C-jun mRNA Levels Research Articles

Expression of RAS-like family members, c-jun and c-myc mRNA levels in neoplastic hemocytes of soft-shell clams Mya arenaria using microsphere-based 8-plex branched DNA assay

The molecular mechanisms by which disseminated neoplasia (DN) is developed in soft shell clams Mya arenaria remain largely unknown. This study aims at quantifying Rho-like GTPase, RAS-Rho, RAS-related C3 botulinum (RAS C3), c-jun as well as c-myc transcript levels in clams sampled at North River (Charlottetown, Prince Edward Island, Canada). The transcripts were quantified using multiplex gene analysis (Quantigene® 2 Plex, Affymetrix) in 3 groups of clams: (1) Group C (healthy clams considered as control) with a low percentage of tetraploid hemocytes (<10%); (2) Group D (disease in development): individuals presenting a percentage of tetraploid cells ranging between 10% and 50%; (3) Group E (established disease): clams with a high percentage of tetraploid hemocytes (>50%). Data showed a down-regulation of Rho-like GTPase, Rho-like subfamily, RAS C3, c-jun and an up-regulation of c-myc gene expression. It is believed that a deregulation of the expression of these genes could partly unravel the molecular mechanisms involved in the development of DN in soft shell clams Mya arenaria. Further investigations should be pursued to determine the role of these gene products in clams' hemocytes.

Auto-activation of c-JUN Gene by Amino Acid Deprivation of Hepatocellular Carcinoma Cells Reveals a Novel c-JUN-mediated Signaling Pathway

Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. This study demonstrates that in hepatocellular carcinoma cells, expression of c-JUN, JUN-B, c-FOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, c-JUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in c-JUN expression was greater in transformed cells compared with nontransformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of c-JUN transcription was ATF4-independent. The increased expression of c-JUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of c-JUN-ATF2-activated heterodimers was increased after AA limitation, and c-JUN or ATF2 knockdown suppressed the induction of c-JUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing c-JUN protein by JNK and subsequent auto-activation of the c-JUN gene by recruitment of c-JUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the c-JUN gene can function as transcriptional amino acid-response elements.

ETS‐1/RhoC signaling regulates the transcription factor c‐Jun in melanoma

Recently, we discovered that the loss of E-cadherin induces c-Jun protein expression, which is a member of the AP-1 transcription factor family and a key player in the processes of cell proliferation and tumor development and also found in elevated levels in melanomas. Notably, the mRNA level of c-Jun was not affected, suggesting that c-Jun is regulated at post-transcriptional level. Here, we present data that suggest that the dynamic cytoskeletal network, linked to E-cadherin, is involved in the regulation of the c-Jun protein and transcriptional activity. In a signaling cascade, the loss of E-cadherin activates the transcriptional regulator ETS-1 and consequently leads to the induction of RhoC expression that stabilizes c-Jun in melanoma. The link between RhoC and c-Jun seems to be indirect via the cytoskeleton. We conclude that the loss of E-cadherin mediated cell-adhesion induces c-Jun protein expression in a multistep process, offering several possibilities for therapeutic intervention.

Activation of the JNK pathway by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.

Distribution and expression analysis of transcription factors in tissues and progenitor cell populations of the goldfish (Carassius auratus L.) in response to growth factors and pathogens

We report on the mRNA levels of a panel of transcription factors in the kidney and spleen tissues, and in the cell populations from the blood, the spleen, and in the sorted kidney progenitor cells. The mRNA levels of cebpα, cjun, cmyb, egr1, gata1, gata2, gata3, lmo2, mafb, pax5, pu.1 and runx1 were assessed in healthy goldfish as well as in fish challenged with two different pathogens, Aeromonas salmonicida A449 or Trypanosoma carassii. Spleen tissue from healthy goldfish showed higher expression of myeloid (cjun), erythroid (gata1) and lymphoid (gata3, pax5) transcription factors, and lower expression of the myeloid transcription factor cebpα when compared to that of kidney. Splenocytes and PBLs had significantly higher mRNA levels of the transcription factors involved in myeloid (pu.1, mafb, cjun, egr1, cebpa), erythroid (gata1, lmo2), and lymphoid pathways (gata3 and pax5) compared to sorted kidney R1 progenitor cells, while R1 progenitor cells had higher mRNA levels of early progenitor transcription factors (runx1 and cmyb). Furthermore, the R1 progenitor cells had higher mRNA levels of the transcription factors involved in early progenitor cells (egr1, gata2) and the lymphoid lineage progenitors (gata3, pax5) compared to those in kidney. The mRNA levels of the transcription factors (gata2, mafb, cjun, gata1, lmo2, gata3, and pax5) in R1 progenitor cells changed during cultivation; they were elevated in day 2 R1 cells and down-regulated by day 6 of cultivation, when compared to those of day 0 R1 cells. Treatment of day 2 R1 progenitor cells with rgCSF-1 resulted in an up-regulation of transcription factors important for myeloid cell development (cjun and egr1). Similarly, rgkitla up-regulated the expressions of myeloid (mafb, egr1 and cebpa) transcription factors. Changes in the expression of transcription factors in the R1 progenitor cells were related to the observed developmental processes of myeloid progenitor cells during cultivation or treatment with recombinant growth factors in vitro. We also observed differential expressions of the transcription factors in R1 progenitor cells following exposure of the goldfish to either prokaryotic (heat-killed A. salmonicida A449) or eukaryotic (T. carassii) pathogens.

STRAP regulates c-Jun ubiquitin-mediated proteolysis and cellular proliferation

STRAP is a ubiquitous WD40 protein that has been implicated in tumorigenesis. Previous studies suggest that STRAP imparts oncogenic characteristics to cells by promoting ERK and pRb phosphorylation. While these findings suggest that STRAP can activate mitogenic signaling pathways, the effects of STRAP on other MAPK pathways have not been investigated. Herein, we report that STRAP regulates the expression of the c-Jun proto-oncogene in mouse embryonic fibroblasts. Loss of STRAP expression results in reduced phospho-c-Jun and total c-Jun but does not significantly reduce the level of two other early response genes, c-Myc and c-Fos. STRAP knockout also decreases expression of the AP-1 target gene, cyclin D1, which is accompanied by a reduction in cell growth. No significant differences in JNK activity or basal c-Jun mRNA levels were observed between wild type and STRAP null fibroblasts. However, proteasomal inhibition markedly increases c-Jun expression in STRAP knockout MEFs and STRAP over-expression decreases the ubiquitylation of c-Jun in 293T cells. Loss of STRAP accelerates c-Jun turnover in fibroblasts and ectopic over-expression of STRAP in STRAP null fibroblasts increases c-Jun expression. Collectively, our findings indicate that STRAP regulates c-Jun stability by decreasing the ubiquitylation and proteosomal degradation of c-Jun.

Post‐transcriptional regulation controlled by E‐cadherin is important for c‐Jun activity in melanoma

A central event in the development of malignant melanoma is the loss of the tumor-suppressor protein E-cadherin. Here, we report that this loss is linked to the activation of the proto-oncogene c-Jun, a key player in tumorigenesis. In vivo, malignant melanomas show strong expression of the c-Jun protein in contrast to melanocytes. Interestingly, c-Jun mRNA levels did not differ in the melanoma cell lines when compared to melanocytes, suggesting that c-Jun could be regulated at the post-transcriptional level. To uncover the link between E-cadherin and c-Jun, we re-expressed E-cadherin in melanoma cells and detected decreased protein expression and activity of c-Jun. Furthermore, c-Jun accumulation is dependent on active E-cadherin-mediated cell-cell adhesion and regulated via the cytoskeleton. Additionally, we determined that, with respect to c-Jun regulation, there are two melanoma subgroups. One subgroup regulates c-Jun expression via the newly discovered E-cadherin-dependent signaling pathway, whereas the other subgroup uses the MAPKinases to regulate its expression. In summary, our data provide novel insights into the tumor-suppressor function of E-cadherin, which contributes to the suppression of c-Jun protein translation and transcriptional activity independent of MAPKinases.

Distinct Effects of Voltage- and Store-dependent Calcium Influx on Stretch-induced Differentiation and Growth in Vascular Smooth Muscle

Stretch of the vascular wall stimulates smooth muscle hypertrophy by activating the MAPK and Rho/Rho kinase (ROK) pathways. We investigated the role of calcium in this response. Stretch-stimulated expression of contractile and cytoskeletal proteins in mouse portal vein was inhibited at mRNA and protein levels by blockade of voltage-dependent Ca(2+) entry (VDCE). In contrast, blockade of store-operated Ca(2+) entry (SOCE) did not affect smooth muscle marker expression but decreased global protein synthesis. Activation of VDCE caused membrane translocation of RhoA followed by phosphorylation of its downstream effectors LIMK-2 and cofilin-2. Stretch-activated cofilin-2 phosphorylation depended on VDCE but not on SOCE. VDCE was associated with increased mRNA expression of myocardin, myocyte enhancer factor (MEF) -2A and -2D, and smooth muscle marker genes, all of which depended on ROK activity. SOCE increased ERK1/2 phosphorylation and c-Fos expression but had no effect on phosphorylation of LIMK-2 and cofilin-2 or on myocardin and MEF2 expression. Knockdown of MEF2A or -2D eliminated the VDCE-induced activation of myocardin expression and increased basal c-Jun and c-Fos mRNA levels. These results indicate that MEF2 mediates VDCE-dependent stimulation of myocardin expression via the Rho/ROK pathway. In addition, SOCE activates the expression of immediate-early genes, known to be regulated by MEF2 via Ca(2+)-dependent phosphorylation of histone deacetylases, but this mode of Ca(2+) entry does not affect the Rho/ROK pathway. Compartmentation of Ca(2+) entry pathways appears as one mechanism whereby extracellular and membrane signals influence smooth muscle phenotype regulation, with MEF2 as a focal point.

Nitric Oxide Is an Essential Mediator for Neuronal Differentiation of Rat Primary Cortical Neuron Cells

Nitric oxide (NO) regulates proliferation, differentiation and survival of neurons. Although NO is reported to involve in NGF-induced differentiation of PC12 cells, the role of NO has not been characterized in primary neuron cells. Therefore, we investigated the role of NO in neuronal differentiation of primary cortical neuron cells. Primary cortical neuron cells were prepared from rat embryos of embryonic day 18 and treated with NMMA (NOS inhibitor) or PTIO (NO scavenger). Neurite outgrowth of neuron cells was counted and the mRNA levels of p21, p27, c-jun and c-myc were measured by RT-PCR. Neurite outgrowth of primary cortical neuron cells was inhibited a little by NOS inhibitor and completely by NO scavenger. The mRNA levels of p21 and p27, differentiation-induced growth arrest genes were increased during differentiation, but they were decreased by NOS inhibitor or NO scavenger. On the other hand, the level of c-jun mRNA was not changed and the level of c-myc mRNA was increased during differentiation differently from previously reported. The levels of these mRNA were reversed in NOS inhibitor- or NO scavenger-treated cells. The level of nNOS protein was not changed but NOS activity was inhibited largely by NOS inhibitor or NO scavenger. These results suggest that NO is an essential mediator for neuronal differentiation of primary cortical neuron cells.

TNF‐α induces matrix metalloproteinase‐9 expression in A549 cells: Role of TNFR1/TRAF2/PKCα‐dependent signaling pathways

Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines, including TNF-alpha and contributes to airway inflammation. However, the mechanisms underlying TNF-alpha-induced MMP-9 expression in human A549 cells remain unclear. Here, we report that TNF-alpha-induced MMP-9 gene expression was mediated through the TNFR1/TRAF2/PKCalpha-dependent signaling pathways in A549 cells, determined by zymographic, RT-PCR, and Western blotting analyses. TNF-alpha-induced MMP-9 expression was reduced by pretreatment with a TNFR Ab. Furthermore, TNF-alpha-induced TNFR1 and TRAF2 complex formation was revealed by immunoprecipitation using an anti-TNFR1 Ab followed by Western blot analysis against an anti-TRAF2 or anti-TNFR1 Ab. In addition, TNF-alpha-induced MMP-9 expression was also reduced by pretreatment with the inhibitor of PKCalpha (Gö6983), c-Src (PP1), EGFR (AG1478), or PI3K (LY294002) or transfection with siRNAs of PKCalpha, Src, EGFR, Akt, p65, p300, and c-Jun. On the other hand, TNF-alpha stimulated the phosphorylation of c-Src, EGFR, Akt, JNK1/2, and c-Jun, which were inhibited by pretreatment with Gö6983. We also showed that TNF-alpha induced Akt translocation and the formation of an Akt/p65/p300 complex. Pretreatment with the inhibitor of JNK1/2 (SP600125) but not the inhibitor of MEK1/2 (U0126), p38 MAPK (SB202190), or PI3K (LY294002), markedly inhibited TNF-alpha-induced c-Jun mRNA levels. Taken together, these data suggest that in A549 cells, TNF-alpha induces MMP-9 expression via the TNFR1/TRAF2/PKCalpha-dependent JNK1/2/c-Jun and c-Src/EGFR/PI3K/Akt pathways.

Dantrolene, a ryanodine receptor 1 inhibitor, mitigates Fusarium mycotoxin nivalenol-caused cytotoxicity in HL60 cells

Effect of glucose on stress-activated protein kinase activity in mesangial cells and diabetic glomeruli.We have reported that hyperglycemia increases c-jun mRNA levels in isolated glomeruli of diabetic rats. The transcriptional activity of c-jun can be modified by phosphorylation of serine residues in the regulatory domain of the protein by stress-activated protein kinases (SAPKs), but the effect of high glucose concentrations on SAPK expression and activity is unknown. Accordingly, we studied p42/44 MAPK, p38 MAPK, and SAPK expression and activity in primary mesangial cells exposed to high glucose concentrations, as well as SAPK expression and activity in glomeruli of normal and streptozotocin-induced diabetic rats.Mesangial cells were incubated in 40 mm glucose for 30 and 60 minutes and 6, 12, 24, and 48 hours, whereas glomeruli of streptozotocin-induced diabetic rats were isolated one day and one and two weeks after the onset of hyperglycemia (blood glucose levels more than 15 mmol/liter), and were compared with age-matched normal rats. Cell lysates were subjected to Western blot analysis of SAPK and phosphorylated SAPK and an in vitro SAPK assay using recombinant c-jun.Western blot analysis revealed that SAPK was expressed, but unphosphorylated, in unstimulated mesangial cells and whole glomerular lysates from normal rats. In accord with these observations, no SAPK activity was detected in lysates from mesangial cells or whole glomeruli from normal rats, although mesangial cell SAPK activity was readily induced in vitro by sorbitol. High glucose concentrations did not increase SAPK activity or lead to detectable phosphorylated SAPK either in vitro or in vivo. In contrast, short-term exposure to 40 mm of glucose activated both p42/44 MAPK and p38 MAPK.We conclude that high glucose concentrations do not activate SAPK in primary cultured mesangial cells or in diabetic glomeruli during the early phase of diabetic renal hypertrophy.

Vitamin C-deficiency stimulates osteoclastogenesis with an increase in RANK expression

The effects of vitamin C (VC) on osteoclastogenesis were studied in vivo using ascorbate-requiring Osteogenic Disorder Shionogi (ODS) rats and in vitro using bone marrow-derived monocyte/macrophage cells (BMMs). The results confirmed previous findings of increases in the number of osteoclasts and in bone resorption at 2 and 3 weeks, but not 1 week, of VC-deficiency in ODS rats. The mRNA and protein levels of receptor activator nuclear factor kappaB (RANK) ligand and osteoprotegerin, and the mRNA level of macrophage-colony stimulating factor (M-CSF) in the proximal tibia of VC-deficient rats did not differ from those in VC-supplemented control rats. However, the mRNA levels of RANK, c-fos and c-jun were significantly increased at as early as 1 week of VC-deficiency. These results suggested that VC-deficiency stimulated osteoclastogenesis by increasing RANK expression. The osteoclastic differentiation of BMMs was suppressed in the presence of VC. The suppressed osteoclastogenesis was associated with decreased levels of RANK, c-fos and c-jun. The pretreatment of BMMs with VC or PD 98059, a specific inhibitor of extracellular signal regulated kinase (ERK)-activating MEK1, decreased the expression of RANK induced by M-CSF. VC inhibited the M-CSF-induced activation of ERK. These results suggested that VC-deficiency increased osteoclastogenesis by increasing RANK expression mediated through the activation of ERK.

Effect of sulfur dioxide on expression of proto‐oncogenes and tumor suppressor genes from rats

Sulfur dioxide (SO(2)) is a ubiquitous air pollutant that is present in low concentrations in the urban air, and in higher concentrations in the working environment. In the present study, male Wistar rats were housed in exposure chambers and treated with 14.00 +/- 1.01, 28.00 +/- 1.77 and 56.00 +/- 3.44 mg m(-3) SO(2) for 6 h/day for 7 days, while control group was exposed to filtered air in the same condition. The mRNA and protein levels of proto-oncogenes (c-fos, c-jun, c-myc, and Ki-ras) and tumor suppressor genes (p53, Rb, and p16) were analyzed in lungs using a real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assay and Western blot analysis. The results showed that mRNA and protein levels of c-fos, c-jun, c-myc, Ki-ras, and p53 in lungs were increased in a dose-dependent manner, while mRNA and protein levels of Rb and p16 were decreased in lungs of rats after SO(2) inhalation. These results lead to a conclusion that SO(2) exposure could activate expressions of proto-oncogenes and suppress expressions of tumor suppressor genes, which might relate to the molecular mechanism of cocarcinogenic properties and potential carcinogenic effects of SO(2). According to previous studies, the results also indicated that promoter genes of apoptosis and tumor suppressor genes could produce apoptotic signals to antagonize the growth signals that arise from oncogenes. Understanding its molecular controls will benefit development of treatments for many diseases.

Abstract 2477: The effect of dissociated glucocorticoids on 12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin inflammation in SENCAR mice

Abstract Glucocorticoids are very effective in preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation when applied to skin together with a carcinogen. Their control of cellular functions is mediated by a well-known transcription factor, glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding and protein-protein interaction between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1), which is independent of DNA binding. We hypothesize that only the transrepression activities of the GR are sufficient to suppress skin tumor development. To test our hypothesis, we have synthesized two glucocorticoids (RU24858 and RU24782) that induce only transrepression activities of the GR. These two compounds are able to bind to the GR with high affinity and repress the AP-1 and NF-κB activity while showing reduced transactivation activities in vitro. Different doses (0.001, 0.01 and 0.1 mg) of RU24858, RU24782, desoxymethasone (DES) and fluocinolone acetonide (FA) or acetone were applied to the dorsal skin of female SENCAR mice 20 min. prior to TPA application (2 μg), two times per week for two weeks. Keratinocytes proliferation rates were estimated by measuring epidermis thickness and BrdU incorporation index. Immunohistochemistry and RT-PCR techniques were also used to evaluate expression level of genes involved in inflammation and cell cycle regulation. All studied compounds did not influence keratinocytes proliferation when applied alone. Application of DES, FA and RU24858 reversed TPA induced hyperplasia, in dose-depended manner, while RU24782 treatment had no effect on epidermis thickness or the BrdU incorporation index in TPA treated skins. All tested compounds were able to decrease c-jun mRNA level both in acetone and TPA-treated skins. DES, FA and RU24858 were also able to decrease elevated by TPA mRNA level of IL-6, COX-2 and iNOS. RU24782 did not influence mRNAs levels of these genes. These findings suggest that RU24782 had no effect on TPA-induced inflammation and hyperplasia in SENCAR mice, while RU24858 was able to reduce all tested inflammation markers, having only slight effect on epidermal hyperplasia. Supported by NIH grants CA 0796065-06 and P30 CA 54174-16S1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2477.

Preventing chemoresistance of human breast cancer cell line, MCF-7 with celecoxib

To investigate the preventive effect of celecoxib, a specific cyclooxygenase-2 (Cox-2) inhibitor, on the development of chemoresistance in breast cancer cell line, MCF-7, and explore the mechanism of the action. Chemoresistance phenotype was established by treating MCF-7 cells with 0.05 μg/ml doxorubicin for 7 days, and then the effect of preventive chemoresistance was investigated by the combination of same dose of doxorubicin with 10 μM celecoxib. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay was used to assess cytostatic efficacy of doxorubicin and 50% inhibiting concentration (IC(50)) of MCF-7 cells. RT-PCR was performed to examine mRNA expression of multidrug resistance gene 1 (MDR1) and its transcription factors c-Jun and NF-κB. Western blotting analysis was performed to detect the expression of protein. Flow cytometry (FCM) was applied to analyze the expression and function of P-glycoprotein (P-gp). Electrophoretic gel mobility shift assay (EMSA) was performed to determine the DNA-binding activity of nuclear transcription factors AP-1 and NF-κB. Compared with sensitive MCF-7 cells, MDR1 and its transcription factors c-Jun and NF-κB were up-regulated at both mRNA level (P < 0.01) and protein level (P < 0.01) by treatment with 0.05 μg/ml doxorubicin for 7 days. After co-incubation with both the same dose of doxorubicin and 10 μM celecoxib for 7 days, both mRNA level and protein level of MDR1, c-Jun and NF-κB up-regulated by doxorubicin were partly reversed (P < 0.01); DNA-binding activity of nuclear transcription factors AP-1 and NF-κB were inhibited; and the function of P-gp was decreased (P < 0.01). When MCF-7 cells were treated with increasing doses of doxorubicin in the presence of 10 μM celecoxib, IC50 value of doxorubicin and doxorubicin plus 10 μM celecoxib was 0.67 ± 0.03 and 0.38 ± 0.04 μg/ml, respectively (P < 0.01). Celecoxib effectively prevents the development of chemoresistance in breast cancer cell line MCF-7 induced by doxorubicin, which was partly involved in inhibiting the expression and DNA-binding activity of nuclear transcription factors AP-1 and NF-κB and downstream expression and function of P-gp. Furthermore, cytostatic efficacy of celecoxib and doxorubicin on MCF-7 cell was synergistic.

Regulation of CMV promoter-driven exogenous gene expression with doxorubicin in genetically modified cells

The regulation of gene expression after the introduction of an exogenous gene is a problematic aspect of gene therapy. The purpose of this study was to use doxorubicin to regulate exogenous gene expression in a vector containing the cytomegalovirus (CMV) promoter. The pQBI25 vector, which encodes the CMV promoter and the cDNA for red-shifted green fluorescent protein (rsGFP), was transfected into a rat skin fibroblast cell line (FR cells). The pEGFP vector, encoding the CMV promoter and enhanced green fluorescent protein (EGFP) cDNA, was transfected into human hepatoma HepG2 cells. FR-pQBI25 cells were then continuously exposed to doxorubicin and methotrexate for 96 and 48 h, respectively; HepG2-pEGFP cells were continuously exposed to doxorubicin for 48 h. The levels of c-fos, c-jun and rsGFP mRNA, as well as the levels of rsGFP protein, in the FR-pQBI25 cells were found to be significantly higher following exposure to doxorubicin. However, the level of rsGFP protein was not changed by exposure to methotrexate. The level of EGFP protein in the HepG2-pEGFP cells was also significantly higher following exposure to doxorubicin. To examine the effect of cessation of doxorubicin exposure, FR-pQBI25 cells that had been exposed to doxorubicin for 48 h were re-plated in fresh medium without doxorubicin for a further 48 h. The increased levels of c-fos, c-jun and rsGFP mRNA and rsGFP protein seen after treatment with doxorubicin had reduced by 48 h after the cessation of exposure to doxorubicin. These findings suggest that CMV-driven exogenous gene expression may be regulated by doxorubicin.

Mechanism of hepatocyte regeneration inhibition in rats with liver fibrosis induced by lipogenic methionine-choline- deficient diet

To obtain the evidence of fibrotic resolution in fatty liver by changing the diet and to clarify the mechanism of hepatocyte proliferation inhibition in rat with fatty liver fibrosis. (1) Nonalcoholic steatohepatitis with advanced fibrosis was induced in rats by giving them a methionine-choline-deficient diet (MCDD) for 10 weeks (group M). A methionine-choline-control diet (MCCD) instead of MCDD was given for the last 2 weeks to the experimental group (group R). (2) Fibrosis and inflammation were determined by tissue staining. The activation of hepatic stellate cells and Kupffer cells were determined by immunostaining, immunoblot or quantitative RT-PCR respectively. (3) Hepatocytic apoptosis and proliferation were determined by TUNEL and BrdU staining respectively. Expressions of IL-6, STAT3, JNK-1, c-Jun, p21, C/EBPalpha, HNF6 and HGF-alpha were evaluated by quantitative RT-PCR and immunoblot to clarify the mechanism of hepatocytic proliferation inhibition. (1) Changing the diet from MCDD to MCCD triggered the reduction of fat in hepatocytes and a decrease in inflammatory response. (2) The regression of fibrosis was accompanied by the disappearance of activated stellate cells and macrophages. (3) Compared with control group (group C), hepatocytic apoptotic number increased significantly in group M (68 +/- 16 vs 40 +/- 8, P < 0.05) and the ratio of hepatocytic proliferation/apoptosis decreased markedly in group M (0.10 +/- 0.03 vs 0.19 +/- 0.03, P < 0.01); compared to group M, hepatocytic apoptotic number decreased significantly in group R (48 +/- 6, P < 0.05) and hepatocytic proliferation number and the ratio of hepatocytic proliferation/apoptosis increased markedly in group R (17.2 +/- 4.4 vs 7.5 +/- 3.0, 0.41 +/- 0.09 vs 0.10 +/- 0.03 respectively, P < 0.01). (4) Compared with group C, the mRNA level of IL-6, JNK-1, c-Jun, C/EBPalpha, p21 and HNF6 mRNA decreased significantly (0.34 +/- 0.18 vs 1.33 +/- 0.44, 0.41 +/- 0.11 vs 0.83 +/- 0.26, 0.19 +/- 0.03 vs 1.53 +/- 1.2, 1.94 +/- 0.64 vs 4.51 +/- 1.15, 0.34 +/- 0.20 vs 1.30 +/- 0.75, 0.47 +/- 0.21 vs 0.92 +/- 0.16 respectively, P < 0.05 or P < 0.01), and protein level of IL-6, STAT3, JNK-1, c-Jun, C/EBPalpha, P21 and HNF6 also decreased significantly in liver fibrotic stage (P < 0.05 or P < 0.01) while only IL-6, JNK-1 and p21 recovered immediately after a changed diet from MCDD to MCCD (P < 0.05 or P < 0.01). Food intake is a very important factor for controlling the fatty status and pathology of liver. Hepatocytic proliferation inhibition is associated with the arrested G(0)-S phasic transition in fatty liver fibrosis and the up-regulated expression of IL-6, JNK-1 and p21. These factors play a very important role in the recovery of fatty liver fibrosis.

Time Course of Expression of “Early” Genes during Long-Term Posttetanic Potentiation in Rat Hippocampal CA1 Field

The expression of egr-1, junB, c-jun, and c-fos genes in rat hippocampal CA1 field was studied by the real-time PCR 30, 60, and 120 min after induction of long-term posttetanic potentiation. The content of egr-1, junB, and c-jun mRNA gradually increased and doubled 120 min after tetanization. The increase in c-jun mRNA level lagged behind the increment of egr-1 and junB. The level of c-fos mRNA increased 30 min after tetanization, returned to the initial level by min 60, and again increased 120 min after induction of long-term posttetanic potentiation.

Differential gene expression in the rat hippocampus during learning of an operant conditioning task

Changes in transcription levels of brain-derived neurotrophic factor (BDNF), cyclic adenosine monophosphate (cAMP) response element binding (CREB), Synapsin I, Ca2+/calmodulin-dependent protein kinase II (CamKII), activity-regulated cytoskeleton-associated protein (Arc), c-jun and c-fos have been associated to several learning paradigms in different brain areas. In this study, we measured mRNA expression in the hippocampus by real time (RT)-PCR mRNA levels of BDNF, CREB, Synapsin I, CamKII, Arc, c-jun and c-fos, during learning and operant conditioning task. Experimental groups were as follows: control (C, the animals never left the bioterium), when the animals reached 50–65% of the expected response (Incompletely Trained, IT), when animals reached 100% of the expected response with a latency time lower than 5 s (Trained, Tr), Box Control of Incompletely Trained (BCIT), animals spent the same time as the IT in the operant conditioning box and Box Control of Trained (BCTr) animals spent the same time as the Tr in the operant conditioning box. All rats were killed at the same time by cervical dislocation 15 min after training and hippocampi were removed and processed. We found increments of mRNA levels of most genes (BDNF, CREB, Synapsin I, Arc, c-jun and c-fos) in IT and Tr groups compared to their box controls, but increments in Tr were smaller compared with IT. These results describe a differential gene expression in the rat hippocampus when the animals are learning and when animals have already learned. Taking together the results presented herein with the known functions of these genes, we propose a link between changes in gene expression in the hippocampus and different degrees of cellular activation and plasticity during learning of an operant conditioning task.

Expression of estrogen receptor and proto-oncogene messenger ribonucleic acids in reproductive tracts of neonatally diethylstilbestrol-exposed female mice with or without post-puberal estrogen administration.

Perinatal treatment of female mice with natural and synthetic estrogens including diethylstilbestrol (DES) results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium. The dynamics of the induction of estrogen receptor (ER), c-jun, c-fos and c-myc mRNAs by 17 beta-estradiol (E2) was examined in the uterus and vagina of neonatally DES-exposed and -unexposed ovariectomized adult mice. In the uterus of neonatally DES-unexposed ovariectomized mice, the expression of ER mRNA increased within 1 h after E2 administration and declined by 12 h thereafter. ER mRNA in the vagina decreased within 1 h after the stimulation and recovered by 12 h thereafter. In the uterus, c-jun and c-fos mRNAs increased in concentration within 1 h after E2 administration, showing a peak 3 h after the stimulation; they decreased with time thereafter. In the vagina, the concentration of c-jun and c-fos mRNAs increased rapidly, reaching a peak within 1 h after the stimulation. However, the expression of c-myc in uterus and vagina was not changed by postpuberal E2. These results suggest that estrogen regulation of ER and proto-oncogene mRNAs in the vagina differs from those in the uterus. In the neonatally DES-exposed ovariectomized adult mice, uterine ER mRNA expression levels were significantly higher than in the unexposed ovariectomized controls; however, vaginal levels were drastically lower than in the controls. Expression of c-jun and c-fos mRNAs was greater in both the uterus (3- and 6-fold, respectively) and the vagina (18- and 4-fold) of neonatally DES-exposed mice than in controls. The ER mRNA and the increased levels of c-fos and c-jun mRNAs in both uterus and vagina of neonatally DES-exposed ovariectomized mice were not further altered by post-puberal E2 and may be related to ovary-independent persistent changes in the genital tract.