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Anthrax toxin receptor 1 (ANTXR1) is a transmembrane protein with an extracellular domain which is deeply associated with the process of bone formation and plays an important role in angiogenesis. However, there have been no reports investigating the effects of ANTXR1 on bone metabolism mediated by the two types of bone cells, osteoclasts, and osteoblasts. The aim of this study is to reveal the role of ANTXR1 in the differentiation and function of osteoclasts and osteoblasts. We found that ANTXR1 positively regulated the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and bone resorption with no effects on osteoblast differentiation by performing gain- and loss-of-function studies. During ANTXR1-mediated regulation of osteoclastogenesis, phosphorylation of early signal transducers such as c-Jun N-terminal kinase (JNK), Akt, inhibitor of kappa B (IκB), and phospholipase C gamma 2 (PLCγ2) was affected, which in turn altered the mRNA and protein levels of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). In addition, genetic manipulation of ANTXR1 in bone marrow macrophages (BMMs) modulated the capillary-like tube formation in HUVECs via secretion of two angiogenic factors, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor-A (VEGF-A). These results elucidated the importance of ANTXR1 in osteoclast differentiation and functional activity, as well as, osteoclast-mediated angiogenesis of endothelial cells. Taken together, we propose that ANTXR1 might be a promising candidate for gene therapy for bone metabolic diseases and further, might potentially serve as an important biomarker in the field of bone metastasis associated with vascularization.

How the expression of immediate early genes (IEGs) is controlled in response to neurotransmissions is unknown. Using cultured rat cortical cells, we investigated the expression of IEGs regulated by Ca(2+) and/or cAMP signals. The expression of c-fos was transiently induced by treatment of cells with high potassium (high K(+)), which evoked depolarization, or forskolin, an adenylate cyclase activator. c-fos expression was persistently and synergistically induced by simultaneous treatment with high K(+) and forskolin via cAMP-response element (CRE). Microarray analysis indicated the expression profiles of IEGs caused by depolarization in the presence or absence of forskolin. When a novel index was included to investigate the profile of IEGs, we found that high K(+)-induced expression of IEGs was stimulatory or negatively changed in the presence of forskolin, suggesting distinct convergent effects of Ca(2+) and cAMP signals on the expression of IEGs.

We aimed at elucidating the molecular basis of c-fos promoter activation in vascular endothelial cells (ECs) in response to shear stress, with emphases on Rho family GTPases (Rho, Cdc42, and Rac) and intracellular calcium. Dominant-negative and constitutively activated mutants of these GTPases were used to block the action of upstream signals and to activate the downstream pathways, respectively. The role of intracellular calcium was assessed with intracellular calcium chelators. Only Rho, but not Cdc42 or Rac, is involved in the shear stress induction of c-fos. This Rho-mediated shear-induction of c-fos is dependent on intracellular calcium, but not on the Rho effector p160ROCK or actin filaments. While the inhibition of p160ROCK and its ensuing disruption of actin filaments decreased the basal c-fos activity in static ECs (no flow), it did not affect the shear-inductive effect. The calcium chelator BAPTA-AM inhibits the shear-induction, as well as the static level, of c-fos activity.

Our previous work has shown that troglitazone (an antidiabetic, thiazolidione drug and a synthetic ligand for peroxisome proliferator-activated receptor gamma, PPARgamma) stimulated basal level of intercellular adhesion molecule-1 (ICAM-1) protein expression in the absence of cytokine stimulation in human vascular endothelial cells. In this study, we examine the molecular mechanism of troglitazone on the basal and TNFalpha-induced ICAM-1 gene expression. Activation of transcription factors, NF-kappaB and AP-1 proteins, known to regulate ICAM-1 gene expression upon external stimulators, was examined. In human vascular endothelial cells (ECV304 cells), troglitazone inhibited TNFalpha-induced ICAM-1 gene expression by suppressing NF-kappaB/DNA binding activity, NF-kappaB transcriptional responses, c-Fos mRNA and protein levels via a ligand-dependent, PPARgamma-activated manner. In contrast, both troglitazone (at 10 microM) and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2), at 15 microM), a natural ligand for PPARgamma, induce c-Jun phosphorylation by activation of c-Jun N-terminal kinase (JNK) through a posttranslational regulation of c-Jun activity, therefore increasing AP-1/DNA binding activity and transcriptional responses as results of increasing basal ICAM-1 gene expression. These findings suggest dual function of troglitazone in the modulation of both basal and stimulated ICAM-1gene expression in human vascular endothelial cells.

Steel factor (SLF) plus GM-CSF induces proliferative synergy in factor-dependent cell line MO7e and hematopoietic progenitor cells. We previously reported ERK1/2 involvement in this synergy, but its downstream signaling molecules are not defined. Here, we investigated activation of the 90-kDa ribosomal S6 kinase (RSK) proteins by measuring the phosphorylation status and in vitro kinase activity in MO7e cells. Both GM-CSF and SLF induced activation of RSK, and the combined stimulation with these two cytokines induced synergistic and persistent activation of RSK. RSK activity was reduced by PI3 kinase inhibitor LY294002 or MEK1 inhibitor PD98059, suggesting that the ERK as well as the PI3 kinase pathways are involved in regulation of RSK activity. Sensitivities of RSK activity to inhibitory drugs correlated well with those of c-fos gene induction. Taken together, synergistic activation of RSK may contribute, at least in part, to the synergistic induction of c-fos after combined stimulation with GM-CSF plus SLF.

Human chymase produces a novel endothelin-1 with 31 amino-acid length {ET-1(1–31)}, which is longer than conventional ET-1, {ET-1(1–21)}. The aim of our study was to investigate the role of ET-1(1–31) on porcine coronary vascular smooth muscle cell (VSMC). Although the increase in [Ca 2+ ] i by ET-1(1–31) was 10 times weaker than that of ET-1(1–21), ET-1(1–31) showed equivalent potency in VSMC proliferation, c-fos / c-myc mRNA expression and cell cycle analysis with ET-1(1–21). ET-1(1–31) significantly induced expression of cyclin D1 but not those of cyclin D2 or D3. These effects were specifically inhibited by BQ485, an ET A receptor antagonist, although that of ET-1(1–21) was not specific to BQ485, suggesting different receptor specificity from ET-1(1–21). These results indicate that ET-1(1–31) also can involve a VSMC proliferation process such as atherosclerosis, although it has weaker vasoconstricting potency and different receptor subtypes on VSMC from those of ET-1(1–21).

We have previously shown that interferon gamma (IFNgamma) synergistically increases PDGF-induced DNA synthesis in mesangial cells. To examine the mechanism, we studied its effect on PDGF-induced c-fos gene transcription using a reporter mesangial cell in which firefly luciferase gene is driven by c-fos promoter. IFNgamma significantly enhanced PDGF-induced c-fos transcription. We have shown previously that PDGF-induced c-fos transcription in mesangial cells is mediated by the ternary complex factor Elk-1. Using a GAL-4 DNA binding-domain-Elk-1 transactivation domain fusion protein-based reporter assay we showed that the increased effect of IFNgamma was not mediated by Elk-1 transactivation. Gel mobility shift assay of lysates of mesangial cells treated with a combination of IFNgamma and PDGF using sis-inducible DNA element (SIE) showed increased STAT1alpha-SIE complex formation as compared to the PDGF alone. To investigate the transcriptional consequences of this observation, stable reporter mesangial cells in which luciferase gene is driven by four copies of SIE was used. IFNgamma and PDGF in combination significantly increased SIE-dependent transcription as compared to PDGF or IFNgamma alone. Using an antibody in the gel mobility shift assay we showed that the PDGF-induced SIE-STAT1alpha complex recruited the transcriptional coactivator CBP. However, the STAT1alpha-SIE complex formed in the presence of IFNgamma and PDGF did not contain CBP. Taken together, our data provide the first evidence that the synergistic effect of IFNgamma on PDGF-induced DNA synthesis may be the result of increased c-fos gene transcription via SIE. This effect occurs in the presence of increased activation of STAT1alpha without recruitment of the transcriptional coactivator CBP.

We have investigated the expression of several growth-related genes in the liver after partial hepatectomy in three experimental models: normal, Dexamethasone-pretreated, and hypophysectomized rats. Dexamethasone and hypophysectomy resulted in a delay in the peak of cell replication in 6 and 18 h, respectively, when compared to the normal animals. TGFalpha mRNA expression was shifted together with the DNA synthesis, but the expression of c-myc, c-fos, c-jun, HGF, TGFbeta1, IL1beta did not delay. This result suggests that liver-derived TGFalpha but not the other factors are important in the timing of the proliferative response after partial hepatectomy.

The human multidrug resistance gene MDR1 encodes a membrane-bound protein, referred to as P-glycoprotein, that acts as a pump to extrude toxins from cells. The 3′ untranslated region (3′UTR) of the human MDR1 mRNA is very AU-rich (70%) and contains AU-rich sequences similar to those shown to confer rapid decay on c- myc, c- fos, and lymphokine mRNAs. We tested the ability of the MDR1 3′UTR to act as an mRNA destabilizing element in the human hepatoma cell line HepG2. The MDR1 mRNA has an intermediate half-life of 8 h in HepG2 cells compared to a half-life of 30 min for c- myc mRNA. The MDR1 mRNA half-life was prolonged to >20 h upon treatment with the protein synthesis inhibitor cycloheximide. We constructed expression vectors containing the human β-globin coding region with the 3′UTR from either MDR1 or c- myc. The c- myc 3′UTR increased the decay of the chimeric mRNA, but the MDR1 3′UTR had no effect. We tested the ability of MDR1 3′UTR sequences to compete for interaction with AU-binding proteins in cell extracts; MDR1 RNA probes had a fivefold lower affinity for AU-binding proteins that interact with the c- myc AU-rich 3′UTR. Overall, our data suggest that the MDR1 3′UTR does not behave as an active destabilizing element in HepG2 cells.

Regulators of G-protein signaling (RGS) are negative regulators of G-protein-coupled receptor (GPCR) signaling. Sepsis is a pathophysiological condition that is induced primarily in response to bacterial infection and is associated with decreased responsiveness to a number of vasoactive GPCR agonists. Using a degenerate RT-PCR screen, we report that RGS1 and RGS16 were amplified from the heart and aorta of septic animals. By Northern blot analysis, RGS1 and RGS16 were upregulated, with their respective levels increasing 6- and 50-fold in septic hearts. Using a yeast-based bioassay, both RGS1 and RGS16 were found to be equipotent in their ability to attenuate GPCR signaling. These results suggest that both RGS1 and RGS16 contribute to the sepsis-mediated decrease in GPCR signaling. Elevated levels of some RGSs may also lead to an increase in Gbetagamma-activated signaling pathways in the absence of GPCR agonists. Using a c-fos luciferase reporter gene that is responsive to Gbetagamma-activated signaling pathways, we observed a respective 8- and 7-fold increase in the basal luciferase in serum-deprived transfected mammalian cells overexpressing RGS1 or RGS16. This suggests that RGSs play a role in promoting the sepsis-mediated increases in the activation of intracellular signal transduction pathways.

The mode of cell death induced by 1 μM 5-fluoro-2′-deoxyuridine (FdUrd) changed in a wild-type F28-7 clone of mouse mammary tumor FM3A cells after a six-month culture. In the original stocked F28-7 clone, FdUrd-induced cell death was accompanied by necrosis-like cell swelling and DNA fragmentation to 100–200 kbp. In subclone F28-7-A isolated from F28-7 cells, which had been cultured for six months, apoptotic bodies and nucleosomal DNA-ladder fragments were observed with the treatment. Furthermore, we investigated the differences in FdUrd-induced intracellular signals between these clones. In F28-7 cells, FdUrd induced increases in caspase-3-like activity, and the mRNA levels of the c-jun,c-fosand c-mycgenes, which were greater and earlier than those in F28-7-A cells. Moreover, intracellular acidification occurred in F28-7-A cells treated with FdUrd, though it was not observed in F28-7 cells. These findings suggest that FdUrd-induced cell death occurred through the death program to cell lysis (necrosis) without apoptosis when the induction of these intracellular signals was very high and when intracellular acidification was deficient. Investigation of the differences in the mode of FdUrd-induced cell death between these clones would be important for elucidating the molecular mechanism of pivotal events guiding cells toward either apoptosis or necrosis.

The protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) activated cell death in androgen-sensitive LNCaP cells but not in androgen-independent DU-145 or PC-3 cells, whose growth was significantly decreased by PKC inhibitors staurosporine and H7. All cell lines had similar levels of total PKC activities which, however, differed on their dependency on Ca2+ ions and lipid and were regulated differently by TPA. Furthermore, expression of the immediate early genes c-fos and c-jun was up-regulated by TPA only in LNCaP and DU-145 cells, whereas PC-3 cells failed to express c-fos mRNA. The regulation of the c-myc mRNA by TPA correlated inversely with activation of cell death being down-regulated in LNCaP cells, and slightly increased in the androgen-independent cell lines. These results suggest that the PKC signal transduction pathway functions differently in androgen-sensitive and insensitive prostatic cells.

Binding of Endothelin-1 (ET-1) to its heterotrimeric G protein-coupled receptors stimulates various signaling cascades involving the activation of phospholipase C-β, phospholipase D, protein kinase C (PKC), tyrosine kinases, Ca 2+ /calmodulin-dependent kinase (CaMKs), and Ras, a small molecular weight G-protein, but, the role of Rho GTPase remains unclear. In this project, we examined whether RhoA contributes to the ET-1-induced signaling cascade to c-fos SRE activation in Rat-2 fibroblast cells. Our results demonstrate that Rho activation is critical for the signal transduction of ET-1 to c-fos SRE.

The obese (ob) gene product, leptin, a peptide hormone, which is synthesized in adipocytes, is a satiety factor and is involved in the control of body weight via the regulation of energy homeostasis. Several alternate spliced isoforms (a-e, as well as others) of the leptin receptor (OBR) have been cloned, all of which, except for OBRe (soluble form), contain a single transmembrane domain. They share the same extracellular domain, with homology to the class I cytokine receptor family. The OBRb, which has longest cytoplasmic domain, is expressed in high levels in the hypothalamus and is thought to be the only isoform capable of signal transmission. Herein, we report the mRNA expression of immediate early genes, c-fos, c-jun and jun-B, which are induced by leptin addition, not only in CHO cells expressing the OBRb, but also in cells expressing one of the short form receptors, OBRa.

6-Thioguanine (thioG) was chemically incorporated into 25-base oligodeoxynucleotides encoding the c-fos serum response element (SRE) at positions corresponding to each guanine of the CArG box, which only slightly impaired DNA binding by the Serum Response Factor (SRF). Upon exposure to long wavelength UV light each thioG-containing SRE could be crosslinked to SRF, with efficiencies ranging from1 to 25% of the complex depending on the position of thioG in the SRE and on the UV source used. Crosslinking was strongest to the 3' side of the CArG box, and to the outer rather than the inner CArG box guanines, consistent with hydrogen bonds formed between SRF and the outer guanines in the crystal structure [Pellegrini et al., Nature 376, 490, 1995]. The crosslinked product was found to be chemically unstable. Possible mechanisms of crosslink formation are discussed.

Although stimulation of N-methyl-D-aspartate receptors or voltage-dependent calcium channels induces both the activation of c-fosand brain-derived neurotrophic factor (BDNF) genes, it is not certain how the activation of these genes is related. Using primary cultures of rat hippocampal neurons, we found that exposing the cells to cycloheximide allowed subsequent activation of BDNF mRNA expression, although activation of AP-1 DNA-binding activity resulting from the c-fosinduction was abolished. Super-induction of BDNF gene was also caused by cycloheximide. The estimated half-life of BDNF mRNA was approximately 2.5 hrs, which was almost identical to that of c-fosmRNA. These results indicate that nascent AP-1 is not required for the activation of BDNF gene, leading to the notion that the BDNF gene can be activated by Ca2+signals as an immediate early gene.

The amino acid sequence of the rat 40S ribosomal subunit protein S3a was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed by the determination of the NH2-terminal sequence by Edman degradation. Ribosomal protein S3a has 263 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and the molecular weight is 29,794. The protein designated S3b has the same amino acid sequence as S3a except that it lacks the carboxyl-terminal 12 residues. We are unable to determine whether there are separate genes for S3a and S3b, or whether there is a single gene and alternate splicing of the precursor to yield separate mRNAs for S3a and S3b, or whether there is a single gene and a single mRNA whose translation yields S3a which is converted by proteolysis, either physiological or fortuitous, to S3b. The mRNA for S3a is about 1000 nucleotides in length. Hybridization of cDNA to digests of nuclear DNA suggests that there are 8-13 copies of the S3a gene. Rat ribosomal protein S3a is identical to the product of the rat Fte-1 gene which encodes the V-Fos transformation effector; S3a is also related to the plant protein cyc07, which is encoded by a cell cycle S-phase specific gene.

4-Hydroxynonenal, an aldehyde produced from lipid peroxidation of cellular membranes, inhibits growth and induces differentiation of HL-60 human leukemic cell line. Since it is highly unstable in the culture medium, its effectiveness is increased when added repeatedly to the cell suspension. We have previously demonstrated that HNE inhibits c-myc but not N-ras expression in HL-60 cells. Here we investigate its effect on the expression of c-myb and c-fos, two early genes involved in the induction of myeloid and monocytic differentiation. Moreover, since c-fos is directly correlated with the intracellular level of cAMP, we also analysed the cAMP concentration after aldehyde treatment. HNE significantly inhibits c-myb expression during and after repeated treatments. A single administration of 1 microM HNE decreases c-myb mRNA at 1 hour whereas 10 microM HNE inhibits c-myb expression from 3 to 6 hours after treatment, and then the expression returns to the control level. By contrast, c-fos expression and intracellular cAMP concentration do not show any significant change after HNE treatments.

To isolate seizure-related genes, we applied differential screening technique to the cDNA library which constructed from primary cultured cerebral cortical cells treated with pentylenetetrazol (PTZ). Northern blotting analysis of mRNA levels in the cerebra after systemic administration of PTZ confirmed the results of differential screening procedure: 6 clones showed increased mRNA level and 3 clones showed decreased expression with PTZ. Interestingly, 4 genes which were isolated by this technique were related to intracellular calcium action. They were cytosolic phospholipase A2, 78 kDa glucose regulated protein, SEZ-15 which has an EF hand motif and PTZ-17 that causes calcium current in Xenopus oocyte with PTZ application. These data and our previous results suggest that intracellular calcium may play an important role for seizure-related pathophysiological changes in neuronal cells.

We have recently developed an experimental model whereby mechanical stimulation induces osteogenesis in the caudal vertebrae of rats. We used this model to assess expression of genes induced by mechanical loading. Bulk preparations of mRNA extracted after loading did not-show >2-fold increases in expression of mRNA for matrix proteins or growth factors in Northern blotting analysis. c-jun was undetectable. However, c-fos showed a 4-fold increase in expression within 60 mins of loading, before returning to control levels by 4 hrs. This increase was associated with intense signals in in situ hybridization, not seen in any nonloaded vertebrae, for c-fos over cortical osteocytes: thus osteocytes respond to mechanical loading with c-fos expression so strongly as to be visible even in the bulk RNA preparations. The results represent persuasive evidence for a role for osteocytes, and for c-fos, in the osteogenic response of bone to mechanical stimulation.

A 52 kDa heregulin secreted by estrogen receptor (ER)-negative human breast cancer cells induced rapid growth of ER-positive MCF-7 breast cancer cells with a stimulatory effect observed at 10(-11)M. This heregulin down-regulated the message for ER in MCF-7 cells within 24 hours after stimulation. Similarly the ER protein was down-regulated within 24 to 48 hours after stimulation of cells. However, this down-regulation occurred without activation of the ER, since the progesterone receptor (PR) level of cells stimulated with the 52 kDa heregulin did not increase over the time period measured. As a control, estradiol down-regulated and activated ER as shown by a pronounced increase in PR content of MCF-7 cells. This finding indicates an important role of this heregulin in the down-regulation of ER in estrogen-dependent human breast cancer cells.

Apigenin, a plant flavonoid, induced the reversion of transformed phenotypes of v-H- ras -transformed NIH 3T3 cells at a quite low concentration of 12.5 μM. In the present study, we have examined the components of this Ras-mediated signaling transduction to determine whether they were involved in the apigenin-induced reversion process. Interestingly, the consitutively activated mitogen activated protein kinase (MAPK) in the ras transformant was inhibited significantly and rapidly by 25 μM apigenin within 30 min, and this reduction continued for more than 4 h. Corroborating these observations, expression of the downstream oncogenes c- jun and c- fos was also dramatically reduced during the first 4 h of treatment. We found that the levels of ras protein and mRNA were not affected by 24 h of treatment with apigenin. These findings indicate that apigenin-induced reversion of v-H- ras -transformed NIH 3T3 cells may occur by inhibiting MAPK activity and its downstream oncogenes rather than by affecting the expression of the ras gene.

Angiogenin is a potent angiogenic molecule in the chick chorioallantoic membrane assay and rabbit corneal assay. However, no angiogenic activity has been reported in in vitro system. In this study,we isolated and purified angiogenin from bovine milk, and the biological effect of the bovine angiogenin on bovine endothelial cells was examined in in vitro angiogenesis models. Bovine angiogenin significantly stimulated both cell migration and formation of tube-like structures in the collagen gel by bovine aortic endothelial cells. Angiogenin at 10-100 ng/ml stimulated about 2-fold higher the tube formation when basic fibroblast growth factor (bFGF) at 10 ng/ml stimulated about 3-fold over the control. Tubular morphogenesis stimulated by bFGF or angiogenin was almost completely blocked in the presence of aprotinin, an inhibitor of serine proteases. Angiogenin upregulated both mRNA level and activity of urokinase type plasminogen activator, a key mediator of angiogenesis. Moreover, both bFGF and bovine angiogenin induced a marked increase of c-fos mRNA level at 30 min after stimulation. These novel effects of bovine angiogenin are to be discussed in relation to its structure specificity.

We presently investigated the effects of pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of nuclear factor kappa B (NF-kappa B), on the induction of nitric oxide synthase (iNOS) and manganese superoxide dismutase (MnSOD) mRNAs by IL-1 beta in insulin-producing RIN cells. PDTC decreased by 90% both IL-1 beta-induced increase in medium nitrite accumulation (an indicator of NO production) and induction of iNOS mRNA expression. However, PDTC did not prevent induction of MnSOD mRNA by IL-1 beta. PDTC induced an early (45 min) expression of c-fos mRNA and potentiated IL-1 beta-induced c-fos expression. Our data suggest that NF-kappa B activation is necessary for iNOS, but not for MnSOD, mRNA expression in insulin producing cells.

Basal and stimulated activity of the c-fos promoter is reduced by triiodothyronine (T3) and retinoic acid (RA) in GH1 cells. We examined the influence of these ligands on the activity of receptor constructs containing the AP-1 site, the serum response element (SRE) and the cyclic AMP responsive element (CRE) of the c-fos promoter under control of an heterologous promoter. T3 and RA decreased the response of AP-1 and SRE sequences to phorbol esters, forskolin or serum but they did not reduce basal or forskolin-stimulated activity mediated by the CRE. Therefore, repression of c-fos gene expression by T3 and RA receptors appears to be exerted through transcriptional interference with the SRE and the AP-1 binding site of the promoter.

The transcription factor AP-1 is an important human mediator of the cellular response to serum, growth factors, and phorbol esters such as 12-O-tetradecanoyl-phorbol-13 acetate (TPA). The AP-1 complex consists of distinct protein heterodimers encoded by the proto-oncogene c-fos and c-jun mRNA whose gene expression can be induced by TPA, cyclic AMP and growth factors. Recent findings suggest an involvement of reactive oxygen species in the pathway of TPA and protein kinase C leading to expression of c-fos and c-jun mRNA. To investigate the role of reactive oxygen species we studied the effects of alpha-lipoic acid and dihydrolipoic acid (natural thiol antioxidants) on the expression of c-fos mRNA in human Jurkat T cells. When cells were preincubated with dihydrolipoic acid (0.2 mM) the expression of c-fos mRNA was suppressed at 30 min after stimulation of TPA (0.5 microM) whereas in the case of preincubation of alpha-lipoic acid (0.2 microM), the expression was enhanced at 30 min. These studies support the idea that superoxide anion radical plays a role in the expression of c-fos mRNA.

Lipoprotein lipase (LPL) is known to be an early marker of adipose cell differentiation. Growth hormone (GH) stimulates in preadipose Ob1771 cells the expression of LPL gene and this effect is mediated at least in part by c-fos protooncogene, the expression of which is transiently activated by a protein kinase C-dependent pathway (Barcellini-Couget et al., Endocrinology, 1993, 132: 55-60). Since GH stimulates the formation of diacylglycerol from phosphatidylcholine independently of Ca2+ mobilization, the role of Ca2+ was studied in regard to LPL gene expression stimulated by GH. The results obtained in the presence of Ca2+ ionophores show that a rise in intracellular free Ca2+ abolishes this expression but has no effect on the expression of various adipose-related genes, including the transient expression of c-fos protooncogene. Therefore, the inability of preadipose cells to mobilize Ca2+ in response to GH, at an early stage of the differentiation process, appears as a prerequisite for the maximal expression of LPL.

Using developing rat cerebellum, we examined the correlation between the turnover of phosphoinositide (PI) and c-myc expression. The 32P incorporation into polyphosphoinositides changed remarkably with advancing age. It reached a maximum value on PND 7, and then decreased gradually until PND 14. Immunostaining by anti-PIP2 antibody changed in parallel. The expression of c-myc mRNA was also changed developmentally, showing a peak on PND 7; whereas c-MYC protein expression showed a peak on PND 10. Together, these results suggest that c-myc expression is regulated by PI turnover during the early developing stage of rat cerebellum.

ER mRNA was detected as 6.0 kb band by Northern blot analysis in vascular smooth muscle cells (VSMC) derived from rat aorta. The presence of ER mRNA in VSMC was confirmed by reverse transcriptase-polymerase chain reaction using specific primers for rat ER cDNA. In addition, the immunocytochemistry of ER was performed in VSMC using a monoclonal anti-ER antibody which recognizes DNA-binding domain of ER. The immunoreactivity was distributed in the cytoplasm as well as in the nuclei. Thus, the expression of ER in VSMC was demonstrated at both the protein and the mRNA level. Furthermore, the expression of c-fos mRNA in VSMC was found up-regulated by 17 beta-estradiol treatment within 30 min. The observation that VSMC possess ER and respond to estrogen supports the idea that estrogen may directly influence vascular cell system through the ER.

We used sodium butyrate to modify the differentiation and growth properties of the Caco-2 colon adenocarcinoma cell line and considered c-fos proto-oncogene expression as a potential target. C-fos is induced by butyric acid very rapidly at a post-transcriptional level and is stimulated transcriptionally at later times. This transcriptional induction does not result in an increase in steady-state mRNA levels. We show by transient transfection assays that the ATF-CRE binding site located between -63 and -54 relative to the c-fos transcriptional start site is a target for butyrate-induced fos transcription. Furthermore, gel retardation assays show an increase in CRE binding activity in cells treated with butyrate. These results demonstrate that butyrate can affect specific transcription factors important for cell growth and differentiation at multiple levels of regulation.

Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, induces differentiation in human MCF-7, AU-565, and MB-231 breast tumor cells. In MCF-7 cells, OA elicited within 5 min an increase in the levels of a set of phosphorylated cellular proteins, within hours expression of the early response genes junB, c-jun, and c-fos, and within days manifestation of differentiation. Differentiation was also induced by two related protein phosphatase inhibitors, but not by an inactive OA derivative or by an inhibitor that penetrates epithelial cells poorly. These results indicate that OA and related agents can induce tumor breast cell differentiation, and this induction is correlated with their ability to inhibit PPH 1 and 2A.

Platelet derived growth factor (PDGF) has been shown to induce c-fos and c-myc protooncogenes. In the present study, we investigated the effects of genistein, a tyrosine kinase inhibitor, and NiCl2, a Ca2+ influx blocker, on PDGF-induced Ca2+ transient and on expression of c-fos and c-myc mRNA. In the presence of extracellular Ca2+, PDGF induced elevation of intracellular Ca2+ concentration ([Ca2+]i) and increases in c-fos and c-myc mRNA, as detected by reverse transcription polymerase chain reaction (RT PCR) and Northern hybridization. PDGF-induced [Ca2+]i elevation was composed of an initial transient increase(first component) followed by steady state elevation (second component). Genistein (10,μ M) blocked the 1st, but not the 2nd, component of [Ca2+]i elevation induced by PDGF. NiCl2 (1mM) and removal of extracellular Ca2+ inhibited the 2nd, but not the 1st, component. In the presence of 10 μM genistein and 1 mM NiCl2, PDGF induced c-fos and c-myc mRNA, although the [Ca2+]i elevation could be completely blocked by these two agents. These results indicate that elevation of [Ca2+]i is not a prerequisite condition for PDGF to induce c-fos and/or c-myc mRNA in rat aortic smooth muscle cells in primary culture.

Treatment of human B lymphocytes with an optimal concentration of okadaic acid, an inhibitor of phosphatases 1 and 2A, resulted in the induction of the transcription factor, AP-1 and a marked increase in NF-kappa B levels. In contrast, no effect on the levels of the octamer binding proteins, Oct-1 or Oct-2, were found. Since both AP-1 and NF-kappa B have been reported to be important in the induction of the tumor necrosis factor-alpha (TNF-alpha) gene we examined the effects of okadaic acid on TNF-alpha mRNA levels. Treatment with okadaic acid resulted in a striking increase in TNF-alpha mRNA transcripts within 1 h of stimulation and large amounts of TNF-alpha were released into the culture media. Although okadaic acid provides a potent inductive signal for AP-1 and NF-kappa B it did not induce either B cell proliferation or immunoglobulin secretion.

To clarify the contribution of c-fos DNA to bone formation, the effect of constitutive expression of the c-fos gene in collagen synthesis was examined by introducing c-fos DNA into osteoblastic MC3T3-E1 cells. The [3H] proline incorporation into the collagenase digestible protein(CDP) and the percent collagen synthesis were significantly decreased in the c-fos transfectants which constitutively express c-fos mRNA as compared with control transfectants. Transcription of type 1(alpha 1) collagen gene was also specifically decreased in the c-fos transfectants. This indicates that constitutive expression of c-fos DNA interferes with bone formation by inhibiting collagen synthesis in osteoblasts.

Expression of murine P-glycoprotein (P-gp), encoded by mdr1 or mdri3, confers a multidrug resistance phenotype. Higher expression of c-fos and c-jun has also been demonstrated in multidrug resistant human and murine cells. We detected increased expression of c-fos early in the derivation of two series of murine erythroleukemia sublines selected for resistance to vincristine or adriamycin which eventually overexpress mdr3. We speculate that early expression of c-fos prepares cells for overexpression of other genes, such as mdr3, that contribute to the multidrug resistance phenotype.

Immunohistochemical analysis revealed the presence of a gut-brain peptide, neuromedin U (NMU), in the suprachiasmatic nucleus (SCN), which is the site of the master circadian oscillator. The expression of NMU mRNA exhibited a circadian rhythm, with the peak expression in the SCN occurring at CT4-8h. The two NMU-binding receptors (NMU-R1 and NMU-R2) were also expressed in the SCN, but their phase angles were different. Intracerebroventricular injection (ICV) of NMU induced the expression of Fos protein in the SCN cells and caused a phase-dependent phase shift of the circadian locomotor activity rhythm. The magnitude of the phase shift was dose dependent. This NMU-induced phase shift was of the nonphotic type. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed increases in the expression in the SCN of immediate early genes, such as c-fos, NGFI-A, NGFI-B, and JunB. Furthermore, ICV injection of NMU increased the expression of Per1, but not Per2, in the SCN. These results indicate that NMU may play some important role in the circadian oscillator by exerting an autocrine or paracrine action in the SCN.

Tissue anoxia occurs early in wound healing. This is accompanied by production of lactate followed by increased hyaluronan and CD44 expression, suggesting a cause and effect relationship. Fibroblasts increased hyaluronan and CD44 when lactate was added to cultures. Increased deposition of hyaluronan correlates with greater turnover. In current models of hyaluronan catabolism, it is tethered to cell surfaces by CD44 in caveolin-enriched invaginations. It is cleaved to 20-kDa fragments by Hyal-2 on the plasma membrane, endocytosed, and delivered ultimately to lysosomes, and further digested by Hyal-1. Sequence analyses of promoter regions of genes for CD44, caveolin-1, Hyal-1, and -2 revealed multiple AP-1 and ets-1 response elements. To test their relevance, RNA from lactate-treated fibroblasts was analyzed by reverse transcriptase-polymerase chain reaction. Increased transcripts of c-fos, c-jun, c-ets, Hyal-1, -2, CD44, and caveolin-1 mRNAs were observed. We have thus identified lactate-activated genes important in the wound healing responses. Similar responses facilitating tumor progression, the Warburg effect, may share such mechanisms.

Hic-5 is a member of LIM family proteins with a striking similarity to paxillin and localizes primarily in the focal adhesion. We recently reported that Hic-5 translocated to the nucleus under oxidative stress and was involved in transcriptional regulation. In the present study, we extended these findings to show that transcription of c-fos gene was up-regulated by overexpression of Hic-5. In clonal stable transformants established from human immortalized fibroblasts by transfection of an expression vector of Hic-5, the constitutive level of c-fos mRNA was well correlated with that of Hic-5. In reporter assays using the luciferase gene under control of the human c-fos 5(')-upstream region from -2.2kb to +1, expression of Hic-5, that was engineered to accumulate in the nucleus, stimulated the transcriptional activity of the c-fos enhancer. From experiments using various deletions and point mutations, it was revealed that multiple sequences including GC/Sp1, Ets, and ERE/AP-1 elements found around the -1.3kb region were responsible for the activation by Hic-5. Hic-5 itself did not bind to these elements in a sequence specific manner, but p300 appeared to be involved in the induction of c-fos. These results suggest that Hic-5 participates in the transcriptional regulation of c-fos as a scaffold in transcriptional complexes.

We find that the activity of a 0.4-kb human c-fos gene promoter (-404/+41), which lacks consensus estrogen-responsive elements (EREs), is regulated by estrogen receptor (ER) ligands in MC3T3-E1 osteoblastic cells through ERs in a manner distinct from ERE-mediated regulation. When ERalpha is coexpressed, both estrogens and antiestrogens upregulate promoter activity. When ERbeta is coexpressed, however, three tested antiestrogens affect c-fos promoter activity, with tamoxifen exerting the greatest effect, while estrogens have no such effect. The tamoxifen agonism through ERbeta is antagonized by 17beta-estradiol, while the 17beta-estradiol agonism through ERalpha is canceled by excess-level coexpression of ERbeta. Deletion analysis revealed that the sequence -206/-110 plays a crucial role in the ERbeta-mediated tamoxifen agonism. Interestingly, there is no ERbeta-mediated tamoxifen agonism when nonosteoblastic cells are tested. Taken together, these results suggest that the transcription of the c-fos gene is regulated by ER ligands possibly through non-ERE elements in ligand structure-, cell type-, and ER subtype-dependent manners.

Ghrelin is a novel acylated peptide that functions in the regulation of growth hormone release and energy metabolism. It was isolated from rat stomach as an endogenous ligand for growth hormone secretagogue receptor. Ghrelin is also localized in the arcuate nucleus of rat hypothalamus. Intracerebroventricular (ICV) administration increases food intake and body weight. We examined the effect of ghrelin on gastric acid secretion in urethane-anesthetized rats and found that ICV administration of ghrelin increased gastric acid output in a dose-dependent manner. Vagotomy and administration of atropine abolished the gastric acid secretion induced by ghrelin. ICV administration of ghrelin also induced c-fos expression in the neurons of the nucleus of the solitary tract and the dorsomotor nucleus of the vagus, which are key sites in the central nervous system for regulation of gastric acid secretion. Our results suggest that ghrelin participates in the central regulation of gastric acid secretion by activating the vagus system.

Effects of vector-averaged gravity on tumor necrosis factor (TNF)-alpha-dependent activation of nuclear factor kappa B (NF-kappa B) in human osteoblastic HOS-TE85 cells were investigated by culturing the cells using clinostat rotation (clinorotation). Cell cultures were rotated for 72 h at 40 rpm in a clinostat. At the end of clinorotation, the cells were treated with TNF-alpha for 30 min under stationary conditions. Electrophoretic mobility shift assays revealed that TNF-alpha-dependent activation of NF-kappa B was markedly reduced in the clinorotated cells when compared with the cells in control stationary cultures or after horizontal rotation (motional controls). The NF-kappa B-dependent transactivation was also impaired in the clinorotated cells, as evidenced by a transient transfection assay with a reporter plasmid containing multimerized NF-kappa B sites. Consistent with these findings, the TNF-alpha-dependent induction of endogenous NF-kappa B-responsive genes p105, I kappa B-alpha, and IL-8, was significantly attenuate in clinorotated cells. These results demonstrate that vector-averaged gravity inhibits the responsiveness of osteoblasts to TNF-alpha by repressing NF-kappa B activation.

Exposure of mesangial cells to ionic Cd(2+) induces the proto-oncogene c-fos, while activating both Erk and stress-activated protein kinase (SAPK) MAP kinase pathways. While we have previously used a pharmacological inhibitor of Erk activation to implicate involvement of this pathway in the induction of c-fos by Cd(2+), the consequences of SAPK activation remained unknown. Here we use dominant negative inhibitors of the SAPK kinases, SEK1 and MKK7, to show that Cd(2+) activates SAPK through MKK7, but that partial inhibition of SAPK alone is insufficient to significantly affect the magnitude of the Cd(2+)-dependent increase in c-fos mRNA. However, inhibition of Erk and SAPK pathways together abrogates the increase, suggesting that these pathways act in concert in the induction of c-fos by this toxic metal.

The effects of 20-min exposure to low-intensity, pulsed ultrasound were investigated in ST2 cells of bone marrow stromal origin. They responded to ultrasound with elevated levels of IGF mRNAs, osteocalcin, and bone sialoprotein mRNAs. The upregulated expression of these messages appeared in a biphasic manner, with the first peak resistant to the protein synthesis inhibitor cycloheximide, and a second peak that was eliminated by NS398, an inhibitor of the inducive prostaglandin G/H synthase (cyclooxygenase-2). A cumulative effect of mechanical loading called the memory effect, which has been observed in vivo, can be explained from such a biphasic anabolic reaction mediated by prostaglandins. The upregulation of IGF or osteocalcin mRNAs can be observed even at 24 h after the initiation of the 20-min exposure to ultrasound. Our results suggest that this low-intensity, pulsed ultrasound, which has been clinically used to accelerate the healing processes of fractured bone, induces a direct anabolic reaction of osteogenic cells, leading to bone matrix formation.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (20) and late passage (65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.

The effect of transient transfection with expression vector of c-Fos on the expression of 12-lipoxygenase in human epidermoid carcinoma A431 cells was studied. Overexpression of c-Fos increased the expression of 12-lipoxygenase mRNA and enzyme activity, and also activated the promoter activity of 12-lipoxygenase gene in a dose-dependent manner. Co-transfection with c-Fos and c-Jun expression vectors in cells synergistically increased the promoter activity of 12-lipoxygenase. With the aid of additional 5′-deletion and site-directed mutagenesis, the downstream and middle Sp1 sites residing at −123 to −114 bp and −158 to −150 bp were found to be critical for the c-Fos response of activating the transcription of human 12-lipoxygenase gene. Furthermore, the specific role of Sp1 in c-Fos response was confirmed by using the reporter plasmid driven by SV40 early promoter. These results indicate that the requirement of Sp1-binding sites in the promoter region of 12-lipoxygenase gene for c-Fos response is similar to that previously observed in EGF response.

Although the neuron-restrictive silencer element (NRSE/Regard) has been shown to function as a negative-acting DNA regulatory element to prevent the expression of neuron-specific genes in non-neuronal cells, little is known about its silencing effect on transcription in primary glial cells nor its effect on transcriptional activation in primary neurons. By DNA transfection in primary cultures of rat cortical neuronal or glial cells, we investigated the effect of NRSE on transcription mediated by the BDNF promoter I or c-fos promoter to which NRSE sequences derived from the SCG10 gene were linked. Transfection of plasmid DNAs to NIH3T3 fibroblasts resulted in a marked repressive effect of NRSE on BDNF promoter I- or c-fos promoter-mediated transcription. In primary neuronal cells, however, NRSE did not repress the basal promoter activities of BDNF and c-fos genes and allowed the transcriptional activation of these genes induced by membrane depolarization although NRSE slightly reduced the magnitude of BDNF promoter I activation. In contrast to neuronal cells, a marked repression of basal promoter activities of both genes was detected in primary glial culture and a two base pair-mutation of NRSE partially recovered the repression. These results indicate that NRSE negatively acts on its linked promoters in primary glial cells and does not interfere an activation of linked promoters in neuronal cells.

AF-6 contains two putative Ras-associating domains (RA domains) which are seen in several Ras effectors such as RalGDS and RIN1. We previously showed that an AF-6 fragment containing the amino-terminal (N-terminal) RA domain directly binds to activated Ras and ZO-1 in vitro. In this study, we showed that a single amino acid mutation in the N-terminal RA domain of AF-6 abolished the interaction of AF-6 with activated Ras and that the sites of this critical amino acid residue were similar to those for Raf-1 and RalGDS. The overexpression of the N-terminal RA domain of AF-6 inhibited the Ras-dependent c- fos promoter/enhancer stimulation in NIH3T3 cells. Endogenous AF-6 was coimmunoprecipitated with activated Ras from Rat1 cells expressing activated Ras. Moreover, we showed that AF-6 was coimmunoprecipitated with ZO-1 from Rat1 cells. Taken together, these results indicate that the Ras-interacting region on AF-6 is structurally similar to that on Raf-1 and on RalGDS and that AF-6 interacts with activated Ras and ZO-1 in vivo.

c- fos mRNA is induced by many types of noxious stimuli that are known to enhance transcription mediated by the heat shock element (HSE). Nonetheless, it has been accepted that the c- fos promoter does not contain an HSE. In this report, we revealed that the human and rodent c- fos promoters do contain HSEs which are highly responsive to heat, arsenite, and cadmium. These HSEs may play important roles in regulating c- fos expression in various pathological and physiological situations.

The expression of the neuropeptide galanin (GAL) is elevated in vivo upon nerve stimulation, injury, and in vitro by phorbol 12-myristate-13-acetate (PMA), suggesting that a signal pathway involving protein kinase C activation may be involved in GAL-gene activation. When plasmids containing a different length of the bovine GAL-promoter fused to luciferase were transfected into the human neuroblastoma cell line (SK-N-SH subclone SH-SY5Y), a PMA-responsive element was identified in the promoter-region -68 to -46 base pairs (bp). Co-transfection experiments with plasmids expressing cJun and cFos revealed that they could act alone, as well as synergistically with PMA to induce luciferase activity. Electrical mobility shift assays revealed that a cAMP response element (CRE)-like sequence (TGACGCGG; -59 to -52 bp) bound PMA-inducible nuclear proteins present in SH-SY5Y cells. These proteins appear to bind mainly as CRE-binding protein/activating-transcription-factor (CREB/ATF) and Jun/ATF heterodimers. In addition, an apparent PMA-inducible protein(s) not recognized by CREB/ATF and Jun antibodies bound to the CRE-like containing probe.