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The c-fos gene is rapidly induced to high levels by various extracellular stimuli. We used a defined in vitro transcription system that utilizes the c-fos promoter to purify a coactivator activity in an unbiased manner. We report here that NF45-NF90 and NF45-NF110, which possess archetypical double-stranded RNA binding motifs, have a direct function as transcriptional coactivators. The transcriptional activities of the nuclear factor (NF) complexes (NF45-NF90 and NF45-NF110) are mediated by both the upstream enhancer and core promoter regions of the c-fos gene and do not require their double-stranded RNA binding activities. The NF complexes cooperate with general coactivators, PC4 and Mediator, to elicit a high level of transcription and display multiple interactions with activators and the components of the general transcriptional machinery. Knockdown of the endogenous NF90/NF110 in mouse cells shows an important role for the NF complexes in inducing c-fos transcription. Chromatin immunoprecipitation assays demonstrate that the NF complexes occupy the c-fos enhancer/promoter region before and after serum induction and that their occupancies within the coding region of the c-fos gene increase in parallel to that of RNAPII upon serum induction. In light of their dynamic occupancy on the c-fos gene as well as direct functions in both transcription and posttranscriptional processes, the NF complexes appear to serve as multifunctional coactivators that coordinate different steps of gene expression to facilitate rapid response of inducible genes.

Rab7 belongs to the Ras superfamily of small GTPases and is a master regulator of early to late endocytic membrane transport. Four missense mutations in the late endosomal Rab7 GTPase (L129F, K157N, N161T, and V162M) cause the autosomal dominant peripheral neuropathy Charcot-Marie-Tooth type 2B (CMT2B) disease. As yet, the pathological mechanisms connecting mutant Rab7 protein expression to altered neuronal function are undefined. Here, we analyze the effects of Rab7 CMT2B mutants on epidermal growth factor (EGF)-dependent intracellular signaling and trafficking. Three different cell lines expressing Rab7 CMT2B mutants and stimulated with EGF exhibited delayed trafficking of EGF to LAMP1-positive late endosomes and lysosomes and slowed EGF receptor (EGFR) degradation. Expression of all Rab7 CMT2B mutants altered the Rab7 activation cycle, leading to enhanced and prolonged EGFR signaling as well as variable increases in p38 and ERK1/2 activation. However, due to reduced nuclear translocation of p38 and ERK1/2, the downstream nuclear activation of Elk-1 was decreased along with the expression of immediate early genes like c-fos and Egr-1 by the disease mutants. In conclusion, our results demonstrate that Rab7 CMT2B mutants impair growth factor receptor trafficking and, in turn, alter p38 and ERK1/2 signaling from perinuclear, clustered signaling endosomes. The resulting down-regulation of EGFR-dependent nuclear transcription that is crucial for normal axon outgrowth and peripheral innervation offers a crucial new mechanistic insight into disease pathogenesis that is relevant to other neurodegenerative diseases.

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

Ras-related nuclear protein (Ran) is a Ras family GTPase, and its documented functions are the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing and exportation, and nuclear protein importation. In this study, we performed detailed mapping of Ran expression during mouse ontogeny using in situ hybridization. High Ran expression was found in various organs and tissues including the thymus cortex and spleen white pulp. Ran was induced in T cells 24 h after their activation. The function of Ran in the immune system was investigated using Ran transgenic (Tg) mice. In Ran Tg T cells, there was compromised activation marker expression, lymphokine secretion, and proliferation upon T cell receptor activation in vitro when compared with wild type T cells. Tg mice also manifested defective delayed type hypersensitivity in vivo. Upon PMA and ionomycin stimulation, Tg T cells were defective in nuclear accumulation of AP-1 factors (c-Jun and c-Fos) but not NF-kappaB family members. Our experiments showed that Ran had important regulatory function in T cell activation. One of the possible mechanisms is that intracellular Ran protein levels control the nuclear retention for selective transcription factors such as c-Jun and c-Fos of AP-1, which is known to be critical in T cell activation and proliferation and lymphokine secretion.

The SWI/SNF chromatin-remodeling complex has been implicated in the activation and proliferation of T cells. After T cell receptor signaling, the SWI/SNF complex rapidly associates with chromatin and controls gene expression in T cells. However, the process by which the SWI/SNF complex regulates peripheral T cell activation has not been elucidated. In this study, we show that the SWI/SNF complex regulates cytokine production and proliferation of T cells. During T cell activation, the SWI/SNF complex is recruited to the promoter of the transcription factor AP-1, and it increases the expression of AP-1. Increased expression of the SWI/SNF complex resulted in enhanced AP-1 activity, cytokine production, and proliferation of peripheral T cells, whereas knockdown of the SWI/SNF complex expression impaired the AP-1 expression and reduced the activation and proliferation of T cells. Moreover, mice that constitutively expressed the SWI/SNF complex in T cells were much more susceptible to experimentally induced autoimmune encephalomyelitis than the normal mice were. These results suggest that the SWI/SNF complex plays a critical role during T cell activation and subsequent immune responses.

Regulation of follicle-stimulating hormone (FSH) synthesis is a central point of convergence for signals controlling reproduction. The FSHbeta subunit is primarily regulated by gonadotropin-releasing hormone (GnRH), gonadal steroids, and activin. Here, we identify elements in the mouse FSHbeta promoter responsible for GnRH-mediated induction utilizing the LbetaT2 cell line that endogenously expresses FSH. The proximal 398 bp of the mouse FSHbeta promoter is sufficient for response to GnRH. This response localizes primarily to an AP-1 half-site (-72/-69) juxtaposed to a CCAAT box, which binds nuclear factor-Y. Both elements are required for AP-1 binding, creating a novel AP-1 site. Multimers of this site confer GnRH induction, and mutation or internal deletion of this site reduces GnRH induction by 35%. The same reduction was achieved using a dominant negative Fos protein. This is the only functional AP-1 site identified in the proximal 398 bp, since its mutation eliminates FSHbeta induction by c-Fos and c-Jun. GnRH regulation of the FSHbeta gene occurs through induction of multiple Fos and Jun isoforms, forming at least four different AP-1 molecules, all of which bind to this site. Mitogen-activated protein kinase activity is required for induction of FSHbeta and JunB protein. Finally, AP-1 interacts with nuclear factor-Y, which occupies its overlapping site in vivo.

POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus. Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. FBI-1 is a BTB/POZ-domain protein that has been shown to modulate HIV-1 Tat trans-activation and to repress transcription of some cellular genes. We have used various viral and cellular FBI-1 binding sites to characterize the interaction of a POZ-domain protein with DNA in detail. We find that FBI-1 binds to inverted sequence repeats downstream of the HIV-1 transcription start site. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible. Indeed, FBI-1 binding sites in the adenovirus 2 major late promoter, the c-fos gene, and the c-myc P1 and P2 promoters reveal variously spaced direct, inverted, and everted sequence repeats with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T) for each repeat.

The duration as well as the magnitude of mitogen-activated protein kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. Activation of ERK1/2 by the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is relatively sustained in alpha T3-1 pituitary gonadotropes and HEK293 cells but is transient in immortalized GT1-7 neurons. Each of these cell types expresses the epidermal growth factor receptor (EGFR) and responds to EGF stimulation with significant but transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation caused by EGFR transactivation was confined to GT1-7 cells and was attenuated by EGFR kinase inhibition. Neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus in GT1-7 cells. In contrast, agonist stimulation of GnRH receptors expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a protein kinase C-dependent but EGFR-independent pathway. GnRH-induced activation of ERK1/2 was attenuated by the selective Src kinase inhibitor PP2 and the negative regulatory C-terminal Src kinase in GT1-7 cells but not in HEK293 cells. In GT1-7 cells, GnRH stimulated phosphorylation and nuclear translocation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1). These results indicate that the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells. Whereas activation of the Gq/protein kinase C pathway in HEK293 cells causes sustained phosphorylation and translocation of ERK1/2 to the nucleus, transactivation of the EGFR by GnRH in GT1-7 cells elicits transient ERK1/2 signals without nuclear accumulation. These findings suggest that transactivation of the tightly regulated EGFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain G protein-coupled receptors.

Heat shock factor 1 (HSF1), in addition to its pivotal role as a regulator of the heat shock response, functions as a versatile gene repressor. We have investigated the structural domains involved in gene repression using mutational analysis of the hsf1 gene. Our studies indicate that HSF1 contains two adjacent sequences located within the N-terminal half of the protein that mediate the repression of c-fos and c-fms. One region (NF) appears to be involved in quenching transcriptional activation factors on target promoters and binds to the basic zipper transcription factor NF-IL6 required for activation of c-fms and IL-1beta. The NF domain encompasses the leucine zipper 1 and 2 sequences as well as the linker domain between the DNA binding and leucine zipper regions. The function of this domain in gene repression is highly specific for HSF1, and the homologous region from conserved family member HSF2 does not restore repressive function in HSF2/HSF1 chimeras. In addition, HSF2 is not capable of binding to NF-IL6. The NF domain, although necessary for repression, is not sufficient, and a second region (REP) occupying a portion of the regulatory domain is required for repression. Neither domain functions independently, and both are required for repression. Furthermore, we constructed dominant inhibitors of c-fos repression by HSF1, which also blocked the repression of c-fms and IL-1beta, suggesting a shared mechanism for repression of these genes by HSF1. Our studies suggest a complex mechanism for gene repression by HSF1 involving the binding to and quenching of activating factors on target promoters. Mapping the structural domains involved in this process should permit further characterization of molecular mechanisms that mediate repression.

Transcriptional regulation of the fos promoter by nitric oxide and cGMP can occur by nuclear translocation of cGMP-dependent protein kinase I (G-kinase I) (Gudi, T., Lohmann, S. M., and Pilz, R. B. (1997) Mol. Cell. Biol. 17, 5244-5254). To identify nuclear targets of G-kinase I, we performed a yeast two-hybrid screen with G-kinase I beta as bait. We found that G-kinase I beta interacted specifically with TFII-I, an unusual transcriptional regulator that associates with multiple proteins to modulate both basal and signal-induced transcription. By using purified recombinant proteins, the interaction was mapped to the N-terminal 93 amino acids of G-kinase I beta and one of six 95-amino acid repeats found in TFII-I. In baby hamster kidney cells, cGMP analogs enhanced co-immunoprecipitation of G-kinase I beta and TFII-I by inducing co-localization of both proteins in the nucleus, but in other cell types containing cytoplasmic TFII-I the G-kinase-TFII-I interaction was largely cGMP-independent. G-kinase phosphorylated TFII-I in vitro and in vivo on Ser(371) and Ser(743) outside of the interaction domain. G-kinase strongly enhanced TFII-I transactivation of a serum-response element-containing promoter in COS7 cells, and this effect was lost when Ser(371) and Ser(743) of TFII-I were mutated. TFII-I by itself had little effect on a full-length fos promoter in baby hamster kidney cells, but it synergistically enhanced transcriptional activation by G-kinase I beta. Binding of G-kinase to TFII-I may position the kinase to phosphorylate and regulate TFII-I and/or factors that interact with TFII-I at the serum-response element.

The Vav family of guanine nucleotide exchange factors for Rho family GTPases plays a critical role in lymphocyte proliferation, gene transcription, and cytoskeleton reorganization following immunoreceptor stimulation. However, its role in immediate early gene activation is unclear. In this study, we have investigated the mechanisms by which Vav1 can regulate c-fos serum response element transcriptional activity. We show that T cell antigen receptor (TCR) stimulation induces the phosphorylation of serum response factor (SRF) on serine 103 and increases the binding of SRF complexes on serum response element in a MEK- and p38-dependent pathway. The physiological relevance of our findings is supported by the inhibition of the interleukin-2 gene transcriptional activity by a dominant negative SRF mutant. Overexpression of Vav1, which partially mimics TCR stimulation, promotes SRF-dependent transcription, and dominant negative Vav1 mutants block SRF activation by TCR. SRF activation by Vav1 occurs through a signaling cascade consisting of Rac1/Cdc42 and the serine/threonine kinases Pak1 and MEK, but independently of the phosphatidylinositol 3-kinase pathway. Interestingly, Vav2 also enhances SRF through Rho GTPases, suggesting that Vav proteins are general regulators of SRF activation in lymphocytes. This report establishes Vav proteins as a direct link between antigen receptors and SRF-dependent early gene expression.

Mutational activation of the Wnt signaling pathway is a common early event in colorectal tumorigenesis, and the identification of target genes regulated by this pathway will provide a better understanding of tumor progression. Gene expression profiling on oligonucleotide microarrays revealed reduced expression of the immediate early genes fos and fosB following stimulation of cells by Wnt-1. Further analysis demonstrated that serum or 12-O-tetradecanoylphorbol-13-acetate activation of several immediate early genes including fos, fosB, junB, and egr1 was inhibited by Wnt signaling. Wnt signaling inhibited transcriptional activation driven by the serum response element without altering the activation of the extracellular signal-regulated kinase cascade or ternary complex formation at the fos serum response element promoter. The Wnt-mediated repression of c-Fos, FosB, and JunB expression was consistent with a decrease in their binding to an AP-1 promoter element and decreased target gene transcription. The expression of fos, fosB, junB, and egr1 was also repressed in human colon tumors relative to patient matched normal tissue. By contrast, the fos family member fra-1 was up-regulated in the human colon tumors, suggesting a compensatory mechanism for the reduction in fos and fosB expression. The results indicate that Wnt signaling can repress the expression of certain immediate early genes, and that this effect is consistent with changes in gene expression observed in human colorectal tumors.

Vav1 and Vav2 are members of the Dbl family of guanine nucleotide exchange factors for the Rho family of small GTPases. Although the role of Vav1 during lymphocyte development and activation is well characterized, the function of Vav2 is still unclear. In this study, we compared the signaling pathways regulated by Vav1 and Vav2 following engagement of the T cell receptor (TCR). We show that Vav2 is tyrosine-phosphorylated upon TCR stimulation and by co-expressed Src and Syk family kinases. Using glutathione S-transferase fusion proteins, we observed that the Src homology 2 domain of Vav2 binds tyrosine-phosphorylated proteins from TCR-stimulated Jurkat T cell lysates, including c-Cbl and SLP-76. Like Vav1, Vav2 cooperated with TCR stimulation to increase extracellular signal-regulated kinase activation and to promote c-fos serum response element transcriptional activity. Moreover, both proteins displayed a similar action in increasing the expression of the early activation marker CD69 in Jurkat T cells. However, in contrast to Vav1, Vav2 dramatically suppressed TCR signals leading to nuclear factor of activated T cells (NF-AT)-dependent transcription and induction of the interleukin-2 promoter. Vav2 appears to act upstream of the phosphatase calcineurin because a constitutively active form of calcineurin rescued the effect of Vav2 by restoring TCR-induced NF-AT activation. Interestingly, the Dbl homology and Src homology 2 domains of Vav2 were necessary for its inhibitory effect on NF-AT activation and for induction of serum response element transcriptional activity. Taken together, our results indicate that Vav1 and Vav2 exert overlapping but nonidentical functions in T cells. The negative regulatory pathway elicited by Vav2 might play an important role in regulating lymphocyte activation processes.

Serum response factor (SRF) plays an important role in regulating smooth muscle cell (SMC) development and differentiation. To understand the molecular mechanisms underlying the activity of SRF in SMCs, the two CArG box-containing elements in the arterial SMC-specific SM22alpha promoter, SME-1 and SME-4, were functionally and biochemically characterized. Mutations that abolish binding of SRF to the SM22alpha promoter totally abolish promoter activity in transgenic mice. Moreover, a multimerized copy of either SME-1 or SME-4 subcloned 5' of the minimal SM22alpha promoter (base pairs -90 to +41) is necessary and sufficient to restrict transgene expression to arterial SMCs in transgenic mice. In contrast, a multimerized copy of the c-fos SRE is totally inactive in arterial SMCs and substitution of the c-fos SRE for the CArG motifs within the SM22alpha promoter inactivates the 441-base pair SM22alpha promoter in transgenic mice. Deletion analysis revealed that the SME-4 CArG box alone is insufficient to activate transcription in SMCs and additional 5'-flanking nucleotides are required. Nuclear protein binding assays revealed that SME-4 binds SRF, YY1, and four additional SMC nuclear proteins. Taken together, these data demonstrate that binding of SRF to specific CArG boxes is necessary, but not sufficient, to restrict transgene expression to SMCs in vivo.

Retinoic acids exert profound effects on many biological processes including cell proliferation, differentiation, and morphogenesis. We previously reported that all-trans-retinoic acid (t-RA) protected mesangial cells from H(2)O(2)-triggered apoptosis by suppressing the activator protein 1 (AP-1) pathway. It was via inhibition of c-fos and c-jun expression and suppression of c-Jun N-terminal kinase (JNK) activation. In this report, we investigated the involvement of retinoic acid receptor (RAR) and retinoid X receptor (RXR) in the antiapoptotic effect of t-RA in H(2)O(2)-exposed cells. We found that pretreatment with RAR pan-antagonist (AGN193109) or RXR pan-antagonist (HX531) attenuated the antiapoptotic effect of t-RA. Similarly, transient transfection with a dominant-negative mutant of RAR or a dominant-negative RXR diminished the antiapoptotic effect of t-RA. Both RAR and RXR antagonists reversed the suppressive effect of t-RA on AP-1 activity. However, the roles of RAR and RXR in the suppression of AP-1 components by t-RA were found to be different. RAR antagonist reversed the suppressive effect of t-RA on both c-fos and c-jun, whereas RXR antagonist reversed the effect of t-RA on c-fos but not c-jun. Furthermore, suppression of JNK activation by t-RA was observed even in the presence of RAR and RXR antagonists. Consistently, suppression of JNK by t-RA was not affected by overexpression of either the dominant-negative RAR or the dominant-negative RXR. These data elucidated that the antiapoptotic effect of t-RA is mediated by both nuclear receptor-dependent and -independent mechanisms.

A prominent pathway of transforming growth factor (TGF)-beta signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-beta1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-beta1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-beta1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [(3)H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-beta1. Lack of Smad2 or Smad3 expression did not affect TGF-beta1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-beta1. Moreover, TGF-beta1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-beta1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-beta-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element)(4)-Lux reporter by TGF-beta1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-beta1 signaling.

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha, although the events through which TNF-alpha induces ROS generation are not yet well characterized. We therefore investigated selected candidates likely to mediate TNF-alpha-induced ROS generation. Consistent with the role of Rac in that process, stable expression of Rac(Asn-17), a dominant negative Rac1 mutant, completely blocked TNF-alpha-induced ROS generation. To understand better the mediators downstream of Rac, we investigated the involvement of cytosolic phospholipase A(2) (cPLA(2)) activation and metabolism of the resultant arachidonic acid (AA) by 5-lipoxygenase (5-LO). TNF-alpha-induced ROS generation was blocked by inhibition of cPLA(2) or 5-LO, but not cyclooxygenase, suggesting that TNF-alpha-induced ROS generation is dependent on synthesis of AA and its subsequent metabolism to leukotrienes. Consistent with that hypothesis, TNF-alpha Rac-dependently stimulated endogenous production of leukotriene B(4) (LTB(4)), while exogenous application of LTB(4) increased levels of ROS. In contrast, application of leukotrienes C(4), D(4), and E(4) or prostaglandin E(2) had little effect. Our findings suggest that LTB(4) production by 5-LO is situated downstream of the Rac-cPLA(2) cascade, and we conclude that Rac, cPLA(2), and LTB(4) play pivotal roles in the ROS-generating cascade triggered by TNF-alpha.

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.

The adipocyte-derived hormone leptin signals the status of body energy stores by activating the long form of the leptin receptor (LRb). Activation of LRb results in the activation of the associated Jak2 tyrosine kinase and the transmission of downstream phosphotyrosine-dependent signals. We have investigated the signaling function of mutant LRb intracellular domains under the control of the extracellular erythropoietin (Epo) receptor. By using this system, we confirm that two tyrosine residues in the intracellular domain of murine LRb become phosphorylated to mediate LRb signaling; Tyr(985) controls the tyrosine phosphorylation of SHP-2, and Tyr(1138) controls STAT3 activation. We furthermore investigated the mechanisms by which LRb controls downstream ERK activation and c-fos and SOCS3 message accumulation. Tyr(985)-mediated recruitment of SHP-2 does not alter tyrosine phosphorylation of Jak2 or STAT3 but results in GRB-2 binding to tyrosine-phosphorylated SHP-2 and is required for the majority of ERK activation during LRb signaling. Tyr(985) and ERK activation similarly mediate c-fos mRNA accumulation. In contrast, SOCS3 mRNA accumulation requires Tyr(1138)-mediated STAT3 activation. Thus, the two LRb tyrosine residues that are phosphorylated during receptor activation mediate distinct signaling pathways as follows: SHP-2 binding to Tyr(985) positively regulates the ERK --c-fos pathway, and STAT3 binding to Tyr(1138) mediates the inhibitory SOCS3 pathway.

In many cell types, increased intracellular calcium gives rise to a robust induction of c-fos gene expression. Here we show that in mouse Ltk(-) fibroblasts, calcium ionophore acts in synergy with either cAMP or PMA to strongly induce the endogenous c-fos gene. Run-on analysis shows that this corresponds to a substantial increase in active polymerases on downstream gene sequences, i.e. relief of an elongation block by calcium. Correspondingly a chimeric gene, in which the human metallothionein promoter is fused to the fos gene, is strongly induced by ionophore alone, unlike a c-fos promoter/beta-globin coding unit chimeric construct. Internal deletions in the hMT-fos reporter localize the intragenic calcium regulatory element to the 5' portion of intron 1, thereby confirming and extending previous in vitro mapping data. Ionophore induced cAMP response element-binding protein phosphorylation on Ser(133) without affecting the extracellular signal-regulated kinase cascade. Surprisingly, induction involved neither CaM-Ks nor calcineurin, while the calmodulin antagonist W7 activated c-fos transcription on its own. These data suggest that a novel calcium signaling pathway mediates intragenic regulation of c-fos expression via suppression of a transcriptional pause site.

CrkL is an SH2 and SH3 domain-containing adaptor protein implicated in pathogenesis of chronic myelogenous leukemia. Here, we demonstrate that overexpression of CrkL enhances the erythropoietin (Epo)- or interleukin (IL)-3-induced activation of Elk-1 and the c-fos gene promoter activity in 32D/EpoR-Wt cells. Moreover, the Epo-induced activation of ERK1 and ERK2 was augmented and prolonged in cells inducibly overexpressing CrkL. A moderate increase in Epo-induced activation of JNK was also observed in cells overexpressing CrkL. Overexpression of C3G enhanced the Elk-1 activation synergistically with CrkL, while a C3G mutant lacking the guanine nucleotide exchange domain showed an inhibitory effect. Studies using a dominant negative Ha-Ras mutant demonstrated that the Elk-1 and ERK2 activation enhanced by CrkL and C3G was dependent on Ras. Consistent with this, the Epo-induced activation of Ras was augmented in cells inducibly overexpressing CrkL. Most importantly, a CrkL mutant defective in the SH2 or N-terminal SH3 domain showed an inhibitory effect on the Epo-induced activation of ERK2. These data indicate that the CrkL-C3G complex plays a role in Epo- or IL-3-induced, Ras-dependent activation of the Raf/ERK pathway leading to the activation of Elk-1 and the c-fos gene transcription.

With increasing size, multicellular prostate tumor spheroids develop regions of quiescent, multidrug-resistant cells expressing the cyclin-dependent kinase inhibitor p27(kip1). Treatment of small (diameter 60 +/- 20 micrometer) spheroids with 200 microM hydrogen peroxide (H(2)O(2)) resulted in cell cycle arrest owing to up-regulation of p27(kip1) and down-regulation of the transcription factor c-Fos. Incubation with 100 nM-1 microM H(2)O(2) led to up-regulation of c-Fos and enhanced tumor growth. Growth stimulation was inhibited by bisindolylmaleimide I, indicating a role for protein kinase C in the signaling cascade that involved the mitogen-activated protein kinase members MEK1,2, ERK1, -2, and c-Jun N-terminal kinase. Changes in Ca(2+) influx underlined the differential effects of H(2)O(2). Incubation with 200 microM H(2)O(2) released [Ca(2+)](i) from intracellular stores followed by prolonged Ca(2+) influx. Inhibition of influx by Ca(2+)-free media or Ni(2+), La(3+), Mn(2+) and SKF-96365 prevented the induction of quiescence and stimulated spheroid growth. Consequently, treatment with 200 microM H(2)O(2) in Ca(2+)-free media down-regulated p27(kip1) and increased Fos protein. ATP exerted effects comparably to those observed with H(2)O(2). Encoding growth stimulation by [Ca(2+)](i) release and induction of cell quiescence by prolonged Ca(2+) influx may provide a general mechanism for the control of tumor growth.

We investigated the extent to which phosphatidylinositol 3-kinase (PI 3-kinase) and Rac, a member of the Rho family of small GTPases, are involved in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha leading to activation of c-fos serum response element (SRE) and c-Jun amino-terminal kinase (JNK) in Rat-2 fibroblasts. Inhibition of PI 3-kinase by LY294002 or wortmannin, two specific PI 3-kinase antagonists, or co-transfection with a dominant negative mutant of PI 3-kinase dose-dependently blocked stimulation of c-fos SRE by TNF-alpha. Similarly, LY294002 significantly diminished TNF-alpha-induced activation of JNK, suggesting that nuclear signaling triggered by TNF-alpha is dependent on PI 3-kinase-mediated activation of both c-fos SRE and JNK. We also found nuclear signaling by TNF-alpha to be Rac-dependent, as demonstrated by the inhibitory effect of transient co-transfection with a dominant negative Rac mutant, RacN17. Our findings suggest that Rac is situated downstream of PI 3-kinase in the TNF-alpha signaling pathway to the nucleus, and we conclude that PI 3-kinase and Rac each plays a pivotal role in the nuclear signaling cascade triggered by TNF-alpha.

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fos and c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.

T cell development and function in complex ganglioside-lacking (GM2/GD2 synthase gene-disrupted) mice were analyzed. GM1, asialo-GM1, and GD1b were representative gangliosides expressed on T cells of the wild type mice and completely deleted on those of the mutant mice. The sizes and cell numbers of the mutant mice spleen and thymus were significantly reduced. Spleen cells from the mutant mice showed clearly reduced proliferation compared with the wild type when stimulated by interleukin 2 (IL-2) but not when treated with concanavalin A or anti-CD3 cross-linking. Expression levels of IL-2 receptor alpha, beta, and gamma were almost equivalent, and up-regulation of alpha chain after T cell activation was also similar between the mutant and wild type mice. Activation of JAK1, JAK3, and SAT5 after IL-2 treatment was reduced, and c-fos expression was delayed and reduced in the mutant spleen cells, suggesting that the IL-2 signal was attenuated in the mutant mice probably due to the modulation of IL-2 receptors by the lack of complex gangliosides.

The effects of UVB irradiation on transcription factor activator protein-1 (AP-1) DNA binding and AP-1 transactivation were studied in a human keratinocyte cell line, HaCaT. UVB-induced AP-1 binding to a consensus AP-1 binding site was observed by gel shift assays with maximum stimulation at 12 h after UVB irradiation. A promoter region of the human collagenase-1 gene containing the same AP-1 binding sequence linked to a luciferase reporter gene was stably transfected into HaCaT cells. UVB irradiation significantly increased luciferase activity in these stably transfected cells, with maximum activity observed at 24 h after UVB irradiation. c-Fos and Jun D were identified by antibody clearing assays as the main components of the bound AP-1 complexes. Inhibition of transcription with actinomycin D and inhibition of protein synthesis with cycloheximide significantly abrogated the effect of UVB on AP-1 DNA binding, indicating that transcription and translation were required for AP-1 activation. Northern and Western analyses revealed a correlation between increased AP-1 activity and accumulation of c-fos mRNA and c-Fos protein after UVB irradiation. UVB irradiation increased c-fos transcription in HaCaT cells stably transfected with a plasmid containing the human c-fos promoter driving a luciferase reporter gene. These results suggest that increased c-fos expression may play an important role in UVB-induced AP-1 activation in HaCaT cells.

Expression of the lactate dehydrogenase A subunit (LDH-A) gene can be controlled by transcriptional as well as posttranscriptional mechanisms. In rat C6 glioma cells, LDH-A mRNA is stabilized by activation and synergistic interaction of protein kinases A and C. In the present study, we aimed to identify the sequence domain which determines and regulates mRNA stability/instability by protein kinase A and focused our attention on the 3'-untranslated region (3'-UTR) of LDH-A mRNA. We have constructed various chimeric globin/lactate dehydrogenase (ldh) genes linked to the c-fos promoter and stably transfected them into rat C6 glioma cells. After their transfection, we determined the half-life of transcribed chimeric globin/ldh mRNAs. The results showed that at least three sequence domains within the LDH-A 3'-UTR consisting of nucleotides 1286-1351, 1453-1471, and 1471-1502 are responsible for the relatively rapid rate of LDH-A mRNA turnover in the cytoplasm. Whereas chimeric globin/ldh mRNAs containing the base sequences 1286-1351 and 1453-1471 were not stabilized by (Sp)-cAMPS, an activator of protein kinase A, instability caused by the 1471-1502 domain was significantly reversed. Additional deletion and mutational analyses demonstrated that the 3'-UTR fragment consisting of the 22 bases 1478-1499 is a critical determinant for the (Sp)-cAMPS-mediated LDH-A mRNA stabilizing activity. Because of its functional characteristics, we named the 22-base region "cAMP-stabilizing region."

The Elav-like proteins are specific mRNA-binding proteins that regulate mRNA stability. The neuronal members of this family (HuD, HuC, and Hel-N1) are required for neuronal differentiation. In this report, using purified HuD protein we have localized a high affinity HuD binding site to a 42-nucleotide region within a U-rich tract in the 3′-untranslated region p21waf1 mRNA. The binding of HuD to this site is readily displaced by an RNA oligonucleotide encoding the HuD binding site of c-fos. The sequence of this binding site is well conserved in human, mouse, and rat p21waf1 mRNA. p21waf1 is an inhibitor of cyclin-dependent kinases and proliferating cell nuclear antigen and induces cell cycle arrest at G1/S, a requisite early step in cell differentiation. The identification of an Elav-like protein binding site in the 3′-untranslated region of p21waf1 provides a novel link between the induction of differentiation, mRNA stability, and the termination of the cell cycle.

Recent evidence indicates that the epidermal growth factor (EGF) receptor mediates a branch of lysophosphatidic acid (LPA)-induced signal transduction pathways that activate mitogen-activated protein (MAP) kinase. However, it is unclear whether the intrinsic tyrosine kinase activity of EGF receptor is involved. We previously showed that reactive oxygen species (ROS) were involved in the LPA-stimulated MAP kinase pathway. Here, we identify tyrosine phosphorylation of EGF receptor as an LPA signaling step that requires ROS. To evaluate the role of the tyrosine kinase activity of EGF receptor in the LPA-stimulated MAP kinase pathway, we examined the effects of an EGF receptor-specific tyrosine kinase inhibitor, PD158780. PD158780 potently inhibited the LPA-stimulated MAP kinase kinase 1/2 (MKK1/2) activation and EGF receptor tyrosine phosphorylation in HeLa cells, while it had no detectable effect on c-Src kinase activity. PD158780 also inhibited LPA-induced MKK1/2 activation and DNA synthesis in NIH 3T3 cells. Furthermore, we compared LPA-stimulated MKK1/2 and MAP kinase activation, transcriptional activity of the c-fos promoter, and DNA synthesis in B82L cells, which lack endogenous EGF receptor, and B82L cells expressing kinase-defective or wild-type human EGF receptor. Results obtained from analysis of these cell lines suggest that the EGF receptor tyrosine kinase contributes to the LPA-stimulated MAP kinase activation, c-fos transcription, and mitogenesis.

The ribotoxic stress response, which is conserved between prokaryotes and eukaryotes, is a cellular reaction to cytotoxic interference with the function of the 3'-end of the large (23 S/28 S) ribosomal RNA. The 3'-end of the large rRNA is directly involved in the three sequential steps of translational elongation: the aminoacyl-tRNA binding, the peptidyl transfer, and the ribosomal translocation. In mammalian cells, the ribotoxic stress response involves activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase and the p38 mitogen-activated protein kinase and transcriptional induction of immediate early genes such as c-fos and c-jun. Active ribosomes are essential mediators of the ribotoxic stress response. We demonstrate here that the transcriptional response of mammalian cells to ultraviolet radiation (UV response) displays the characteristics of a ribotoxic stress response, inasmuch as (i) the activation of stress kinases and gene expression in response to UV requires the presence of active ribosomes at the moment of irradiation; (ii) UV irradiation inhibits protein synthesis; and (iii) irradiation of cells with UV causes specific damage to the 3'-end of the 28 S rRNA. In contrast, the activation of the stress kinases by hyperosmolarity, by the DNA-cross-linking agent diepoxybutane, or by growth factors and cytokines does not depend on the presence of active ribosomes. Our results identify UV as a potential ribotoxic stressor and support the notion that some of the cellular signaling cascades in response to UV might be generated in the ribosome, possibly triggered by damage to rRNA.

The mechanisms by which HMG-I proteins regulate cell growth are unknown, and their effects on gene expression have only been partially elucidated. We explored the potential interaction between HMG-I proteins and serum-response factor (SRF), a member of the MADS-box family of transcription factors. In cotransfection experiments, HMG-I(Y) potentiated SRF-dependent activation (by more than 5-fold) of two distinct SRF-responsive promoters, c-fos and the smooth muscle-specific gene SM22alpha. This effect was also observed with a heterologous promoter containing multiple copies of the CC(A/T)6GG (CArG) box. HMG-I proteins bound specifically to the CArG boxes of c-fos and SM22alpha in gel mobility shift analysis and enhanced binding of SRF to these CArG boxes. By chelating peptide-immobilized metal affinity chromatography, we mapped the domain of HMG-I(Y) that interacts with SRF to amino acids 50-81, a region that does not bind specifically to DNA in electrophoretic mobility shift assays even though it includes the third AT-hook DNA-binding domain. Surprisingly, HMG-I(Y) mutants that failed to bind DNA still enhanced SRF binding to DNA and SRF-dependent transcription. In contrast, deletion of the HMG-I(Y) 50-81 domain that bound SRF prevented enhancement of transcription. To our knowledge, this is the first report of an HMG-I protein interacting with a MADS-box transcription factor. Our observations suggest that members of the HMG-I family play an important role in SRF-dependent transcription and that their effect is mediated primarily by a protein-protein interaction.

Retinoids, including retinol and retinoic acid derivatives, inhibit the growth of normal human bronchial epithelial (HBE) cells. The signaling pathways through which retinoids mediate this effect have not been defined. Normal HBE cell growth is stimulated by treatment with a variety of growth factors that increase mitogen-activated protein (MAP) activity. In this study, we examined MAP kinase-dependent pathways as potential targets of retinoid signaling and the role of MAP kinases in retinoid-induced c-fos gene regulation. All-trans-retinoic acid (t-RA) inhibited Jun N-terminal kinase (JNK) and, to a lesser extent, extracellular signal-regulated kinase activity in normal HBE cells. t-RA reduced c-fos mRNA and protein levels by decreasing c-fos gene transcription. The c-fos promoter was activated by co-transfection with a constitutively active JNK kinase (SEK)-1 and suppressed by a dominant negative JNK kinase kinase (MEKK)-1. Furthermore, c-fos expression was inhibited by agonists of retinoic acid receptors (RARs) or retinoid X receptors (RXRs), and suppression of c-fos promoter activity by t-RA was abrogated by treatment with antagonists of RAR-alpha or of all the RXRs. These findings provide the first evidence that t-RA inhibits JNK activity and demonstrate a potential role of JNK-dependent pathways in the suppression of c-fos expression by t-RA. Furthermore, c-fos expression was inhibited through activation of RAR- and RXR-dependent signaling pathways. In light of the growth activation induced by JNK/SEK-dependent pathways in a variety of cells, these data support further investigation into the role of JNK-dependent signaling in the growth-suppressive effects of retinoids.

The Src homology 2 (SH2)-containing protein tyrosine phosphatase 1, SHP-1, is highly expressed in all hematopoietic cells as well as in many non-hematopoietic cells, particularly in some malignant epithelial cell lines. In hematopoietic cells, SHP-1 negatively regulates multiple cytokine receptor pathways. The precise function and the targets of SHP-1 in non-hematopoietic cells, however, are largely unknown. Here we demonstrate that SHP-1 associates with both the tyrosine-phosphorylated platelet-derived growth factor (PDGF) receptor and the p85 subunit of phosphatidylinositol 3-kinase in MCF-7 and TRMP cells. Through the use of mutant PDGF receptors and performing peptide competition for immunoprecipitation, it was determined that SHP-1 independently associates with the PDGF receptor and p85 and that its N-terminal SH2 domain is directly responsible for the interactions. Overexpression of SHP-1 in TRMP cells transfected with the PDGF receptor markedly inhibited PDGF-induced c-fos promoter activation, whereas the expression of three catalytically inactive SHP-1 mutants increased the c-fos promoter activation in response to PDGF stimulation. These results indicate that SHP-1 might negatively regulate PDGF receptor-mediated signaling in these cells. Identification of the association of SHP-1 with the PDGF receptor and p85 in MCF-7 and TRMP cells furthers our understanding of the function of SHP-1 in non-hematopoietic cells.

Heat shock factor 1, the critical molecular regulator of the stress response is conserved throughout eukaryotic organisms and activates the transcription of heat shock genes. We now show that heat shock factor 1 inhibits the expression of c-fos, an immediate early gene that controls responses to extracellular stimuli for growth and differentiation. Heat shock factor 1 inhibits the transcription of the c-fos gene and antagonizes the activating effects of the signal transducing protein Ras on the c-fos promoter and on the promoter of another Ras responsive gene uPA. This property was specific for heat shock factor 1; c-fos repression was not seen with the structurally related protein heat shock factor 2. Repression involved different molecular mechanisms compared with those involved in transcriptional activation by heat shock factor 1 and specifically did not require binding to the c-fos promoter. Thus, in addition to its known role as a transcriptional activator of the cellular heat shock response, heat shock factor 1 also antagonizes the expression of Fos, a key component of the ubiquitous AP-1 transcription factor complex and as such could influence multiple aspects of cell regulation.

Tec is the prototype of a recently emerging subfamily among nonreceptor type protein-tyrosine kinases and is known to become tyrosine-phosphorylated and activated by a wide range of cytokine stimulations in hematopoietic cells. Although Tec was recently shown to be involved in the cytokine-driven activation mechanism of c-fos transcription, it is yet obscure how Tec relays the signals from cell surface receptors to the nucleus. To identify signaling molecules acting downstream of Tec, we have looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid system. Here we report the identification and characterization of a novel protein, TIP3, which has been simultaneously identified by other groups as SOCS-1, JAB, or SSI-1. TIP3 carries one Src homology 2 domain with a sequence similarity to that of CIS. In 293 cells, TIP3 associates with Tec and suppresses its kinase activity. Interestingly, TIP3 can also down-regulate the activity of Jak2 but not that of Lyn. We propose that SOCS-1/JAB/SSI-1/TIP3 is a novel type of negative regulator to a subset of protein-tyrosine kinases.

It is well established that mitogens inhibit differentiation of skeletal muscle cells, but the insulin-like growth factors (IGFs), acting through a single receptor, stimulate both proliferation and differentiation of myoblasts. Although the IGF-I mitogenic signaling pathway has been extensively studied in other cell types, little is known about the signaling pathway leading to differentiation in skeletal muscle. By using specific inhibitors of the IGF signal transduction pathway, we have begun to define the signaling intermediates mediating the two responses to IGFs. We found that PD098059, an inhibitor of mitogen-activated protein (MAP) kinase kinase activation, inhibited IGF-stimulated proliferation of L6A1 myoblasts and the events associated with it, such as phosphorylation of the MAP kinases and elevation of c-fos mRNA and cyclin D protein. Surprisingly, PD098059 caused a dramatic enhancement of differentiation, evident both at a morphological (fusion of myoblasts into myotubes) and biochemical level (elevation of myogenin and p21 cyclin-dependent kinase inhibitor expression, as well as creatine kinase activity). In sharp contrast, LY294002, an inhibitor of phosphatidylinositol 3-kinase, and rapamycin, an inhibitor of the activation of p70 S6 kinase (p70(S6k)), completely abolished IGF stimulation of L6A1 differentiation. We found that p70(S6k) activity increased substantially during differentiation, and this increase was further enhanced by PD098059. Our results demonstrate that the MAP kinase pathway plays a primary role in the mitogenic response and is inhibitory to the myogenic response in L6A1 myoblasts, while activation of the phosphatidylinositol 3-kinase/p70(S6k) pathway is essential for IGF-stimulated differentiation. Thus, it appears that signaling from the IGF-I receptor utilizes two distinct pathways leading either to proliferation or differentiation.

Lysophosphatidic acid (LPA) utilizes a heterotrimeric guanine nucleotide regulatory (G) protein-coupled receptor to activate the mitogen-activated protein kinase pathway and induce mitogenesis in fibroblasts and other cells. A single cell assay system was used to examine the functional interaction of the LPA receptor with G proteins in intact mouse fibroblasts, by measuring LPA-stimulated induction of the immediate-early gene, c-fos, as read out by a stably expressed fos-lacZ reporter gene. Pretreatment of these cells with pertussis toxin at 100 ng/ml almost completely abolished LPA-stimulated c-fos induction. Western blotting revealed that two pertussis toxin (PTX)-sensitive G proteins, G alpha i2 and G alpha i3, were present in membranes prepared from these cells, and Northern blotting confirmed the absence of message for other PTX-sensitive subunits. Microinjection of an alpha il/alpha i2-specific antibody into living cells decreased LPA-stimulated induction of c-fos by 60%, whereas introduction of antibodies to either alpha i3 or alpha 16, a subtype not present in these cells but used as a control, decreased LPA-stimulated c-fos induction by only 19%. In contrast, the alpha i1/alpha i2-specific antibody had no effect on insulin-induced c-fos expression, which is thought to utilize a G protein-independent mechanism of signaling. In addition, cellular expression of an epitope-tagged PTX-resistant mutant of G alpha i2, but not PTX-resistant G alpha i3, restored LPA-stimulated c-fos induction in cells in which endogenous G protein a subunits were uncoupled from the receptor by pretreatment with PTX. Together, these results provide conclusive in vivo evidence that G alpha i2 is the PTX-sensitive G protein a subunit which mediates LPA-stimulated c-fos induction and perhaps mitogenesis in these cells.

HuD is a human neuronal specific RNA-binding protein. In this study we have purified HuD and examined its RNA binding properties in detail. HuD binds to mRNAs that contain an AU-rich element with high affinity. In the case of the c-fos AU-rich element, HuD binds to a 27-nucleotide core element comprising AUUUA, AUUUUA, and AUUUUUA motifs. Mutation in any two of these motifs abrogates binding. HuD contains two tandem RNA recognition motifs (RRM), a basic domain, and a third RRM. Deletion analysis has shown that only the first and second RRMs are essential for RNA binding. Thus, these specific RNA binding properties support the idea that the HuD regulates gene expression at the posttranscriptional level.

Interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to activate JAK2 in various cells, but the role of JAK2 in IL-3 or GM-CSF receptor signal transduction is largely unknown. We have now examined the role of JAK2 in GM-CSF-induced signaling events in BA/F3 cells. In BA/F3 cells expressing hGMR, activation of JAK2 by hGM-CSF requires the box1 region of hGMR beta. Dominant negative JAK2 (delta JAK2), which lacked the kinase domain suppressed mIL-3 or hGM-CSF-induced c-fos promoter activation as well as c-myc promoter activation/cell proliferation, thereby suggesting that JAK2 is involved in the signaling of both pathways. Further analyses of the role of JAK2 in c-fos gene activation in BA/F3 cells expressing hGMR revealed that delta JAK2 inhibited hGM-CSF-induced phosphorylation of Shc and protein tyrosine phosphatase 1D. Within hGMR beta, the several tyrosine residues which exist are related to activation of Shc or protein tyrosine phosphate 1D, and are phosphorylated in response to hGM-CSF stimulation. In addition, we observed that delta JAK2 inhibited hGM-CSF-induced phosphorylation of hGMR beta. Taken together, our results suggest that JAK2 activated by the box1 region of hGMR mediates hGM-CSF-induced c-fos promoter activation through phosphorylation of hGMR.

We studied the effect of ionizing radiation on the activation of the AP-1 transcription factors and the regulation of basic fibroblast growth factor (bFGF) gene expression in drug-sensitive human breast carcinoma (MCF-7) cells and its drug-resistant variant (MCF-7/ADR) cells. Northern blot and gel mobility shift assays showed that 135 cGy of ionizing radiation induced c-jun and c-fos gene expression, AP-1 binding activity, as well as bFGF gene expression in MCF-7/ADR cells. In MCF-7 cells, however, we observed little/no induction of bFGF gene expression and AP-1 binding activity after the stress. Nevertheless, MCF-7 cells transfected with plasmids containing c-jun gene contain high levels of bFGF protein. H-7 (60 micrograms/ml), a potent protein kinase C (PKC) inhibitor, inhibited the stress-induced AP-1 binding activity and bFGF gene expression in MCF-7/ADR cells. Corroborating this observation, overexpression of PKC alpha induced bFGF gene expression in MCF-7 cells. Taken together, these results suggest that stress-induced bFGF gene expression is mediated through the activation of PKC and AP-1 transcription factors. Differences in the levels of PKC activity and AP-1 binding factors may be responsible for differential expression of bFGF among breast cancer cell lines. Although there are large differences in response to ionizing radiation between MCF-7 and MCF-7/ADR cell lines, we observed no significant differences in radiocytotoxicity between them.

It has been demonstrated that Ras is involved in interleukin 3 (IL-3)-stimulated signal transduction in various hematopoietic cultured cells (Satoh, T., Nakafuku, M., Miyajima, A., and Kaziro, Y.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3314-3318; Duronio, V., Welham, M. J., Abraham, S., Dryden, P., and Schrader, J. W.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1587-1591). However, it has not been fully understood which of IL-3-promoted cellular responses, i.e. proliferation, survival, and differentiation, requires Ras function. We employed a system of inducible expression of the dominant-negative (S17N) or dominant-active (G12V) mutant of Ras in BaF3 mouse pro-B cell line to analyze the role of Ras in IL-3-stimulated signal transduction. Induction of the dominant-negative Ras(S17N) effectively inhibited the IL-3induced activation of c-Raf-1 and mitogen-activated protein kinase (MAPK). Furthermore, the activation of fos gene promoter following IL-3 stimulation was almost completely abolished when Ras(S17N) was induced. Under these conditions, Ras(S17N) exhibited no inhibitory effect on IL-3-dependent proliferation assessed by the increase of cell numbers and a mitochondrial enzyme activity. The results indicate that Ras-dependent pathways, including the Raf/MAPK/Fos pathway, are dispensable for IL-3-induced growth stimulation. When BaF3 cells were treated with a tyrosine kinase inhibitor, herbimycin A, IL-3-dependent proliferation of the cells was impaired, suggesting that tyrosine kinase-mediated pathways are critical for growth promotion. On the other hand, apoptotic cell death caused by deprivation of IL-3 was prevented by the induction of the activated mutant Ras(G12V), although the rate of cell number increase was markedly reduced. Thus, it is likely that Ras-independent pathways play important roles to facilitate the proliferation although they may not be essential for IL-3-stimulated antiapoptotic signal transduction.

We recently reported that gastrin and glycine-extended progastrin processing intermediates (G-Gly) exert growth-promoting effects on AR4-2J cells (derived from rat pancreas) via interaction with distinct receptors. In this study we sought to investigate the mechanisms by which gastrin and G-Gly stimulate cell proliferation. While gastrin increased [Ca2+]i in AR4-2J cells, G-Gly had no effect. Similarly, G-Gly had no effect either on basal and 10(-7) M vasoactive intestinal polypeptide-stimulated cAMP generation, although gastrin is known to inhibit cAMP generation. Gastrin dose dependently stimulated AR4-2J cell mRNA content of both c-fos and c-jun, two genes known to function in regulating cell proliferation, but G-Gly had no effect. Gastrin also induced the expression of luciferase in AR4-2J cells transfected with a construct consisting of a luciferase reporter gene coupled to the serum response element of the c-fos gene promoter. In similar fashion, gastrin stimulated the activity of mitogen-activated protein kinase, an enzyme known to mediate the induction of the c-fos serum response element in response to growth factor stimulation. Although G-Gly had none of these effects of gastrin in AR4-2J cells, it stimulated activity of c-Jun amino-terminal kinase, an enzyme known to phosphorylate and transcriptionally activate c-Jun. These data support the notion that gastrin stimulates cell proliferation by inducing c-fos and c-jun gene expression, while G-Gly acts by post-translationally regulating early gene transcriptional activation. Our studies represent a novel model in which both the precursor and the product of a key processing reaction, peptide alpha-amidation, act cooperatively to stimulate cell proliferation via distinct receptors linked to different signal transduction pathways.

We have demonstrated previously that dexamethasone treatment of HepG2 cells caused an enhancement of insulin's metabolic effects (Kosaki, A., and Webster, N. J.(1993) J. Biol. Chem. 268, 21990-21996). This correlated with increased expression of the mRNA encoding the B isoform of the insulin receptor (IR). In the present study, we have demonstrated that dexamethasone treatment caused in addition an enhancement in insulin-stimulated immediate-early gene expression (c-fos and egr-1). Dexamethasone treatment caused an increase in in vivo IR autophosphorylation and insulin receptor substrate-1 (IRS-1) phosphorylation both early events in the insulin signaling pathway. Furthermore, the IRS-1 phosphorylation was distinctly left shifted, although the level of IRS-1 protein was unchanged. Total cellular tyrosine phosphatase activity was unaltered when assayed with 32P-labeled IR and IRS-1. Studies in vitro on wheat-germ agglutinin-purified receptors showed that the B isoform of the IR had increased kinase activity both toward itself and the exogenous substrates poly-glu4:tyr1 and recombinant IRS-1 protein. In addition, two-dimensional tryptic phosphopeptide maps indicated that the B isoform has an additional phosphopeptide that is not seen for the A isoform. In conclusion, it appears that the B isoform of the IR signals more efficiently than the A isoform in HepG2 cells.

Insulin stimulates the activity of mitogen-activated protein kinase (MAPK) via its upstream activator, MAPK kinase (MEK), a dual specificity kinase that phosphorylates MAPK on threonine and tyrosine. The potential role of MAPK activation in insulin action was investigated with the specific MEK inhibitor PD98059. Insulin stimulation of MAPK activity in 3T3-L1 adipocytes (2.7-fold) and L6 myotubes (1.4-fold) was completely abolished by pretreatment of cells with the MEK inhibitor, as was the phosphorylation of MAPK and pp90Rsk, and the transcriptional activation of c-fos. Insulin receptor autophosphorylation on tyrosine residues and activation of phosphatidylinositol 3'-kinase were unaffected. Pretreatment of cells with PD98059 had no effect on basal and insulin-stimulated glucose uptake, lipogenesis, and glycogen synthesis. Glycogen synthase activity in extracts from 3T3-L1 adipocytes and L6 myotubes was increased 3-fold and 1.7-fold, respectively, by insulin. Pretreatment with 10 microM PD98059 was without effect. Similarly, the 2-fold activation of protein phosphatase 1 by insulin was insensitive to PD98059. These results indicate that stimulation of the MAPK pathway by insulin is not required for many of the metabolic activities of the hormone in cultured fat and muscle cells.

Epidermal growth factor (EGF) acutely inhibits acid secretion; however, prolonged administration of EGF has been reported to increase acid production. We undertook these studies to examine whether the physiological effects of EGF on acid secretion are mediated by regulation of gastric H+,K+-ATPase, the principle enzyme responsible for acid secretion. EGF in concentrations equivalent to those in plasma increased H+,K(+)-ATPase alpha-subunit mRNA levels. Using H+,K(+)-ATPase-luciferase constructs transfected into primary cultured parietal cells, a significant step up in EGF inducibility was observed between bases -162 and -156 (5'-GACATGG-3') relative to the cap site. This EGF response element (ERE) conferred EGF inducibility when linked to homologous and heterologous promoters. The ERE is homologous to the 3' half-site of the c-fos serum response element to which rNFIL-6, rE12, and SRE-ZBP bind. Electrophoretic mobility shift assays using an ERE probe and parietal cell nuclear extracts revealed a specific DNA-protein complex, the formation of which was changed by neither E12 and NFIL-6 consensus oligonucleotides nor antibodies for NFIL-6, SRE-ZBP, and E12. Our studies indicate that EGF induces gastric H+,K(+)-ATPase alpha-subunit gene expression via an interaction between a specific ERE and a novel transcriptional factor and that this may be a physiologic mechanism by which EGF regulates acid secretion.

The angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors belong to the seven transmembrane receptor superfamily. Previous studies have suggested that the AT1R couples to a Gq signaling pathway, whereas the AT2R does not associate with Gq. To identify the role that individual intracellular domains play in AT1R function, AT1R/AT2R chimeric receptors were prepared by substitution of intracellular loops. CHO cells expressing these chimeras were used to test angiotensin II-induced c-fos expression and Ca2+ mobilization which are involved in the AT1R signaling pathway through Gq coupling. Substitution of the second intracellular loop (IC2) and the cytoplasmic tail between the two receptors did not affect AT1R function. However, exchange of the third intracellular loop (IC3) resulted in the loss of function in the AT1R and conferred to the AT2R the ability to constitutively activate the fos promoter. These findings suggest that the third intracellular loop of the AT1R is critical for Gq coupling. Substitution of discrete amino acid sequences of the third intracellular loop indicate that its N-terminal and C-terminal portions, especially the seven amino acids 219-225 in the N-terminal portion, are important for AT1R function, and that the intermediate portion of this loop is not required for Gq coupling.

Growth hormone (GH) treatment of cells promotes activation of JAK2, a GH receptor (GHR)-associated tyrosine kinase. We now explore JAK2 regions required for GHR-induced signaling. Wild-type (WT) JAK2 and JAK2 molecules with deletions of the amino terminus (JAK2ATD), carboxyl terminus (JAK2CTD), or kinase-like domain (JAK2PKD) were each transiently coexpressed in COS-7 cells with the rabbit GHR. The following responses were assayed: GH-induced transactivation of a luciferase reporter governed by a c-fos enhancer element; GH-induced shift in the molecular mass of a cotransfected epitope-tagged extracellular signal-regulated kinase molecule; and GH-induced antiphosphotyrosine immunoprecipitability of the transfected JAK2 form. In each assay, WTJAK2 and JAK2PKD allowed GH-induced signaling, whereas JAK2ATD and JAK2CTD did not. Anti-GHR serum coimmunoprecipitated WTJAK2, JAK2PKD, and JAK2CTD, but not JAK2ATD. Finally, a chimera in which the JAK2 kinase domain replaced the GHR cytoplasmic domain signaled GH-induced transactivation. We conclude: 1) kinase-like domain deletion eliminates neither physical nor functional interaction between JAK2 and the GHR; 2) kinase domain deletion eliminates functional but not physical coupling of JAK2 to the GHR; 3) interaction with the GHR appears dependent on the NH2-terminal one-fifth of JAK2; and 4) a GH-responsive signaling unit can include as little as the GHR external and transmembrane domains and the JAK2 kinase domain.

A mechanism by which voltage-sensitive Ca2+ channel (VSCC) activation triggers c-fos transcription has been characterized. Ca2+ influx through VSCCs stimulates phosphorylation of the transcription factor cAMP response element-binding protein (CREB) on serine 133 leading to an increase in the formation of transcription complexes that can elongate through a transcription pause site within the c-fos gene. Ca(2+)-stimulated CREB serine 133 phosphorylation is mediated by a Ca(2+)-activated kinase and is not dependent on the cAMP-dependent protein kinase (PKA). While necessary for c-fos transcriptional induction following VSCC opening, CREB serine 133 phosphorylation is not sufficient for transcriptional activation. A second, PKA-dependent event is required. Following induction, c-fos transcription is rapidly down-regulated. Dephosphorylation of CREB serine 133 parallels and likely mediates the transcriptional shut-off event. These results suggest that the phosphorylation and dephosphorylation of CREB controls its ability to regulate transcription in membrane-depolarized cells and that multiple pathways contribute to Ca(2+)-activated gene expression.

Experiments were designed to clarify the role of several proteins, junB, retinoblastoma protein (RB), and the transforming growth factor-beta (TGF-beta) receptors that are potential intermediates in TGF-beta activation of the alpha 2(I) collagen promoter. Treatment of NIH-3T3 cells with TGF-beta increased the activity of a transiently transfected murine alpha 2(I) collagen promoter (nucleotides -350 to +54) fused to a luciferase reporter gene 9-fold. Cotransfection of a junB stimulated the basal activity of the alpha 2(I) collagen promoter 93-fold, respectively. Expression of antisense junB RNA attenuated the effect of TGF-beta. Simian virus 40 large T antigen, an inhibitor RB function, did not prevent TGF-beta effects on the alpha 2(I) collagen promoter. A chimeric receptor containing the extracellular domain of the colony-stimulating factor-1 receptor and the intracellular domain of the type I TGF-beta receptor enhanced alpha 2(I) collagen promoter activity 4.8-fold, whereas a similar chimera containing the type II receptor intracellular domain had much weaker effects. Similar results were obtained with a plasminogen activator inhibitor-1 promoter, previously shown to be activated by TGF-beta through AP-1 elements. We conclude that TGF-beta activates the alpha 2(I) collagen and plasminogen activator inhibitor-1 promoters in NIH-3T3 cells through junB and the type I TGF-beta receptor kinase domain.

Studying Swiss 3T3 fibroblasts, we report that arachidonic acid strongly stimulates mRNA levels of the growth-associated immediate early genes c-fos and Egr-1. Structurally related compounds like arachidonic acid methyl ester, arachidonyl alcohol, or eicosatetraynoic acid are ineffective, indicating a specific role of free unesterified arachidonic acid or an arachidonic acid metabolite in c-fos and Egr-1 mRNA accumulation. Blocking the conversion of arachidonic acid to prostaglandins by inhibiting cyclooxygenase abolishes arachidonic acid-induced accumulation of c-fos and Egr-1 mRNA. Inhibition of the lipoxygenase or cytochrome P-450 epoxygenase pathways has no significant effect on arachidonic acid-induced c-fos and Egr-1 mRNA levels, indicating that prostaglandin synthesis is necessary for the increase in c-fos and Egr-1 mRNA. Reversed phase high performance liquid chromatography revealed prostaglandin E2 (PGE2) as the major arachidonic acid metabolite in Swiss 3T3 fibroblasts. When added to the cells, PGE2 stimulates c-fos and Egr-1 mRNA levels to the same degree as arachidonic acid. Also, the inhibition of arachidonic acid-stimulated c-fos and Egr-1 mRNA accumulation by indomethacin is reversed by PGE2. Contrary to reports that PGE2 caused an increase in cAMP levels in Swiss 3T3 fibroblasts, we found that arachidonic acid and PGE2 only minimally increase cAMP levels as compared with untreated cells. In contrast, inhibition of protein kinase C by calphostin C and chelerythrine or down-regulation with phorbol 12-myristate 13-acetate drastically reduces PGE2 and arachidonic acid-induced c-fos and Egr-1 mRNA levels. These data indicate that arachidonic acid exerts its stimulatory effect on c-fos and Egr-1 mRNA via synthesis of PGE2 and subsequent activation of protein kinase C, probably through a PGE2 receptor coupled to phospholipase C.