1: Cancer Res. 2005 Nov 1;65(21):9883-90. Rac3-mediated transformation requires multiple effector pathways. Keller PJ, Gable CM, Wing MR, Cox AD. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7512, USA. Our initial characterization of Rac3, a close relative of the small GTPase Rac1, established its ability to promote membrane ruffling, transformation, and activation of c-jun transcriptional activity. The finding that Rac3 is transforming, and its similarity to Rac1, a protein that has a well-established connection to many processes important for cancer progression, prompted further investigation into Rac3 transformation. We used effector domain mutants (EDMs) to explore the relationship among Rac signaling, transformation, and effector usage. All Rac3 EDMs tested (N26D, F37L, Y40C, and N43D) retained the ability to promote membrane ruffling and focus formation. In contrast, only the N43D mutant promoted anchorage independence. This differs from Rac1, where both N26D and N43D mutants were impaired in both types of transformation. To learn more about the signaling pathways involved, we did luciferase reporter assays and glutathione S-transferase pull-down assays for effector binding. We found evidence for a functional link between activation of phospholipase Cbeta2 by Rac3 and signaling to the serum response factor (SRF). Surprisingly, we also found that Rac3 binds poorly to the known Rac1 effectors mixed lineage kinases 2 and 3 (MLK2 and MLK3). Transcription of cyclin D1 was the only pathway that correlated with growth in soft agar. Our experiments show that activation of membrane ruffling and transcriptional activation of c-jun, SRF, or E2F are not sufficient to promote anchorage-independent growth mediated by Rac3. Instead, multiple effector pathways are required for Rac3 transformation, and these overlap partially but not completely with those used by Rac1. PMID: 16267012 [PubMed - in process] --------------------------------------------------------------- 2: Biochem Soc Trans. 2005 Oct;33(Pt 5):891-5. Rho GTPases and the control of cell behaviour. Hall A. MRC Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, Cancer Research UK Oncogene and Signal Transduction Group, University College London, Gower Street, London WC1E 6BT, U.K. Rho, Rac and Cdc42, three members of the Rho family of small GTPases, each control a signal transduction pathway linking membrane receptors to the assembly and disassembly of the actin cytoskeleton and of associated integrin adhesion complexes. Rho regulates stress fibre and focal adhesion assembly, Rac regulates the formation of lamellipodia protrusions and membrane ruffles, and Cdc42 triggers filopodial extensions at the cell periphery. These observations have led to the suggestion that wherever filamentous actin is used to drive a cellular process, Rho GTPases are likely to play an important regulatory role. Rho GTPases have also been reported to control other cellular activities, such as the JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) cascades, an NADPH oxidase enzyme complex, the transcription factors NF-kappaB (nuclear factor kappaB) and SRF (serum-response factor), and progression through G(1) of the cell cycle. Thus Rho, Rac and Cdc42 can regulate the actin cytoskeleton and gene transcription to promote co-ordinated changes in cell behaviour. We have been analysing the biochemical contributions of Rho GTPases in cell movement and have found that Rac controls cell protrusion, while Cdc42 controls cell polarity. PMID: 16246005 [PubMed - in process] --------------------------------------------------------------- 3: Circ Res. 2005 Sep 2;97(5):427-33. Epub 2005 Aug 4. Comment in: Circ Res. 2005 Sep 2;97(5):409-10. Depletion of serum response factor by RNA interference mimics the mitogenic effects of platelet derived growth factor-BB in vascular smooth muscle cells. Kaplan-Albuquerque N, Van Putten V, Weiser-Evans MC, Nemenoff RA. Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA. Promoters of many smooth muscle-specific genes (SM-genes) contain multiple CArG boxes, which represent a binding site for serum response factor (SRF). Transcriptional control through these regions involves interactions with SRF and specific coactivators such as myocardin. We have previously reported that suppression of SM-gene expression by platelet derived growth factor (PDGF) is associated with redistribution of SRF, leading to lower intra-nuclear levels, and a reduction in SRF transactivation. To further assess the role of SRF depletion on VSMC phenotype, the current study used RNA interference (RNAi). Two SRF-specific sequences constructed as hairpins were stably expressed in rat VSMC. Clones expressing SRF RNAi had no detectable SRF expression by immunoblotting, and showed diminished levels of SM alpha-actin protein and promoter activity. Unexpectedly, depletion of VSMC resulted in increased rates of proliferation and migration. Several genes whose expression is increased by PDGF stimulation, including c-Jun, were similarly induced in cells lacking SRF. Effects of SRF depletion were not attributable to altered PDGF receptor activity or alterations in activation of Akt. These data indicate that loss of SRF transactivation in VSMC, in this case through suppression via RNAi, induces biological responses similar to that seen with PDGF. PMID: 16081871 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Oncogene. 2005 Jun 16;24(26):4193-205. Mitogen regulated induction of FRA-1 proto-oncogene is controlled by the transcription factors binding to both serum and TPA response elements. Adiseshaiah P, Peddakama S, Zhang Q, Kalvakolanu DV, Reddy SP. Department of Environmental Health Sciences, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA. FRA-1, a member of the FOS family of transcription factors, is overexpressed in a variety of human tumors, and contributes to tumor progression. In addition to mitogens, various toxicants and carcinogens persistently induce FRA-1 expression in vitro and in vivo. Although the mitogen induced expression of c-FOS is relatively well understood, it is poorly defined in the case of FRA-1. Our recent analysis of the FRA-1 promoter has shown a critical role for a TRE located at -318 in mediating the TPA-induced expression. The -379 to -283 bp promoter segment containing a critical TRE (-318), however, is insufficient for the induction of FRA-1 promoter. Here, we show that a 40-bp (-276/-237) segment, comprising a TCF binding site and the CArG box (collectively known as serum response element, SRE), and an ATF site, is also necessary for the FRA-1 induction by TPA and EGF. Interestingly, the -283 to +32 bp FRA-1 promoter fragment containing an SRE and an ATF site alone was also insufficient to confer TPA sensitivity to a reporter gene. However, in association with the -318 TRE, the SRE and ATF sites imparted a strong TPA-inducibility to the reporter. Similarly, EGF also required these motifs for the full induction of this gene. Using ChIP assays we show that, in contrast to c-Jun, SRF, Elk1, ATF1 and CREB proteins bind to SRE and ATF sites of the FRA-1 promoter, constitutively. RNAi-mediated knockdown of endogenous SRF, ELK1 and c-JUN protein expression significantly reduced TPA-stimulated FRA-1 promoter activity. Thus, a bipartite enhancer formed by an upstream TRE and the downstream SRE and ATF sites and the cognate factors is necessary and sufficient for the regulation of FRA-1 in response to mitogens. PMID: 15806162 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Cell Signal. 2005 Apr;17(4):461-71. TIM, a Dbl-related protein, regulates cell shape and cytoskeletal organization in a Rho-dependent manner. Xie X, Chang SW, Tatsumoto T, Chan AM, Miki T. Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892-4255, USA. The Dbl-like guanine nucleotide exchange factors (GEFs) have been implicated in direct activation of the Rho family of small GTPases. We previously isolated transforming immortalized mammary (TIM) as a Dbl-like protein. Here, we show that, when expressed in cells, TIM was a potent activator of RhoA. Like activated Rho proteins, expression of TIM potentiated the serum response factor (SRF)- and AP-1-regualted transcriptional activities and activated the SAPK/JNK signaling pathway. In NIH 3T3 cells, TIM induced transforming foci, which was inhibited by the ROCK inhibitor Y-27632 or the dominant negative mutants of Rho proteins. Expression of TIM led to pronounced changes in cell shape and organization of the actin cytoskeleton, including the formation of thick stress fibers at the cell periphery and cell rounding. TIM also promoted redistribution of vinculin-enriched focal adhesions at the cell periphery and increased the phosphorylation of myosin light chain (MLC). These results, taken together, suggest that TIM acts as an upstream regulator for the RhoA/ROCK-mediated cellular functions. PMID: 15601624 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Mol Pharmacol. 2005 Mar;67(3):789-97. Epub 2004 Nov 18. Regulation of serum response factor-dependent gene expression by proteasome inhibitors. Sandbo N, Qin Y, Taurin S, Hogarth DK, Kreutz B, Dulin NO. Section of Pulmonary and Critical Care Medicine, The University of Chicago Department of Medicine, 5841 S. Maryland Ave., MC 6076, Chicago, IL 60637, USA. Serum response factor (SRF) is activated by contractile and hypertrophic agonists, such as endothelin-1 (ET1) to stimulate expression of cytoskeletal proteins in vascular smooth muscle cells (VSMCs). While studying the regulation of smooth muscle alpha-actin (SMA) expression at the level of protein stability, we discovered that inhibition of proteasome-dependent protein degradation by N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) or lactacystin (LC) did not enhance the levels of SMA, but, unexpectedly, attenuated SMA expression in response to ET1, without affecting the viability of VSMCs. Down-regulation of SMA protein by MG132 or LC occurred at the level of SMA transcription and via the inhibition of SRF activity. By contrast, MG132 and LC potentiated the activity of activator protein-1 transcription factor. Regulation of SRF by MG132 was not related to inhibition of nuclear factor-kappaB, an established target of proteasome inhibitors, and was not mediated by protein kinase A, a powerful regulator of SRF activity. Signaling studies indicate that inhibition of ET1-induced SRF activity by MG132 occurs at the level downstream of heterotrimeric G proteins Gq/11 and G13, of small GTPase RhoA, and of actin dynamics but at the level of SRF-DNA binding. MG132 treatment did not result in ubiquitination or accumulation of SRF. By contrast, the levels of c-Jun were rapidly increased upon incubation of cells with MG132, and ectopic overexpression of c-Jun mimicked the effect of MG132 on SRF activity. Together, these data suggest that inhibition of proteasome results in down-regulation of SMA expression via up-regulation of c-Jun and repression of SRF activity at the level of DNA binding. PMID: 15550677 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Cell Biochem. 2003 Apr 15;88(6):1129-44. Progression of chondrogenesis in C3H10T1/2 cells is associated with prolonged and tight regulation of ERK1/2. Seghatoleslami MR, Roman-Blas JA, Rainville AM, Modaressi R, Danielson KG, Tuan RS. Division of Rheumatology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. reza.seghatoleslami@mexil.tju.edu Close contact of mesenchymal cells in vivo and also in super dense micromass cultures in vitro results in cellular condensation and alteration of existing cellular signaling required for initiation and progression of chondrogenesis. To investigate chondrogenesis related changes in the activity of ubiquitous cell signaling mediated by mitogen-activated protein kinases (MAP kinase), we have compared the effect of cell seeding of pluripotent C3H10T1/2 mesenchymal cells as monolayers (non-chondrogenic culture) or high density micromass cultures (chondrogenic) on the regulation and phosphorylation state of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and also on regulation of ERK1/2 nuclear targets, namely, activation protein-1 (AP-1) and serum response factor (SRF). Increasing cell density resulted in reduced DNA binding as well as activity of AP-1. SRF activity, on the other hand, was up-regulated in confluent monolayer cultures but like AP-1 was inhibited in micromass cultures. Low levels of PD 98059 (5 microM), a specific inhibitor of ERK1/2, resulted in delayed induction of AP-1 and SRF activity whereas higher concentrations of this inhibitor (10-50 microM) conferred an opposite effect. Increasing concentrations of the PD 98059 inhibitor in long term monolayer or micromass cultures (2.5 day) resulted in differential regulation of c-Fos and c-Jun protein levels as well as total expression and phosphorylation levels of ERK1/2. PD 98059 treatment of C3H10T1/2 micromass cultures also resulted in up-regulation of type IIB collagen and Sox9 gene expression. While high expression of aggrecan and type IIB collagen genes were dependent on BMP-2 signaling, ERK inhibition of BMP-2 treated micromass cultures resulted in reduced activity of both genes. Our findings show that the activity of ERK1/2 in chondrogenic cultures of C3H10T1/2 cells is tightly controlled and can cross interact with other signaling activities mediated by BMP-2 to positively regulate chondrogensis. PMID: 12647296 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: J Biol Chem. 2003 Apr 18;278(16):13847-54. Epub 2003 Feb 7. Expression of angiogenic factor Cyr61 during neuronal cell death via the activation of c-Jun N-terminal kinase and serum response factor. Kim KH, Min YK, Baik JH, Lau LF, Chaqour B, Chung KC. Department of Biology, Yonsei University College of Sciences, Seoul 120-749, Korea. The immediate early gene, cyr61, is transcriptionally activated within minutes by serum and serum growth factors. The encoded Cyr61 protein is secreted into the extracellular matrix and promotes cell adhesion and migration. In this study, we sought to examine the expression profile of cyr61 gene during neuronal cell death induced by various toxic stimuli and the mechanisms involved. Our data show that toxic stimuli, such as etoposide, significantly increased cyr61 mRNA levels in immortalized hippocampal progenitor (H19-7) cells. Cyr61 transcriptional activation was corroborated at the protein level as well. To identify the upstream signaling cascades involved in cyr61 gene induction, the blocking effect of either JNK or p38 kinase-signaling pathway on cyr61 induction in response to etoposide was tested. Transfection of the cells with a kinase-deficient mutant MEKK, an upstream activator of JNK, significantly decreased the cyr61 expression induced by etoposide. In contrast, cyr61 mRNA levels did not change after pretreatment with SB203580, the p38 kinase inhibitor. When the induction of cyr61 was tested by using several of its deleted promoters driving the expression of reporter gene, the promoter activation occurred primarily within the region containing an SRE-like CArG box. In addition, the SRF, which binds to the CArG site, was directly phosphorylated by active JNK. Furthermore, the blockade of cyr61 gene expression using an antisense encoding cyr61 sequence significantly inhibited the cell death induced by etoposide. Overall, these results suggest that the induction of the immediate early gene, cyr61, is important for neuronal cell death in the central nervous system hippocampal progenitor cells, and JNK activation, but not of p38, as well as the subsequent SRF phosphorylation are involved in cyr61 gene induction. PMID: 12576482 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Mol Cell Biol. 2002 Jul;22(14):5203-11. Novel transcription coactivator complex containing activating signal cointegrator 1. Jung DJ, Sung HS, Goo YW, Lee HM, Park OK, Jung SY, Lim J, Kim HJ, Lee SK, Kim TS, Lee JW, Lee YC. Department of Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea. Human activating signal cointegrator 1 (hASC-1) was originally isolated as a transcriptional coactivator of nuclear receptors. Here we report that ASC-1 exists as a steady-state complex associated with three polypeptides, P200, P100, and P50, in HeLa nuclei; stimulates transactivation by serum response factor (SRF), activating protein 1 (AP-1), and nuclear factor kappaB (NF-kappaB) through direct binding to SRF, c-Jun, p50, and p65; and relieves the previously described transrepression between nuclear receptors and either AP-1 or NF-kappaB. Interestingly, ectopic expression of Caenorhabditis elegans ASC-1 (ceASC-1), an ASC-1 homologue that binds P200 and P100, like hASC-1, while weakly interacting only with p65, in HeLa cells appears to replace endogenous hASC-1 from the hASC-1 complex and exerts potent dominant-negative effects on AP-1, NF-kappaB, and SRF transactivation. In addition, neutralization of endogenous P50 by single-cell microinjection of a P50 antibody inhibits AP-1 transactivation; the inhibition is relieved by coexpression of wild-type P50, but not of P50DeltaKH, a mutant form that does not interact with P200. Overall, these results suggest that the endogenous hASC-1 complex appears to play an essential role in AP-1, SRF, and NF-kappaB transactivation and to mediate the transrepression between nuclear receptors and either AP-1 or NF-kappaB in vivo. PMID: 12077347 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Am J Physiol Heart Circ Physiol. 2001 Apr;280(4):H1782-92. Cardiomyopathy in transgenic mice with cardiac-specific overexpression of serum response factor. Zhang X, Azhar G, Chai J, Sheridan P, Nagano K, Brown T, Yang J, Khrapko K, Borras AM, Lawitts J, Misra RP, Wei JY. Department of Medicine, Beth Israel Deaconess Medical Center, and Division on Aging, Harvard Medical School, Boston, Massachusetts 02215, USA. Serum response factor (SRF), a member of the MCM1, agamous, deficiens, SRF (MADS) family of transcriptional activators, has been implicated in the transcriptional control of a number of cardiac muscle genes, including cardiac alpha-actin, skeletal alpha-actin, alpha-myosin heavy chain (alpha-MHC), and beta-MHC. To better understand the in vivo role of SRF in regulating genes responsible for maintenance of cardiac function, we sought to test the hypothesis that increased cardiac-specific SRF expression might be associated with altered cardiac morphology and function. We generated transgenic mice with cardiac-specific overexpression of the human SRF gene. The transgenic mice developed cardiomyopathy and exhibited increased heart weight-to-body weight ratio, increased heart weight, and four-chamber dilation. Histological examination revealed cardiomyocyte hypertrophy, collagen deposition, and interstitial fibrosis. SRF overexpression altered the expression of SRF-regulated genes and resulted in cardiac muscle dysfunction. Our results demonstrate that sustained overexpression of SRF, in the absence of other stimuli, is sufficient to induce cardiac change and suggest that SRF is likely to be one of the downstream effectors of the signaling pathways involved in mediating cardiac hypertrophy. PMID: 11247792 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Oncogene. 2000 Sep 28;19(41):4685-94. Rit, a non-lipid-modified Ras-related protein, transforms NIH3T3 cells without activating the ERK, JNK, p38 MAPK or PI3K/Akt pathways. Rusyn EV, Reynolds ER, Shao H, Grana TM, Chan TO, Andres DA, Cox AD. Department of Radiation Oncology, Lineberger Cancer Center, University of North Carolina at Chapel Hill, 27599, USA. The biological functions of Rit (Ras-like protein in tissues) and Rin (Ras-like protein in neurons), members of a novel branch of Ras-related GTP-binding proteins that are approximately 50% identical to Ras, have not been characterized. Therefore, we assessed their activity in growth control, transformation and signaling. NIH cells stably expressing a constitutively activated mutant of Rit [Rit(79L)] (analogous to the oncogenic mutant H-Ras(61L)) demonstrated strong growth transformation, proliferating rapidly in low serum and forming colonies in soft agar and tumors in nude mice. Although Rit(79L) alone did not promote morphologically transformed foci, it cooperated with both Raf and Rho A to form Rac/Rho-like foci. Rin [Rin(78L)] cooperated only with Raf. Rit(79L) but not Rin(78L) stimulated transcription from luciferase reporter constructs regulated by SRF, NF-kappaB, Elk-1 and Jun. However, neither activated ERK, JNK or p38, or PI3-K/Akt kinases in immune complex kinase assays. Interestingly, although Rit lacks any known recognition signal for C-terminal lipidation, Rit-transformed cell growth and survival in low serum is dependent on a farnesylated protein, as treatment with farnesyltransferase inhibitors caused apoptosis. Rin cooperated with Raf in focus assays but did not otherwise function in these assays, perhaps due to a lack of appropriate effector pathways in NIH3T3 fibroblasts for this neural-specific Ras family member. In summary, although Rit shares most core effector domain residues with Ras, our results suggest that Rit uses novel effector pathways to regulate proliferation and transformation. PMID: 11032018 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Life Sci. 2000 Jun 16;67(4):335-64. Molecular mechanisms associated with long-term consolidation of the NMDA signals. Platenik J, Kuramoto N, Yoneda Y. Department of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Kanazawa University, Ishikawa, Japan. The N-methyl-D-aspartate (NMDA) subtype of glutamate receptors in the mammalian brain plays a central role in synaptic plasticity underlying refinement of neuronal connections during development, or processes like long-term potentiation (LTP), learning and memory. On the other hand, over-activation of glutamate receptors leading to neurodegeneration has been implicated in major areas of brain pathology. Any sustained effect of a transient NMDA receptor activation is likely to involve signaling to the nucleus and coordinated changes in gene expression. Classically, a set of immediate-early genes is induced first; some of them are themselves transcription factors that control expression of other target genes. This review deals with the induction of Fos, Jun and Egr (Krox) transcription factors in response to NMDA or non-NMDA (AMPA/kainate) ionotropic receptor agonists in vivo or in neuronal cultures in vitro. In addition, the mechanism of induction of a model immediate-early gene c-fos in response to Ca2+ influx through activated NMDA receptors or voltage-sensitive calcium channels is discussed. Both modes of calcium entry induce c-fos via activation of multiple signaling pathways that converge on constitutive transcription factors cAMP-response element-binding protein (CREB), serum response factor (SRF) and a ternary complex factor (TCF), such as Elk-1. In contrast to the traditional view of the NMDA receptor as a ligand-gated calcium channel, whose activation leads to calcium influx and activation of Ca2+/calmodulin-dependent kinases, recent evidence highlights involvement of the Ras/ mitogen-activated protein kinase (MAPK) pathway in the NMDA signaling to the nucleus. Publication Types: Review PMID: 11003045 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Mol Biol Cell. 2000 Aug;11(8):2513-28. Critical activities of Rac1 and Cdc42Hs in skeletal myogenesis: antagonistic effects of JNK and p38 pathways. Meriane M, Roux P, Primig M, Fort P, Gauthier-Rouviere C. Centre de Recherche de Biochimie Macromoleculaire, Centre National de la Recherche Scientifique Unite Propre de Recherche, Montpellier, France. The Rho family of GTP-binding proteins plays a critical role in a variety of cellular processes, including cytoskeletal reorganization and activation of kinases such as p38 and C-jun N-terminal kinase (JNK) MAPKs. We report here that dominant negative forms of Rac1 and Cdc42Hs inhibit the expression of the muscle-specific genes myogenin, troponin T, and myosin heavy chain in L6 and C2 myoblasts. Such inhibition correlates with decreased p38 activity. Active RhoA, RhoG, Rac1, and Cdc42Hs also prevent myoblast-to-myotube transition but affect distinct stages: RhoG, Rac1, and Cdc42Hs inhibit the expression of all muscle-specific genes analyzed, whereas active RhoA potentiates their expression but prevents the myoblast fusion process. We further show by two different approaches that the inhibitory effects of active Rac1 and Cdc42Hs are independent of their morphogenic activities. Rather, myogenesis inhibition is mediated by the JNK pathway, which also leads to a cytoplasmic redistribution of Myf5. We propose that although Rho proteins are required for the commitment of myogenesis, they differentially influence this process, positively for RhoA and Rac1/Cdc42Hs through the activation of the SRF and p38 pathways, respectively, and negatively for Rac1/Cdc42Hs through the activation of the JNK pathway. PMID: 10930450 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Brain Res Mol Brain Res. 2000 Jun 23;79(1-2):138-49. Chronic DeltaFosB expression and increased AP-1 transcription factor binding are associated with the long term plasticity changes in epilepsy. Morris TA, Jafari N, DeLorenzo RJ. Department of Neurology, Medical College of Virginia at Virginia Commonwealth University, Richmond 23298, USA. NMDA receptor activation during status epilepticus (SE) has previously been shown to be required for epileptogenesis as well as the persistent upregulation of serum response factor (SRF) in the in vivo pilocarpine model of epilepsy. SRF is established as a regulator of the FosB gene which expresses FosB and DeltaFosB components of the AP-1 transcription factor complex. Therefore we investigated whether DeltaFosB expression and AP-1 DNA binding were also persistently elevated in pilocarpine-treated rats which chronically displayed spontaneous seizures. Using hippocampal nuclear extracts, DeltaFosB expression and AP-1 DNA binding were significantly elevated for up to one year in the epileptic animals. The expression of other fos and jun proteins was not persistently altered in epilepsy. Neuronal upregulation of DeltaFosB was correlated with regions of the brain that were involved in seizure generation and propagation. The increase in AP-1 DNA binding was shown to be dependent on NMDA receptor activation during SE. Hippocampal DeltaFosB immunostaining was seen predominately in the neuronal nuclei as opposed to other cell types. The data indicate that recurrent seizures which persistently occur in this model were not responsible for the increased DeltaFosB expression. Chronic DeltaFosB expression in epilepsy may be playing a role in the altered expression of other genes in this model and may be involved in some of the neuronal plasticity changes associated with epileptogenesis. PMID: 10925151 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Mol Endocrinol. 2000 Jun;14(6):915-25. Activating protein-1, nuclear factor-kappaB, and serum response factor as novel target molecules of the cancer-amplified transcription coactivator ASC-2. Lee SK, Na SY, Jung SY, Choi JE, Jhun BH, Cheong J, Meltzer PS, Lee YC, Lee JW. Center for Ligand and Transcription, Department of Biology, Chonnam National University, Kwangju, Korea. ASC-2 was recently discovered as a cancer-amplified transcription coactivator molecule of nuclear receptors, which interacts with multifunctional transcription integrators steroid receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP)/p300. Herein, we report the identification of three mitogenic transcription factors as novel target molecules of ASC-2. First, the C-terminal transactivation domain of serum response factor (SRF) was identified among a series of ASC-2-interacting proteins from the yeast two-hybrid screening. Second, ASC-2 specifically interacted with the activating protein-1 (AP-1) components c-Jun and c-Fos as well as the nuclear factor-kappaB (NFkappaB) components p50 and p65, as demonstrated by the glutathione S-transferase pull-down assays as well as the yeast two-hybrid tests. In cotransfection of mammalian cells, ASC-2 potentiated transactivations by SRF, AP-1, and NFkappaB in a dose-dependent manner, either alone or in conjunction with SRC-1 and p300. In addition, ASC-2 efficiently relieved the previously described transrepression between nuclear receptors and either AP-1 or NFkappaB. Overall, these results suggest that the nuclear receptor coactivator ASC-2 also mediates transactivations by SRF, AP-1, and NFkappaB, which may contribute to the putative, ASC-2-mediated tumorigenesis. PMID: 10847592 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Curr Top Cell Regul. 2000;36:201-16. Antioxidant regulation of genes encoding enzymes that detoxify xenobiotics and carcinogens. Dhakshinamoorthy S, Long DJ 2nd, Jaiswal AK. Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030, USA. Antioxidants are substances that delay or prevent the oxidation of cellular oxidizable substrates. The various antioxidants exert their effect by scavenging superoxide or by activating a battery of detoxifying/defensive proteins. In this chapter, we have focused on the mechanisms by which antioxidants induce gene expression. Many xenobiotics (e.g., beta-naphthoflavone) activate genes similar to those activated by antioxidants. The promoters of these genes contain a common cis-element, termed the antioxidant response element (ARE), which contains two TRE (TPA response element) or TRE-like elements followed by GC box. Mutational studies have identified GTGAC***GC as the core of the ARE sequence. Many transcription factors, including Nrf, Jun, Fos, Fra, Maf, YABP, ARE-BP1, Ah (aromatic hydrocarbon) receptor, and estrogen receptor bind to the ARE from the various genes. Among these factors, Nrf-Jun heterodimers positively regulate ARE-mediated expression and induction of genes in response to antioxidants and xenobiotics. This Nrf-Jun heterodimerization and binding to the ARE requires unknown cytosolic factors. The mechanism of signal transduction from antioxidants and xenobiotics includes several steps: (1) Antioxidants and xenobiotics undergo metabolism to generate superoxide and related reactive species, leading to the generation of a signal to activate expression of detoxifying/defensive genes. (2) The generation of superoxide and related reactive species is followed by activation of yet to be identified cytosolic factors by unknown mechanism(s). (3). Activated cytosolic factors catalyze modification of Nrf and/or Jun proteins, which bind to the ARE in promoters of the various detoxifying/defensive genes. (4) The transcription of genes encoding detoxifying/defensive proteins is increased. The unknown cytosolic factors are significant molecules because they represent the oxidative sensors within the cells. Identification of the cytosolic factors will be of considerable importance in the field of antioxidants and gene regulation research. Future studies will also be required to completely understand the molecular mechanism of signal transduction from antioxidants and xenobiotics to Nrf-Jun. In addition to the Nrf-Jun pathway, mammalian cells also contain other pathways that activate gene expression in response to oxidative stress. These include NF-KB-, HIF-1-, Mac-1-, and SRF-mediated pathways. It is expected that collectively these pathways increase transcription of more than four dozen genes to protect cells against oxidative stress. Publication Types: Review Review, Tutorial PMID: 10842753 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: J Biol Chem. 2000 Jul 21;275(29):22537-43. Induction of smooth muscle alpha-actin in vascular smooth muscle cells by arginine vasopressin is mediated by c-Jun amino-terminal kinases and p38 mitogen-activated protein kinase. Garat C, Van Putten V, Refaat ZA, Dessev C, Han SY, Nemenoff RA. Departments of Medicine and Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. Exposure of vascular smooth muscle cells to arginine vasopressin (AVP) increases smooth muscle alpha-actin (SM-alpha-actin) expression through activation of the SM- alpha-actin promoter. The goal of this study was to determine the role of the mitogen-activated protein kinase (MAP kinase) family in regulation of SM-alpha-actin expression. AVP activated all three MAP kinase family members: ERKs, JNKs, and p38 MAP kinase. Inhibition of JNKs or p38 decreased AVP-stimulated SM-alpha-actin promoter activity, whereas inhibition of ERKs had no effect. A 150-base pair region of the promoter containing two CArG boxes was sufficient to mediate regulation by vasoconstrictors. Mutations in either CArG box decreased AVP-stimulated promoter activity. Electrophoretic mobility shift assays using oligonucleotides corresponding to either CArG box resulted in a complex of similar mobility whose intensity was increased by AVP. Antibodies against serum response factor (SRF) completely super-shifted this complex, indicating that SRF binds to both CArG boxes. Overexpression of SRF increased basal promoter activity, but activity was still stimulated by AVP. AVP stimulation rapidly increased SRF phosphorylation. These data indicate that both JNKs and p38 participate in regulation of SM- alpha-actin expression. SRF, which binds to two critical CArG boxes in the promoter, represents a potential target of these kinases. PMID: 10807920 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: J Biol Chem. 2000 Apr 28;275(17):12470-4. Silencing mediator of retinoic acid and thyroid hormone receptors, as a novel transcriptional corepressor molecule of activating protein-1, nuclear factor-kappaB, and serum response factor. Lee SK, Kim JH, Lee YC, Cheong J, Lee JW. Center for Ligand and Transcription, Chonnam National University, Kwangju 500-757, Korea. Silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is known to interact with Sin3 and recruit the histone deacetylases (HDACs) that lead to hypoacetylation of histones and transrepression of target transcription factors. Herein, we found that coexpression of SMRT significantly repressed transactivations by activating protein-1 (AP-1), nuclear factor-kappaB (NFkappaB), and serum response factor (SRF) in a dose-dependent manner, but not in the presence of trichostatin A, a specific inhibitor of HDAC. Similarly, coexpression of HDAC1 and mSin3A also showed repressive effects. Consistent with these results, the C-terminal region of SMRT directly interacted with SRF, the AP-1 components c-Jun and c-Fos, and the NFkappaB components p50 and p65, as demonstrated by the yeast and mammalian two hybrid tests as well as the glutathione S-transferase pull down assays. Thus, we concluded that SMRT serves to recruit Sin3/HDACs to SRF, NFkappaB, and AP-1 in vivo and modulate their transactivation. PMID: 10777532 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: J Mol Biol. 2000 Mar 24;297(2):437-49. Crystal structure of MEF2A core bound to DNA at 1.5 A resolution. Santelli E, Richmond TJ. Institut fur Molekularbiologie und Biophysik, ETH Zurich, Zurich, CH, Switzerland. Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors bind to and activate transcription through A+T-rich DNA sequences found primarily, but not exclusively, in the promoters of muscle-specific genes. Their importance has been established for myogenic development and in activation of the immediate-early gene, c-jun, and recently further functional roles in the immune system have emerged. The MEF2 factors belong to the MADS-box superfamily, sharing homology in a 58 amino acid domain that mediates DNA binding and dimerization. The structures of two MADS-box proteins, SRF and MCM1, bound to their cognate DNA have been previously reported and shown to share extensive similarity in their mode of DNA binding. We have solved the structure of MEF2A 2-78 bound to its DNA consensus sequence at 1.5 A resolution. It reveals how the absence of amino acids N-terminal to the MADS-box contributes to the DNA binding properties of MEF2 proteins and shows that the MEF domain C-terminal to the MADS-box adopts a conformation considerably different from the same region in SRF and MCM1. Copyright 2000 Academic Press. PMID: 10715212 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Cancer Res. 1999 Aug 1;59(15):3795-802. Transformation blocks differentiation-induced inhibition of serum response factor interactions with serum response elements. Ding W, Witte MM, Scott RE. Department of Pathology, University of Tennessee Medical Center, Memphis 38163, USA. The differentiation of nontransformed 3T3T mesenchymal stem cells is a multistep process that is associated with the progressive repression of mitogenic responsiveness to serum growth factors that ultimately results in expression of the terminally differentiated adipocyte phenotype. The repression of serum-induced mitogenesis by differentiation correlates with repression of the serum-inducible transcription of junB and c-fos. In contrast, the differentiation of neoplastically transformed cells does not repress mitogenic responsiveness or junB or c-fos inducibility. Because the junB and c-fos promoters both contain serum response elements (SREs), the current studies tested the possibility that differentiation might repress the ability of serum response factor (SRF) to bind to the SRE in normal cells but not in transformed cells. We now report that differentiation represses SRE serum inducibility using nontransformed cells transiently transfected with pjunB SRE thymidine kinase/chloroamphenicol acetyltransferase (SREtk/CAT) or pc-fos SREtk/CAT containing an intact SRF-binding domain. Adipocyte differentiation of nontransformed cells also markedly represses the ability of SRF to bind to the junB SRE, the c-fos SRE, and other SREs, as determined by mobility shift and gel supershift assays, without affecting the DNA binding characteristics of the nuclear protein SP-1. By comparison, the ability of SRF to bind SRE is not repressed by the differentiation of SV40 large T antigen-transformed 3T3T cells. The results further establish that adipocyte differentiation blocks the nuclear localization of SRF, thus preventing its interaction with SREs in nontransformed cells but not in transformed cells. PMID: 10446998 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Oncogene. 1999 Jul 1;18(26):3831-45. Cellular functions of TC10, a Rho family GTPase: regulation of morphology, signal transduction and cell growth. Murphy GA, Solski PA, Jillian SA, Perez de la Ossa P, D'Eustachio P, Der CJ, Rush MG. Department of Biochemistry, New York University Medical Center, New York 10016, USA. The small Ras-related GTPase, TC10, has been classified on the basis of sequence homology to be a member of the Rho family. This family, which includes the Rho, Rac and CDC42 subfamilies, has been shown to regulate a variety of apparently diverse cellular processes such as actin cytoskeletal organization, mitogen-activated protein kinase (MAPK) cascades, cell cycle progression and transformation. In order to begin a study of TC10 biological function, we expressed wild type and various mutant forms of this protein in mammalian cells and investigated both the intracellular localization of the expressed proteins and their abilities to stimulate known Rho family-associated processes. Wild type TC10 was located predominantly in the cell membrane (apparently in the same regions as actin filaments), GTPase defective (75L) and GTP-binding defective (31N) mutants were located predominantly in cytoplasmic perinuclear regions, and a deletion mutant lacking the carboxyl terminal residues required for post-translational prenylation was located predominantly in the nucleus. The GTPase defective (constitutively active) TC10 mutant: (1) stimulated the formation of long filopodia; (2) activated c-Jun amino terminal kinase (JNK); (3) activated serum response factor (SRF)-dependent transcription; (4) activated NF-kappaB-dependent transcription; and (5) synergized with an activated Raf-kinase (Raf-CAAX) to transform NIH3T3 cells. In addition, wild type TC10 function is required for full H-Ras transforming potential. We demonstrate that an intact effector domain and carboxyl terminal prenylation signal are required for proper TC10 function and that TC10 signals to at least two separable downstream target pathways. In addition, TC10 interacted with the actin-binding and filament-forming protein, profilin, in both a two-hybrid cDNA library screen, and an in vitro binding assay. Taken together, these data support a classification of TC10 as a member of the Rho family, and in particular, suggest that TC10 functions to regulate cellular signaling to the actin cytoskeleton and processes associated with cell growth. PMID: 10445846 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Brain Res Mol Brain Res. 1999 Jun 8;69(2):232-41. Co-expression of c-Jun and ATF-2 characterizes the surviving retinal ganglion cells which maintain axonal connections after partial optic nerve injury. Kreutz MR, Bien A, Vorwerk CK, Bockers TM, Seidenbecher CI, Tischmeyer W, Sabel BA. Department of Neurochemistry/Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany. kreutz@ifn-magdeburg.de The expression of c-fos, c-jun, jun-b, jun-d, srf and pc4 mRNA was examined after partial optic nerve crush in the adult rat retina by in situ hybridization. Optic nerve injury led exclusively to the upregulation of c-jun, with cellular label indicative for c-jun mRNA in the retinal ganglion cell layer after two days, three days and one week post-injury. This expression pattern was in accordance with the appearance of c-Jun immunoreactivity in retinal flat mounts. Injection of an antisense but not a missense oligonucleotide against c-jun after partial crush resulted in a reduced number of connected retinal ganglion cells (RGCs) as shown by retrograde labeling. Prelabeling of RGCs with fluorogold before optic nerve section and subsequent antisense targeting against c-jun, however, led to a slightly higher number of surviving but axotomized RGCs. C-Jun antibody staining of retinal whole mounts pre- or postlabeled after crush by intracollicular administration of fluorogold showed strong c-Jun immunoreactivity in connected RGCs and also in a population of disconnected RGCs. Double labeling with an antibody directed against the transcription factor ATF-2 revealed strong co-expression of c-Jun and ATF-2 in connected RGCs but not in axotomized cells. Taken together these data indicate that both RGCs in continuity and those in discontinuity with the superior colliculus respond both equally to the noxious stimulus with c-Jun expression. Moreover, the co-expression of c-Jun with high levels of ATF-2 appears to be essential for either the continuity or survival of RGCs which remain connected with their target. In disconnected RGCs, however, low levels of ATF-2 and the co-expression of c-Jun may be related to cell death. Copyright 1999 Elsevier Science B.V. PMID: 10366744 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: J Biol Chem. 1999 Feb 5;274(6):3772-80. C3 toxin activates the stress signaling pathways, JNK and p38, but antagonizes the activation of AP-1 in rat-1 cells. Beltman J, Erickson JR, Martin GA, Lyons JF, Cook SJ. ONYX Pharmaceuticals, Richmond, California 94806, USA. Lysophosphatidic acid (LPA) stimulates the c-Fos serum response element (SRE) by activating two distinct signal pathways regulated by the small GTPases, Ras and RhoA. Ras activates the ERK cascade leading to phosphorylation of the transcription factors Elk-1 and Sap1a at the Ets/TCF site. RhoA regulates an undefined pathway required for the activation of the SRF/CArG site. Here we have examined the role of the Ras and RhoA pathways in activation of the SRE and c-Fos expression in Rat-1 cells. Pertussis toxin and PD98059 strongly inhibited LPA-stimulated c-Fos expression and activation of a SRE:Luc reporter. C3 toxin completely inhibited RhoA function, partially inhibited SRE:Luc activity, but had no effect on LPA-stimulated c-Fos expression. Thus, in a physiological context the Ras-Raf-MEK-ERK pathway, but not RhoA, is required for LPA-stimulated c-Fos expression in Rat-1 cells. C3 toxin stimulated the stress-activated protein kinases JNK and p38 and potentiated c-Jun expression and phosphorylation; these properties were shared by another cellular stress agonist the protein kinase C inhibitor Ro-31-8220. However, C3 toxin alone or in combination with growth factors did not stimulate AP-1:Luc activity and actually antagonized the synergistic activation of AP-1:Luc observed in response to co-stimulation with growth factors and Ro-31-8220. These data indicate that C3 toxin is a cellular stress which antagonizes activation of AP-1 at a point downstream of stress-activated kinase activation or immediate-early gene induction. PMID: 9920930 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Mol Cell Biol. 1999 Feb;19(2):1313-24. Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Soh JW, Lee EH, Prywes R, Weinstein IB. Department of Biochemistry & Molecular Biophysics, College of Physicians & Surgeons, Columbia University, New York, New York 10032, USA. Protein kinase C (PKC) is a multigene family of enzymes consisting of at least 11 isoforms. It has been implicated in the induction of c-fos and other immediate response genes by various mitogens. The serum response element (SRE) in the c-fos promoter is necessary and sufficient for induction of transcription of c-fos by serum, growth factors, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). It forms a complex with the ternary complex factor (TCF) and with a dimer of the serum response factor (SRF). TCF is the target of several signal transduction pathways and SRF is the target of the rhoA pathway. In this study we generated dominant-negative and constitutively active mutants of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta to determine the roles of individual isoforms of PKC in activation of the SRE. Transient-transfection assays with NIH 3T3 cells, using an SRE-driven luciferase reporter plasmid, indicated that PKC-alpha and PKC-epsilon, but not PKC-delta or PKC-zeta, mediate SRE activation. TPA-induced activation of the SRE was partially inhibited by dominant negative c-Raf, ERK1, or ERK2, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of Elk-1. TPA-induced activation of the SRE was also partially inhibited by a dominant-negative MEKK1. Furthermore, TPA treatment of serum-starved NIH 3T3 cells led to phosphorylation of SEK1, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of c-Jun, a major substrate of JNK. Constitutively active mutants of PKC-alpha and PKC-epsilon could also induce a mutant c-fos promoter which lacks the TCF binding site, and they also induce transactivation activity of the SRF. Furthermore, rhoA-mediated SRE activation was blocked by dominant negative mutants of PKC-alpha or PKC-epsilon. Taken together, these findings indicate that PKC-alpha and PKC-epsilon can enhance the activities of at least three signaling pathways that converge on the SRE: c-Raf-MEK1-ERK-TCF, MEKK1-SEK1-JNK-TCF, and rhoA-SRF. Thus, specific isoforms of PKC may play a role in integrating networks of signal transduction pathways that control gene expression. PMID: 9891065 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Brain Res Brain Res Rev. 1998 Dec;28(3):370-490. Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Herdegen T, Leah JD. Institute of Pharmacology, University of Kiel, Hospitalstrasse 4, 24105, Kiel, Germany.t.herdegen@pharmakologie.uni-kiel.de This article reviews findings up to the end of 1997 about the inducible transcription factors (ITFs) c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, Fra-2, Krox-20 (Egr-2) and Krox-24 (NGFI-A, Egr-1, Zif268); and the constitutive transcription factors (CTFs) CREB, CREM, ATF-2 and SRF as they pertain to gene expression in the mammalian nervous system. In the first part we consider basic facts about the expression and activity of these transcription factors: the organization of the encoding genes and their promoters, the second messenger cascades converging on their regulatory promoter sites, the control of their transcription, the binding to dimeric partners and to specific DNA sequences, their trans-activation potential, and their posttranslational modifications. In the second part we describe the expression and possible roles of these transcription factors in neural tissue: in the quiescent brain, during pre- and postnatal development, following sensory stimulation, nerve transection (axotomy), neurodegeneration and apoptosis, hypoxia-ischemia, generalized and limbic seizures, long-term potentiation and learning, drug dependence and withdrawal, and following stimulation by neurotransmitters, hormones and neurotrophins. We also describe their expression and possible roles in glial cells. Finally, we discuss the relevance of their expression for nervous system functioning under normal and patho-physiological conditions. Copyright 1998 Elsevier Science B.V. Publication Types: Review PMID: 9858769 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: J Biol Chem. 1998 Aug 7;273(32):20636-43. p38 Mitogen-activated protein kinase mediates the transcriptional induction of the atrial natriuretic factor gene through a serum response element. A potential role for the transcription factor ATF6. Thuerauf DJ, Arnold ND, Zechner D, Hanford DS, DeMartin KM, McDonough PM, Prywes R, Glembotski CC. Department of Biology and Molecular Biology, Institute, San Diego State University, San Diego, California 92182, USA. In various cell types certain stresses can stimulate p38 mitogen-activated protein kinase (p38 MAPK), leading to the transcriptional activation of genes that contribute to appropriate compensatory responses. In this report the mechanism of p38-activated transcription was studied in cardiac myocytes where this MAPK is a key regulator of the cell growth and the cardiac-specific gene induction that occurs in response to potentially stressful stimuli. In the cardiac atrial natriuretic factor (ANF) gene, a promoter-proximal serum response element (SRE), which binds serum response factor (SRF), was shown to be critical for ANF induction in primary cardiac myocytes transfected with the selective p38 MAPK activator, MKK6 (Glu). This ANF SRE does not possess sequences typically required for the binding of the Ets-related ternary complex factors (TCFs), such as Elk-1, indicating that p38-mediated induction through this element may take place independently of such TCFs. Although p38 did not phosphorylate SRF in vitro, it efficiently phosphorylated ATF6, a newly discovered SRF-binding protein that is believed to serve as a co-activator of SRF-inducible transcription at SREs. Expression of an ATF6 antisense RNA blocked p38-mediated ANF induction through the ANF SRE. Moreover, when fused to the Gal4 DNA-binding domain, an N-terminal 273-amino acid fragment of ATF6 was sufficient to support trans-activation of Gal4/luciferase expression in response to p38 but not the other stress kinase, N-terminal Jun kinase (JNK); p38-activating cardiac growth promoters also stimulated ATF6 trans-activation. These results indicate that through ATF6, p38 can augment SRE-mediated transcription independently of Ets-related TCFs, representing a novel mechanism of SRF-dependent transcription by MAP kinases. PMID: 9685422 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Neurosci Lett. 1998 Jan 30;241(2-3):87-90. Differential time course of angiotensin-induced AP-1 and Krox proteins in the rat lamina terminalis and hypothalamus. Blume A, Seifert K, Lebrun CJ, Mollenhoff E, Gass P, Unger T, Herdegen T. Department of Pharmacology, University of Kiel, Germany. We studied the time course of expression of the inducible transcription factors (ITF) c-Fos, FosB, c-Jun, JunB, JunD, Krox-20 and Krox-24, induced by a single intracerebroventricular injection of angiotensin II, in the subfornical organ (SFO), median preoptic nucleus (MnPO) paraventricular nucleus (PVN) and supraoptic nucleus (SON). c-Fos and Krox-24 were expressed rapidly in neurons of all four areas but completely disappeared after 4 h. FosB showed a delayed but persistent expression between 4 h and 24 h in the MnPO and PVN. c-Jun was induced in the MnPO, SFO and PVN after 1.5 h and in the SON after 4 h. JunB was selectively expressed in the MnPO and SFO and the level of JunD did not change. The expression of the pre-existing transcription factors SRF, CREB and ATF-2 which contribute to the transcriptional control of jun, fos and krox genes, was not affected by Ang II. Thus, we could show for the first time that an acute stimulation of AT receptors results in continual changes in ITF expression over 24 h. PMID: 9507927 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Mol Cell Biol. 1998 Mar;18(3):1225-35. Mas oncogene signaling and transformation require the small GTP-binding protein Rac. Zohn IE, Symons M, Chrzanowska-Wodnicka M, Westwick JK, Der CJ. Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill 27599-7038, USA. The Mas oncogene encodes a novel G-protein-coupled receptor that was identified originally as a transforming protein when overexpressed in NIH 3T3 cells. The mechanism and signaling pathways that mediate Mas transformation have not been determined. We observed that the foci of transformed NIH 3T3 cells caused by Mas were similar to those caused by activated Rho and Rac proteins. Therefore, we determined if Mas signaling and transformation are mediated through activation of a specific Rho family protein. First, we observed that, like activated Rac1, Mas cooperated with activated Raf and caused synergistic transformation of NIH 3T3 cells. Second, both Mas- and Rac1-transformed NIH 3T3 cells retained actin stress fibers and showed enhanced membrane ruffling. Third, like Rac, Mas induced lamellipodium formation in porcine aortic endothelial cells. Fourth, Mas and Rac1 strongly activated the JNK and p38, but not ERK, mitogen-activated protein kinases. Fifth, Mas and Rac1 stimulated transcription from common DNA promoter elements: NF-kappaB, serum response factor (SRF), Jun/ATF-2, and the cyclin D1 promoter. Finally, Mas transformation and some of Mas signaling (SRF and cyclin D1 but not NF-kappaB activation) were blocked by dominant negative Rac1. Taken together, these observations suggest that Mas transformation is mediated in part by activation of Rac-dependent signaling pathways. Thus, Rho family proteins are common mediators of transformation by a diverse variety of oncogene proteins that include Ras, Dbl family, and G-protein-coupled oncogene proteins. PMID: 9488437 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Exp Neurol. 1998 Jan;149(1):161-74. BDNF restores the expression of Jun and Fos inducible transcription factors in the rat brain following repetitive electroconvulsive seizures. Hsieh TF, Simler S, Vergnes M, Gass P, Marescaux C, Wiegand SJ, Zimmermann M, Herdegen T. II. Institute of Physiology, University of Heidelberg, Germany. The expression of inducible transcription factors was studied following repetitive electroconvulsive seizures (ECS), c-Fos, c-Jun, JunB, and JunD immunoreactivities were investigated following a single (1 x ECS) or repetitive ECS evoked once per day for 4, 5, or 10 days (4 x ECS, 5 x ECS, or 10 x ECS). Animals were killed 3 or 12 h following the last ECS. Three hours after 1 x ECS, c-Fos was expressed throughout the cortex and hippocampus. After 5 x ECS and 10 x ECS, c-Fos was reexpressed in the CA4 area, but was completely absent in the other hippocampal areas and cortex. In these areas, c-Fos became only reinducible when the time lag between two ECS stimuli was 5 days. In contrast to c-Fos, intense JunB expression was inducible in the cortex and hippocampus, but not CA4 subfield, after 1 x ECS, 5 x ECS, and 10 x ECS. Repetitive ECS did not effect c-Jun and JunD expression. In a second model of systemic excitation of the brain, repetitive daily injection of kainic acid for 4 days completely failed to express c-Fos, c-Jun, and JunB after the last application whereas injection of kainic acid once per week did not alter the strong expressions compared to a single application of kainic acid. In order to study the maintenance of c-Fos expression during repetitive seizures, brain-derived neurotrophic factor (BDNF) was applied in parallel for 5 or 10 days via miniosmotic pumps and permanent cannula targeted at the hippocampus or the parietal cortex. Infusion of BDNF completely reinduced c-Fos expression during 5 x ECS or 10 x ECS in the cortex ipsilaterally to the cannula and, to a less extent, also increased the expression of c-Jun and JunB when compared to saline-treated controls. BDNF had no effect on the expression patterns in the hippocampus. ECS with or without BDNF infusion did not change the expression patterns of the constitutive transcription factors ATF-2, CREB, and SRF. These data demonstrate that various transcription factors substantially differ in their response to acute and chronic neural stimulation. Repetitive pathophysiological excitation decreases the transcriptional actions of neurons over days in the adult brain, and this decrement can be prevented by BDNF restoring the neuroplasticity at the level of gene transcription. PMID: 9454625 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Oncogene. 1997 Nov 27;15(22):2675-85. Identification and characterization of R-ras3: a novel member of the RAS gene family with a non-ubiquitous pattern of tissue distribution. Kimmelman A, Tolkacheva T, Lorenzi MV, Osada M, Chan AM. The Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029, USA. Members of the Ras subfamily of GTP-binding proteins, including Ras (H-, K-, and N-), TC21, and R-ras have been shown to display transforming activity, and activating lesions have been detected in human tumors. We have identified an additional member of the Ras gene family which shows significant sequence similarity to the human TC21 gene. This novel human ras-related gene, R-ras3, encodes for a protein of 209 amino acids, and shows approximately 60-75% sequence identity in the N-terminal catalytic domain with members of the Ras subfamily of GTP-binding proteins. An activating mutation corresponding to the leucine 61 oncogenic lesion of the ras oncogenes when introduced into R-ras3, activates its transforming potential. R-ras3 weakly stimulates the mitogen-activated protein kinase (MAPK) activity, but this effect is greatly potentiated by the co-expression of c-raf-1. By the yeast two-hybrid system, R-ras3 interacts only weakly with known Ras effectors, such as Raf and RalGDS, but not with RglII. In addition, R-ras3 displays modest stimulatory effects on trans-activation from different nuclear response elements which bind transcription factors, such as SRF, ETS/TCF, Jun/Fos, and NF-kappaB/Rel. Interestingly, Northern blot analysis of total RNA isolated from various tissues revealed that the 3.8 kilobasepair (kb) transcript of R-ras3 is highly restricted to the brain and heart. The close evolutionary conservation between R-ras3 and Ras family members, in contrast to the significant differences in its biological activities and the pattern of tissue expression, raise the possibility that R-ras3 may control novel cellular functions previously not described for other GTP-binding proteins. PMID: 9400994 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Neuroscience. 1997 Nov;81(1):199-212. Expression of activating transcription factor-2, serum response factor and cAMP/Ca response element binding protein in the adult rat brain following generalized seizures, nerve fibre lesion and ultraviolet irradiation. Herdegen T, Blume A, Buschmann T, Georgakopoulos E, Winter C, Schmid W, Hsieh TF, Zimmermann M, Gass P. Department of Pharmacology, University of Kiel, Germany. The expression of the constitutive transcription factors activating transcription factor-2 (ATF-2), serum response factor (SRF) and cAMP/Ca response element binding factor (CREB), and the phosphorylation of SRF and CREB were studied in the untreated adult rat nervous system and following seizure activities and neurodegenerative stimuli. In the untreated rat, intense nuclear SRF immunoreactivity was present in the vast majority of neurons in the forebrain, cortex, striatum, amygdala and hippocampus, and in some scattered neurons in the medulla and spinal cord. In contrast, SRF immunoreactivity was absent in the midline areas of the forebrain, e.g., the globus pallidum and septum, and in the hypothalamus, thalamus, mesencephalon and motoneurons. Nuclear ATF-2 was expressed at high levels in apparently all neurons, but not glial cells, throughout the neuraxis except for those neuronal populations which exhibit a high basal level of c-Jun, i.e. dentate gyrus and the motoneurons of cranial and somatosensory neurons. CREB immunoreactivity was present at a rather uniform intensity in all neuronal and glial cells throughout the neuraxis. Two hours, but not 5 h or 24 h, following systemic application of kainic acid, an increase in SRF was detectable by western blot analysis in hippocampal and cortical homogenates whereas the expression of ATF-2 and CREB did not change. Phosphorylation of CREB at serine 133 and of SRF at serine 103 were studied with specific antisera. In untreated rats, intense phosphoCREB and phosphoSRF immunoreactivities labelled many glial cells and/or neurons with the highest levels in the dentate gyrus, the entorhinal cortex and the retrosplenial cortex. Following kainate-induced seizures, phosphoSRF-IR but not phosphoCREB-IR transiently increased between 0.5 h and 2 h. Following transection of peripheral or central nerve fibres such as optic nerve, medial forebrain bundle, vagal and facial nerve fibres, ATF-2 rapidly decreased in the axotomized neurons during that period when c-Jun was rapidly expressed. SRF remained unchanged and CREB disappeared in some axotomized subpopulations. Similar to axotomy, c-Jun increased and ATF-2 decreased in cultured adult dorsal root ganglion neurons following ultraviolet irradiation. The distribution of SRF and ATF-2 suggests that their putative target genes c-fos, junB, krox-24 and c-jun can be independently regulated from SRF and ATF-2. The suppression of ATF-2 and the expression of c-Jun following axotomy and ultraviolet irradiation might be part of a novel neuronal stress response in the brain that strongly resembles the stress response characterized in non-neuronal cells. PMID: 9300412 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Mol Cell Biol. 1997 Mar;17(3):1324-35. Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways. Westwick JK, Lambert QT, Clark GJ, Symons M, Van Aelst L, Pestell RG, Der CJ. Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, 27599-7038, USA. Rac1 and RhoA are members of the Rho family of Ras-related proteins and function as regulators of actin cytoskeletal organization, gene expression, and cell cycle progression. Constitutive activation of Rac1 and RhoA causes tumorigenic transformation of NIH 3T3 cells, and their functions may be required for full Ras transformation. The effectors by which Rac1 and RhoA mediate these diverse activities, as well as the interrelationship between these events, remain poorly understood. Rac1 is distinct from RhoA in its ability to bind and activate the p65 PAK serine/threonine kinase, to induce lamellipodia and membrane ruffling, and to activate the c-Jun NH2-terminal kinase (JNK). To assess the role of PAK in Rac1 function, we identified effector domain mutants of Rac1 and Rac1-RhoA chimeric proteins that no longer bound PAK. Surprisingly, PAK binding was dispensable for Rac1-induced transformation and lamellipodium formation, as well as activation of JNK, p38, and serum response factor (SRF). However, the ability of Rac1 to bind to and activate PAK correlated with its ability to stimulate transcription from the cyclin D1 promoter. Furthermore, Rac1 activation of JNK or SRF, or induction of lamellipodia, was neither necessary nor sufficient for Rac1 transforming activity. Finally, the signaling pathways that mediate Rac1 activation of SRF or JNK were distinct from those that mediate Rac1 induction of lamellipodia. Taken together, these observations suggest that Rac1 regulates at least four distinct effector-mediated functions and that multiple pathways may contribute to Rac1-induced cellular transformation. PMID: 9032259 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: J Biol Chem. 1996 May 3;271(18):10827-33. Serum response factor mediates AP-1-dependent induction of the skeletal alpha-actin promoter in ventricular myocytes. Paradis P, MacLellan WR, Belaguli NS, Schwartz RJ, Schneider MD. Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA. "Fetal" gene transcription, including activation of the skeletal alpha-actin (SkA) promoter, is provoked in cardiac myocytes by mechanical stress and trophic ligands. Induction of the promoter by transforming growth factor beta or norepinephrine requires serum response factor (SRF) and TEF-1; expression is inhibited by YY1. We and others postulated that immediate-early transcription factors might couple trophic signals to this fetal program. However, multiple Fos/Jun proteins exist, and the exact relationship between control by Fos/Jun versus SRF, TEF-1, and YY1 is unexplained. We therefore cotransfected ventricular myocytes with Fos, Jun, or JunB, and SkA reporter genes. SkA transcription was augmented by Jun, Fos/Jun, Fos/JunB, and Jun/JunB; Fos and JunB alone were neutral or inhibitory. Mutation of the SRF site, SRE1, impaired activation by Jun; YY1, TEF-1, and Sp1 sites were dispensable. SRE1 conferred Jun activation to a heterologous promoter, as did the c-fos SRE. Deletions of DNA binding, dimerization, or trans-activation domains of Jun and SRF abolished activation by Jun and synergy with SRF. Neither direct binding of Fos/Jun to SREs, nor physical interaction between Fos/Jun and SRF, was detected in mobility-shift assays. Thus, AP-1 factors activate a hypertrophy-associated gene via SRF, without detectable binding to the promoter or to SRF. PMID: 8631897 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Oncogene. 1995 Apr 6;10(7):1361-70. Two serum response elements mediate transcriptional repression of human smooth muscle alpha-actin promoter in ras-transformed cells. Bushel P, Kim JH, Chang W, Catino JJ, Ruley HE, Kumar CC. Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA. The mechanism by which activated ras oncogene expression leads to repression of genes encoding specific actin filament proteins is not understood. However, these changes associated with loss of organized actin filaments, are necessary to maintain the transformed phenotype. The human smooth muscle (sm) alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cell lines. In this study, we demonstrate that two serum response elements (SREs) present in the alpha-actin promoter are required for transcriptional repression in ras-transformed cells and the two SREs act synergistically to repress heterologous promoters in a ras-transformation dependent manner. Serum response factor (SRF), which can bind to the sm alpha-actin SREs, restores alpha-actin promoter activity in ras-transformed cells. c-Fos, c-Jun and YY1 also repress alpha-actin promoter through SREs, suggesting that these transcription factors may play a role in repressing alpha-actin promoter in ras-transformed cells. PMID: 7731687 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Trends Biochem Sci. 1992 Jul;17(7):251-6. The ets gene family. Macleod K, Leprince D, Stehelin D. CNRS, Institut Pasteur, Lille, France. Ets proteins have a conserved DNA-binding domain and regulate transcriptional initiation from a variety of cellular and viral gene promoter and enhancer elements. Some members of the Ets family, Ets-1 and Ets-2, cooperate in transcription with the AP-1 transcription factor, the product of the proto-oncogene families, fos and jun, while others, Elk-1 and SAP-1, form ternary complexes with the serum response factor (SRF). Certain ets gene family members possess transforming activity while others are activated by proviral integration in erythroleukaemias. Publication Types: Review Review, Tutorial PMID: 1502727 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Mol Biol Rep. 1991 May;15(2):87-92. A set of 4 nuclear proteins binds to a DNA sequence within the FOS promoter region. Radtke J, Dooley S, Blin N, Unteregger G. Institute of Human Genetics, University of the Saar, Hamburg, Germany. We investigated DNA-protein-interactions occurring in the promoter region of c-fos using two-dimensional electrophoresis and south-western-blotting. When nuclear extracts from the human glioblastoma cell line HeRoSV were tested for their DNA-binding behaviour to a 650 bp-fragment within the promoter region of c-fos, we found 4 proteins designated as 120/6.6, 75/5.4, 65/6.4 55/5.0 interacting with this fragment. An additional protein 60/6.0 was detected by using a digoxygenine-labelled probe. These observations let us to assume that beside the well characterized SRF and FOS-JUN proteins additional factors recognize the promoter sequence and may play a role in c-fos regulation. PMID: 1660958 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: Neuron. 1990 Apr;4(4):603-14. Differential expression of immediate early genes in the hippocampus and spinal cord. Wisden W, Errington ML, Williams S, Dunnett SB, Waters C, Hitchcock D, Evan G, Bliss TV, Hunt SP. Molecular Neurobiology Unit, MRC Centre, Cambridge, England. We have demonstrated that immediate early genes can be differentially activated within the central nervous system. We examined the effects of tetanic stimulation in the hippocampus and of noxious sensory stimulation of the spinal cord on the expression of eight immediate early genes. Induction of long-term potentiation (LTP) in the dentate gyrus resulted in an increase in mRNA and protein for NGFI-A (also termed Zif/268, Egr-1, or Krox 24), and less consistently for jun-B mRNA. No increase was seen for c-fos, NGFI-B, c-jun, jun-D, SRF, or PC4 mRNAs. Blockade of the NMDA receptor prevented the induction of both LTP and NGFI-A mRNA in the dentate gyrus. However, commissural stimulation, which prevented the induction of LTP, resulted in bilateral activation of all the genes examined, including NGFI-A. No change was seen in animals trained in a water maze. These results suggest that no simple relationship exists between LTP, spatial learning, and immediate early gene induction. Stimulation of sensory fibers resulted in an increase in mRNA for NGFI-A, c-fos, SRF, NGFI-B, and c-jun in spinal cord neurons. Blockade of the NMDA receptor had no effect on immediate early gene induction in the spinal cord. PMID: 2108708 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: Genes Dev. 1990 Feb;4(2):255-68. The inner core of the serum response element mediates both the rapid induction and subsequent repression of c-fos transcription following serum stimulation. Rivera VM, Sheng M, Greenberg ME. Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115. Serum stimulation of quiescent fibroblasts results in a dramatic increase in c-fos transcription that peaks by 15 min and is then rapidly repressed to basal levels within 60 min. Using a nuclear run-on assay to follow directly the kinetics of transcription of mutant c-fos constructs, we demonstrate that the serum response element (SRE) is the site of regulation of both the induction and repression events. This is indicated by the ability of the SRE to mediate c-fos kinetics of induced transcription when fused to a heterologous gene and in the absence of a recognizable TATA element. Functions of the inner core and the outer palindromic arms of the SRE have been determined by mutagenesis. The 14-bp inner core binds the serum response factor (SRF) and is, itself, sufficient to mediate both the induction and shutoff of serum-stimulated transcription. Therefore, SRF and any other factors that regulate the transient kinetics of c-fos transcription require no more than these 14 nucleotides to function. The palindromic outer arms of the SRE stabilize the binding of SRF and thereby enhance the transcriptional response to serum. Autoregulation by the c-fos gene product is not affected by the direct interaction of Fos/Jun complexes with the c-fos promoter and is likely to be mediated by either a novel function of the Fos protein or by an effect of Fos on the expression of another gene. PMID: 2110922 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------