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Structural maintenance of chromosome (SMC) protein complexes are the key organizers of the spatiotemporal structure of chromosomes. The condensin SMC complex has recently been shown to be a molecular motor that extrudes large loops of DNA, but the mechanism of this unique motor remains elusive. Using atomic force microscopy, we show that budding yeast condensin exhibits mainly open 'O' shapes and collapsed 'B' shapes, and it cycles dynamically between these two states over time, with ATP binding inducing the O to B transition. Condensin binds DNA via its globular domain and also via the hinge domain. We observe a single condensin complex at the stem of extruded DNA loops, where the neck size of the DNA loop correlates with the width of the condensin complex. The results are indicative of a type of scrunching model in which condensin extrudes DNA by a cyclic switching of its conformation between O and B shapes.

Ataxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder with multisystem involvement and cancer predisposition, caused by mutations in the A-T mutated (ATM) gene. To study genotype-phenotype correlations, we evaluated the clinical and laboratory data of 51 genetically proven A-T patients, and additionally measured ATM protein expression and kinase activity. Patients without ATM kinase activity showed the classical phenotype. The presence of ATM protein, correlated with slightly better immunological function. Residual kinase activity correlated with a milder and essentially different neurological phenotype, absence of telangiectasia, normal endocrine and pulmonary function, normal immunoglobulins, significantly lower X-ray hypersensitivity in lymphocytes, and extended lifespan. In these patients, cancer occurred later in life and generally consisted of solid instead of lymphoid malignancies. The genotypes of severely affected patients generally included truncating mutations resulting in total absence of ATM kinase activity, while patients with milder phenotypes harbored at least one missense or splice site mutation resulting in expression of ATM with some kinase activity. Overall, the phenotypic manifestations in A-T show a continuous spectrum from severe classical childhood-onset A-T to a relatively mild adult-onset disorder, depending on the presence of ATM protein and kinase activity. Each patient is left with a tremendously increased cancer risk., (© 2011 Wiley Periodicals, Inc.)

The protein products of the recombination activating genes (RAG1 and RAG2) initiate the formation of immunoglobulin (Ig) and T-cell receptors, which are essential for B- and T-cell development, respectively. Mutations in the RAG genes result in severe combined immunodeficiency disease (SCID), generally characterized by the absence of mature B and T lymphocytes, but presence of natural killer (NK) cells. Biochemically, mutations in the RAG genes result either in nonfunctional proteins or in proteins with partial recombination activity. The mutated RAG genes of 9 patients from 7 families were analyzed for their recombination activity using extrachromosomal recombination substrates, rearrangement of endogenous Ig loci in RAG gene-transfected nonlymphoid cells, or the presence of Ig gene rearrangements in bone marrow (BM). Recombination activity was virtually absent in all 6 patients with mutations in the RAG core domains, but partial activity was present in the other 3 RAG-deficient patients, 2 of them having Omenn syndrome with oligoclonal T lymphocytes. Using 4-color flow cytometry, we could define the exact stage at which B-cell differentiation was arrested in the BM of 5 RAG-deficient SCID patients. In 4 of 5 patients, the absence of recombination activity was associated with a complete B-cell differentiation arrest at the transition from cytoplasmic (Cy) Igmu(-) pre-B-I cells to CyIgmu(+) pre-B-II cells. However, the fifth patient showed low frequencies of precursor B cells with CyIgmu and surface membrane IgM, in line with the partial recombination activity of the patient's mutated RAG gene and the detection of in-frame Ig gene rearrangements in BM.

Myeloid differentiation is a highly regulated process governed by various cytokines, such as EPO, TPO, G-CSF, IL-3, IL-5 and GM-CSF. These cytokines act in part through activation of the STAT transcription factor family. In particular, various isoforms of STAT3 and STAT5 are activated during myeloid differentiation in a cell-type and maturation-state dependent fashion. In vitro studies have shown that STAT proteins are essential for cytokine-regulated processes such as cellular proliferation, differentiation as well as survival. Similarly, various STAT knock-outs have highlighted the role of STATs in myeloid differentiation in vivo. STATs also appear to play an important role in various myeloid malignancies, which are characterized by arrested maturation and cytokine-independent proliferation of myeloid progenitors. Constitutive activation of STAT3 and/or STAT5 resulting in enhanced transcription of anti-apoptotic- cell-cycle progression genes is likely to contribute to the pathogenesis of various myeloid leukemia's. Oncogene (2000).

Signal transducers and activators of transcription (STATs) are a family of transcription factors that were originally identified as mediators of cytokine-induced gene expression. We and others have recently shown that STAT5 also plays a major role in cellular transformation by the Bcr-Abl oncogene. Here we show that the antiapoptotic bcl-xL gene product and the cell cycle regulator cyclin D1 are targets of STAT5 in Bcr-Abl-transformed cells. In the CML cell line K562 and in BaF3 cells ectopically expressing Bcr-Abl, both the cyclin D1 and bcl-x promoters are highly active. The activity of these promoters can be strongly repressed by cotransfection of a dominant negative (DN) mutant of STAT5. Moreover, the cyclin D1 and bcl-x promoters contain STAT binding sites to which STAT5 constitutively binds in Bcr-Abl transformed cells. These results suggest that STAT5 contributes to transformation by Bcr-Abl by induction of cyclin D1 and bcl-xL expression., (Copyright 2000 Academic Press.)

Introduction: Activation of the signal transducer and activator of transcription protein STAT3 is a crucial step in granulocyte colony-stimulating factor (G-CSF)-mediated cell cycle exit and subsequent neutrophilic differentiation of myeloid precursor cells. We have recently demonstrated that this is mediated, at least in part, by upregulation of the cyclin-dependent kinase inhibitor p27(Kip1). The splice variant STAT3beta, that lacks a C-terminal serine residue implicated in the transcriptional activity of STAT3, has been shown to inhibit STAT3-mediated transcription in certain situations. STAT3beta is known to be expressed in hematopoietic cells, but its role in controlling the balance between proliferation and differentiation has not been established., Materials and Methods: We ectopically introduced STAT3beta in differentiation-competent 32D cell transfectants expressing human wild type (WT) G-CSF receptors and studied the consequences for G-CSF-mediated responses., Results: Overexpression of STAT3beta did not alter the kinetics of G-CSF-mediated neutrophilic differentiation or p27 induction in 32D/G-CSF-R WT cells. In addition, we found that p27(Kip1) promoter activity was not inhibited by STAT3beta, while inhibition of p27 transactivation by a dominant-negative STAT3 mutant could in fact be alleviated by coexpression of the beta form., Conclusion: These findings argue against a role of STAT3beta as a negative regulator of G-CSF-induced expression of p27 and myeloid differentiation.

The eosinophil-derived neurotoxin (EDN), a member of the mammalian ribonuclease family, is found in the large specific granules of human eosinophilic leukocytes. We have investigated the role of the C/EBP transcription factor family in the regulation of EDN promoter activity. Here we show that the C/EBP family is involved in intrinsic regulation of EDN promoter activity. We have identified a C/EBP binding site located at -124 in the proximal promoter of the EDN gene. Mutation of this C/EBP site results in a decrease of promoter activity in HL-60-eos cells as well as in eosinophils differentiated in vitro from CD34+ cells. Different C/EBP proteins are able to bind to the C/EBP site as shown by gel shift assay. Our results indicate the importance of the C/EBP family in the regulation of the EDN gene in eosinophils.

The common beta-chain (beta c) is the main signaling component of the heterodimeric receptors for IL-3, IL-5, and GM-CSF and is primarily expressed on myeloid cells. The proximal beta c promoter is regulated by GGAA binding proteins, including PU.1, a hemopoietic specific member of the Ets family. However, it is not likely that PU.1 alone accounts for the myeloid-restricted expression of the beta c subunit. Here we describe the identification of a C/EBP binding enhancer that is located 2 kb upstream of the transcription start site. The enhancer contains two elements that bind C/EBP alpha and -beta in U937 cells, while C/EBP epsilon is also bound in extracts of HL-60 cells. Importantly, deletion of the enhancer or mutation of either of one of the C/EBP sites results in a complete loss of promoter activity in cell lines as well as in primary cells, showing the importance of C/EBP members in beta c gene activation. We further show that PU.1 has to cooperate with C/EBP proteins to induce beta c transcription. Since the beta c is already expressed on CD34+ cells, these results demonstrate that both C/EBP and PU.1 are not only important for the myeloid-specific gene regulation at later stages of myeloid differentiation.

Signal transducers and activators of transcription (STATs) belong to a family of transcription factors that were originally identified as mediators of cytokine-induced gene expression. Recent evidence, however, has shown that certain members of the STAT family, including STAT3, are also involved in cellular transformation. Here we show that STAT5 also plays a role in cellular transformation by the BCR-Abl oncogene. In BCR-Abl transformed K562 cells, STAT5A and 5B are constitutively phosphorylated on tyrosine and are transcriptionally active. Moreover, expression of a dominant negative form of STAT5 shows that active STAT5 is necessary for the growth in soft agar of these cells. These results show that besides STAT3, STAT5 can also be involved in cellular transformation.

Interleukin-5 (IL-5), IL-3, and granulocyte macrophage colony-stimulating factor (GM-CSF) are hematopoietic cytokines which signal through a common beta subunit (betac) of a heterodimeric receptor. Among the intracellular signaling pathways activated via betac is the JAK/STAT pathway. We show that different STAT5 isoforms are activated by IL-5 and GM-CSF in eosinophils, neutrophils, and differentiated eosinophilic HL-60 cells. Whereas IL-5 activated the wild-type STAT5A and STAT5B proteins in HL60-eos cells, a carboxyl-terminally truncated 80-kDa STAT5 isoform was activated in mature eosinophils and neutrophils. Surprisingly, while both isoforms bind strongly to an element from the beta-casein promoter, only p80 STAT5 binds to the ICAM1-IRE. Consequently, a carboxyl-terminal truncated STAT5 is capable of blocking STAT3-mediated transcription of an IREtkCAT reporter construct. The cell type-specific expression of these functionally distinct STAT5 isoforms might contribute to the pleiotropic effects of IL-5 and GM-CSF on different target cells.

Binding of interferon-gamma (IFN-gamma) to its heterodimeric receptor induces activation of the tyrosine kinases JAK1 and JAK2 followed by tyrosine phosphorylation of STAT1alpha. Selective activation of STAT1alpha at the IFN-gamma receptor is achieved by specific interaction between a cytosolic tyrosine motif including Y440 in the IFN-gamma receptor alpha-chain and the SH2 domain of STAT1alpha. We demonstrate that, in addition to STAT1alpha, STAT3 is also activated by IFN-gamma in human neutrophils. The activation of STAT3 was not found in human eosinophils, monocytes, and HL-60 cells, although the STAT3 protein was expressed in these cells. The cell type-specific activation of STAT3 by IFN-gamma was also observed in neutrophils that are differentiated in vitro from human CD34+ hematopoietic stem cells. These results indicate that a single cytokine receptor can activate different STAT family members in a cell-specific manner, which might result in cell-specific gene transcription.

Interleukin-1beta (IL-1beta) is a potent inflammatory cytokine involved in type 1 diabetes and acts through defined IL-1beta signaling pathways. In the present work we describe induction of DNA binding activity to signal transducer and activator of transcription (STAT) in response to IL-1beta in clonal insulin-secreting cells. Moreover, IL-1beta activates a short isoform of STAT-3 that potently stimulates transcription. Immunoprecipitation studies reveal an interaction between the activated STAT-3 and the IL-1 receptor accessory protein indicating an association between the two signaling pathways. This may be a novel point of transduction cross talk and an additional mechanism utilised by IL-1beta in the pancreatic beta-cell during the process of type 1 diabetes.

While signal transducers and activators of transcription (STATs) were originally discovered as intracellular effectors of normal signaling by cytokines, increasing evidence also points to a role for STAT transcription factors in oncogenesis. Previous studies have demonstrated that one STAT family member, Stat3, possesses constitutively elevated tyrosine phosphorylation and DNA-binding activity in fibroblasts stably transformed by the Src oncoprotein. To determine if this Stat3 activation by Src could induce Stat3-mediated gene expression, luciferase reporter constructs based on synthetic and authentic promoters were transfected into NIH 3T3 cells. Activation of endogenous cellular Stat3 by the Src oncoprotein induced gene expression through a Stat3-specific binding element (TTCCCGAA) of the C-reactive protein gene promoter. A naturally occurring splice variant of human Stat3 protein, Stat3beta, with a deletion in the C-terminal transactivation domain abolished this gene induction in a dominant negative manner. Expression of Stat3beta did not have any effect on a reporter construct based on the c-fos serum response element, which is not dependent on Stat3 signaling, indicating that Stat3beta does not nonspecifically inhibit other signaling pathways or Src function. Transfection of vectors expressing Stat3beta together with Src blocked cell transformation by Src as measured in a quantitative focus formation assay using NIH 3T3 cells. By contrast, Stat3beta had a much less pronounced effect on focus formation induced by the Ras oncoprotein, which does not activate Stat3 signaling. In addition, three independent clones of NIH 3T3 cells stably overexpressing Stat3beta were generated and characterized, demonstrating that Stat3beta overexpression does not have a toxic effect on cell viability. These Stat3beta-overexpressing clones were shown to be deficient in Stat3-mediated signaling and refractory to Src-induced cell transformation. We conclude that Stat3 activation by the Src oncoprotein leads to specific gene regulation and that Stat3 is one of the critical signaling pathways involved in Src oncogenesis. Our findings provide evidence that oncogenesis-associated activation of Stat3 signaling is part of the process of malignant transformation.

Interleukin-5 (IL-5) and granulocyte macrophage-colony stimulating factor (GM-CSF) are important cytokines for the proliferation, differentiation, and activation of myeloid lineages. The JAK/STAT pathway is one of the signaling pathways implicated in mediating biological responses induced by these cytokines. Previous studies have demonstrated that these cytokines predominantly activate an 80 kDa STAT5 isoform in mature granulocytes. To better understand the role of STAT proteins during growth and differentiation of granulocytes, we evaluated differentiation of human CD34+ hematopoietic stem cells ex vivo toward eosinophils and neutrophils. Bandshift experiments showed that in an early stage of both differentiation pathways (14 days), the 94 kDa STAT5B protein was activated by both IL-5 (eosinophil lineage) and GM-CSF (neutrophil lineage). However, during maturation of both lineages (days 21 and 28), increased expression of a functionally distinct 80 kDa STAT5 isoform was observed, resulting in heterodimer DNA-binding complexes containing both the 94 and 80 kDa STAT5 proteins. The finding that functionally distinct isoforms of STAT5 are activated during the early and late differentiation stages of granulocytes suggests that they might be involved in regulating different biological functions in these cells.

In contrast to the general model of cytokine-induced JAK/STAT signaling, tyrosine phosphorylation of the IL-5R beta chain seems to be dispensable for STAT activation in cells overexpressing exogenous STAT proteins. In this study we expressed IL-5 receptor mutants in 293 cells and studied IL-5-induced endogenous STAT-dependent transcription. Our results indicate that: (a) tyrosine phosphorylation of the IL-5R beta chain is required for endogenous STAT5 activation, (b) multiple tyrosine residues are phosphorylated upon IL-5 stimulation, including Tyr577, Tyr612, Tyr695, and Tyr750, and (c) Tyr612, Tyr695, and Tyr750 are all capable of inducing activation of STAT5, demonstrating a high level of functional redundancy within the IL-5R beta chain.

The 89-kDa STAT3 protein is a latent transcription factor which is activated in response to cytokines (interleukin (IL)-5 and -6) and growth factors (epidermal growth factor). Binding of IL-5 to its specific receptor activates JAK2 which leads to the tyrosine phosphorylation of STAT3 proteins. Here we report the cloning of a cDNA encoding a variant of the transcription factor STAT3 (named STAT3beta) which was isolated by screening an eosinophil cDNA library. Compared to wild-type STAT3, STAT3beta lacks an internal domain of 50 base pairs located near the C terminus. This splice product is a naturally occurring isoform of STAT3 and encodes a 80-kDa protein. We found by reconstitution of the human IL-5R in COS cells that like STAT3, STAT3beta is phosphorylated on tyrosine and binds to the pIRE from the ICAM-1 promoter after IL-5 stimulation. However, STAT3beta fails to activate a pIRE containing promoter in transient transfection assays. Instead, co-expression of STAT3beta inhibits the transactivation potential of STAT3. These results suggests that STAT3beta functions as a negative regulator of transcription.

The receptor for interleukin-5 (IL-5R) is composed of a unique alpha chain (IL-5R alpha) expressed on eosinophils and basophils, associated with a beta c subunit, which is shared by the receptors for IL-3 and granulocyte macrophage-colony stimulating factor. One of the molecular events activated via the IL-5R is the JAK/STAT signaling pathway. Recent reports have shown that IL-5 induces tyrosine phosphorylation of JAK2 followed by the subsequent cell type-specific activation of either STAT1 alpha or STAT5. To identify additional STAT proteins activated by IL-5, we co-transfected the IL-5R with STAT cDNAs in COS cells. We found that IL-5 induces binding of STAT3 to the intercellular adhesion molecule-1 pIRE, and activates STAT3-dependent transcription. Moreover, endogenous STAT3 was tyrosine phosphorylated and activated in human IL-5-stimulated BaF3 cells ectopically expressing the human IL-5R (BaF3/IL5R). These data imply that multiple STAT proteins are involved in gene regulation by IL-5 in a cell type-specific manner. We further demonstrate using C-terminal truncations of the alpha and beta c subunits of the IL-5R that the membrane-proximal STAT activation. Interestingly, a beta c receptor mutant lacking intracellular tyrosine residues is able to mediate STAT3 activation, suggesting that tyrosine phosphorylation of the beta c receptor is not essential for STAT3 activation.

Activation of the adenylyl cyclase signaling pathway elicits the induction of genes via activators binding to cAMP-responsive elements (CREs). Nuclear factor CRE modulator (CREM) is activated by PKA-mediated phosphorylation on a serine at position 117. We show that Ser-117 is also phosphorylated by the mitogen-activated p70 S6 kinase (p70S6K) in vitro. Activation of cellular p70S6K by serum factors enhances Ser-117 phosphorylation and CREM transactivation. Coexpression of p70S6K significantly increases transactivation by a GAL4-CREM fusion. The macrolide rapamycin, a potent and specific inhibitor of p70S6K in vivo, completely blocks CREM activation induced by serum and by p70S6K. Thus, CREM constitutes a target for mitogenic signaling through p70S6K and may acts as a nuclear effector in which transduction pathways may converge and cross-talk.

DNA bending is postulated to be a major determinant of gene expression and has been shown to be specifically induced by some regulatory proteins with DNA-binding properties. Here we show that nuclear factors which naturally bind to CREs (cAMP-responsive elements) are able to induce bending in the sequences flanking this recognition site. In our assays we used a permutated binding site/gel retardation assay and bacterially generated nuclear factors. We have been studying the cAMP-responsive-element modulator (CREM) gene, which encodes both repressors (CREM alpha, beta and gamma) and an activator (CREM tau) of cAMP-responsive transcription by alternative splicing. In addition, two alternative DNA-binding domains can be encoded in different CREM isoforms. No differences in induction of DNA bending by the CREM proteins with the two DNA binding domains were detected. The activator CREB induced DNA bending in a fashion similar to CREM. Importantly, we show that phosphorylation of CREM or CREB alters their mobilities in a regular gel shift assay as well as enhances the angle of DNA bending induced by these proteins.

Transcription factors that bind to cAMP-responsive elements (CREs) regulate the expression of target genes in response to activation of the adenylyl cyclase pathway. It is generally thought that activation is obtained through direct phosphorylation by the cAMP-dependent protein kinase-A. We have isolated the gene CRE modulator (CREM), which encodes multiple members of the CRE-binding protein family, by cell-specific alternative splicing. Various isoforms have been characterized, encoding both repressors (CREM alpha, -beta, and -gamma) as well as activators (CREM tau). Here we show that the function of the activator CREM tau is regulated by the p34cdc2 kinase. Multiple serine and threonine residues are phosphorylated in vivo as well as in vitro by p34cdc2. Although there is no effect of p34cdc2-mediated phosphorylation on CREM tau DNA binding, we observed a dramatic effect on the trans-regulatory function. Coexpression of a constitutively active p34cdc2 mutant shows that the trans-activation potential of CREM tau is strongly reduced by p34cdc2. This represents the first example of negative regulation of a transcription factor of this class by p34cdc2.

Phosphorylation is one of the major mechanisms by which the activity of transcription factors can be regulated. We have investigated the role of phosphorylation in the regulation of the transcription factor CREM. We show that the CREM tau activator is phosphorylated on multiple serine and threonine residues in vivo. Stimulation of various signal transduction pathways by forskolin, TPA or Ca2+ ionophore leads to enhanced phosphorylation of serine 117, concomitant with an increase in the transactivation potential of CREM tau. We have identified multiple kinases that can also phosphorylate S117 in vitro. Moreover, we show that casein kinase I and II cooperatively phosphorylate CREM tau on multiple residues, eliciting enhanced DNA binding. Cooperative phosphorylation is also observed with other kinases. These results show that the activity of CREM tau is regulated by multiple phosphorylation events, suggesting that CREM could be considered as a nuclear effector where signalling pathways may converge and/or cross-talk.

The cAMP-responsive-element modulator (CREM) gene encodes both antagonists (CREM alpha/beta/gamma) and an activator (CREM tau) of cAMP-responsive transcription by alternative splicing. In adult mouse brain a predominant 21-kDa protein, not corresponding to any previously characterized transcript, is detected with specific CREM antibodies. A developmental switch occurs in brain as expression changes at birth from CREM alpha/beta to the 21-kDa protein. We show that the 21-kDa protein corresponds to S-CREM (short CREM), a protein produced by the use of an internal AUG initiation codon in the CREM tau transcript. S-CREM shares with the other CREM proteins the basic DNA-binding and leucine-zipper dimerization domain. S-CREM functions as a transcriptional repressor of cAMP-induced transcription. Thus, two proteins with opposite functions are generated by alternative translation using two AUG codons within the same reading frame.

The genes of the Jun family encode components of the TPA-inducible transcription factor AP-1. These genes are induced by a wide variety of extracellular stimuli, such as growth factors, phorbol esters and activators of protein kinase A. We have previously shown that the adenovirus type 5 E1A protein (E1A5) induces c-jun and junB expression in a number of different cell types. In this paper we show that the third member of the Jun family, junD, is also strongly induced by E1A5. Multiple sequences in the junB and junD promoters are responsible for the effects of E1A5. By contrast, adenovirus type 12 E1A (E1A12), like retinoic acid (RA), strongly induces c-jun expression, while expression of junB and junD is not altered. Interestingly, E1A12 expression leads to complete differentiation of P19 EC cells, comparable to the effect of RA on these cells, while E1A5-expressing cells are only partially differentiated.

The product of the junD gene belongs to the Jun/Fos family of nuclear DNA binding transcription factors. This family regulates the expression of TPA responsive genes by binding to the TPA responsive element (TRE). Unlike its counterparts c-jun and junB, junD expression is hardly inducible by growth factors and phorbol esters. In fact, junD is constitutively expressed at high levels in a wide variety of cells. To unravel the molecular mechanisms underlying constitutive junD expression, we have cloned and characterized the mouse junD promoter. We show that the high constitutive expression is caused by multiple cis-acting elements in its promoter, including an SP1 binding site, an octamer motif, a CAAT box, a Zif268 binding site and a TRE-like sequence. The octamer motif is the major determinant of junD promoter activity, while somewhat smaller contributions are made by the TRE and Zif268 binding site. The SP1 and CAAT box are shown to be of minor importance. The junD TRE is in its behavior indistinguishable from previously identified TREs. However, the junD promoter is not TPA inducible due to the presence of the octamer motif.

The proto-oncogene c-jun, a major component of transcription factor AP-1, is expressed at very low levels in undifferentiated embryonal carcinoma (EC) end embryonic stem (ES) cells. Retinoic acid (RA) induced differentiation causes a strong increase in the levels of c-jun mRNA. In this paper we report the cloning and characterization of the mouse c-jun promoter. Our results show that RA treatment causes a strong enhancement in c-jun promoter activity, an effect probably mediated by the RA-receptor beta (RAR beta). Sequences located between -329 and -293 are responsible for the observed RA effect, and bind at least five different protein complexes, of which three are decreased upon RA treatment. These protein binding sites do not resemble RA-responsive elements (RARE's) found in the promoters of retinoic acid receptor beta (RAR beta) and laminin B1. Furthermore, we could not detect a direct interaction of RAR alpha and RAR beta to these sequences, indicating that RA-induced c-jun expression is an indirect effect of RAR action.

Although the structure of several members of the GH receptor family has been defined, signal transduction following GH binding to its receptor has not been elucidated. Mouse osteoblasts were used to study the effect of GH on immediate early gene expression and, subsequently, the cellular signal(s) mediating this expression were analysed. GH rapidly and transiently induced the expression of c-jun and jun B in concert with the already reported expression of c-fos. The GH-induced expression of c-fos was completely blocked by the protein kinase inhibitors staurosporine and H7, indicating that the action of GH is mediated by one or several protein kinases. We next analysed the identity of the putative protein kinases in more detail by using a more specific protein kinase inhibitor, namely the ether-lipid 1-O-alkyl-2-O-methylglycerol, understood to be an inhibitor of protein kinase C (PKC). Data obtained from these studies revealed that GH-induced expression of c-fos is mediated by PKC. In addition, we observed a profound increase in formation of the PKC activator diacyglycerol upon addition of GH, a natural activator of PKC. In conclusion, upon binding of GH to mouse osteoblasts, the receptor-mediated cellular signal involves diacyglycerol formation and activation of PKC, leading to the induction of oncogene expression. Finally, the expression of c-fos, c-jun and jun B results in an increased binding of protein complexes to AP-1 binding sites.

The product of the junB gene, a gene homologous to the proto-oncogene c-jun, is a component of transcription factor AP-1. JunB expression is modulated by a wide variety of extracellular stimuli, such as serum, growth factors, phorbol esters (TPA) and activators of protein kinase A (PKA). In order to study the molecular basis of this complex regulation, we have cloned the mouse junB gene from a genomic testis library, and characterized the junB promoter. Here we show that the junB promoter is activated by serum, TPA, and activated PKA. Sequences located between -91 and -44 are necessary for induction. These sequences contain a CAAT box, a G-C rich region and a previously undescribed inverted repeat (IR). The IR element can mediate induction by TPA and PKA when coupled to a heterologous promoter, and specifically binds a protein of 110 kD.

Proteins encoded by the adenovirus E1A oncogene are capable of positive and negative transcriptional regulation of both viral and cellular genes. E1A regulatory function is commonly thought to involve modifications of specific cellular factors that interact with responsive promoters. In this report we present evidence that E1A induces the activity of the jun/AP-1 transcription factor in three different cell types: P19, JEG-3, and HeLa. AP-1 binds to 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs); therefore, E1A might modulate a specific signal transduction pathway normally induced by activation of the protein kinase C. Binding of jun/AP-1 to a TRE is induced in all cell types studied when E1A is expressed. We observe that the expression of endogenous c-jun and jun B genes is induced by E1A, which directly transactivates the promoters of c-fos, c-jun, and jun B. Similar inducibility is obtained by treatment with retinoic acid and differentiation of P19-embryonal carcinoma cells. The E1A 13S product transactivates TRE sequences and cooperates with c-jun in the transcriptional stimulation. The 12S E1A product does not activate a TRE sequence, but cotransfection with c-jun circumvents this lack of stimulation. Coexpression of c-fos and E1A 12S, however, blocks the transactivation by c-jun, suggesting an important role for fos in determining the dominance of the 12S or 13S protein.

Epidermal growth factor (EGF) activates a well characterized signal transduction system in human A431 epidermoid carcinoma cells, which leads to rapid and transient expression of the c-fos proto-oncogene. In order to investigate the influence of altered gravity on EGF-induced signal transduction, we have studied the EGF-induced c-fos expression under simulated hypo- and hypergravity conditions. In this report we show that EGF-induced fos expression is decreased under simulated hypogravity conditions, while hypergravity has a stimulatory effect on EGF-induced fos expression. These results show that the EGF-activated signal transduction system is influenced by gravity, and that gravity exerts its effects already in the early phases of the signal transduction cascade.

The jun and fos gene families encode DNA binding proteins involved in transcriptional regulation of genes containing a TPA responsive element (TRE). To study their role in gene regulation during early mammalian development, expression and transcription regulatory properties of their gene products were investigated during retinoic acid (RA) induced differentiation of P19 embryonal carcinoma (EC) cells. Our results show, that c-jun is expressed at low but detectable levels in undifferentiated P19 EC cells and at elevated levels in its RA differentiated derivatives, corresponding with increased expression of Jun and TRE binding activity. Jun D is constitutively expressed at constant levels in both undifferentiated and differentiated P19 cells, while jun B and c-fos are not expressed. Addition of TPA to undifferentiated P19 cells does not result in induction of c-jun, jun B and c-fos, while these genes are transiently induced in RA-differentiated P19 cells. In addition, TPA treatment resulted in expression of Fos and Jun protein in RA-differentiated, but not in undifferentiated P19 cells. Addition of TPA to P19 EC cells expressing low levels of TRE binding proteins is neither followed by transcriptional activation of the TRE reporter gene nor by induction of c-jun, previously shown to be autoregulated by its own gene product. By contrast, in P19 cells differentiated by RA that contain elevated levels of TRE binding proteins, TRE dependent transcription is enhanced upon TPA treatment.

The product of the c-jun proto-oncogene, a major component of the transcription factor AP1, has been implicated in both positive and negative transcriptional control as well as in transformation. The role of c-jun in early development and differentiation processes, however, is still largely unknown. In this paper we show that ectopic expression of exogenous c-jun sequences leads to differentiation and loss of the transformed phenotype of P19 embryonal carcinoma (EC) cells. The mixed populations of endoderm- and mesoderm-like cells that were obtained after the introduction of c-jun morphologically and biochemically resembled P19 cells differentiated in monolayer by retinoic acid (RA). Furthermore, we provide evidence that direct effects of the c-jun gene product on transcription of the retinoic acid receptor beta (RAR beta) gene may be responsible for the differentiation-promoting potential of c-jun.

Transforming growth factor beta 1 (TGF beta 1) is a multifunctional regulator of growth and differentiation. However, both cytoplasmatic and nuclear signal transduction mechanisms leading to the biological effects of TGF beta 1 are largely unknown. In this report we show, that TGF beta 1 induces the expression of the immediate early genes c-jun and jun B, that encode trans-acting factors regulating transcription of a variety of genes in response to growth factors and phorbol esters. The jun genes are induced by TGF beta 1 in a protein synthesis independent fashion both in quiescent mouse 3T3 fibroblasts, which are growth stimulated by TGF beta 1, as well as in mink lung CCL64 (ML-CCL64) epithelial cells, which are growth inhibited by TGF beta 1. The PDGF inducible JE gene was induced by TGF beta 1 in 3T3, but not in ML-CCL64 cells. Furthermore, we show that chimaeric reporter-CAT constructs containing the TPA responsive element (TRE) or the dyad symmetry element (DSE) are activated by TGF beta 1 in transient transfection assays in both growth inhibited and growth stimulated cells. These results show that the early genomic responses to TGF beta 1 resemble changes in gene expression induced by serum, growth factors and phorbol esters, suggesting common mechanisms of transcriptional activation.