jun N terminal kinase (JNK) inhibits multiple pathological processes and causes regression of established AAA. Thus, emerging evidence indicates that pharmacological intervention targeting pro-inflammatory signaling and abnormal ECM metabolism is a promising strategy for treatment of AAA.

Critical role for matrix metalloproteinase‐9 in platelet‐activating factor‐induced experimental tumor metastasis

In this study, the roles of matrix metalloproteinase (MMP)-2 and MMP-9 in platelet-activating factor (PAF)-induced experimental pulmonary metastasis of the murine melanoma cell, B16F10, were investigated. An injection of PAF resulted in increases in mRNA expression, protein levels and the activities of both MMP-2 and MMP-9 in the lungs. The overall expression of MMP-9 was stronger than that of MMP-2. The increased MMP-9 expression was inhibited by both NF-kappaB and AP-1 inhibitors, whereas the increased MMP-2 expression was inhibited by only AP-1 inhibitors. Immunohistochemical analysis revealed that MMP-9 was expressed in bronchial epithelial cells as well as in the walls of blood vessels, whereas MMP-2 expression was observed only in bronchial epithelial cells. PAF significantly enhanced the pulmonary metastasis of B16F10, which was inhibited by both NF-kappaB and c-jun inhibitors. MMP-9 inhibitor, but not that of MMP-2, completely inhibited PAF-induced B16F10 metastasis. These data indicate that MMP-9, the expression of which was regulated by NF-kappaB and AP-1, plays a critical role in PAF-induced enhancement of pulmonary melanoma metastasis.

C-Jun NH2-Terminal Kinase/c-Jun Signaling Promotes Survival and Metastasis of B Lymphocytes Transformed byTheileria

Theileria parasites infect and transform bovine lymphocytes resulting in tumors with metastatic/invasive potential. Importantly, cellular transformation is reversed upon drug-induced parasite death, and the infected lymphocyte dies of apoptosis within 48 hours. Theileria-dependent transformation leads to the constitutive activation of c-Jun NH2-terminal kinase (both JNK1 and JNK2) and permanent induction of activator protein-1. Inactivation of JNK (following transfection of dominant-negative mutants, or treatment with a JNK-specific inhibitor) leads to lymphocyte apoptosis, suggesting an antiapoptotic role for JNK activation in Theileria-induced B cell transformation. Theileria-induced JNK activation also leads to constitutive c-Jun phosphorylation, and inhibition of c-Jun and activator protein-1 transactivation following the expression of a dominant-negative mutant of c-Jun sensitizes Theileria-transformed B cells to apoptosis, but does not significantly affect their proliferation. Thus, JNK activation and c-Jun induction have overlapping, but nonidentical antiapoptotic roles in Theileria-induced B cell transformation. Increased sensitivity to apoptosis may be related to the fact that the expression levels of antiapoptotic proteins such as Mcl-1 and c-IAP are reduced upon c-Jun inhibition. In addition, decreased c-Jun expression correlates with the impaired ability of transfected B cells to degrade synthetic matrix in vitro, and their injection into lymphoid mice gives rise to significantly less and smaller tumors. Combined, these data argue for a role for JNK and c-Jun induction in the survival and metastasis of Theileria-transformed B cells. The similarity between Theileria-transformed B cells with human B lymphomas argues that exploiting the reversible nature of Theileria-induced transformation could throw light on the mechanisms underlying human malignancies.

Neuroprotection by Histone Deacetylase-Related Protein

The expression of histone deacetylase-related protein (HDRP) is reduced in neurons undergoing apoptosis. Forced reduction of HDRP expression in healthy neurons by treatment with antisense oligonucleotides also induces cell death. Likewise, neurons cultured from mice lacking HDRP are more vulnerable to cell death. Adenovirally mediated expression of HDRP prevents neuronal death, showing that HDRP is a neuroprotective protein. Neuroprotection by forced expression of HDRP is not accompanied by activation of the phosphatidylinositol 3-kinase-Akt or Raf-MEK-ERK signaling pathway, and treatment with pharmacological inhibitors of these pathways fails to inhibit the neuroprotection by HDRP. Stimulation of c-Jun phosphorylation and expression, an essential feature of neuronal death, is prevented by HDRP. We found that HDRP associates with c-Jun N-terminal kinase (JNK) and inhibits its activity, thus explaining the inhibition of c-Jun phosphorylation by HDRP. HDRP also interacts with histone deacetylase 1 (HDAC1) and recruits it to the c-Jun gene promoter, resulting in an inhibition of histone H3 acetylation at the c-Jun promoter. Although HDRP lacks intrinsic deacetylase activity, treatment with pharmacological inhibitors of histone deacetylases induces apoptosis even in the presence of ectopically expressed HDRP, underscoring the importance of c-Jun promoter deacetylation by HDRP-HDAC1 in HDRP-mediated neuroprotection. Our results suggest that neuroprotection by HDRP is mediated by the inhibition of c-Jun through its interaction with JNK and HDAC1.

The Janus role of c-Jun: Cell death versus survival and regeneration of neonatal sympathetic and sensory neurons

We investigated the functional outcome of c-Jun activation in sympathetic and sensory neurons of neonatal rat superior cervical ganglion (SCG) and dorsal root ganglion (DRG), respectively. Distinctly different roles of c-Jun activation have been suggested for these two types of neurons. In dissociated sympathetic neurons, c-Jun has been demonstrated to promote apoptosis, whereas in sensory neurons it stimulates axonal outgrowth. In organ-cultured ganglia, we found that c-Jun was activated within 24 h of explantation in both types of neurons, and that the JNK inhibitor SP600125 could mitigate this response. In both types of neurons, c-Jun activation was also reduced by NGF treatment. Inhibition of c-Jun activation did not affect the viability of sympathetic neurons, whereas the number of apoptotic sensory neurons increased. Furthermore, inhibition of c-Jun reduced axonal outgrowth from both SCG and DRG. Thus, in organ culture, c-Jun activation may be required for axonal outgrowth and, at least in sensory neurons, it promotes survival. The role of ATF3, a neuronal marker of injury and a c-Jun dimerization partner, was also examined. We found an ATF3 induction in both SCG and DRG neurons, a response, which was reduced by JNK inhibition. The reduction of ATF3 upon JNK inhibition was much larger in DRG than in SCG, a result which might account for the higher number of apoptotic neurons in JNK inhibitor exposed DRG. Taken together, and contrary to our expectations, neonatal sympathetic and sensory neurons seem to respond to axonal injury similarly with respect to c-Jun activation, and in no case was this activation pro-apoptotic.

DNA Topoisomerase I Is a Cofactor for c-Jun in the Regulation of Epidermal Growth Factor Receptor Expression and Cancer Cell Proliferation

DNA topoisomerase I (Topo I) is a molecular target for the anticancer agent topotecan in the treatment of small cell lung cancer and ovarian carcinomas. However, the molecular mechanisms by which topotecan treatment inhibits cancer cell proliferation are unclear. We describe here the identification of Topo I as a novel endogenous interaction partner for transcription factor c-Jun. Reciprocal coimmunoprecipitation analysis showed that Topo I and c-Jun interact in transformed human cells in a manner that is dependent on JNK activity. c-Jun target gene epidermal growth factor receptor (EGFR) was identified as a novel gene whose expression was specifically inhibited by topotecan. Moreover, Topo I overexpression supported c-Jun-mediated reporter gene activation and both genetic and chemical inhibition of c-Jun converted cells resistant to topotecan-elicited EGFR downregulation. Topotecan-elicited suppression of proliferation was rescued by exogenously expressed EGFR. Furthermore, we demonstrate the cooperation of the JNK-c-Jun pathway, Topo I, and EGFR in the positive regulation of HT-1080 cell proliferation. Together, these results have identified transcriptional coactivator Topo I as a first endogenous cofactor for c-Jun in the regulation of cell proliferation. In addition, the results of the present study strongly suggest that inhibition of EGFR expression is a novel mechanism by which topotecan inhibits cell proliferation in cancer therapy.

Site-dependent inhibition of neuronal c-jun in the brainstem elicited by imidazoline I 1 receptor activation: Role in rilmenidine-evoked hypotension

Clonidine (a mixed α 2-adrenoceptor and imidazoline I 1 receptor agonist)-evoked hypotension was associated with dissimilar reductions in c-jun gene expression in the rostral ventrolateral medulla (RVLM) and the nucleus tractus solitarius (NTS) in normotensive rats. In the present study, we investigated the relative contribution of the α 2-adrenoceptor vs. the imidazoline I 1 receptor to the reduction in c-jun gene expression in these two brainstem areas. In conscious spontaneously hypertensive rats (SHRs), equihypotensive doses of three centrally acting hypotensive drugs with different selectivity for the two receptors were administered intracisternally (4 μl) to limit their actions to the brain. As a control, a similar hypotensive response was elicited by i.v. hydralazine. Clonidine (0.5 μg), or α-methylnorepinephrine (α-MNE, 4 μg), a highly selective α 2-adrenoceptor agonist, similarly reduced c-jun mRNA expression in the NTS and rostral ventrolateral medulla. In contrast, a similar hypotensive response (−37 ± 3.5 mm Hg) caused by the selective imidazoline I 1 receptor agonist rilmenidine (25 μg) was associated with reduction in c-jun mRNA expression in the rostral ventrolateral medulla, but not in the NTS. Further, intra-rostral ventrolateral medulla rilmenidine (40 nmol) reduced c-Jun protein expression in rostral ventrolateral medulla and blood pressure and both responses were antagonized by selective imidazoline I 1 receptor (efaroxan, 4 nmol), but not α 2-adrenoceptor (SK&F 86466, 10 nmol) blockade. These results suggest: (1) the c-jun containing neurons in the brainstem are involved in the centrally mediated hypotension elicited by centrally acting antihypertensive agents, and (2) the α 2-adrenoceptor modulates c-jun gene expression in the NTS and rostral ventrolateral medulla implicated in centrally mediated hypotension, and (3) the imidazoline I 1 receptor mediated inhibition of c-jun gene expression in the rostral ventrolateral medulla, but not in the NTS, contributes to the centrally mediated hypotension by the second generation drugs.