Transcriptional Activation Of C-fos Research Articles

Fucoidan from Laminaria cichorioides inhibits AP-1 transactivation and cell transformation in the mouse epidermal JB6 cells.

Fucoidan, a sulfated polysaccharide extracted from brown seaweeds, has anticoagulant and antithrombotic activities. Unlike heparine, fucoidan is known to exhibit anticarcinogenic activities. However, the underlying molecular mechanisms of the chemopreventive activities of fucoidan are not understood. Here we report that fucoidan from Laminaria cichorioides inhibited the epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic cell transformation, but had less cytotoxic effects on JB6 mouse epidermal cells. The EGF-induced phosphorylation of extracellular signal-regulated kinases 1/2 and c-Jun N-terminal kinases, and c-Jun was inhibited by fucoidan, resulting from the inhibition of phosphorylation of epidermal growth factor receptor (EGFR). Fucoidan dose-dependently attenuated the c-fos or c-jun transcriptional activity, and thereby inhibited the associated activator protein-1 (AP-1) transactivation activity. In vitro binding assay revealed that fucoidan directly interacted with EGF, suggested that antitumor promoting effect of fucoidan might be due to preventing the binding of EGF to its cell surface receptor (EGFR). These findings are the first to reveal a molecular basis for the anticarcinogenic action of fucoidan and may partially account for the reported chemopreventive effects of brown seaweeds.

Heterodimerization with Jun Family Members Regulates c-Fos Nucleocytoplasmic Traffic

c-Fos proto-oncoprotein forms AP-1 transcription complexes with heterodimerization partners such as c-Jun, JunB, and JunD. Thereby, it controls essential cell functions and exerts tumorigenic actions. The dynamics of c-Fos intracellular distribution is poorly understood. Hence, we have combined genetic, cell biology, and microscopic approaches to investigate this issue. In addition to a previously characterized basic nuclear localization signal (NLS) located within the central DNA-binding domain, we identified a second NLS within the c-Fos N-terminal region. This NLS is non-classic and its activity depends on transportin 1 in vivo. Under conditions of prominent nuclear localization, c-Fos can undergo nucleocytoplasmic shuttling through an active Crm-1 exportin-independent mechanism. Dimerization with the Jun proteins inhibits c-Fos nuclear exit. The strongest effect is observed with c-Jun probably in accordance with the relative stabilities of the different c-Fos:Jun dimers. Retrotransport inhibition is not caused by binding of dimers to DNA and, therefore, is not induced by indirect effects linked to activation of c-Fos target genes. Monomeric, but not dimeric, Jun proteins also shuttle actively. Thus, our work unveils a novel regulation operating on AP-1 by demonstrating that dimerization is crucial, not only for active transcription complex formation, but also for keeping them in the compartment where they exert their transcriptional function.

Sequential actions of ERK1/2 on the AP‐1 transcription factor allow temporal integration of metabolic signals in pancreatic β cells

The AP-1 transcription factor composed of fos and jun gene products mediates transcriptional responses to hormonal and metabolic stimulations of pancreatic beta cells. Here, we investigated the mechanisms that dynamically control expression of AP-1 subunit proteins. In MIN6 cells, glucose and GLP-1 raised c-FOS protein with biphasic kinetics, an initial peak being followed by a plateau that persisted as long as stimuli were maintained. ERK1/2 activation paralleled c-FOS expression. Whereas initial induction of c-FOS protein required ERK1/2-dependent activation of c-fos transcription and de novo protein synthesis, persistent accumulation of c-FOS under sustained stimulation did not. Indeed, dependent on ERK1/2 activation, c-FOS accumulated in its hyperphosphorylated form protected from degradation through the proteasome pathway. The implication of ERK1/2 in the accumulation of c-FOS protein was confirmed in rat primary beta cells, and the functional consequences of this mechanism were demonstrated with DNA-binding and reporter assays. Altogether these findings reveal a sequential regulation of AP-1 by ERK1/2, which initially increases transcription of c-fos and, if stimulation persists, stabilizes freshly synthesized c-FOS protein to efficiently activate the transcription of AP-1-regulated genes. This ERK1/2-AP-1 module can function as a temporal integrator converting metabolic stimuli of different durations into differential transcriptional outputs.

ERK1/2 regulates ANG II-dependent cell proliferation via cytoplasmic activation of RSK2 and nuclear activation of elk1

In a concurrently submitted article, we show that ANG II-induced ERK1/2 activation is mediated by both c-Src/Yes/Fyn and heterotrimeric G protein/PKCzeta-dependent signaling. Furthermore, we show that heterotrimeric G protein/PKCzeta-activated ERK1/2 is destined for the nucleus while ERK1/2 activated by c-Src/Yes/Fyn-dependent signaling remains in the cytoplasm. Interestingly, both mechanisms of activation are required for maximum ANG II-induced cell proliferation. In this study, we sought to determine the mechanisms by which ERK1/2 facilitate cell proliferation via these distinct nuclear and cytoplasmic events, using cells that were lacking either c-Src/Yes/Fyn or heterotrimeric G protein/PKCzeta-dependent ERK1/2 activation. A loss of c-Src/Yes/Fyn blocked ANG II-dependent RSK2 activation, RSK2 nuclear translocation, serum-response factor (SRF) phosphorylation, a portion of c-fos transcriptional activity and c-Fos phosphorylation. Blocking ANG II-induced heterotrimeric G protein/PKCzeta activity resulted in a loss of ERK1/2 nuclear translocation, elk1 phosphorylation, and the remaining portion of c-fos transcriptional activity not dependent on c-Src/Yes/Fyn. Inhibition of RSK with the potent and selective inhibitor, SL0101, attenuated ANG II-induced cell proliferation, and, in combination with a PKCzeta pseudosubstrate, completely attenuated cell proliferation. Thus we conclude that ERK1/2 mediate ANG II-dependent cell proliferation via distinct cytoplasmic and nuclear signaling events, which are in turn governed by c-Src/Yes/Fyn and heterotrimeric G protein/PKCzeta-dependent signaling, respectively.

Transcriptional activation of c-Fos by constitutively active Gα 16QL through a STAT1-dependent pathway

Hematopoietic restrictive Gα 16 has long been known to stimulate phospholipase Cβ (PLCβ) and induce mitogen-activated protein kinase (MAPK) phosphorylation. Recently, we have demonstrated that Gα 16 is capable of inducing the phosphorylation and transcriptional activation of transcription factors, such as signal transducer and activator of transcription 3 (STAT3) and nuclear factor κB (NFκB). However, the downstream signaling regulation by Gα 16 has not yet been documented. In the present study, we have determined the signaling mechanism by which constitutively active Gα 16 mediates c-Fos transcriptional activation in human embryonic kidney (HEK) 293 cells. Overexpression of constitutively active Gα 16, Gα 16QL, resulted in the stimulation of c-Fos transcriptional activation in HEK 293 cells. The participation of PLCβ, c-Src/Janus kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signaling pathways in Gα 16QL-induced c-Fos transcriptional activation was demonstrated by the use of their specific inhibitors. However, c-Jun N terminal kinase (JNK), p38 MAPK and phosphatidylinositol-3 kinase (PI3K) were not required. Interestingly, the dominant negative mutant of STAT1, but not STAT3, suppressed c-Fos transcriptional activation induced by Gα 16QL, implying that STAT1 was involved in this signaling mechanism. To further examine the role of STAT1 in the signaling pathway of Gα 16, we demonstrated that Gα 16QL was able to induce STAT1 activation. Also, stimulation of adenosine A1 receptor-coupled Gα 16 was shown to induce ERK and STAT1 phosphorylations in a concentration-dependent manner. Using selective inhibitors, PLCβ, c-Src/JAK and ERK, but not JNK, p38 MAPK and PI3K, were shown to be involved in Gα 16QL-induced STAT1 activation. Collectively, our results demonstrate for the first time that stimulation of Gα 16 can lead to STAT1-dependent c-Fos transcriptional activation via PLCβ, c-Src/JAK and ERK pathways.

Expression of Insulin-Like Growth Factor Receptor (IGF-1R), c-Fos, and c-Jun in Uveal Melanoma: An Immunohistochemical Study

The immunoreactivity of insulin-like growth factor receptor type 1 (IGF-1R), c-Fos, and c-Jun by immunohistochemistry was studied in three groups of uveal melanomas and was correlated clinicopathologically. Immunoanalysis was correlated with cell types, largest tumor diameter, tumor-infiltrating lymphocytes, mitosis, nuclear grade, and extrascleral extension/liver metastasis. In group C (n = 6), tumors with liver metastasis showed higher expressions of IGF-1R (p = 0.0001), c-Fos (p = 0.004), and c-Jun (p = 0.018) compared with the tumors with no extension/extrascleral extension without liver metastasis (groups A-45 and B-9). Further studies are required to elucidate the role of sequential upregulation of these proteins and the transcriptional activity of c-Fos and c-Jun in uveal melanomas with liver metastasis.

Phosphorylation and Isomerization

Activation of the mitogen-activated protein kinase (MAPK) cascade stimulates the activity and increases the abundance of the transcription factor AP-1, which is composed of two subunits, Jun and Fos. Monje et al. used transfected cell lines to show that the prolyl isomerase Pin1 enhanced AP-1-mediated reporter gene expression in response to growth factors. In cells overexpressing c-Jun or c-Fos, forced expression of Pin1 increased the growth factor-stimulated transcription activation of the reporter gene. Further analysis of the c-Fos and Pin1 relationship in vitro demonstrated that the interaction between Pin1 and the transactivation domain of c-Fos required phosphorylation of c-Fos by extracellular signal-regulated kinase (ERK). The interaction between c-Fos and Pin1 was also observed by coimmunoprecipitation from cells overexpressing the proteins and in glutathione- S -transferase (GST) pull-down experiments with GST-Pin1. Nonphosphorylatable mutants of c-Fos did not interact with Pin1. Thus, Pin1 appears to contribute to activation of AP-1 through interactions with both subunits--this interaction with c-Fos and an interaction with c-Jun, which had been reported previously. P. Monje, J. Hernández-Losa, R. J. Lyons, M. D. Castellone, J. Silvio Gutkind, Regulation of the transcriptional activity of c-Fos by ERK: A novel role for the prolyl isomerase Pin1. J. Biol. Chem. 280 , 35081-35084 (2005). [Abstract] [Full Text]

Down-Regulation of c-Fos/c-Jun AP-1 Dimer Activity by Sumoylation

The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. We report here that lysine 265 of c-Fos is conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 and that c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, we show that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. We show here that it also inhibits c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally we report that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation.

Interference of CREB‐dependent transcriptional activation by expanded polyglutamine stretches – augmentation of transcriptional activation as a potential therapeutic strategy for polyglutamine diseases

On the basis of the hypothesis that the interaction of mutant proteins with expanded polyglutamine stretches with transcriptional co-activator, TAFII130, leads to transcriptional dysregulation, the transcriptional activation of c-Fos and its suppression by expanded polyglutamine stretches was investigated. The phosphorylation of cAMP-responsive element binding protein (CREB) and induction of c-Fos in response to cAMP were strongly suppressed in Neuro2a cells expressing expanded polyglutamine. The suppression of CREB-dependent transcriptional activation was reversibly rescued by increasing the concentration of cAMP. Expanded polyglutamine-induced cytotoxicity was also substantially suppressed by augmenting CREB-dependent transcriptional activation with a high concentration of cAMP. FR901228, a histone deacetylase inhibitor, was also demonstrated as rescuing the expanded polyglutamine-induced suppression of CREB phosphorylation and c-Fos expression. Furthermore, nuclear fragmentation was significantly suppressed by FR901228. The co-expression of dominant-negative CREB vectors considerably abrogated the suppressive effect of cAMP and FR901228 on the expanded polyglutamine-induced nuclear fragmentation, suggesting that these compounds suppress polyglutamine-induced cytotoxicity, largely, via the enhancement of CREB-dependent transcriptional activation. These findings suggest that the interference of CREB-dependent transcriptional activation by expanded polyglutamine stretches is involved in the pathogenetic mechanisms underlying neurodegeneration, and that the augmentation of CREB-dependent transcriptional activation is a potential strategy in treating polyglutamine diseases.

Gonadotropin Releasing Hormone and Transforming Growth Factor β Activate Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase and Differentially Regulate Fibronectin, Type I Collagen, and Plasminogen Activator Inhibitor-1 Expression in Leiomyoma and Myometrial Smooth Muscle Cells

GnRH analog (GnRHa) and TGF-beta act directly on leiomyoma/myometrial smooth muscle cells (LSMCs and MSMCs) regulating diverse activities resulting in leiomyoma growth and regression. Because GnRH and TGF-beta receptor signaling is in part mediated through the MAPK pathway, we determined whether the contribution of MAPK/ERK and transcriptional activation of c-fos and c-jun, result in differential regulation of type I collagen, fibronectin, and plasminogen activator inhibitor 1 (PAI-1) gene expression, whose products are known to influence extracellular matrix turnover, which is critical in leiomyoma growth and GnRHa-induced regression. We found that GnRHa and TGF-beta in a dose- and time-dependent manner increased the level of phosphorylated ERK1/2 (pERK1/2) in LSMCs and MSMCs. GnRHa and TGF-beta increased ERK1/2 nuclear accumulation resulting in differential regulation of c-fos and c-jun mRNA expression via downstream signaling from MAPK kinase (MEK)1/2, because pretreatment with U0126, a synthetic inhibitor of MEK1/2, abolished basal and GnRHa- and TGF-beta-induced pERK1/2 and the expression of c-fos and c-jun. LSMCs and MSMCs also express fibronectin, type I collagen, and PAI-1 mRNA, and GnRHa and TGF-beta altered their expression in a cell-specific manner through MEK1/2. We concluded that GnRHa and TGF-beta acting through a MAPK/ERK pathway and transcriptional activation of c-fos/c-jun results in differential regulation of specific genes whose products may in part influence the outcome of leiomyoma growth and regression.

UVA irradiation-induced activation of activator protein-1 is correlated with induced expression of AP-1 family members in the human keratinocyte cell line HaCaT.

To determine whether the transcription factor activator protein-1 (AP-1) could be modulated by ultraviolet A (UVA) exposure, we examined AP-1 DNA-binding activity and transactivation after exposure to UVA in the human immortalized keratinocyte cell line HaCaT. Maximal AP-1 transactivation was observed with 250 kJ/m2 UVA between 3 and 4 h after irradiation. DNA binding of AP-1 to the target 12-O-tetradecanoylphorbol-13-acetate response element sequence was maximally induced 1-3 h after irradiation. Both de novo transcription and translation contributed to the UVA-induced AP-1 DNA binding. c-Fos was implicated as a primary component of the AP-1 DNA-binding complex. Other components of the complex included Fra-2, c-Jun, JunB and JunD. UVA irradiation induced protein expression of c-Fos, c-Jun, Fra-1 and Fra-2. Phosphorylated forms of these induced proteins were determined at specific time points. A strong correlation existed between UVA-induced AP-1 activity and accumulation of c-Fos, c-Jun and Fra-1 proteins. UVA irradiation also induced c-fos and c-jun mRNA expression and transcriptional activation of the c-fos gene promoter. These results demonstrate that UVA irradiation activates AP-1 and that c-fos induction may play a critical role in the response of these human keratinocytes to UVA irradiation.

Cyclic-adenosine 3′,5′-monophosphate-stimulated c-fos gene transcription involves distinct calcium pathways in single β-cells

In β-cells activation of the cyclic AMP (cAMP)-signaling cascade stimulates c-fos mRNA expression, which involves cAMP- and Ca 2+-mediated mechanisms. To delineate potential crosstalk between both pathways at the transcriptional level we simultaneously measured c-fos promoter-driven enhanced green fluorescent protein (EGFP) expression and cytosolic free calcium ([Ca 2+] i ) in single β-cells (HIT-T15). Forskolin stimulated a rapid rise in cellular cAMP and in [Ca 2+] i through activation of voltage-sensitive Ca 2+-influx and enhanced wild-type c-fos promoter-driven EGFP (pF711d2EGFP) expression about 4-fold after 6 h. The voltage-sensitive Ca 2+ channel (VSCC)-blocker nifedipine, which completely blocked the forskolin-induced rise in [Ca 2+] i , partially inhibited the forskolin-induced increase in pF711d2EGFP expression, while it was completely abolished in Ca 2+-free medium. VSCC-dependent Ca 2+-influx per se when stimulated by K + (45 mM) increased pF711d2EGFP expression only minimally. No correlations could be delineated between the forskolin-induced amplitude of the Ca 2+ signal and the expression of pF711d2EGFP at the single cell level, which may indicate that small rises in [Ca 2+] i are sufficient to fully activate the Ca 2+-dependent pathways required for cAMP-dependent c-fos promoter regulation. In experiments with various deletion constructs of the c-fos promoter, it could be shown that cAMP-mediated activation of the c-fos promoter involves both the cAMP-responsive element (CRE) and the serum-responsive element (SRE). While nifedipine completely abrogated the cAMP-dependent activation of c-fos transcription via the SRE, the CRE-mediated effect of cAMP on the c-fos promoter remained unaffected by nifedipine. Thus, cAMP and Ca 2+ are required for full c-fos promoter activation by the cAMP-signaling pathway in β-cells. cAMP-dependent Ca 2+-influx through VSCC is crucial for c-fos gene transcription via the SRE, whereas cAMP-mediated activation of the CRE demands Ca 2+-influx, which is distinct from voltage-sensitive Ca 2+-influx. This indicates a complex interplay between cAMP and Ca 2+ in controlling c-fos gene transcription and suggests that the mode of Ca 2+ entry may differentially act on signaling pathways leading to gene transcription in β-cells.

Dynamics of c-fos and ICER mRNA expression in rat forebrain following lithium chloride injection

Lithium is commonly used as a treatment for affective disorders in humans and as a toxin to produce conditioned taste aversions in rats. LiCl administration in rats has been correlated with activation of c-fos and cAMP-mediated gene transcription in many brain regions; however, little is known about the timing or duration of gene activation. We hypothesized that c-fos gene transcription is rapidly stimulated by LiCl, followed later by the expression of the inducible cAMP early repressor (ICER) transcription factor, a negative modulator of cAMP-mediated gene transcription. By in situ hybridization, we analyzed the timecourse of c-fos and ICER mRNA expression in the central nucleus of the amygdala (CeA), the paraventricular nucleus of the hypothalamus (PVN) and the supraoptic nucleus (SON) at seven time points (0, 0.3, 1, 3, 6, 9 and 12 h) after intraperitoneal LiCl injection (0.15 M, 12 ml/kg, 76 mg/kg). Expression of c-fos mRNA peaked between 20 min and 1 h and returned to baseline by 3 h in the CeA, PVN and SON. ICER mRNA was detected in these regions at 20 min, peaked at 1–3 h and returned to nearly baseline 9 h following LiCl injection. The time lag between c-fos mRNA expression and ICER mRNA expression within the same regions is consistent with ICER terminating c-fos gene transcription. However, no refractory period was detected for restimulation of c-fos transcription by a second injection of LiCl during the period of peak ICER mRNA expression, suggesting the involvement of other transcriptional modulators.

Signaling Pathways Leading to Transcription and Translation Cooperatively Regulate the Transient Increase in Expression of c-Fos Protein

The mechanisms by which growth factors trigger signal transduction pathways leading to the regulation of c-Fos expression are of great interest. In this study we investigated the effect of hepatocyte growth factor (HGF/SF) and epidermal growth factor (EGF) on the expression of c-fos and its product, c-Fos, in human epithelial cell line MKN74. The expression level of c-Fos protein in HGF/SF-stimulated cells was 5--10-fold higher than that in EGF-stimulated cells, whereas the level of c-fos mRNA induced by HGF/SF was similar to that by EGF. The hyperphosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), indicative of an increased number of translation initiation complexes, was detected only in HGF/SF-induced MKN74 cells. Activation of phosphatidylinositol-3'-OH kinase and FKBP12-rapamycin associated mammalian target of rapamycin (FRAP/mTOR) was observed after the treatment with HGF/SF. Pretreatment with an inhibitor of either one, i.e. LY294002 for phosphatidylinositol-3'-OH kinase or rapamycin for FRAP/mTOR, completely inhibited 4E-BP1 phosphorylation and decreased the c-Fos synthesis induced by HGF/SF down to the level found in EGF-induced cells. These results suggest that the phosphorylation of 4E-BP1 is stimulated by HGF/SF in a manner requiring both phosphatidy-linositol-3'-OH kinase-dependent and FRAP/mTOR-dependent pathways, thereby stimulating c-fos mRNA translation. Regulation of the translation process of c-fos mRNA in addition to the immediate activation of c-fos transcription is necessary for the transient increase in the level of c-Fos protein to stimulate cell proliferation.

Cadmium-induced cell transformation and tumorigenesis are associated with transcriptional activation of c-fos, c-jun, and c-myc proto-oncogenes: role of cellular calcium and reactive oxygen species.

The molecular mechanisms of carcinogenesis by cadmium were studied using BALB/c-3T3 cell transformation and nude mouse tumorigenesis models. BALB/c-3T3 cells transformed with cadmium chloride were subcutaneously injected into nude mice to develop tumors and the cell lines derived from these tumors were used in the present study. The proto-oncogenes c-fos and c-jun were overexpressed in 100% (10 out of 10) of the cell lines, while a statistically significant overexpression of c-myc was observed in 40% (4 out of 10) of the cell lines. Analysis of tumor cells stained with fluorescent dyes specific for reactive oxygen species revealed that these cells possessed markedly higher levels of superoxide anion and hydrogen peroxide compared with the nontransformed cells. Similarly, the intracellular calcium level was higher in the tumor cells compared with the nontransformed cells. Overexpression of the proto-oncogenes in these cells was blocked by treating the cells with superoxide dismutase, catalase, and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra acetoxy methyl ester (BAPTA/AM), which are scavengers of superoxide anion, hydrogen peroxide, and calcium, respectively. This confirmed that the overexpression of the proto-oncogenes in the tumor cells required elevated intracellular levels of reactive oxygen species and calcium. In addition to the scavengers of reactive oxygen species and calcium, inhibitors specific for transcription (actinomycin D), protein kinase C (RO-31-8220), and MAP kinase (PD 98059) also blocked the cadmium-induced overexpression of the proto-oncogenes in the tumor cells. Exposure of the nontransformed BALB/c-3T3 cells to 20 microM cadmium chloride for 1 h caused elevated intracellular levels of superoxide anion, hydrogen peroxide, and calcium, with corresponding increases in the expression levels of c-fos, c-jun, and c-myc. As in the case of the tumor cells, treating the nontransformed cells with the various modulators prior to their exposure to cadmium chloride resulted in inhibition in the expression of the proto-oncogenes. Based on these data, we conclude that the cadmium-induced overexpression of cellular proto-oncogenes is mediated by the elevation of intracellular levels of superoxide anion, hydrogen peroxide, and calcium. Further, the cadmium-induced overexpression of the proto-oncogenes is dependent on transcriptional activation as well as on pathways involving protein kinase C and MAP kinase.

Robust mRNA transcription in chicken DT40 cells depleted of TAF(II)31 suggests both functional degeneracy and evolutionary divergence.

We have employed gene targeting coupled with conditional expression to construct a chicken DT40 cell line in which a tetracycline (Tet)-repressible promoter is exclusively responsible for expression of cTAF(II)31, a histone-like TAF(II) residing in both the transcription factor TFIID and the histone acetylase complex PCAF/SAGA. Tet addition resulted in rapid loss of cTAF(II)31 mRNA and protein, eventually leading to apoptotic cell death. Significantly, five of six other TAF(II)s tested were also rapidly depleted, but levels of the TATA binding protein and subunits of PCAF/SAGA were at most modestly compromised. Strikingly, pulse-labeling experiments indicate that total poly(A)(+) mRNA transcription was not significantly reduced after cTAF(II)31 depletion, and steady-state levels of several specific transcripts remained the same or decreased only mildly. Moreover, activation of c-fos transcription following serum starvation occurred efficiently in the absence of cTAF(II)31. These data, which contrast with comparable studies in yeast, strongly suggest that cTAF(II)31 and perhaps other TAF(II)s are not essential for general mRNA transcription in DT40 cells. We propose that this is due to extensive functional degeneracy in the highly complex metazoan transcriptional machinery.

Significance of Nuclear Relocalization of ERK1/2 in Reactivation of c-fos Transcription and DNA Synthesis in Senescent Fibroblasts

Two of mitogen-activated protein kinases (MAPK), p44(mapk)/p42(mapk) extracellular signal-regulated kinases (ERK1/2), translocate into nuclei following activation and play critical roles in connecting the signal to gene expression and allowing cell-cycle entry. Here we found that the nuclear translocation of ERK1/2 in response to growth stimuli was significantly inhibited in senescent cells that were irreversibly growth arrested, compared with presenescent cells. The activation step of these enzymes was not impaired, since ERK1/2 were phosphorylated and activated in senescent cells as efficiently as in presenescent cells. By elaborately localizing ERK2 in the nuclei of senescent cells, we could restore c-fos transcriptional activity upon growth stimuli, which was repressed in senescent cells. Furthermore, the nuclear localization of ERK1/2 has been suggested to potentiate the proliferative activity of the senescent cells in collaboration with adenovirus E1A protein. More importantly, SV40 large T antigen, the strong inducer of DNA synthesis, had the inherent ability to restore nuclear relocalization of active ERK1/2 in senescent cells, which was essentially required for the reinitiation of DNA synthesis. Thus, manipulating the relocalization of ERK1/2 into nuclei was expected to open the way to overcome some of the senescent phenotypes.

Activation of ERK and p38 MAP Kinases in Human Fibroblasts during Collagen Matrix Contraction

Studies were carried out to characterize changes in MAP kinase activation during contraction of collagen matrices by fibroblasts under isometric tension. We found that both ERK and p38 MAP kinases were activated during contraction, as determined by immunoblotting and in vitro kinase assays. ERK activation was biphasic, with peaks at 10 min and 2 h; whereas p38 activation was monophasic, with a single peak at 10 min. Activation of ERK, but not p38, appeared to depend at least in part on the Gi class of heterotrimeric G proteins. The results show that ERK and p38 cooperate in contraction-stimulated activation of c-fos transcription.

Retinoic acid inhibits transformation by preventing phosphatidylinositol 3-kinase dependent activation of the c-fos promoter.

Retinoic acid inhibits transformation of cells by polyoma virus middle T oncoprotein. Inhibition of transformation results from a retinoic acid-dependent failure of cells to fully express the c-fos proto-oncogene. Retinoic acid prevents transactivation of the c-fos promoter by disrupting signaling between tyrosine kinases at the plasma membrane and trans-acting factors at the c-fos promoter. We used complementary genetic, biochemical and molecular approaches to demonstrate that: (1) phosphatidylinositol 3-kinase signaling is the principle mechanism of polyoma virus middle T oncoprotein activation of c-fos expression; (2) middle T/phosphatidylinositol 3-kinase transactivation of the c-fos promoter and transformation of cells requires activation of both the small GTP-binding protein Rac and Jun N-terminal kinase; (3) retinoic acid inhibits activation of Jun N-terminal kinase, thereby preventing c-fos transactivation and transformation; and (4) middle T activation of c-fos transcription requires both the serum response element and the promoter proximal cyclic AMP response element. These studies identify a novel target through which retinoids prevent oncogenic transformation.

Signal transduction pathways activated during fibroblast contraction of collagen matrices.

Fibroblasts cultured in collagen matrices have been used to develop in vitro models of the wound contraction process. Contraction of floating matrices resembles initial wound contraction, contraction of attached matrices resembles granulation tissue formation, and contraction of stressed matrices resembles granulation tissue contraction and regression at the end of repair. Studies in our laboratory have focused on contraction of stressed matrices. The basal component of stressed matrix contraction results in plasma-membrane ectocytosis, actin cytoskeletal disruption, Ca2+ uptake, stimulation of cyclic adenosine monophosphate (cAMP) and mitogen-activated protein (MAP) kinase signaling pathways, and transcriptional activation of c-fos and other immediate early genes. The stimulated component of stressed matrix contraction (stimulated by serum or lysophosphatidic acid) results in actin cytoskeletal retraction and growth-factor desensitization. Subsequently, the cells become quiescent and regress through apoptosis.