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Tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) is a Rel protein that activates transcription of osmoprotective genes at high extracellular NaCl. Other Rel proteins NFAT1-4 and NF-kappaB complex with activator protein-1 (AP-1) to transactivate target genes through interaction at composite NFAT/NF-kappaB.AP-1 sites. TonEBP/OREBP target genes commonly have one or more conserved AP-1 binding sites near TonEBP/OREBP cognate elements (OREs). Also, TonEBP/OREBP and the AP-1 proteins c-Fos and c-Jun are all activated by high NaCl. We now find, using an ORE.AP-1 reporter from the target aldose reductase gene or the same reporter with a mutated AP-1 site, that upon stimulation by high extracellular NaCl, 1) the presence of a wild type, but not a mutated, AP-1 site contributes to TonEBP/OREBP-dependent transcription and 2) AP-1 dominant negative constructs inhibit TonEBP/OREBP-dependent transcription provided the AP-1 site is not mutated. Using supershifts and an ORE.AP-1 probe, we find c-Fos and c-Jun present in combination with TonEBP/OREBP. Also, c-Fos and c-Jun coimmunoprecipitate with TonEBP/OREBP, indicating physical association. Small interfering RNA knockdown of either c-Fos or c-Jun inhibits high NaCl-induced increase of mRNA abundance of the TonEBP/OREBP target genes AR and BGT1. Furthermore, a dominant negative AP-1 also reduces high NaCl-induced increase of TonEBP/OREBP transactivating activity. Inhibition of p38, which is known to stimulate TonEBP/OREBP transcriptional activity, reduces high NaCl-dependent transcription of an ORE.AP-1 reporter only if the AP-1 site is intact. Thus, AP-1 is part of the TonEBP/OREBP enhanceosome, and its role in high NaCl-induced activation of TonEBP/OREBP may require p38 activity.

Cysteinyl-leukotrienes are involved in inflammation and act on at least two G-protein-coupled receptors, CysLT1 and CysLT2. However, the role of the CysLT2 receptor as well as its signaling remain poorly understood. Here we show that leukotriene (LT)C(4) induced the production of the chemokine interleukin (IL)-8 in endothelial cells. To further study the signaling cascade involved, HEK293 cells were stably transfected with CysLT2 and used to study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with increasing concentrations of LTC(4) resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-kappaB, AP-1, or NF-IL-6 binding elements revealed an almost total requirement for NF-kappaB and AP-1 elements, and a lesser requirement for the NF-IL-6 element. Overexpression of dominant-negative IkappaBalpha prevented the IL-8 transactivation induced by LTC(4). LTC(4) stimulation induced NF-kappaB and AP-1 DNA binding, which involved the formation of a p50/p65 and a c-JUN.c-FOS complex, respectively. Transfection of the cells with a dominant negative (dn) form of PKCepsilon prevented p65 phosphorylation, whereas dnPKCdelta prevented AP-1 binding. Moreover, dnPKCdelta, dnPKCepsilon, and dnPKCzeta prevented LTC(4)-induced IL-8 transcription in response to LTC(4). Our data show for the first time that LTC(4) can act via the CysLT2 receptor to transcriptionally activate chemokine production through induction of NF-kappaB and AP-1 transcription factors. These findings suggest the potential implication of CysLT2 in the inflammatory response through the modulation of chemokine gene transcription.

Arginine vasotocin (VT) is the ortholog in all nonmammalian vertebrates of arginine vasopressin (AVP) in mammals. We have previously cloned an amphibian V1atype vasotocin receptor (VT1R) that exhibited higher sensitivity for VT than AVP, while the mammalian V1a type receptor (V1aR) responded better to AVP than VT. In the present study, we identified the amino acid residues that confer differential ligand selectivity for AVP and VT between rat V1aR and bullfrog VT1R (bfVT1R). A chimeric rat V1aR having transmembrane domain (TMD) VI to the carboxyl-terminal tail (C-tail) of bfVT1R showed a reverse ligand preference for AVP and VT, whereas a chimeric VT1R with TMD VI to the C-tail of rat V1aR showed a great increase in sensitivity for AVP. A single mutation (Ile(315(6.53)) to Thr) in TMD VI of V1aR increased the sensitivity for VT, while a single mutation (Phe(313(6.51)) to Tyr or Pro(334(7.33)) to Thr) reduced sensitivity toward AVP. Interestingly the triple mutation (Phe(313(6.51)) to Tyr, Ile(6.53) to Thr, and Pro(7.33) to Thr) of V1aR increased sensitivity to VT but greatly reduced sensitivity to AVP, behaving like bfVT1R. Further, like V1aR, a double mutant (Tyr(306(6.51)) to Phe and Thr(327(7.33)) to Pro) of bfVT1R showed an increased sensitivity to AVP. These results suggest that Phe/Tyr(6.51), Ile/Thr(6.53), and Pro/Thr(7.33) are responsible for the differential ligand selectivity between rat V1aR and bfVT1R. This information regarding the molecular interaction of VT/AVP with their receptors may have important implications for the development of novel AVP analogs.

Tissue factor is critically important for initiating the activation of coagulation zymogens leading to the generation of thrombin. Quiescent endothelial cells do not express tissue factor on their surface, but many stimuli including cytokines and coagulation proteases can elicit tissue factor synthesis. We challenged human endothelial cells simultaneously with tumor necrosis factor alpha (TNFalpha) and thrombin because many pathophysiological conditions, such as sepsis, diabetes, and coronary artery disease, result in the concurrent presence of circulating inflammatory mediators and activated thrombin. We observed a remarkable synergy in the expression of tissue factor by thrombin plus TNFalpha. This was due to altered regulation of the transcription factors c-Jun and c-Fos. The activation of c-Jun was greater and more sustained than that obtained with either thrombin or TNFalpha alone. Thrombin-stimulated expression of c-Fos was both enhanced and prolonged by the concurrent presence of TNFalpha. These changes support the increased availability of c-Jun/c-Fos AP-1 complexes for mediating transcription at the tissue factor promoter. Transcription factors downstream of the extracellular signal-regulated kinases as well as changes in NFkappaB regulation were not involved in the synergistic increase in tissue factor expression by thrombin and TNFalpha. Thus, concurrent exposure of vascular endothelial cells to cytokines and procoagulant proteases such as thrombin can result in greatly enhanced tissue factor expression on the endothelium, thereby perpetuating the prothrombotic phenotype of the endothelium.

Mitogenic activation of expression of immediate-early genes, such as c-fos, is controlled through signal-induced phosphorylation of constitutively bound transcription factors that is correlated with a nucleosomal response that involves inducible chromatin modifications, such as histone phosphorylation and acetylation. Here we have explored a potential role for the transcription factor NF-kappaB and its associated signaling components in mediating induction of c-fos gene expression downstream of epidermal growth factor (EGF)-dependent signaling. Here we show that EGF treatment of quiescent fibroblast does not induce the classical pathway of NF-kappaB activation through IkappaB kinase (IKK)-directed IkappaBalpha phosphorylation. Interestingly, efficient induction of c-fos transcription requires IKKalpha, one of the subunits of the IkappaB kinase complex. The NF-kappaB subunit, p65/RelA, is found constitutively associated with the c-fos promoter, and knock-out of this transcription factor significantly reduces c-fos gene expression. Importantly, EGF induces the recruitment of IKKalpha to the c-fos promoter to regulate promoter-specific histone H3 Ser(10) phosphorylation in a manner that is independent of p65/RelA. Collectively, our data demonstrate that IKKalpha and p65/RelA contribute significantly to EGF-induced c-fos gene expression in a manner independent of the classical, IkappaBalpha degradation, p65/RelA nuclear accumulation response pathway.

Cytoplasmic export of the RNA-binding protein HuR, a process that critically regulates its function, was recently shown to be inhibited by the AMP-activated protein kinase (AMPK). In the present investigation, treatment of human fibroblasts with AMPK activators such as 5-amino-imidazole-4-carboxamide riboside, antimycin A, and sodium azide inhibited cell growth and lowered the expression of proliferative genes. As anticipated, AMPK activation also decreased both the cytoplasmic HuR levels and the association of HuR with target radiolabeled transcripts encoding such proliferative genes. HuR function was previously shown to be implicated in the maintenance of a "young cell" phenotype in models of replicative cellular senescence. We therefore postulated that AMPK activation in human fibroblasts might contribute to the implementation of the senescence phenotype through mechanisms that included a reduction in HuR cytoplasmic presence. Indeed, AMP:ATP ratios were 2-3-fold higher in senescent fibroblasts compared with young fibroblasts. Accordingly, in vitro senescence was accompanied by a marked elevation in AMPK activity. Evidence that increased AMPK activity directly contributed to the implementation of the senescent phenotype was obtained through two experimental approaches. First, use of AMPK activators triggered senescence characteristics in fibroblasts, such as the acquisition of senescence-associated beta-galactosidase (beta-gal) activity and increased p16INK4a expression. Second, infection of cells with an adenoviral vector that expresses active AMPK increased senescence-associated beta-gal activity, whereas infection with an adenovirus that expresses dominant-negative AMPK decreased senescence-associated beta-gal activity. Together, our results indicate that AMPK activation can cause premature fibroblast senescence through mechanisms that likely involve reduced HuR function.

The c-fos gene was one of the earliest vertebrate genes shown to be transcriptionally induced by growth factors. Intensive study of the promoter of c-fos (-325 to -80) by transient or permanent transfections of synthetic DNA constructs has repeatedly shown the importance of several sequence elements and the resident nuclear proteins that bind them (e.g. ternary complex factor/ELK1; serum response factor, cAMP response element-binding protein/amino-terminal fragment/AP-1). However these studies have left unanswered numerous questions about the role of these proteins in the regulation of the native chromosomal gene. In particular, the role of a site in this enhancer that binds STATs has been controversial. We present evidence here that STAT3 and not STAT1 accumulates on the chromosomal c-fos promoter and provides a boost to transcription without the activation of resident nuclear proteins through serine kinases. Also, when resident nuclear proteins such as ELK1 are activated to varying extents by mitogen-activated protein kinase pathways, STAT3 activation provides a 2-fold boost regardless of the final level of activated transcription. Thus the several proteins that interact with the c-fos enhancer apparently can act either in a cooperative or independent manner to achieve very different levels of transcription.

Although epidemiological studies have long established that inorganic arsenic is a potent human carcinogen, the underlying mechanisms are still poorly understood. Recent studies suggest that inorganic arsenic may act as a tumor promoter by perturbing key signaling transduction pathways. We have shown previously that arsenite can potently activate the mitogen-activated protein kinase cascades and induce the expression of proliferation-associated genes, including proto-oncogenes c-jun and c-fos. In order to elucidate further the molecular mechanisms underlying its tumor-promoting properties, we investigated the signaling events involved in arsenite-mediated induction of c-fos and c-jun. We found that induction of both c-fos and c-jun by arsenite can be substantially inhibited by the MEK- selective inhibitor U0126, suggesting that the ERK pathway is critically involved in their up-regulation. Interestingly, arsenite dramatically induced the phosphorylation and acetylation of histone H3 preceding the induction of mRNAs encoding c-fos and c-jun. Finally, chromatin immunoprecipitation assays revealed that arsenite treatment markedly induced the phosphorylation/acetylation of histone H3 associated with the c-fos and c-jun genes through an ERK-dependent pathway. Our results strongly suggest that arsenic-triggered alterations in chromatin structure perturb specific gene transcription, including that of proto-oncogenes c-jun and c-fos, and may thereby contribute to the carcinogenic process.

In this study, we have identified novel regulatory steps involved in the cross-talk between protein kinase B (PKB) and MAPK signaling pathways. We found that PKB down-regulates the Ras-Raf-MEK-ERK pathway by reducing the activity of ERK, which leads to inactivation of the transcription factor Elk1. In addition, PKB is able to reduce protein levels of Elk1. Both events lead to suppression of serum response element (SRE)-dependent transcription and a consequent decrease in the transcription of SRE-containing genes, such as c-fos. Because activation of the Ras/MAPK cascade is reported to increase c-fos transcription before apoptosis, our results are consistent with a specific role for PKB in promoting cell survival. Decrease in c-Fos protein levels in glioblastoma cells with constitutively active PKB provides further support for our observations. Therefore, our findings delineate a novel mechanism regulating immediate-early transcription, which may be involved in the initial steps in PKB-induced oncogenic transformation.

Steroid hormones exert genotropic actions through members of the nuclear receptor family. Here, we have demonstrated genotropic actions of testosterone that are independent of intracellular androgen receptors (iAR). Through plasma membrane androgen receptors (mAR), testosterone induces a rapid rise in the intracellular free Ca(2+) concentration of iAR-free murine RAW 264.7 macrophages. This nongenomic testosterone signaling, which is independent of both iAR and estrogen receptors, does not in itself activate either the mitogen-activated protein kinase (MAPK) families ERK1/2, p38, and JNK/SAPK, the stably and transiently transfected c-fos promoter, or NO production. In the context of lipopolysaccharide (LPS) signaling, however, testosterone attenuates LPS activation of the c-fos promoter and NO production, which is abolished by the intracellular Ca(2+) chelator BAPTA. Testosterone also attenuates the LPS activation of p38 but not that of ERK1/2 and JNK/SAPK, and this attenuation is abrogated by BAPTA. Moreover, the p38 inhibitor, SB 203580, largely reduces LPS activation of the c-fos promoter and NO production, and the remaining levels are no longer regulated by testosterone. This study is the first to provide information on genotropic actions of mAR-mediated nongenomic testosterone Ca(2+) signaling by cross-talk with the LPS signaling pathway through p38 MAPK with impact on cell function.

Gender is an important determinant of clinical outcome across a broad spectrum of kidney diseases, but the mechanism(s) responsible for the protective effect of female gender have not been fully elucidated. Remnant kidney glomerular injury is limited in female rats compared with male rats despite similar elevations in glomerular capillary pressure. In vitro, mechanical strain leads to the activation of p44/42 mitogen-activated kinase (p44/42 MAPK) and Jun N-terminal kinase/stress-activated protein kinase (SAPK) in glomerular mesangial cells (MC). Accordingly, we studied the effect of 17beta-estradiol on mechanical strain-induced signal transduction in MC. Exposure of MC to mechanical strain increased p44/42 MAPK activation (3-fold) and SAPK activation (2.5-fold), and kinase activation was inhibited by pretreatment with 17beta-estradiol (10(minus sign8) to 10(minus sign11) m) for 24 h in a dose-dependent manner. Mechanical strain-induced nuclear translocation of p44/42 MAPK and SAPK and nuclear protein binding to AP-1 were also attenuated by 17beta-estradiol. The inhibitory effects of 17beta-estradiol were not reproduced by the cell-impermeable estrogen, BSA/17beta-estradiol, nor did preincubation with 17beta-estradiol lead to actin cytoskeleton disassembly or impaired stress fiber formation. However, 17beta-estradiol did increase base-line levels of the dual specificity phosphatase MKP-1. The inhibitory effects of 17beta-estradiol on p44/42 MAPK activation and SAPK activation, translocation, and AP-1 binding were all abrogated by the estrogen receptor antagonist, ICI-182,780. We conclude that attenuation of mechanical strain-induced MAPK activation by 17beta-estradiol is dependent on intracellular estrogen receptor. The attenuation of stretch-induced kinase activation may be due, at least in part, to an effect of 17beta-estradiol on MKP-1 expression. Together, these findings add insight into the protective effect of gender on renal disease progression.

Macrophages exposed to receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*) demonstrate increased DNA synthesis and cell division. In the current study, we have probed the role of cytosolic phospholipase A(2) (cPLA(2)) activity in the cellular response to alpha(2)M*. Ligation of the alpha(2)M* signaling receptor by alpha(2)M*, or its receptor binding fragment, increased cPLA(2) activity 2-3-fold in a concentration and time-dependent manner. This activation required a pertussis toxin-insensitive G protein. Cellular binding of alpha(2)M* also induced transient translocation of cPLA(2) activity to nuclei and membrane fractions. Inhibition of protein kinase C activity or chelation of Ca(2+) inhibited alpha(2)M*-induced increased cPLA(2) activity. Binding of alpha(2)M* to macrophages, moreover, increased phosphorylation of MEK 1/2, ERK 1/2, p38 MAPK, and JNK. Incubation of macrophages with inhibitors of MEK 1/2 or p38 MAPK before stimulation with alpha(2)M* profoundly decreased phosphorylation of MAPKs, blocking cPLA(2) activation. alpha(2)M*-induced increase in [(3)H]thymidine uptake and cell proliferation was completely abolished if activation of cPLA(2) was prevented. The response of macrophages to alpha(2)M* requires transcription factors nuclear factor kappaB, and cAMP-responsive element-binding protein as well as expression of the proto-oncogenes c-fos and c-myc. These studies indicate that the activation of cPLA(2) plays a crucial role in alpha(2)M*-induced mitogenesis and cell proliferation.

Proliferation of mesangial cells requires platelet-derived growth factor receptor beta (PDGFR)-mediated signal transduction. We have previously shown that activation of phosphatidylinositol (PI) 3-kinase is necessary for PDGFR-induced DNA synthesis in these cells. The mechanism by which PI 3-kinase stimulates DNA synthesis is not known. One target of PI 3-kinase, Akt serine threonine kinase, regulates survival of many cells by inhibiting the actions of certain proapoptotic proteins. In this study, we investigated the role of Akt in PDGF-induced DNA synthesis in mesangial cells. PDGF increased Akt serine threonine kinase activity in a time- and PI 3-kinase-dependent manner. Expression of dominant negative Akt by adenovirus-mediated gene transfer blocked PDGF-induced activation of endogenous Akt in mesangial cells, resulting in complete inhibition of DNA synthesis. On the other hand, inhibition of MAPK attenuated PDGF-induced DNA synthesis only partially. Inhibition of Akt also attenuated PDGF-induced c-fos gene transcription, with concomitant inhibition of Elk-1-dependent transcription, indicating positive regulation of this early response gene by Akt. To further determine the role of Akt in PDGF-induced DNA synthesis, we investigated its effect on cyclin-dependent kinase 2 (CDK2). PDGF stimulated CDK2 activity in mesangial cells and decreased the level of p27(kip1) cyclin kinase inhibitor protein. Expression of dominant negative Akt increased p27(kip1) protein and resulted in inhibition of CDK2 activity. The increase in p27(kip1) expression in response to Akt kinase inhibition was due to increased transcription of the p27(kip1) gene. p27(kip1) transcription similarly was decreased by expression of constitutively active Akt kinase in mesangial cells. These data provide the first evidence that Akt kinase regulates PDGF-induced DNA synthesis by regulating CDK2 activity and define Akt-mediated inhibition of transcription of p27(kip1) as one of the mechanisms for PDGF-induced DNA synthesis in mesangial cells.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

Serum response factor (SRF) is a MADS box transcription factor that regulates muscle-specific and growth factor-inducible genes by binding the consensus sequence CC(A/T)6GG, known as a CArG box. Because SRF expression is not restricted solely to muscle, its expression alone cannot account for the muscle specificity of some of its target genes. To understand further the role of SRF in muscle-specific transcription, we created transgenic mice harboring lacZ transgenes linked to tandem copies of different CArG boxes with flanking sequences. CArG boxes from the SM22 and skeletal alpha-actin promoters directed highly restricted expression in developing smooth, cardiac, and skeletal muscle cells during early embryogenesis. In contrast, the CArG box and flanking sequences from the c-fos promoter directed expression throughout the embryo, with no preference for muscle cells. Systematic swapping of the core and flanking sequences of the SM22 and c-fos CArG boxes revealed that cell type specificity was dictated in large part by sequences immediately flanking the CArG box core. Sequences that directed widespread embryonic expression bound SRF more strongly than those that directed muscle-restricted expression. We conclude that sequence variations among CArG boxes influence cell type specificity of expression and account, at least in part, for the ability of SRF to distinguish between growth factor-inducible and muscle-specific genes in vivo.

RNA splicing generates two products in equal molar amounts, mature mRNAs and spliced introns. Although the mechanism of RNA splicing and the fate of the spliced mRNA products have been well studied, very little is known about the fate and stability of most spliced introns. Research in this area has been hindered by the widely held view that most vertebrate introns are too unstable to be detectable. Here, we report that we are able to detect all three spliced introns from the coding region of the Pem homeobox gene. By using a tetracycline (tet)-regulated promoter, we found that the half-lives of these Pem introns ranged from 9 to 29 min, comparable with those of short lived mRNAs such as those encoding c-fos and c-myc. The half-lives of the Pem introns correlated with both their length and 5' to 3' orientation in the Pem gene. Subcellular fractionation analysis revealed that spliced Pem introns and pre-mRNA accumulated in the nuclear matrix, high salt-soluble, and DNase-sensitive fractions within the nucleus. Surprisingly, we found that all three of the spliced Pem introns were also in the cytoplasmic fraction, whereas Pem pre-mRNAs, U6 small nuclear RNA, and a spliced intron from another gene were virtually excluded from this fraction. This indicates either that spliced Pem introns are uniquely exported to the cytoplasm for degradation or they reside in a unique soluble nuclear fraction. Our study has implications for understanding the regulation of RNA metabolism, as the stability of introns and the location of their degradation may dictate the following: (i) the stability of nearby mRNAs that compete with spliced introns for rate-limiting nucleases, (ii) the rate at which free nucleotides are available for further rounds of transcription, and (iii) the rate at which splicing factors are recycled.

Recent studies have demonstrated that sphingosine 1-phosphate (SPP) plays a functional role as a signaling molecule in gene expression in several kinds of cells. The present study demonstrates selective expression by ceramide of retinoic acid receptor-alpha (RAR-alpha) and retinoic X receptor-alpha (RXR-alpha) in osteoblastic MC3T3-E1 cells and a functional role of SPP-mediated AP-1 in the signaling mechanism of ligand-dependent transcriptional activity of heterodimers of these receptors in the cells. C(2)- and C(6)-ceramides selectively stimulated the expression of RAR-alpha and RXR-alpha genes, but not that of beta and gamma isoform genes of RAR and RXR, in the cells. The C(2)-ceramide-induced stimulation was clearly inhibited by dl-threo-dihydrosphingosine, an inhibitor of sphingosine kinase. SPP also selectively stimulated the expression of both receptors and increased the specific binding of the nuclear proteins to direct repeat 5 (DR-5), a consensus sequence of RAR-RXR. In addition, SPP markedly stimulated transient chloramphenicol acetyltransferase (CAT) activity of retinoic acid-dependent transcriptional activity in the cells transfected with a DR-5-CAT reporter gene. The SPP stimulation was activation protein-1 (AP-1)-dependent, because the SPP stimulatory action toward these nuclear gene expressions and the transient CAT activity were inhibited by antisense c-fos and c-jun oligonucleotides. We observed that SPP actually stimulated AP-1 transcriptional activity in the cells. This study suggests an important role of SPP-mediated AP-1 in the selective expression of RAR-alpha and RXR-alpha in osteoblastic cells via the sphingosine pathway.

The carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3'-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an alpha-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*Delta5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of alpha-amanitin. Moreover, pol II harboring LS*Delta5 failed to transcribe up to the promoter-proximal pause sites in the hsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA.

The role of IL-6 in collagen production and tissue remodeling is controversial. In Rat-1 fibroblasts, we measured the effect of IL-6 on matrix metalloproteinase-13 (MMP-13), c-jun, junB, and c-fos gene expression, binding of activator protein 1 (AP1) to DNA, amount of AP1 proteins, immunoreactive MMP-13 and TIMP-1 proteins, and Jun N-terminal kinase activity. We show that IL-6 increased MMP-13-mRNA and MMP-13 protein. These effects were exerted by acting on the AP1-binding site of the MMP-13 promoter, as shown by transfecting cells with reporter plasmids containing mutations in this element. Mobility shift assays demonstrated that IL-6 induced the DNA binding activity of AP1. This effect was accompanied by a marked increase in c-Jun, JunB, and c-Fos mRNA, as well as in c-Jun protein and its phosphorylated form. The latter is not due to increased Jun N-terminal kinase activity but to a decreased serine/threonine phosphatase activity. We conclude that IL-6 increases interstitial MMP-13 gene expression at the promoter level. This effect seems to be mediated by the induction of c-jun, junB, and c-fos gene expression, by the binding of AP1 to DNA, by increasing phosphorylated c-Jun, and by the inhibition of serine/threonine phosphatase activity. These effects of IL-6 might contribute to remodeling connective tissue.

Platelet-derived growth factor (PDGF) stimulates transcription of an immediate-early gene set in Balb/c 3T3 cells. One cohort of these genes, typified by c-fos, is induced within minutes following activation of PDGF receptors. A second cohort responds to PDGF only after a significant time delay, although induction is still a primary response to receptor activation as shown by "superinduction" in the presence of the protein synthesis inhibitor cycloheximide. PDGF-receptor activated signaling pathways for the "slow" immediate-early genes are poorly resolved. Using gain-of-function mutations together with small molecule inhibitors of kinase activity, we show that activation of PI 3-kinase is both necessary and sufficient for the induction of the prototype slow immediate-early gene, monocyte chemoattractant-1 (MCP-1). Following activation of PDGF receptors, MCP-1 mRNA does not begin to accumulate for at least 90 min. However, only a brief (10 min) interval of PI 3-kinase activity is required to trigger this delayed response. The serine/threonine protein kinase, Akt/PKB, likely functions as a downstream affector of PI 3-kinase for this induction.

Bone morphogenetic proteins (BMPs) play an important role in nephrogenesis. The biologic effect and mechanism of action of these proteins in the adult kidney has not yet been studied. We investigated the effect of BMP2, a member of these growth and differentiation factors, on mitogenic signal transduction pathways induced by platelet-derived growth factor (PDGF) in glomerular mesangial cells. PDGF is a growth and survival factor for these cells in vitro and in vivo. Incubation of mesangial cells with increasing concentrations of BMP2 inhibited PDGF-induced DNA synthesis in a dose-dependent manner with maximum inhibition at 250 ng/ml. Immune complex tyrosine kinase assay of PDGF receptor beta immunoprecipitates from lysates of mesangial cells treated with PDGF showed no inhibitory effect of BMP2 on PDGF receptor tyrosine phosphorylation. This indicates that the inhibition of DNA synthesis is likely due to postreceptor events. However, BMP2 significantly inhibited PDGF-stimulated mitogen-activated protein kinase (MAPK) activity that phosphorylates the Elk-1 transcription factor, a component of the ternary complex factor. Using a fusion protein-based reporter assay, we also show that BMP2 blocks PDGF-induced Elk-1-mediated transcription. Furthermore, we demonstrate that BMP2 inhibits PDGF-induced transcription of c-fos gene, a natural target of Elk-1 that normally forms a ternary complex that activates the serum response element of the c-fos gene. These data provide the first evidence that in mesangial cells, BMP2 signaling cross-talks with MAPK-based transcriptional events to inhibit PDGF-induced DNA synthesis. One target for this inhibition is the early response gene c-fos.

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7) has often been used in combination with protein kinase inhibitor (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide) (HA1004) to assess the contribution of protein kinase C (PKC) to cellular processes, including the induction of gene expression. This use of H7 and HA1004 is based upon the fact that H7 inhibits PKC more potently than HA1004 in in vitro assays. Thus, although both compounds are broad spectrum protein kinase inhibitors, inhibition by H7, but not by HA1004, has often been interpreted as evidence for the involvement of PKC in the cellular process under study. Here we describe experiments that show that this interpretation is not correct with regard to the induction of two immediate-early genes, zif268 and c-fos, in PC12D cells. In these studies we confirmed that H7, but not HA1004, potently blocks the induction of zif268 and c-fos mRNA by nerve growth factor, carbachol, phorbol ester, Ca2+ ionophore, or forskolin. Surprisingly, however, H7 has no effect on the ability of these agents to activate mitogen-activated protein kinase (MAPK), an upstream activator of zif268 and c-fos gene expression. H7 also does not inhibit preactivated MAPK in vitro. Taken together, these results suggest that H7 blocks gene expression by acting at a site downstream from MAPK. H7 has previously been shown to block transcription in vitro by blocking the phosphorylation of the carboxyl-terminal domain of RNA polymerase II (Yankulov, K., Yamashita, K., Roy, R., Egly, J.-M., and Bentley, D. L.(1995) J. Biol. Chem. 270, 23922-23925). In this study, we show that pretreating PC12D cells with H7, but not with HA1004, significantly reduces levels of phosphorylated RNA polymerase II in vivo. These results suggest that H7 blocks gene expression by inhibiting the phosphorylation of RNA polymerase II, a step required for progression from transcription initiation to mRNA chain elongation.

The adipocyte hormone leptin activates signal transducer and activator of transcription 3 (STAT3) in the hypothalamus, mediating increased satiety and increased energy expenditure. To date, leptin-mediated activation of the STAT pathway in vivo has not been established in tissues other than hypothalamus. We now describe leptin receptor expression and in vivo signaling in discrete regions of the mouse gastrointestinal tract. Expression of the functional isoform leptin receptor (OB-Rb) is restricted to the jejunum and is readily detected by RT-PCR in isolated enterocytes from this site. Intravenous injection of leptin rapidly induced nuclear STAT5 DNA binding activity in jejunum of +/+ and obese (ob/ob) mice but had no effect in the diabetic (db/db) mouse that lacks the OB-Rb isoform. In addition, an induction of the immediate-early gene c-fos is observed in jejunum in vivo. Leptin-mediated induction of a number of immediate-early genes and activation of STAT3 and STAT5 in a human model of small intestine epithelium, CACO-2 cells, corroborate this effect. Furthermore, intravenous leptin administration caused a significant 2-fold reduction in the apolipoprotein AIV transcript levels in jejunum 90 min after a fat load. Our results suggest that the epithelium of jejunum is a direct target of leptin action, and this activity is dependent on the presence of OB-Rb. Lack of leptin or resistance to leptin action in this site may contribute to obesity and its related syndromes by directly affecting lipid handling.

Many lines of evidence have suggested that angiotensin II (AngII) plays an important role in the development of cardiac hypertrophy through AngII type 1 receptor (AT1). To determine whether AngII is indispensable for the development of mechanical stress-induced cardiac hypertrophy, we examined the activity of mitogen-activated protein kinase (MAPK) family and the expression of the c-fos gene as hypertrophic responses after stretching cultured cardiac myocytes of AT1a knockout (KO) mice. When cardiac myocytes were stretched by 20% for 10 min, extracellular signal-regulated protein kinases (ERKs) were strongly activated in KO cardiomyocytes as well as wild type (WT) myocytes. Both basal and stimulated levels of ERKs were higher in cardiomyocytes of KO mice than in those of WT mice. Activation of another member of the MAPK family, p38(MAPK), and expression of the c-fos gene were also induced by stretching cardiac myocytes of both types of mice. An AT1 antagonist attenuated stretch-induced activation of ERKs in WT cardiomyocytes but not in KO cardiomyocytes. Down-regulation of protein kinase C inhibited stretch-induced ERK activation in WT cardiomyocytes, whereas a broad spectrum tyrosine kinase inhibitor (genistein) and selective inhibitors of epidermal growth factor receptor (tyrphostin, AG1478, and B42) suppressed stretch-induced activation of ERKs in KO cardiac myocytes. Epidermal growth factor receptor was phosphorylated at tyrosine residues by stretching cardiac myocytes of KO mice. These results suggest that mechanical stretch could evoke hypertrophic responses in cardiac myocytes that lack the AT1 signaling pathway possibly through tyrosine kinase activation.

The present study demonstrates 1alpha,25-dehydroxyvitamin D3 (1alpha-25-(OH)2D3) synergism toward transforming growth factor (TGF)-beta1-induced activation protein-1 (AP-1) activity in mouse osteoblastic MC3T3-E1 cells via the nuclear receptor of the vitamin. 1alpha-25-(OH)2D3 synergistically stimulated TGF-beta1-induced expression of the c-jun gene in the cells but not that of the c-fos gene. We actually showed by a gel mobility shift assay 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced AP-1 binding to the 12-(O-tetradecanoylphorbol-13-acetate response element (TRE). 1alpha-25-(OH)2D3 markedly stimulated the transient activity of TGF-beta1-induced AP-1 in the cells transfected with a TRE-chloramphenicol acetyltransferase (CAT) reporter gene. Also, a synergistic increase in TGF-beta1-induced CAT activity was observed in the cells cotransfected with an expression vector encoding vitamin D3 receptor (VDR) and the reporter gene. However, the synergistic CAT activity was inhibited by pretreatment with VDR antisense oligonucleotides. In addition, in a Northern blot assay, we observed 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced expression of the c-jun gene in the cells transfected with the VDR expression vector and also found that the synergistic action was clearly blocked by VDR antisense oligonucleotide pretreatment. The present study strongly suggests a novel positive regulation by 1alpha-25-(OH)2D3 of TGF-beta1-induced AP-1 activity in osteoblasts via "genomic action."

CD43, the most abundant membrane protein of T lymphocytes, is able to initiate signals that lead to Ca2+ mobilization and interleukin-2 production, yet the molecular events involved in signal transduction pathway of the CD43 molecule are only beginning to be understood. We have shown recently that cross-linking CD43 on the cell surface of human T lymphocytes with the anti-CD43 monoclonal antibody L10 leads to CD43-Fyn kinase interactions and to Fyn phosphorylation on tyrosine residues. This interaction seems to be mediated by the SH3 domain of Fyn and a proline-rich sequence located in the cytoplasmic domain of CD43. Here we show that CD43-specific activation of human T lymphocytes induced tyrosine phosphorylation of the adaptor protein Shc and of the guanine exchange factor Vav, as well as the formation of a macromolecular complex that comprises Shc, GRB2, and Vav. CD43 ligation resulted in enhanced formation of Vav.SLP-76 complexes and in the activation and nuclear translocation of ERK2. Cross-linking of the CD43 molecule in 3T3-CD43(+) cells induced luciferase activity from a construct under the control of the Fos serum responsive element. Altogether, these data suggest that the mitogen-activated protein kinase pathway is involved in CD43-dependent interleukin-2 gene expression.

Phosphatidylinositol (PI) 3-kinase is known to be activated by cytokine stimulation through different types of receptors to transduce intracellular responses. We have previously reported that leukemia inhibitory factor (LIF) induces the activation of Janus kinase signal transducer and activator of transcription (JAK-STAT) and mitogen-activated protein (MAP) kinase pathways through glycoprotein (gp) 130 in cardiac myocytes. However, whether PI 3-kinase is involved in regulation of gp130 signaling and the activation mechanisms by which it associates with other tyrosine-phosphorylated proteins remain unknown. We found that LIF induced the activation of PI 3-kinase in cardiac myocytes. Moreover, JAK1 binds to PI 3-kinase, and LIF stimulation increases the PI 3-kinase activity in JAK1 immunoprecipitates. Activation of MAP kinase and protein kinase B by LIF was attenuated by wortmannin. LIF-induced p70 S6 kinase activation, protein synthesis, and c-fos mRNA expression were inhibited by wortmannin and rapamycin. Both inhibitors failed to appreciably affect the phosphorylation of STAT3. In conclusion, PI 3-kinase is activated with LIF in cardiac myocytes, and JAK1 is found to associate with this enzyme. PI 3-kinase provides a crucial link between gp130, MAP kinase, protein kinase B, and p70 S6 kinase in cardiac myocytes.

Interleukin-1 (IL-1) is an important inflammatory mediator and plays a central role in the destruction of connective tissue matrices in diseases such as arthritis and periodontitis. It is well established that IL-1 activation of the mitogen-activated protein (MAP) kinase pathway and induction of c-fos expression is a required step in the induction of matrix metalloproteinase expression involved in tissue degradation. Previous studies in our laboratory showed that IL-1-induced calcium flux is dependent on focal adhesion formation, suggesting a matrix-dependent restriction system for IL-1 signaling. Therefore, in the present study, we examined the consequences of this restriction on IL-1-mediated activation of the MAP kinase family and on c-fos expression. Treatment of human gingival fibroblasts with IL-1 activated extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase activity and induced c-fos expression in a dose- and time-dependent fashion. Plating cells on poly-l-lysine prevented focal adhesion formation, eliminated IL-1-induced calcium influx, abolished ERK stimulation, and blocked c-fos expression. Cells in suspension and hence with no suitable substratum for focal adhesion formation also showed no ERK activation or enhanced c-fos expression in response to IL-1. In contrast, eliminating focal adhesion formation or calcium depletion in cells plated on fibronectin had no effect on IL-1 stimulation of JNK and p38 kinases, demonstrating that their activation was mediated through pathways independent of focal adhesions and calcium. Calcium depletion abolished IL-1-induced calcium uptake, ERK activation, and c-fos expression. The focal adhesion dependence of IL-1-induced ERK activation and c-fosexpression could be circumvented in cells plated on poly-l-lysine by simultaneous incubation with IL-1 and the calcium ionophore ionomycin. In transfection studies, IL-1 stimulation of serum responsive element (SRE) transcriptional activity was dependent on the presence of extracellular calcium. This is consistent with a requirement for calcium in the activation of ERKs and their involvement in the induction of c-fos expression through the SRE site on the 5′ promoter of the c-fos gene. Our results demonstrate that in cells attached to substrates by focal adhesions, IL-1-mediated calcium flux is required for ERK activation and c-fos expression but not for JNK or p38 activation. We conclude that cellular interactions with the extracellular matrix play an important role in restricting ERK and c-fos-dependent processes.

The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and MIP (macrophage inflammatory protein)-1alpha have been implicated in regulating T cell functions. RANTES-induced T cell activation is apparently mediated via two distinct signal transduction cascades: one linked to recruitment of pertussis toxin-sensitive G proteins and the other linked to protein-tyrosine kinase activation. In this report, we identified that the transcription factors Stat1 and Stat3 (for signal transducers and activators of transcription) are rapidly activated in T cells in response to RANTES and MIP-1alpha. Nuclear extracts from MOLT-4 and Jurkat T cells treated with RANTES or MIP-1alpha contain tyrosine-phosphorylated Stat1:1 and Stat1:3 dimers that exhibit DNA-binding activity. We demonstrated that RANTES and MIP-1alpha treatment of Jurkat cells resulted in transcriptional activation of a Stat-inducible gene, c-fos, with kinetics consistent with Stat activation by these chemokines. RANTES and MIP-1alpha mediate their effects via shared chemokine receptors (CCRs): CCR1, CCR4, and CCR5. Our data revealed a concordance between chemokine-induced Stat activation and c-fos induction and CCR4 and CCR5 expression. These findings indicate that chemokine-mediated activation of G-protein-coupled receptors leads to signal transduction that invokes intracellular phosphorylation intermediates used by other cytokine receptors.

Cadmium is mitogenic under some circumstances and has been shown to cause accumulation of transcripts for several proto-oncogenes in a variety of cells, but the mechanism(s) remain to be delineated. Here we show that CdCl2 causes an increase in c-fos mRNA within 30 min of exposure of mesangial cells. At 10 microM Cd2+, this increase persists for at least 8 h in both rat and human cells. The half-life of c-fos mRNA is the same whether it accumulates following 4 h of treatment with Cd2+ or is induced transiently by phorbol ester. Cycloheximide, which stabilizes the transcript, causes a synergistic increase when administered with CdCl2. Nuclear run-on analysis confirms that Cd2+ causes transcriptional activation of the c-fos gene. Calmodulin and Ca2+/calmodulin-dependent kinase, and classical protein kinase C (PKC) isoforms represent two Ca2+-dependent signaling pathways that can lead to induction of c-fos, and Cd2+ has been shown to activate both calmodulin and PKC in vitro, possibly by virtue of the similar ionic radii of Cd2+ and Ca2+. Therefore, we investigated the effect of Cd2+ on these pathways in vivo. 10 microM CdCl2 did not increase total PKC activity or Ca2+/calmodulin-dependent kinase II activity and inhibited the latter at higher concentrations, ruling out either pathway in the Cd2+-dependent induction of c-fos. However, Cd2+ did lead to a sustained activation of the Erk family mitogen-activated protein kinases (MAPK) that correlated with induction of c-fos. A specific inhibitor of the MAPK kinases, PD98059, partially inhibited the induction of c-fos by Cd2+. We conclude that Cd2+ induces c-fos at least in part by causing a sustained activation of MAPK independent of its ability to activate PKC and calmodulin in vitro.

Caveolae are plasma membrane-attached vesicular organelles. Caveolin-1, a 21–24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Both caveolae and caveolin are most abundantly expressed in terminally differentiated cells: adipocytes, endothelial cells, and muscle cells. Conversely, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes such as v-abl and H-ras(G12V); caveolae are absent from these cell lines. However, its remains unknown whether down-regulation of caveolin-1 protein and caveolae organelles contributes to their transformed phenotype. Here, we have expressed caveolin-1 in oncogenically transformed cells under the control of an inducible-expression system. Regulated induction of caveolin-1 expression was monitored by Western blot analysis and immunofluorescence microscopy. Our results indicate that caveolin-1 protein is expressed well using this system and correctly localizes to the plasma membrane. Induction of caveolin-1 expression in v-Abl-transformed and H-Ras (G12V)-transformed NIH 3T3 cells abrogated the anchorage-independent growth of these cells in soft agar and resulted in the de novo formation of caveolae as seen by transmission electron microscopy. Consistent with its antagonism of Ras-mediated cell transformation, caveolin-1 expression dramatically inhibited both Ras/MAPK-mediated and basal transcriptional activation of a mitogen-sensitive promoter. Using an established system to detect apoptotic cell death, it appears that the effects of caveolin-1 may, in part, be attributed to its ability to initiate apoptosis in rapidly dividing cells. In addition, we find that caveolin-1 expression levels are reversibly down-regulated by two distinct oncogenic stimuli. Taken together, our results indicate that down-regulation of caveolin-1 expression and caveolae organelles may be critical to maintaining the transformed phenotype in certain cell populations.

Using murine AtT20 pituitary cells transfected with a rat pro-opiomelanocortin (POMC) promoter (-706/+64) linked to the luciferase reporter, we showed leukemia inhibitory factor (LIF) to strongly potentiate corticotropin-releasing hormone (CRH) induction of POMC gene expression. We therefore tested mechanisms for molecular interactions between LIF and CRH. Although LIF and CRH synergized to induce an 8-fold induction of POMC transcription, CRH alone (but not LIF) induced cAMP response element-binding protein phosphorylation (5-fold) or an increase of c-fos mRNA levels (>100-fold), suggesting that these pathways are not implicated in LIF transcriptional synergistic effects. Using a DNase I footprint assay, POMC promoter regions protected by AtT20 cell nuclear extracts were identified (-121/-109, and -143/-134, and -173/-160). The protected -173/-160 element fused to a heterologous promoter conferred LIF-CRH synergy (6.5-fold induction of POMC) and formed a specific complex with AtT20 cell nuclear extracts. This complex was supershifted by an anti-phosphoserine antibody, and a serine/threonine kinase inhibitor also altered both this complex and LIF-CRH transcriptional synergy on the POMC promoter-luciferase reporter construct, indicating that these events depend on post-translational serine phosphorylations. LIF-CRH synergy on POMC transcription is therefore mediated at least in part by -173/-160 sequences conferring confluent transcriptional activity of both peptides.

Ral GDP dissociation stimulator-like (RGL) has been identified to be a possible effector protein of Ras. RGL shares 50% amino acid identity with Ral GDP dissociation stimulator and contains the CDC25-like domain in the central region and the Ras-interacting domain in the C-terminal region. Since the modes of activation and action of RGL have not yet been clarified, in this paper we have analyzed the functions of RGL. In COS cells, RGL interacted with RasG12V/E37G (a Ras mutant in which Gly-12 and Glu-37 were changed to Val and Gly, respectively) which failed to bind to Raf, but not with RasG12V/T35S which bound to Raf. Raf did not inhibit the binding of RGL to RasG12V/E37G under the condition that Raf inhibited that of RGL to RasG12V. Expression of either RGL or Raf into NIH3T3 cells slightly activated c-fos promoter, while coexpression of both proteins greatly stimulated the c-fos promoter activity. RGL stimulated the GDP/GTP exchange of Ral and this action was enhanced by the post-translational modification of Ral. However, RGL was not active on Ras, Rac, CDC42, Rap, or Rho. Furthermore, this action of RGL to stimulate the GDP/GTP exchange of Ral was dependent on Ras in COS cells. These results suggest that RGL constitutes another Ras-signaling pathway which is distinct from the Raf pathway and indicate that the RGL pathway regulates the c-fos promoter activity and the GDP/GTP exchange of Ral.

The cellular redox state is thought to play an important role in a wide variety cellular signaling pathways. Here, we investigated the involvement of redox regulation in the nerve growth factor (NGF) signaling pathway and neuronal differentiation in PC12 cells. N-acetyl-L-cysteine (NAC), which acts as a reductant in cells both by its direct reducing activity and by increasing the synthesis of the cellular antioxidant glutathione, inhibited neuronal differentiation induced by NGF or by the expression of oncogenic ras in PC12 cells. NAC suppressed NGF-induced c-fos gene expression and AP-1 activation. These results suggest that neuronal differentiation and NGF signaling are subject to regulation by the cellular redox state. NAC also suppressed the NGF-induced activation of mitogen-activated protein kinases (MAPKs) and decreased the amount of tyrosine phosphorylation of MAPKs. The suppression of MAPK by NAC was independent of glutathione synthesis. In parallel with the suppression of MAPK, the activation of MAPK kinase kinase activity was also suppressed in the presence of NAC. In contrast, NGF-induced activation of Ras was not inhibited by NAC. The inhibitory effect of NAC on the MAPK cascade was independent of transcription and translation. Thus, NAC suppresses NGF-induced neuronal differentiation by uncoupling the signal transduction from Ras to the MAP kinase cascade in PC12 cells.

Many parathyroid hormone (PTH)-mediated events in osteoblasts are thought to require immediate early gene expression. PTH induces the immediate early gene, c-fos, in this cell type through a cAMP-dependent pathway. The present work investigated the nuclear mechanisms involved in PTH regulation of c-fos in the osteoblastic cell line, UMR 106-01. By transiently transfecting c-fos promoter 5′ deletion constructs into UMR cells, we demonstrated that PTH induction of the c-fos promoter requires the major cAMP response element (CRE). Point mutations created in the major CRE within the largest construct inhibited both PTH-stimulated and basal expression. This element, therefore, performs concerted basal and PTH-responsive cis-acting functions. Gel retardation and Western blotting techniques revealed that CRE-binding protein (CREB) constitutively binds the major CRE but becomes phosphorylated at its cAMP-dependent protein kinase consensus recognition site following PTH treatment. CREB was functionally implicated in c-fos regulation by coexpressing a dominant CREB repressor, KCREB (killer CREB), with the c-fos promoter constructs. KCREB suppressed both basal and PTH-mediated c-fos induction. We conclude that PTH activates c-fos in osteoblasts through cAMP-dependent protein kinase-phosphorylated CREB interaction with the major CRE in the promoter region of the c-fos gene.

Heparin suppresses mitogenic responses in renal mesangial cells, and when quiescent mesangial cells are stimulated with serum, heparin blocks the induction of c-fos seen at 15 min. Because heparin is taken up by cells over a much longer time course, we addressed mechanisms whereby extracellular heparin might suppress c-fos induction at such early times. Quiescent cells were treated with serum, 12-O-tetradecanoylphorbol-13-acetate, or low concentrations of Ca2+ ionophores that produced increases in intracellular Ca2+ concentration ([Ca2+]i) in the physiological range. Each treatment caused an increase in c-fos mRNA, but they did so by different mechanisms. Serum activated mitogen-activated protein kinase (MAPK) and increased [Ca2+]i without affecting protein kinase C. Activation of protein kinase C with phorbol ester activated MAPK without much effect on [Ca2+]i. Ionophores increased [Ca2+]i without affecting basal levels of protein kinase C or MAPK. Heparin (1 microg/ml) suppressed the induction of c-fos initiated by all three treatments. It did not affect the activity of protein kinase C, but inhibited activation of MAPK by either serum or phorbol ester, suggesting a common site of action at or below the probable convergence of the induced signals at Ras/Raf-1 activation. Heparin also inhibited the serum-stimulated entry of extracellular Ca2+ to the same extent as verapamil, consistent with the ability of verapamil to block L-type Ca2+ channels and the known presence of these channels in mesangial cells. However, this effect does not appear to be related to heparin's ability to inhibit induction of c-fos. First, verapamil had no effect on induction of c-fos by serum. Second, heparin had no effect on changes in [Ca2+]i achieved by ionophores. We conclude that heparin suppresses induction of c-fos in mesangial cells by blocking at least two different points in signal transduction cascades, one upstream of MAPK and the other independent of MAPK, but dependent on intracellular Ca2+.

Bradykinin and platelet-derived growth factor (PDGF) are inflammatory mediators important in the response to vascular injury. Based upon the known effect of oncogenic Ras to increase bradykinin receptor expression and the ability of PDGF to stimulate Ras, we examined whether PDGF regulates bradykinin B2 receptor expression in cultured arterial smooth muscle cells. Treatment with PDGF (AB and BB, but not AA) produced a dose- and time-dependent increase in both mRNA (6-7-fold increase at 2-4 h) and cell surface receptors (2-4-fold at 6-12 h) for the B2 receptor. There was a 60-min delay between exposure to PDGF and the initial increase in B2 receptor mRNA. Transcriptional inhibitors, actinomycin D or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, completely blocked the increase in B2 receptor mRNA when added up to 60 min after stimulation with PDGF. However, protein synthesis was not required, as treatment with cycloheximide did not block but rather superinduced the PDGF-induced increase in B2 receptor mRNA. Comparison with the immediate early response gene c-fos demonstrated that the increase in B2 receptor mRNA was similarly inhibited by the tyrosine kinase inhibitor, tyrphostin, as well as staurosporine. However, stimulation of c-fos was slightly more sensitive to genistein, while the B2 receptor mRNA was more sensitive to inhibition by the protein kinase C inhibitor, calphostin C. The increase in cell surface B2 receptors were functionally coupled to an increase in phosphoinositide-specific phospholipase C, and the effects of PDGF were selective as there was no increase in either angiotensin II- or arginine vasopressin-induced inositol phosphate formation or intracellular calcium release. Taken together, these results demonstrate that the B2 receptor is a delayed early response gene for PDGF in vascular smooth muscle cells.

Previously, our laboratory has shown that lactosylceramide (LacCer) can serve as a mitogenic agent in the proliferation of aortic smooth muscle cells "a hallmark in the pathogenesis of atherosclerosis" (Chatterjee, S. (1991) Biochem. Biophys. Res. Commun. 181, 554-561). Here we report a novel aspect of LacCer-mediated signal transduction. We demonstrate that LacCer (10 microM) can stimulate the phosphorylation of mitogen-activated protein (MAP) kinase p44MAPK to phosphorylated p44MAPK in aortic smooth muscle cells from rabbit or human origin. Western immunoblot assays and direct measurement of activity in immunoprecipitated MAP kinase revealed that within 5 min of incubation of cells with LacCer there was a 3.5-fold increase in the activity of p44MAPK. This continued up to 10 min of incubation; thereafter, the MAP kinase activity decreased in these cells. Phosphoamino acid analysis revealed that the tyrosine and threonine moieties of p44MAPK was phosphorylated by LacCer. Incubation of cells with ceramide and glucosylceramide did not significantly stimulate p44MAPK activity. Preincubation with tyrphostin (20 microM; a potent and specific inhibitor of tyrosine kinase) markedly inhibited the LacCer mediated stimulation in p44MAPK activity. Next we investigated the upstream and downstream parameters in MAP kinase signaling pathways. We found that lactosylceramide stimulated (7-fold) the loading of GTP on Ras. Concomitantly, LacCer stimulated the phosphorylation of MAP kinase kinases (MEK) and Raf within 2.5 min. Lactosylceramide specifically induced c-fos mRNA expression (3-fold) in these cells as compared to control. In summary, one of the biochemical mechanisms in LacCer mediated induction in the proliferation of aortic smooth muscle cells may involve Ras-GTP loading, activation of the kinase cascade (MEK, Raf, p44MAPK), and c-fos expression.

PTP1C, an SH2 domain-containing protein-tyrosine phosphatase, is predominantly expressed in hematopoietic cells, in which it negatively regulates cellular signaling. However, this enzyme is also expressed in many non-hematopoietic cells. We demonstrate here that in non-hematopoietic 293 cells, overexpression of a catalytically inactive mutant of PTP1C strongly suppressed the stimulatory effects of the epidermal growth factor or serum on cell proliferation, early gene transcription, and DNA synthesis. Similarly, the phosphorylation of the mitogen-activated protein kinase and mitogen-activated protein kinase kinase activity was markedly inhibited by overexpression of mutant PTP1C. The inhibitory effect of mutant PTP1C was overcome by cotransfection with wild-type PTP1C, but not with the structurally related PTP2C. Furthermore, expression of the mutant phosphatase resulted in hyperphosphorylation on tyrosine of a 95-kDa protein that was co-immunoprecipitated with the mutant, but not with the wild-type protein. These results suggest that, unlike in hematopoietic cells, PTP1C in 293 cells plays a positive role in epidermal growth factor- or serum-activated mitogenesis. Thus, PTP1C participates in multiple signaling pathways, where the enzyme, depending on its target molecules, may function as either a positive or negative mediator.

Pancreatic beta cells and neuronal cells show a large number of similarities. For example, functional receptors for nerve growth factor are present in beta cells. Here we investigate whether TrkC, a neuronal high affinity receptor for neurotrophin-3, is expressed in the insulin-secreting cell line INS-1. We demonstrate the expression in INS-1 cells of mRNAs coding for TrkC identical in size to those found in the brain. As in neuronal cells, different alternatively spliced forms of TrkC mRNA, differing by the insertion of an alternative exon in their kinase domain, were expressed in INS-1 cells. TrkC protein is also expressed in INS-1 cells and is functional. Indeed, when INS-1 cells were treated with neurotrophin-3, TrkC became phosphorylated on tyrosine residues, and the expression of early response genes was induced. This activation of the receptor was paralleled by a rapid and transient increase in cytosolic free calcium due to an influx of extracellular calcium. Functional receptors for NT-3 are thus expressed in INS-1 cells. This cell line provides a new model for the study of NT-3 signal transduction and should be useful in the understanding of the role of neurotrophins in insulin-secreting cells.

Cardiotrophin-1 (CT-1) was recently isolated by expression cloning based on its ability to induce an increase in cell size in neonatal rat ventricular cardiomyocytes. Sequence similarity data suggested that CT-1 is a novel member of a family of structurally related cytokines sharing the receptor component gp130. The present study documents that gp130 is required for CT-1 signaling in cardiomyocytes, by demonstrating that a monoclonal anti-gp130 antibody completely inhibits c-fos induction by CT-1. Similarly, a leukemia inhibitory factor receptor subunit beta (LIFRbeta) antagonist effectively blocks the CT-1 induction of c-fos, indicating a requirement for LIFRbeta in the hypertrophic response, as well. Upon stimulation with CT-1, both gpl30 and the LIFRbeta are tyrosine-phosphorylated, providing further evidence that CT-1 signals through the gp130/LIFRbeta heterodimer in cardiomyocytes. CT-1 induces a hypertrophic response in cardiomyocytes that is distinct from the phenotype seen after alpha-adrenergic stimulation, both with regard to cell morphology and gene expression pattern. Stimulation with CT-1 results in an increase in cardiac cell size that is characterized by an increase in cell length but no significant change in cell width. Confocal laser microscopy of CT-1 stimulated cells reveals the assembly of sarcomeric units in series rather than in parallel, as seen after alpha-adrenergic stimulation. CT-1 induces a distinct pattern of immediate early genes, and up-regulates the atrial natriuretic factor (ANF) gene, but does not affect skeletal alpha-actin or myosin light chain-2v expression. As evidenced by nuclear run-on transcription assays, both CT-1 and alpha-adrenergic stimulation lead to an increase in ANF gene transcription. Transient transfection analyses document that, in contrast to alpha-adrenergic stimulation, the CT-1 responsive cis-regulatory elements are located outside of the proximal 3 kilobase pairs of the ANF 5'-flanking region. These studies indicate that CT-1 can activate a distinct form of myocardial cell hypertrophy, characterized by the promotion of sarcomere assembly in series, via gpl30/LIFRbeta-dependent signaling pathways.

The neuronal-specific Elav-like proteins (HuD, Hel-N, and HuC) contain three RNP-type concensus motifs and bind to AU-rich elements. We have identified and cloned a fourth member of this family (HuR) that is expressed in a wide variety of cell types. The purified recombinant protein binds avidly to the AU-rich element in c-fos and interleukin-3 mRNAs. In the case of the c-fos AU-rich element, HuR binds to a core element of 27 nucleotides that contain AUUUA, AUUUUA, and AUUUUUA motifs. Mutational analysis has shown that all three AU motifs are required for maximal binding.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) provokes a proliferative response and induction of early-response genes such as c-fos in target cells. It also induces rapid tyrosine phosphorylation of cellular proteins, including the beta subunit (betac) of its functional receptor. However, locations and functions of phosphorylated tyrosine residues within the betac are unclear. To elucidate the mechanism of the human GM-CSF receptor signal transduction, mutational analyses were made of the cytoplasmic domain of the beta-c, using murine BA/F3 cells. Deletion of the conserved box 1 motif resulted in loss of tyrosine phosphorylation of the betac, thereby indicating an essential role for this motif in activating the tyrosine kinase which phosphorylates betac. A C-terminal truncated mutant at position 589 activated the c-fos promoter, and this activation was diminished by a substitution at tyrosine 577 (Tyr577). However, the same substitution in the full-length betac did not completely abrogate the c-fos promoter activation, hence, redundant signaling pathways probably exist. When we analyzed signaling molecules functioning downstream of the beta-c we found that Tyr577 is essential for Shc phosphorylation, while tyrosine phosphorylation of PTP1D was mediated through Tyr577 as well as through other site(s). We suggest that GM-CSF stimulates at least two modes of signals leading to Ras activation, an event which ultimately gives rise to promoter activation of c-fos.

Transforming growth factor-beta (TGF-beta), a growth regulator of fetal hepatocytes in primary culture, also regulates death of these cells. Dose-response analysis showed that the TGF-beta concentration needed to induce hepatocyte death (2.5 ng/ml) was 5 times that needed to inhibit growth in these cells (0.5 ng/ml). In response to TGF-beta, hepatocytes induced DNA fragmentation and the appearance of nuclei with a DNA content lower than 2C (diploid content), typical of a programmed cell death model. TGF-beta-induced apoptosis in fetal hepatocytes was preceded by an induction of reactive oxygen species production and a decrease in the glutathione intracellular content, indicating that this factor induces oxidative stress in fetal hepatocytes. Studies performed to analyze levels of c-fos mRNA, a gene whose expression is modulated by redox state, demonstrated that only high, apoptotic concentrations of TGF-beta (2.5 ng/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of tert-butyl hydroperoxide. Gel mobility shift assays showed that the c-fos-induced expression was coincident with an increase in AP-1 activity. Finally, cell death induced by TGF-beta in fetal hepatocytes was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-beta in these cells. In summary, the results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-beta in fetal hepatocytes.

The genetic locus for the TrkC/neurotrophin 3 (NT-3) receptor tyrosine kinase encodes multiple isoforms including receptors with inserts in the catalytic domain. This study examines the signaling capabilities of TrkC and related kinase insert isoforms TrkC14 and TrkC25. We show that in PC12 cells expressing both TrkC and TrkA/nerve growth factor (NGF) receptors, different morphological changes occur upon addition of NGF or NT-3. NT-3-treated cells exhibit longer neurites and larger cell bodies as compared to NGF-treated cells. Both TrkC and TrkA mediate qualitatively similar increases in the tyrosine phosphorylation of phospholipase C (PLC)-1, Shc, SNT, and MAPK and the transcription of the c-fos, c-jun, NGFI-A, and NGFI-B immediate early genes. However, the TrkC kinase insert forms fail to stimulate these events. Furthermore, TrkC14 and TrkC25 have only a low intrinsic tyrosine kinase activity, and insertion of the TrkC14 kinase insert into TrkA at an equivalent position results in a dramatic reduction of the kinase activity and signaling capabilities of TrkA. The TrkC14 and −25 isoforms may fail to transmit signals due to their low intrinsic kinase activity and failure to activate and/or tyrosine phosphorylate targets shown to be involved in neurotrophin signal transduction pathways.

The cAMP/cAMP-dependent protein kinase (A-kinase) and Ca2+/calmodulin-dependent protein kinase (Cam-kinase) signal transduction pathways are well known to regulate gene transcription, but this has not been demonstrated directly for the cGMP/cGMP-dependent protein kinase (G-kinase) signal transduction pathway. Here we report that transfection of G-kinase into G-kinase-deficient cells causes activation of the human c-fos promoter in a strictly cGMP-dependent manner. The effect of G-kinase appeared to be mediated by several sequence elements, most notably the serum response element (SRE), the AP-1 binding site (FAP), and the cAMP response element (CRE). The magnitude of G-kinase transactivation of the fos promoter was similar to that of A-kinase, but there were significant differences between G-kinase and A-kinase activation of single enhancer elements and of a chimeric Gal4-CREB transcription factor. Our results indicate that G-kinase transduces signals to the nucleus independently of A-kinase or Ca2+, although it may target some of the same transcription factors as A-kinase and Cam-kinase.

The mitogen-activated protein kinase (MAPK) family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. In this study we investigated the factors controlling MAPK activation by H2O2 and explored the impact of altering the pathways to kinase activation on cell survival following H2O2 exposure. Potent activation (10-20-fold) of extracellular signal-regulated protein kinase (ERK2) occurred within 10 min of H2O2 treatment, whereupon rapid inactivation ensued. H2O2 activated ERK2 in several cell types and also moderately activated (3-5-fold) both c-Jun N-terminal kinase and p38/RK/CSBP. Additionally, H2O2 increased the mRNA expression of MAPK-dependent genes c-jun, c-fos, and MAPK phosphatase-1. Suramin pretreatment completely inhibited H2O2 stimulation of ERK2, highlighting a role for growth factor receptors in this activation. Further, ERK2 activation by H2O2 was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol, indicating that metal-catalyzed free radical formation mediates the initiation of signal transduction by H2O2. H2O2-stimulated activation of ERK2 was abolished in PC12 cells by inducible or constitutive expression of the dominant negative Ras-N-17 allele. Interestingly, PC12/Ras-N-17 cells were more sensitive than wild-type PC12 cells to H2O2 toxicity. Moreover, NIH 3T3 cells expressing constitutively active MAPK kinase (MEK, the immediate upstream regulator of ERK) were more resistant to H2O2 toxicity, while those expressing kinase-defective MEK were more sensitive, than cells expressing wild-type MEK. Taken together, these studies provide insight into mechanisms of MAPK regulation by H2O2 and suggest that ERK plays a critical role in cell survival following oxidant injury.

In response to changes in vascular homeostasis, endothelial cells secrete endothelin-1 (ET-1), which in turn regulates gene expression and phenotype in underlying vascular cells. We characterized a nuclear signaling cascade in which Src protein-tyrosine kinases link the ET-1 receptor to induction of c-fos transcription. A dominant negative SrcK- kinase mutant blocked ET-1-stimulated c-fos transcription. Expression of the COOH-terminal Src kinase (Csk), which represses Src kinases, also blocked induction of c-fos transcription by ET-1. Activation of the c-fos promoter by ET-1 required both the CArG DNA sequence of the c-fos serum response element and the Ca2+/cAMP response element. In contrast, Src-induced c-fos transcription required only the CArG cis-element, demonstrating a divergence in signals regulating c-fos transcription. Thus, Src kinases contribute to a nuclear signaling cascade linking an ET-1 receptor to the CArG element of the c-fos serum response element. A Src-based pathway might play a more general role to propagate ET-1 nuclear signals that regulate cell growth and development. In addition, these results point to a widening role for nonreceptor protein-tyrosine kinases in propagating signals from G protein-coupled receptors.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine that positively or negatively regulates the proliferation of various types of cells. In this study we have examined whether or not the activation of the mitogen-activated protein (MAP) kinases is involved in the transduction of cell growth modulation signals of TGF-beta 1, as MAP kinase activity is known to be closely associated with cell cycle progression. Although TGF-beta 1 stimulated the growth of quiescent Balb 3T3 and Swiss 3T3 cells, it failed to detectably stimulate the tyrosine phosphorylation and activation of the 41- and 43-kDa MAP kinases at any time point up to the reinitiation of DNA replication. TGF-beta 1 also failed to stimulate the expression of the c-fos gene. Furthermore, TGF-beta 1 synergistically enhanced the mitogenic action of epidermal growth factor (EGF) without affecting EGF-induced MAP kinase activation in these fibroblasts, and it inhibited the EGF-stimulated proliferation of mouse keratinocytes (PAM212) without inhibiting EGF-induced MAP kinase activation. Thus, the ability of TGF-beta 1 to modulate cell proliferation is apparently not associated with the activation of MAP kinases. In this respect, TGF-beta 1 is clearly distinct from the majority, if not all, of peptide growth factors, such as platelet-derived growth factor and EGF, whose ability to modulate cell proliferation is closely associated with the activation of MAP kinases. These results also suggest that the activation of MAP kinases is not an absolute requirement for growth factor-stimulated mitogenesis.

Exposure of primary cultures of embryonic rat striatal neurons to agents releasing nitric oxide (NO), including sin-1 molsidomine, S-nitroso-n-acetyl-penicillamine (SNAP), and S-nitrosoglutathione, resulted in an increase in the levels of expression of the immediate early genes c-fos and zif/268 in the cultured neurons. The membrane-permeable cGMP analogue, 8-bromo-cGMP, did not significantly affect c-fos and zif/268 mRNA levels, and the highly selective inhibitor of cGMP-dependent protein kinase, KT5823, was unable to inhibit the elevation in c-fos and zif/268 mRNA levels induced by SNAP. The induction of c-fos by the calcium ionophore A23187 was reduced by treatment with SNAP or 8-bromo-cGMP. Inhibitors of ADP-ribosyltransferases attenuated the stimulation of c-fos expression by SNAP. These results demonstrate for the first time that NO can induce immediate early gene expression in neurons, suggesting that NO may act as a mediator of neuronal plasticity via alterations in the expression of downstream genes. In addition, the results suggest that NO may exert these effects through a pathway that does not involve guanylate cyclase and cGMP-dependent protein kinase.