Downregulation Of c-Jun Expression Research Articles

Regulation of FGF2-induced proliferation by inhibitory GPCR in normal pituitary cells.

Intracellular communication is essential for the maintenance of the anterior pituitary gland plasticity. The aim of this study was to evaluate whether GPCR-Gαi modulates basic fibroblast growth factor (FGF2)-induced proliferative activity in normal pituitary cell populations. Anterior pituitary primary cell cultures from Wistar female rats were treated with FGF2 (10ng/mL) or somatostatin analog (SSTa, 100nM) alone or co-incubated with or without the inhibitors of GPCR-Gαi, pertussis toxin (PTX, 500nM), MEK inhibitor (U0126, 100µM) or PI3K inhibitor (LY 294002, 10 μM). FGF2 increased and SSTa decreased the lactotroph and somatotroph BrdU uptak2e (p<0.05) whereas the FGF2-induced S-phase entry was prevented by SSTa co-incubation in both cell types, with these effects being reverted by PTX, U0126 or LY294002 pre-incubation. The inhibition of lactotroph and somatotroph mitosis was associated with a downregulation of c-Jun expression, a decrease of phosphorylated (p) ERK and pAKT. Furthermore, SSTa was observed to inhibit the S-phase entry induced by FGF2, resulting in a further increase in the number of cells in the G1 phase and a concomitant reduction in the number of cells in the S phases (p< 0.05), effects related to a decrease of cyclin D1 expression and an increase in the expression of the cell cycle inhibitors p27 and p21. In summary, the GPCR-Gαi activated by SSTa blocked the pro-proliferative effect of FGF2 in normal pituitary cells via a MEK-dependent mechanism, which acts as a mediator of both anti and pro-mitogenic signals, that may regulate the principal effectors of the G1 to S-phase transition.

Stimulation of Fas/FasL-mediated apoptosis by luteolin through enhancement of histone H3 acetylation and c-Jun activation in HL-60 leukemia cells.

Luteolin (3',4',5,7-tetrahydroxyflavone), which exists in fruits, vegetables, and medicinal herbs, is used in Chinese traditional medicine for treating various diseases, such as hypertension, inflammatory disorders, and cancer. However, the gene-regulatory role of luteolin in cancer prevention and therapy has not been clarified. Herein, we demonstrated that treatment with luteolin resulted in a significant decrease in the viability of human leukemia cells. In the present study, by evaluating fragmentation of DNA and poly (ADP-ribose) polymerase (PARP), we found that luteolin was able to induce PARP cleavage and nuclear fragmentation as well as an increase in the sub-G0 /G1 fraction. In addition, luteolin also induced Fas and Fas ligand (FasL) expressions and subsequent activation of caspases-8 and -3, which can trigger the extrinsic apoptosis pathway, while knocking down Fas-associated protein with death domain (FADD) prevented luteolin-induced PARP cleavage. Immunoblot and chromatin immunoprecipitation (ChIP) analyses revealed that luteolin increased acetylation of histone H3, which is involved in the upregulation of Fas and FasL. Moreover, both the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways are involved in luteolin-induced histone H3 acetylation. Finally, luteolin also activated the c-Jun signaling pathway, which contributes to FasL, but not Fas, gene expression and downregulation of c-Jun expression by small interfering RNA transfection which resulted in a significant decrease in luteolin-induced PARP cleavage. Thus, our results demonstrate that luteolin induced apoptosis of HL-60 cells, and this was associated with c-Jun activation and histone H3 acetylation-mediated Fas/FasL expressions.

A co-delivery nanosystem of chemotherapeutics and DNAzyme overcomes cancer drug resistance and metastasis

Multidrug resistance (MDR) constitutes a major problem in the management of cancer and cancer metastasized from primary-source tumor causes cancer-related deaths. Our new approach is the co-delivery of chemotherapy drugs with a transcription-factor-targeting genetic agent to simultaneously inhibit the growth and metastasis of cancer cells. C-Jun is a transcription factor that regulates multidrug resistance-associated protein 1 (MRP1) pump efflux transcription and tumor metastasis. In this work, we reported that mesoporous silica nanoparticles (MSNs) can be functionalized to co-deliver doxorubicin (Dox) and DNAzyme (Dz) to increase cancer cell killing in an additive fashion. The MSNs were sequentially conjugated with Dox into the MSNs’ nanochannels and Dz onto the MSNs’ outermost surface to target c-Jun as the Dox@MSN-Dz co-delivery system. The Dox-resistant PC-3 cells treated with Dox@MSN-Dz efficiently enhanced the intracellular Dox concentration due to the abrogation of Dox-induced MRP1 expression through the downregulation of c-Jun expression by Dz. Additionally, significant reductions in invasion and migration related to metastasis were also observed in cells treated with Dox@MSN-Dz. Therefore, our results contribute new insight to the treatment of MDR combined metastatic cancer cells, worthwhile for studying its potential for development in clinical translation.

Differential Regulation of c-Jun Protein Plays an Instrumental Role in Chemoresistance of Cancer Cells

The chemotherapeutic drug cisplatin (cis-diamminedichloroplatinum(II) (CDDP)) is widely used in the treatment of human cancers. However, the mechanism underlying intrinsic tumor resistance to CDDP remains elusive. Here, we demonstrate that treatment with CDDP resulted in down-regulation of c-Jun expression via caspase-9-dependent cleavage of c-Jun at Asp-65 and MEKK1-mediated ubiquitylation and degradation of c-Jun in CDDP-sensitive cancer cells. In contrast, activation of JNK2 (but not JNK1) phosphorylated and up-regulated the expression of c-Jun in CDDP-resistant cells. Activated c-Jun bound to the promoter regions of the MDR1 gene and promoted the expression of MDR1. Expression of a cleavage-resistant c-Jun mutant (D65A) suppressed CDDP-induced apoptosis of CDDP-sensitive cells, whereas depletion of JNK2, c-Jun, or MDR1 in CDDP-resistant cancer cells promoted apoptosis upon CDDP treatment. In addition, mammary gland tumors induced by polyomavirus middle T antigen in JNK2(-/-) mice were more sensitive to CDDP compared with those in JNK2(+/+) mice. These findings highlight the instrumental role of c-Jun in the resistance of tumors to treatment with CDDP and indicate that c-Jun is a molecular target for improving cancer therapy.

Pleiotropic Action of AP-1 in v-Src-Transformed Cells

The activation of AP-1 is a hallmark of cell transformation by tyrosine kinases. In this study, we characterize the role of AP-1 proteins in the transformation of chicken embryo fibroblasts (CEF) by v-Src. In normal CEF, the expression of a dominant negative mutant of c-Jun (TAM67) induced senescence. In contrast, three distinct phenotypes were observed when TAM67 was expressed in v-Src-transformed CEF. While senescent cells were also present, the inhibition of AP-1 caused apoptosis in a fraction of the v-Src-transformed cells. In addition, cells containing lipid-rich vesicles accumulated, suggesting that a subpopulation of the v-Src-transformed cells underwent differentiation in response to the inhibition of AP-1. JunD and Fra-2 were the main components of this factor, while c-Jun accounted for a minor fraction of AP-1 in v-Src-transformed CEF. The downregulation of c-Jun expression by short hairpin RNA (shRNA) induced senescence in normal and v-Src-transformed cells. In contrast, a high incidence of apoptosis was caused by the downregulation of JunD, suggesting that it is required for the survival of v-Src-transformed CEF. Levels of the p53 tumor suppressor were elevated under conditions of JunD inhibition. Repression of p53 by shRNA enhanced the survival and anchorage-independent proliferation of v-Src-transformed CEF with JunD/AP-1 inhibition. The inhibition of Fra-2 had no visible phenotype in normal CEF but caused the appearance of lipid-rich vesicles in v-Src-transformed CEF. Therefore, AP-1 facilitated transformation by acting as a survival factor, by inhibiting premature entry into senescence, and by blocking the differentiation of v-Src-transformed CEF.

Interleukin-1 Regulates the Expression of Sphingosine Kinase 1 in Glioblastoma Cells

Chronic inflammation and inflammatory cytokines have recently been implicated in the development and progression of various types of cancer. In the brain, neuroinflammatory cytokines affect the growth and differentiation of both normal and malignant glial cells, with interleukin 1 (IL-1) shown to be secreted by the majority of glioblastoma cells. Recently, elevated levels of sphingosine kinase 1 (SphK1), but not SphK2, were correlated with a shorter survival prognosis for patients with glioblastoma multiforme. SphK1 is a lipid kinase that produces the pro-growth, anti-apoptotic sphingosine 1-phosphate, which can induce invasion of glioblastoma cells. Here, we show that the expression of IL-1 correlates with the expression of SphK1 in glioblastoma cells, and neutralizing anti-IL-1 antibodies inhibit both the growth and invasion of glioblastoma cells. Furthermore, IL-1 up-regulates SphK1 mRNA levels, protein expression, and activity in both primary human astrocytes and various glioblastoma cell lines; however, it does not affect SphK2 expression. The IL-1-induced SphK1 up-regulation can be blocked by the inhibition of JNK, the overexpression of the dominant-negative c-Jun(TAM67), and the down-regulation of c-Jun expression by small interference RNA. Activation of SphK1 expression by IL-1 occurs on the level of transcription and is mediated via a novel AP-1 element located within the first intron of the sphk1 gene. In summary, our results suggest that SphK1 expression is transcriptionally regulated by IL-1 in glioblastoma cells, and this pathway may be important in regulating survival and invasiveness of glioblastoma cells.

QS292. Signal Transduction in Macrophages Through Carcinoembryonic Antigen (CEA) and Its Receptor hnRNP M

Introduction: Secretion of carcinoembryonic antigen (CEA) is an indicator of poor prognosis for colorectal cancer. Circulating CEA is removed by receptor mediated endocytosis by Kupffer cells and this results in an inflammatory response characterized by a cytokine cascade (IL-1, IL-6, IL-10 and TNF-α) that increases the metastatic potential of the cancer cells. The transcription factor c-jun has been shown to be a binding partner with the CEA receptor hnRNP M. There is also activation of the AP-1 transcription factor in Kupffer cells exposed to CEA. We have used differentiated THP-1 macrophages as a model for CEA induced macrophage activation and have demonstrated hnRNP M on the surface of these terminally differentiated cells. Exposure to CEA results in production of cytokines and these influence the ability of CEA producing cancers to implant and grow in the liver. Methods: THP-1 a human monocyte cell line was differentiated overnight to produce a macrophage phenotype with phorbol myristate acetate (PMA) (200ng/ml). Differentiated THP-1 cells were treated with CEA (2μg?ml) and the cells harvested at a number of time points between 0 and 4 hours. Cells were extracted following treatment at these various times, with RIPA buffer containing a cocktail of protease and phosphatase inhibitors. These extracts were run on one or 2-D SDS-PAGE and transferred to membranes for Western blotting. Membranes were probed with antibodies against c-jun, phospho c-jun, JNK 1,2 and 3, p38, phospho-tyrosine and phospho serine/threonine. Results: We showed a significant down regulation of c-jun expression on treatment of the differentiated cells with CEA while an increase in c-jun phosphorylation was observed. JNK-1, and 2 expressions also increase with CEA treatment that is consistent with phosphorylation of c-jun. JNK-3 levels however, decrease following CEA binding. There was also a down regulation of the JNK pathway protein p38. These data point to role for the JNK/c-jun/AP-1 system in signaling by hnRNP-M4. Because hnRNP M4 has a tyrosine phosphorylation motif in its N-terminal domain we have examined the effect of CEA binding on phosphorylation at this locus. Use of western blotting showed increased tyrosine phosphorylation of both isoforms of hnRNP M (M3 and M4). Using 2D electrophoresis we have shown changes in the isoelectric point of the receptor isoforms that would be consistent with phosphorylation. Use of phospho-serine/threonine antibodies showed phosphorylation of 3 proteins distinct from hnRNP M in differentiated THP-1 cells. CEA treatment causes a decline in one band and an increase in phosphorylation of the other two. We are presently attempting to identify these proteins. Conclusions: There is involvement of the c-jun pathway in the initiation of macrophages by CEA to produce cytokines and involves up regulation of JNK-1 and 2. Activation of macrophages by CEA probably involves the transcription factor AP-1. Both isoforms of the receptor appear to be phosphorylated on the consensus tyrosine located in the N-terminal domain. These pathways may be potential targets for intervention to prevent implantation of CEA producing colorectal cancer cells in the liver.

A Comparative Study of Behavioral and immunohistological Changes after Spinal Cord Injury between Young and Adult Rats

Purpose: The aim of this study was to compare the behavioral and immunohistochemical changes in a spinal cord injury (SCI) between young and adult rats and to clarify the differences in the mechanism underlying the changes in a SCI between young and adult rats. Materials and Methods: A total of 25 young and 25 adult male Sprague-Dawley rats (5 weeks and 16 weeks old) were used. The rats were anesthetized with pentobarbital and laminectomies were carried out at the level of the 11th and 12th thoracic vertebra. Using a modified New York University Impactor, a SCI was induced by dropping a 10 gm weight at a height of 20 mm. The bladders were emptied manually twice a day to prevent urinary problems. The animals that received no surgery were used as the normal controls. Behavior tests were performed using the Basso-Beatti-Bresnahan (BBB) scoring system, 1 and 7 days after the weight drop injury. The difference in the BBB score between the young and adult rats were analyzed by a paired t-test with a p value Results: The hind limbs of young rats were paralyzed 1 day after surgery, but had recovered partially 7 days after surgery. However, the adult rats remained in the paralyzed status 7 days after surgery. The c-Jun expression level increased in the gray matter up to 7 days after the weight-drop injury in adult rats. The c-Jun expression level increased significantly in the gray matter 1 day after the injury in the young rats. However, the c-Jun expression level decreased significantly in the gray matter 7 days after the injury in the young rats compared with that of the 1 day post-injury. The GFAP expression level in the gray matter increased 1 day after the weigh-drop injury in the adult rats. However, it decreased in the necrotic region 7 days after the injury in the adult rats. GFAP expression in the gray matter increased gradually up to 7 days after the injury in the young rats. Conclusion: These results suggest that lack of recovery from a SCI in adult rats may be related to the continuous upregulation of c-Jun expression and/or the downregulation of GFAP after the weight-drop injury. In contrast, the upregulation of GFAP expression and/or the downregulation of c-Jun expression in the spinal cord might be related to a partial recovery in young rats after a weight-drop injury.

Downregulation of c-Jun expression by transcription factor C/EBPalpha is critical for granulocytic lineage commitment.

The transcription factor C/EBPalpha is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and C/EBPalpha can block 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation of bipotential myeloid cells. In C/EBPalpha knockout mice, no mature granulocytes are present. A dramatic increase of c-Jun mRNA in C/EBPalpha knockout mouse fetal liver was observed. c-Jun, a component of the AP-1 transcription factor complex and a coactivator of the transcription factor PU.1, is important for monocytic differentiation. Here we report that C/EBPalpha downregulates c-Jun expression to drive granulocytic differentiation. An ectopic increase of C/EBPalpha expression decreases the c-Jun mRNA level, and the human c-Jun promoter activity is downregulated eightfold in the presence of C/EBPalpha. C/EBPalpha and c-Jun interact through their leucine zipper domains, and this interaction prevents c-Jun from binding to DNA. This results in downregulation of c-Jun's capacity to autoregulate its own promoter through the proximal AP-1 site. Overexpression of c-Jun prevents C/EBPalpha-induced granulocytic differentiation. Thus, we propose a model in which C/EBPalpha needs to downregulate c-Jun expression and transactivation capacity for promoting granulocytic differentiation.

Regulation of c-jun/AP-1 expression in rat L6 myoblasts.

Myogenic differentiation of skeletal myoblasts in culture is triggered by withdrawal of serum mitogens. Expression of one of the serum-inducible, immediate early genes, the protooncogene c-jun, is maintained under low-serum conditions during myogenic differentiation of L6 myoblasts. In this report we have used agents that modulate protein kinases and Ca2+ levels to investigate how the expression of c-jun and myogenin mRNA and also the activator protein 1 (AP-1) DNA-binding activity are regulated in differentiating L6 cells. Our results show that expression of c-jun and myogenin are regulated independent of each other. Furthermore, down regulation of c-jun expression does not cause an increase in myogenin expression, suggesting that c-jun does not suppress myogenin expression in these cells. Electrophoretic mobility shift and ultraviolet cross-linking analyses revealed that the AP-1 complexes of proliferating myoblasts and differentiating myotubes are formed of similar set of polypeptides, and the AP-1 binding activity is probably modulated by posttranslational modifications in differentiating L6 cells.