Critical role of Smads and AP-1 complex in transforming growth factor-beta -dependent apoptosis.

Transforming growth factor-beta1 (TGF-beta1) induces not only cell growth inhibition but also apoptosis in hepatocytes, myeloid cells, and epithelial cells. Although Smad proteins are identified as key signal transducers in TGF-beta1-dependent growth inhibition, their roles in the induction of apoptosis are unclear. We show here that both Smad proteins and AP-1 complex are involved in TGF-beta1 signaling for apoptosis. Overexpression of a dominant-negative Smad3 mutant or Smad7, both of which impair Smad-mediated signal transduction, inhibits TGF-beta1-dependent apoptosis. Only the JunD. FosB form of the AP-1 complex is markedly activated during TGF-beta1-dependent apoptosis. FosB substantially enhances Smad3. Smad4-dependent transcription, and dominant-negative FosB blocks TGF-beta1-dependent apoptosis but not growth inhibition. Expression of JunD.FosB enhances induction of apoptosis by TGF-beta1. Moreover, JunD.FosB binds to the 12-O-tetradecanoyl-13-acetate-responsive gene promoter element and recruits Smad3.Smad4 to form a multicomponent complex. These results suggest that Smad proteins and AP-1 complex synergize to mediate TGF-beta1-dependent apoptosis.

Tyrosine residues within the intracellular domain of the erythropoietin receptor mediate activation of AP-1 transcription factors.

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) initiates a signaling cascade resulting in tyrosine phosphorylation of several proteins and induction of AP-1 transcription factor(s). While Epo is known to activate c-fos gene expression, the mechanism of AP-1 activation is unknown. Here we show that AP-1 activation by Epo requires tyrosine kinase activity and also de novo protein synthesis. Using a mutant EpoR containing no cytosolic tyrosine residues, and a set of eight mutants containing a single cytosolic tyrosine residue, we show that multiple EpoR tyrosines, thought to activate multiple intracellular signal transduction proteins, can mediate AP-1 activation. An EpoR containing only tyrosine 343 or tyrosine 464 supports a maximal level of AP-1 activation. We also show that AP-1 activation does not require maximal STAT5 activation and may occur via a STAT5-independent signaling pathway.

Regulatory mechanisms involved in activator-protein-1 (AP-1)-mediated activation of glutathione-S-transferase gene expression by chemical agents.

Induction of murine glutathione-S-transferase (GST) Ya gene expression by a variety of chemical agents is mediated by a regulatory element, EpRE, composed of an Ets and two adjacent activator protein-1 (AP-1)-like sites and activated by the Fos/Jun heterodimeric complex (AP-1). The mechanism of this induction was examined in the present study. We find that the regulation of EpRE-mediated GST Ya gene expression by 3-methylcholanthrene, tert-butylhydroquinone and beta-naphthoflavone is associated with an induction of AP-1 DNA-binding activity and that the AP-1 complex induced in hepatoma cells by these chemicals contains members of the Fos and Jun protein families. We show that tert-butylhydroquinone induces c-fos gene expression and indicate the formation of a transcriptionally active AP-1 complex that contains Fos/Jun heterodimer. In F9 cells, which are considered to lack AP-1 complex, a careful examination reveals that tert-butylhydroquinone induces a low level of an AP-1-related activity responsible for the enhanced expression of EpRE as well as of AP-1 reporter constructs. We find that protein phosphorylations mediate the activation of the GST Ya gene by chemical agents since okadaic acid, an inhibitor of protein phosphatases, can mimic this activation while protein kinase inhibitors abolish it. Evidence is presented that 3-methylcholanthrene, tert-butylhydroquinone and beta-naphthoflavone use a signal transduction pathway to Fos/Jun-dependent GST Ya gene expression via Ras and protein-tyrosine kinase activity. Furthermore, we find that activation by phorbol 12-myristate 13-acetate, which uses both protein kinase C and protein-tyrosine kinase activities, may share a common pathway with these chemicals downstream of Ras.

Signaling pathways in the induction of c-fos and c-jun proto-oncogenes by 3-methylcholanthrene.

3-methylcholanthrene (MC), a potent promutagen and procarcinogen, is also an inducer of mammalian CYPIAI (cytochrome P1-450) gene. The CYPIAI enzyme is responsible for the detoxification of MC and its oxidation into reactive epoxide intermediates. Through its epoxide metabolites, MC functions also as an inducer of drug-metabolizing enzyme glutathione S-transferase (GST) gene expression. Induction of murine GST Ya gene by MC and a variety of other chemical agents is mediated by a regulatory element composed of two adjacent AP-1-like sites, and activated by the Fos/Jun heterodimeric complex (AP-1). In cultured cells, MC causes the induction of AP-1 activity, which is the result of an increased expression of c-Fos and c-Jun proteins. The mechanisms involved in MC activation of c-fos and c-jun gene expression were examined in the present study. Evidence is presented that stimulation of c-fos transcription by MC involves a signal transduction pathway, which includes activation of the small G protein Ras, Raf-1 kinase, and the mitogen-activated protein (MAP) kinases, ERK1 and ERK2. Furthermore, we find that phorbol 12-myristate 13-acetate, which uses both protein kinase C and protein-tyrosine kinase activities to induce c-fos promoter, may share a common pathway with MC downstream of Ras. The signal transduction pathway induced by MC to stimulate c-jun promoter involves Ras activation and the JNK group of MAP-kinases.

Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5.

Signaling through the erythropoietin receptor (EPO-R) is crucial for proliferation, differentiation, and survival of erythroid progenitor cells. EPO induces homodimerization of the EPO-R, triggering activation of the receptor-associated kinase JAK2 and activation of STAT5. By mutating the eight tyrosine residues in the cytosolic domain of the EPO-R, we show that either Y343 or Y401 is sufficient to mediate maximal activation of STAT5; tyrosine residues Y429 and Y431 can partially activate STAT5. Comparison of the sequences surrounding these tyrosines reveals YXXL as the probable motif specifying recruitment of STAT5 to the EPO-R. Expression of a mutant EPO-R lacking all eight tyrosine residues in the cytosolic domain supported a low but detectable level of EPO-induced STAT5 activation, indicating the existence of an alternative pathway for STAT5 activation independent of any tyrosine in the EPO-R. The kinetics of STAT5 activation and inactivation were the same, regardless of which tyrosine residue in the EPO-R mediated its activation or whether the alternative pathway was used. The ability of mutant EPO-Rs to activate STAT5 did not directly correlate with their mitogenic potential.

Cooperative interaction between Ets and AP-1 transcription factors regulates induction of glutathione S-transferase Ya gene expression.

Induction of mouse glutathione S-transferase (GST) Ya gene expression by a variety of chemical agents is mediated by a regulatory element, EpRE, composed of an Ets and two adjacent AP-1-like binding sites. In this report we present evidence that the basal and inducible activity of EpRE is mediated by AP-1 transcription factor and that the cooperative interaction between AP-1 and an Ets protein contributes to enhance the EpRE inducibility. We also show that EpRE, similar to a single AP-1 site, when ligated to GST Ya gene promoter, is transactivated by c-Fos/c-Jun or c-Fos/Jun-B heterodimer and that c-Jun/c-Jun homodimer is an activator of an AP-1 site only in the context of collagenase gene promoter.

Induction of AP-1 (Fos/Jun) by chemical agents mediates activation of glutathione S-transferase and quinone reductase gene expression.

A regulatory element, EpRE, was found to be responsible for the induction of mouse glutathione S-transferase (GST) Ya gene expression by a variety of chemical agents such as planar aromatic hydrocarbons, diphenols, phorbol ester, phenobarbital and electrophilic compounds. The EpRE is composed of two adjacent AP-1-like binding sites and was recently found to be activated by Fos/Jun heterodimeric complex (AP-1). In this report we show that regulatory elements ARE, previously demonstrated to mediate the chemical induction of rat GST Ya and quinone reductase genes, have a similar structure with EpRE and are activated by Fos/Jun complex. The activation of GST Ya and quinone reductase genes by a variety of chemical inducers is found to be associated with an increase in AP-1 binding activity. We present evidence that chemical agents induce expression of c-fos and c-jun proto-oncogenes and an enhanced synthesis of protein components of AP-1 complex. We suggest that the increased synthesis of AP-1 complex followed by an AP-1-mediated transcriptional activation of GST Ya and quinone reductase genes may provide a molecular mechanism for the induction of these drug-metabolizing enzymes by chemical agents.

Intracellular glutathione levels regulate Fos/Jun induction and activation of glutathione S-transferase gene expression.

Induction of glutathione S-transferase Ya and NAD(P)H:quinone reductase gene expression by a variety of chemical agents is mediated by regulatory elements, EpRE and ARE, composed of two adjacent AP-1-like binding sites and activated by Fos/Jun heterodimeric complex (AP-1). Recent studies show that chemical induction of glutathione S transferase Ya and quinone reductase gene expression is associated with an induction of c-fos and c-jun gene expression and AP-1 binding activity. In this report we present evidence that the AP-1 binding activity and the expression of chloramphenicol acetyltransferase activity from an EpRE Ya-cat gene construct are induced by an increase in intracellular oxidant levels. We observe that lowering the glutathione levels with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, or diamide, a thiol-oxidizing agent, stimulates both basal and chemical-inducible expression of chloramphenicol acetyltransferase activity from EpRE Ya-cat and the AP-1 binding activity. Furthermore, we observe that the induction of these activities by a variety of chemical agents is inhibited by thiol compounds N-acetylcysteine and glutathione. These findings suggest that diverse chemicals that induce the AP-1 complex, leading to the AP-1-mediated transcriptional activation of glutathione S-transferase Ya gene expression, may act through a common mechanism involving the production of reactive oxygen species and depletion of reduced glutathione.

Phenobarbital induction of AP-1 binding activity mediates activation of glutathione S-transferase and quinone reductase gene expression.

Phenobarbital is an inducer of xenobiotic-metabolizing enzymes, such as cytochrome P-450, glutathione S-transferases (GSTs) and NAD(P)H:quinone reductase, as well as being a promoter of hepatocarcinogenesis. The molecular mechanisms regulating these biological activities are, however, unknown. In this paper we show that induction by phenobarbital of GST Ya and quinone reductase gene expression is mediated by regulatory elements, EpRE and ARE respectively, which are composed of two adjacent AP-1-like binding sites. EpRE was recently found to be activated by a Fos/Jun heterodimeric complex (AP-1). Here we show that phenobarbital induces an increase in AP-1 binding activity in nuclear extracts of cultured hepatoma cells. Furthermore, we observe that the induction of chloramphenicol acetyltransferase (CAT) activity from an EpRE Ya-cat gene construct and of AP-1 binding activity by phenobarbital is inhibited by the thiol compounds N-acetyl-L-cysteine and glutathione. These results suggest that the phenobarbital induction of AP-1 activity, leading to the AP-1-mediated transcriptional activation of the GST Ya and quinone reductase genes, may involve production of reactive oxygen species and an increase in intracellular oxidant levels, which is prevented by thiol compounds. In view of the involvement of AP-1 in the control of cell proliferation and transformation, the induction by phenobarbital of AP-1 binding activity observed here provides a possible molecular mechanism for the tumour-promoting activity of this drug.

Two adjacent AP-1-like binding sites form the electrophile-responsive element of the murine glutathione S-transferase Ya subunit gene.

An electrophile-responsive element (EpRE) in the 5' flanking region of the mouse glutathione S-transferase Ya subunit gene was recently found to be responsible for the induction of gene expression by xenobiotics that contain or acquire by metabolism an electrophilic center. We now find that this EpRE is composed of two adjacent 9-base-pair motifs related in sequence to the AP-1 binding site, a transcriptional enhancer originally identified as the phorbol 12-myristate 13-acetate (PMA) response element and known to be regulated by the binding of protein products of c-jun and c-fos genes. Synthetic oligonucleotides representing each of the AP-1-like binding sites of the EpRE and the AP-1 site consensus sequence were prepared and assayed for their enhancer activity and inducibility by tert-butylhydroquinone, beta-naphthoflavone, and PMA. Single AP-1-like sequences showed a lower enhancer activity than an AP-1 consensus sequence and no inducibility. Two adjacent AP-1-like sites were found to act synergistically and to confer inducibility beyond that observed for a single AP-1 consensus sequence. Examination of the PMA-responsive region of a number of genes shows the presence of adjacent AP-1-like sites and indicates that the structure of the EpRE found in the Ya gene may occur more generally and may be important in regulating the magnitude of the electrophilic response. The present study demonstrates the binding and transactivation of the EpRE by Jun and Fos and indicates that the AP-1 site is part of the EpRE. The induction by PMA or tert-butylhydroquinone appears to be independent of protein kinase C activity since it is not affected by inhibitors of this enzyme.