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PubMed2006

Functional characterization of the promoter of the vitellogenin gene, AsVg1, of the malaria vector, Anopheles stephensi.

Some genetic strategies for controlling transmission of mosquito-borne diseases call for the introgression of antipathogen effector genes into vector populations. Endogenous mosquito promoter and other cis-acting DNA sequences are needed to direct the expression of the effector molecules to maximize their efficacy. Vitellogenin (Vg)-encoding gene control sequences are candidates for driving tissue-, stage- and sex-specific expression of exogenous genes. One of the Anopheles stephensi Vg genes, AsVg1, was cloned and a full-length cDNA, as well as 850 base pairs adjacent to the 5'-end, were sequenced and characterized. Expression of AsVg1 is restricted to the fat body tissues of blood-fed females, and the amino acid sequence of the conceptual translation product is >85% identical to those of other anopheline Vgs. These characteristics support the conclusion that AsVg1 is a Vg-encoding gene. Functional analyses of the AsVg1 putative cis-regulatory sequences were performed using transgenic mosquitoes. The results showed that DNA fragments encompassing the 850 base pairs immediately adjacent to the 5'-end of the gene and the 3'-end untranslated region are sufficient to direct sex-, stage- and tissue-specific expression of a reporter gene. These data indicate that the AsVg1 promoter is a good candidate for controlling the expression of anti-pathogen effector molecules in this malaria vector mosquito.

Animals↗
PubMed2026

Systematic Identification and Functional Characterisation of Colletotrichum fructicola Effectors During Camellia oleifera Colonisation.

Camellia oleifera is an important woody oil crop in southern China, but its production is severely threatened by anthracnose caused by Colletotrichum fructicola. C. fructicola deploys secreted effector proteins to establish infection. However, systematic identification and functional characterisation of C. fructicola effector genes upregulated during infection remains largely unexplored. Here, we integrated genome-wide secretome prediction with RNA-seq data from C. oleifera leaves inoculated with C. fructicola to identify candidate effectors induced during infection, followed by functional screening, targeted gene deletion, complementation and pathogenicity assays. Five novel effectors required for C. fructicola full virulence were identified, all of which suppressed Bax-induced cell death in Nicotiana benthamiana. Targeted deletion of the corresponding genes in C. fructicola reduced lesion areas by 47%-78% on C. oleifera leaves and by up to 67% on apple fruits, whereas complementation restored their virulence to wild-type levels. Transcriptomic profiling of infected postharvest C. oleifera fruits identified differentially expressed genes enriched in GO terms related to copper ion response, as well as in KEGG pathways associated with phenylpropanoid biosynthesis, taurine and hypotaurine metabolism, and plant-pathogen interactions. In addition, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities, as well as malondialdehyde content, were altered in C. oleifera leaves inoculated with the effector-deletion mutants compared with those infected with the wild-type strain, suggesting that these effectors may contribute to the modulation of host oxidative stress responses. Taken together, our findings provide genetic and physiological evidence that these effectors contribute to C. fructicola virulence, offering potential targets for anthracnose control.

Colletotrichum↗
PubMed1996

Molecular characterization of the 71E late puff in Drosophila melanogaster reveals a family of novel genes.

Early metamorphic development in Drosophila melanogaster is initiated by pulses of the steroid hormone ecdysone, which are transduced into tissue-specific transcriptional cascades. This process begins with the hormone-dependent activation of a set of transcription factors (early genes) that, in turn, activate set of tissue-specific effector genes (late genes). The 71E cytogenetic region of the salivary gland polytene genome contains several ecdysone-regulated transcription units. Molecular techniques were used to analyze these genes, their transcriptional program and their evolutionary relatedness. We find that this region contains a cluster of ten coordinately regulated late genes (L71 genes) that are organized as five divergently transcribed gene pairs. Maximum parsimony analysis suggests that an ancestral L71 gene duplicated to form the first gene pair which was, in turn, duplicated to form the set of gene pairs. The L71 gene products form a family of small, chemically basic proteins with a conserved backbone of cysteine residues. In addition, the 71E region contains another gene (I71-1) with the regulatory and biochemical characteristics of the salivary gland intermolt glue proteins.

Amino Acid Sequence↗
PubMed2002

Investigation of bax, bcl-2, bcl-x and p53 gene polymorphisms in multiple sclerosis.

Clinical course, outcome, radiological features, severity, and histopathology are heterogenous in multiple sclerosis (MS). Since MS is considered to be a polygenic disease, the genetic background may at least partly be responsible for this variability. Some MS cases are histopathologically characterized by a dramatic oligodendrocyte loss that is in part caused by apoptosis. A dysregulated apoptotic elimination of self-reactive T cells may also contribute to disease susceptibility. To analyze genetic differences in the apoptosis regulating factors bcl-2, bax, bcl-x and p53 we investigated polymorphisms of these genes in 105 patients with a relapsing remitting disease course and 99 controls by PCR-SSCP and direct sequencing. We identified so far unpublished sequence alterations in the promotor region of the bxl-x gene, in exon 7 of the p53 gene, and in exon 1 of the bax gene. No differences were observed between MS patients and controls. Additional known polymorphisms were found in intron 3 of the bax gene and in exon 6 of the p53 gene. No significant differences in the frequency of gene sequence variations were found between MS patients and controls. The apoptosis genes studied here therefore appear less likely to be important effector genes in MS.

Apoptosis↗
PubMed1999

Sensitization components of post-traumatic stress disorder: implications for therapeutics.

Behavioral sensitization to psychomotor stimulants and stressors involves increasing reactivity to repetition of the same dose (or intensity) of the stimulus over time. In cocaine-induced behavioral sensitization, conditioned components are prominent and the underlying pharmacological and neuroanatomical substrates differ as a function of stage of its evolution. Since there is cross-sensitization between cocaine-induced behavioral sensitization and stress-induced behavioral sensitization and, as well, a high comorbidity of stimulant abuse with post-traumatic stress disorder, many of the mechanisms and principles of cocaine-induced behavioral sensitization may be pertinent to post-traumatic stress disorder. The extent and duration of cocaine-induced behavioral sensitization/stress-induced behavioral sensitization are a function of age, genetic strain, and gender of the organism, as well as magnitude, frequency, duration, quality, and environmental contingencies of the inducing stimuli. These effect a spatiotemporal evolution of cascades of alterations in gene expression involving a host of immediate early and late effector genes, including neurotrophic and apoptotic factors. They may change the physiology, biochemistry, and neuroanatomy of the developing brain and, in adults, substrates critical for learning and memory. Suggestions are made for differential strategies of therapeutic intervention as primary prevention, in the immediate post-traumatic period, and in the late phases of post-traumatic stress disorder evolution.

Animals↗
PubMed1997

A dominant negative mutant of jun blocking 12-O-tetradecanoylphorbol-13-acetate-induced invasion in mouse keratinocytes.

We previously reported that induced activator protein-1 (AP-1) transcriptional activity appears to be required for tumor promoter-induced transformation in mouse epidermal JB6 cells. To extend this investigation to a keratinocyte culture model and a transgenic mouse model, we constructed K14TAM67, a keratin 14 promoter-controlled version of the dominant negative jun mutant to directly block AP-1 activity and possibly indirectly block NF kappa B activity in basal squamous epithelia. This study was directed at characterizing TAM67 expression and biological activity in the mouse cell line 308, a keratinocyte model for studying carcinogenesis. Cotransfection of K14TAM67 with luciferase plasmid reporter DNAs produced inhibition of basal and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 and NF kappa B activity but had no effect on p53-dependent transcriptional activity. In an in vitro invasion assay, stable expression of TAM67 in 308 cells blocked TPA-induced Matrigel invasion. This suggests that blocking TPA-induced AP-1- or NF kappa B-regulated gene expression by TAM67 inhibits TPA-induced progression. Recombinant tissue inhibitor of metalloproteinase 1 reduced TPA-induced in vitro invasion, thus implicating metalloproteinases at least in part in the transcription factor-dependent process. Analysis of mRNA levels for members of the matrix metalloproteinase (MMP) family, however, revealed that the expression of any single MMP family member did not correlate with regulation of AP-1 or NF kappa B activity. However, the combination of substantial levels of mRNA for stromelysin-1, stromelysin-2, collagenase, membrane type 1 MMP, and gelatinase A occurred only in TPA-treated cells in the absence of TAM67. These results suggest that the action of the dominant negative jun mutant on AP-1 and NF kappa B gene regulation results in complex alterations in the levels of downstream effector genes, such as the metalloproteinases, that effect TPA-induced cellular invasion.

Animals↗
PubMed2005

cAMP induced modifications of HOX D gene expression in prostate cells allow the identification of a chromosomal area involved in vivo with neuroendocrine differentiation of human advanced prostate cancers.

The acquisition of epithelial-neuroendocrine differentiation (ND) is a peculiarity of human advanced, androgen-independent, prostate cancers. The HOX genes are a network of transcription factors controlling embryonal development and playing an important role in crucial adult eukaryotic cell functions. The molecular organization of this 39-gene network is unique in the genome and probably acts by regulating phenotype cell identity. The expression patterns of the HOX gene network in human prostate cell phenotypes, representing different stages of prostate physiology and prostate cancer progression, make it possible to discriminate between different human prostate cell lines and to identify loci and paralogous groups harboring the HOX genes mostly involved in prostate organogenesis and cancerogenesis. Exposure of prostate epithelial phenotypes to cAMP alters the expression of lumbo-sacral HOX D genes located on the chromosomal region 2q31-33 where the cAMP effector genes CREB1, CREB2, and cAMP-GEFII are present. Interestingly, this same chromosomal area harbors: (i) a global cis-regulatory DNA control region able to coordinate the expression of HOX D and contiguous phylogenetically unrelated genes; (ii) a prostate specific ncRNA gene associated with high-risk prostate cancer (PCGEM1); (iii) a series of neurogenic-related genes involved with epithelial-neuronal cell conversion. We report the expression of neurexin 1, Neuro D1, dlx1, and dlx2 in untreated and cAMP treated epithelial prostate cells. The in vivo expression of Neuro D1 in human advanced prostate cancers correlate with the state of tumor differentiation as measured by Gleason score. Thus, we suggest that the chromosomal area 2q 31-33 might be involved in the epithelial-ND characteristic of human advanced prostate cancers.

Bucladesine↗
PubMed1996

Peripheral nerve stimulation increases Fos immunoreactivity without affecting type II Ca2+/calmodulin-dependent protein kinase, glutamic acid decarboxylase, or GABAA receptor gene expression in cat spinal cord.

Expression patterns of the immediate early gene c-fos and of other genes including those for the alpha-subunit of type II Ca2+/calmodulin-dependent protein kinase (CaMKII alpha), 67-kDa glutamic acid decarboxylase (GAD), and the alpha 1-, beta 2-, and gamma 2-subunits of the GABAA receptor were described in the spinal cord of normal cats and following peripheral nerve stimulation. As revealed by in situ hybridization histochemistry, CaMKII alpha messenger RNA (mRNA) is normally distributed only in cells of Rexed's laminae I-IV, whereas GAD mRNA is expressed by subpopulations of cells in all laminae, with the heaviest hybridization signal found in laminae I-III and medial parts of laminae V and VI. The three GABAA receptor subunits have varying expression patterns in the laminae. All of them are expressed by many cells located in the base of the dorsal horn and the intermediate zone, but only the gamma 2-subunit is intensely expressed by motoneurons. Single-pulse, electrical stimulation of the sciatic or median and ulnar nerve of anesthetized cats at a pulse rate of 1/s for 6-8 h failed to induce observable changes in gene expression for CaMKII alpha, GAD, or for the three subunits of the GABAA receptor; although immunoreactivity for the protein products of c-fos (or c-fos-related genes) was markedly upregulated in some neurons of the dorsal horn and the intermediate zone. Therefore, under the present experimental conditions, upregulation of the immediate early gene c-fos (or c-fos-related genes) is not associated with changes in expression of late-effector genes potentially involved in central nervous system plasticity.

Animals↗
PubMed1996

Isolation and developmental expression analysis of Enx-1, a novel mouse Polycomb group gene.

Members of the Polycomb group (Pc-G) of genes encode transcriptional regulators that control the expression of key developmental effector genes in Drosophila melanogaster. Although multiple Pc-G genes have been identified and characterized in Drosophila, information about these important regulatory proteins in vertebrates, including their precise expression patterns, has remained scarce. We report here the cloning of Enx-1, a novel vertebrate Pc-G gene, which encodes the murine homolog of the Drosophila Enhancer of zeste (E(z)) gene. Drosophila E(z) controls the expression of several homeobox genes as well as some segmentation genes and its disruption causes multiple phenotypes in Drosophila development. Analysis of the primary structure of murine Enx-1 reveals the conservation of several regions, including the previously described SET domain and a newly defined CXC domain. In addition, we find the SET domain to be conserved in evolutionarily distant species ranging from vertebrates to plants and fungi. The expression pattern analysis of Enx-1 reveals ubiquitous expression throughout early embryogenesis, while in later embryonic development Enx-1 expression becomes restricted to specific sites within the central and peripheral nervous system and to the major sites of fetal hematopoiesis. In adult stages we also find Enx-1 expression to be restricted to specific tissues, including spleen, testis and placenta.

Amino Acid Sequence↗
PubMed2000

VisN and VisR are global regulators of chemotaxis, flagellar, and motility genes in Sinorhizobium (Rhizobium) meliloti.

The known 41 flagellar, chemotaxis, and motility genes of Sinorhizobium (Rhizobium) meliloti contained in the "flagellar regulon" are organized as seven operons and six transcription units that map to a contiguous 45-kb chromosomal region. By probing gene expression on Western blots and with lacZ fusions, we have identified two master regulatory genes, visN and visR, contained in one operon. The gene products probably form a heterodimer, VisNR, acting as a global transcription activator of other flagellar genes. The related 27-kDa VisN and VisR proteins are LuxR-type proteins with typical ligand- and DNA-binding domains. The vis operon itself is constitutively transcribed; however, to activate flagellar genes, VisNR seemingly requires the binding of a yet-unknown effector. Gene expression in tester strains with known deficiencies revealed a hierarchy of three classes of flagellar genes: class I comprises visN and visR; class II, controlled by VisNR, comprises flagellar assembly (class IIA) and motor (class IIB) genes; and class III comprises flagellin and chemotaxis genes that require functional class I and class IIA genes for expression. In contrast to their enterobacterial counterparts, mot genes belong to class II without exerting control over class III genes. While the general hierarchy of gene expression resembles the enterobacterial scheme, the assignment of mot genes to class IIB and the global control by a LuxR-type VisNR activator are new features distinguishing the S. meliloti flagellar gene system.

Amino Acid Sequence↗
PubMedDate not supplied

The effect of von hippel-lindau gene transfer on human vascular smooth muscle cell proliferation and apoptosis.

Von Hippel-Lindau (VHL) gene is a tumor suppressor gene that plays a genome "gatekeeper'' role and controls several downstream effector genes. We have previously demonstrated that both in vivo and in vitro adenovirus-mediated gene transfer of tumor suppressor genes into the vascular endothelium is effective in decreasing neointimal hyperplasia and abnormal cell proliferation. The degree of apoptosis induced by these genes is critical in mediating the in vivo responses to gene therapy and the maintenance of the crucial balance between cell death and viability. Since VHL gene is known to regulate vascular endothelial growth factor (VEGF) as well as other angiogenic factors, it may exhibit a greater potential in the attenuation of vascular disorders in comparison to other tumor suppressor genes. This study focused on whether adenovirus-mediated VHL gene transfer into human vascular smooth muscle cells has an effect on cell proliferation and induction of apoptosis. Human aortic smooth muscle cells (HASMC) were grown as monolayers and transfected with varying titers of adenovirus containing the VHL cDNA (AdVHL). The negative controls were adenovirus containing green fluorescent protein (AdGFP), vector alone (AdNull), and virus-free infection medium. Adenovirus encoding wild-type p53 (Adp53) was used as positive control. Cell viability and proliferation were determined by using trypan blue exclusion and MTS-based CellTiter 96 AQ Proliferation Assay. Apoptosis was evaluated by TUNEL assay, morphologic changes, and nucleosomal DNA degradation. Following AdVHL transfection HASMCs demonstrated a dose-dependent decrease in viability as compared to negative controls (p < 0.05). AdVHL-transfected cells exhibited a decrease in their proliferative ability by 40.21 +/-1.66 (SEM)%. In cultures transfected with the positive control, Adp53, the cell viability as well as proliferation was highly reduced (p < 0.001). AdGFP and AdNull did not increase HASMC apoptosis above baseline levels. The cells exposed to adenoviruses expressing tumor suppressor genes underwent apoptosis, with Adp53 demonstrating a very high magnitude of cell death (75.27 +/-3.52 [SEM]%). AdVHL expression caused 45.36 +/-2.55 (SEM)% apoptosis in HASMC. Recombinant adenovirus-mediated VHL expression is efficacious in limiting vascular smooth muscle cell growth in vitro. Overexpression of VHL suppresses HASMC proliferation and regulates apoptosis. Further experiments are indicated to examine whether VHL may be a useful adjunct in limiting myointimal hyperplasia.

Adenoviridae↗
PubMed2002

Identification of genes that are downregulated in the absence of the POU domain transcription factor pou3f1 (Oct-6, Tst-1, SCIP) in sciatic nerve.

Despite the importance of myelinating Schwann cells in health and disease, little is known about the genetic mechanisms underlying their development. The POU domain transcription factor pou3f1 (Tst-1, SCIP, Oct-6) is required for the normal differentiation of myelinating Schwann cells, but its precise role requires identification of the genes that it regulates. Here we report the isolation of six genes whose expression is reduced in the absence of pou3f1. Only one of these genes, the fatty acid transport protein P2, was known previously to be expressed in Schwann cells. The LIM domain proteins cysteine-rich protein-1 (CRP1) and CRP2 are expressed in sciatic nerve and induced by forskolin in cultured Schwann cells, but only CRP2 requires pou3f1 for normal expression. pou3f1 appears to require the claw paw gene product for activation of at least some of its downstream effector genes. Expression of the novel Schwann cell genes after nerve injury suggests that they are myelin related. One of the genes, tramdorin1, encodes a novel amino acid transport protein that is localized to paranodes and incisures. Our results suggest that pou3f1 functions to activate gene expression in the differentiation of myelinating Schwann cells.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Identification of Erwinia amylovora genes induced during infection of immature pear tissue.

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In this study, we used a modified in vivo expression technology system to identify E. amylovora genes that are activated during infection of immature pear tissue, a process that requires the major pathogenicity factors of this organism. We identified 394 unique pear fruit-induced (pfi) genes on the basis of sequence similarity to known genes and separated them into nine putative function groups including host-microbe interactions (3.8%), stress response (5.3%), regulation (11.9%), cell surface (8.9%), transport (13.5%), mobile elements (1.0%), metabolism (20.3%), nutrient acquisition and synthesis (15.5%), and unknown or hypothetical proteins (19.8%). Known virulence genes, including hrp/hrc components of the type III secretion system, the major effector gene dspE, type II secretion, levansucrase (lsc), and regulators of levansucrase and amylovoran biosynthesis, were upregulated during pear tissue infection. Known virulence factors previously identified in E. (Pectobacterium) carotovora and Pseudomonas syringae were identified for the first time in E. amylovora and included HecA hemagglutinin family adhesion, Peh polygalacturonase, new effector HopPtoC(EA), and membrane-bound lytic murein transglycosylase MltE(EA). An insertional mutation within hopPtoC(EA) did not result in reduced virulence; however, an mltE(EA) knockout mutant was reduced in virulence and growth in immature pears. This study suggests that E. amylovora utilizes a variety of strategies during plant infection and to overcome the stressful and poor nutritional environment of its plant hosts.

Erwinia amylovora↗
PubMed2006

Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference.

Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with non-pathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.

Animals↗
PubMed2005

The accumulation of specific mRNAs following multiple blood meals in Anopheles gambiae.

One approach to genetic control of transmission of the parasites that cause human malaria is based on expressing effector genes in mosquitoes that disable the pathogens. Endogenous mosquito promoter and other cis-acting DNA sequences are needed to direct the optimal tissue-, stage- and sex-specific expression of the effector molecules. The mRNA accumulation profiles of eight different genes expressed specifically in the midgut, salivary glands or fat body tissues of the malaria vector, Anopheles gambiae, were characterized as a measure of their suitability to direct the expression of effector molecules designed to disable specific stages of the parasites. RT-PCR techniques were used to determine the abundance of the gene products and their duration following multiple blood meals. Transcription from the midgut-expressed carboxypeptidase-encoding gene, AgCP, follows a cyclical, blood-inducible expression pattern with maximum accumulation every 3 h post blood meal. Other midgut-expressed genes encoding a trypsin and chymotrypsin, Antryp2 and Anchym1, respectively, and the fat body-expressed genes, Vg1 and Cathepsin, also show a blood-inducible pattern of expression with maximum accumulation 24 h after every blood meal. Expression of the Lipophorin gene in the fat body and apyrase and D7-related genes (AgApy and D7r2) in the salivary glands is constitutive and not significantly affected by blood meals. Promoters of the midgut- and fat body-expressed genes may lead to maximum accumulation of antiparasite effector molecule transcripts after multiple blood meals. The multiple feeding behaviour of An. gambiae thus can be an advantage to express high levels of antiparasite effector molecules to counteract the parasites throughout most of adult development.

Animals↗
PubMed2003

Functional genomics as a window on radiation stress signaling.

Exposure to ionizing radiation, as well as other stresses, results in the activation of complex signal transduction pathways, which eventually shape the response of cells and organisms. Some of the important pathways responding to radiation include the ATM/P53 pathway, MAPK cascades and NF-kappaB activation, as well as signaling events initiated at the cell membrane and within the cytoplasm. Alterations in gene expression play roles both as intermediaries in signaling and as downstream effector genes. Differences in cell type, interindividual genetic differences and crosstalk occurring between signaling pathways may help to channel radiation stress signals between cell cycle delay, enhanced DNA repair, and apoptosis. These differences may in turn help determine the likelihood of late effects of radiation exposure, including carcinogenesis and fibrosis. The tools of the postgenomic era enable high-throughput studies of the multiple changes resulting from the interplay of radiation signaling pathways. Gene expression profiling, in particular shows great promise, both in terms of insight into basic molecular mechanisms and for the future hope of biomarker development and individual tailoring of cancer therapy.

Animals↗
PubMed2002

Rottlerin inhibits tonicity-dependent expression and action of TonEBP in a PKCdelta-independent fashion.

Novel protein kinase C (PKC) isoforms PKCdelta and PKCepsilon have recently been implicated in signaling by hypertonic stress. We investigated the role of the putative PKCdelta inhibitor rottlerin on tonicity-dependent gene regulation. In the renal medullary mIMCD3 cell line, rottlerin blocked tonicity-dependent transcription of a tonicity enhancer (TonE)-driven luciferase reporter gene, as well as tonicity-dependent transcription of the physiological tonicity effector gene aldose reductase, but not urea-dependent transcription. Consistent with these data, rottlerin inhibited tonicity-dependent expression of TonE binding protein (TonEBP) at the mRNA and protein levels. Another inhibitor of both novel and conventional PKC isoforms, GF-109203X, suppressed TonEBP-dependent transcription but failed to influence tonicity-inducible TonEBP expression. Global PKC downregulation with protracted phorbol ester treatment, however, failed to influence tonicity-dependent signaling, arguing against a PKCdelta-dependent mechanism of rottlerin action in this model. In addition, hypertonic stress failed to induce phosphorylation of PKCdelta. Furthermore, in a PC-12 cell model with a comparable degree of tonicity-dependent transcription, constitutive overexpression of dominant negative-acting PKCdelta or PKCepsilon effectively decreased tonicity signaling to extracellular signal-regulated kinase activation, as expected, but failed to influence TonE-dependent transcription. TonE-dependent transcription, however, remained rottlerin sensitive in this PC-12 cell model. In the aggregate, these data indicate that rottlerin dramatically inhibits tonicity-dependent TonEBP expression and TonE-dependent transcription but, despite its reputed mode of action, does so through a PKCdelta-independent pathway.

Acetophenones↗
PubMed1991

Acquisition of mature functional responsiveness in T cells: programming for function via signaling.

The results discussed here provide strong evidence that different T-cell effector gene programs are activated by different signals, and that in several cases their responses to the same exogenous stimuli shift during the development and antigen responses of the cells. T-cell responses are thus conditional and plastic at the individual cell level. In the formalism of the introductory section, the results support elements of Models 2 and 3, and suggest a fusion between them as differentiation is explained in terms of alteration in the relative strengths of different intracellular signaling pathways. Returning to an initial question, how are different functional capabilities assigned nonrandomly to cells with different antigen recognition specificities? This question has not been answered, but it can be reformulated. If all virgin T cells can transiently make IL-2, then we must ask what features of cell biology explain the preferential preservation of IL-2 inducibility in CD4+ cells as opposed to CD8+ cells. If the capacity to induce IL-4 expression is not acquired in the thymus, then we may ask whether the initial opening of this locus depends on a CD4-transmitted signal. Similarly, the CD8 molecule itself might participate in inducing the initial differentiation events that render CTL-p inducible for granzyme C and perforin. This would be in accord with a large literature showing that CD8 engagement is much more important in the initial induction of CTL activity than in the exercise of function by pre-primed CTL effectors. The subtext of each of these "questions", however, is that intrathymic events may not directly affect the genes used by terminal effectors for function at all. They may instead bias a cell's complement of triggering receptors, thus rendering it differentially sensitive to particular signals generated during antigen reception. This view is extreme, and will probably turn out to be an overstatement. But it does inspire a unique set of investigations into the basis of T-cell function. It lends urgency to the question of whether CD4+ and CD8+ cells differ in their G proteins, kinases, or inducible proto-oncogenes. If they do, we can then ask whether such differences themselves arise in the periphery, or whether they can be traced back to thymocytes fresh from positive selection--or before.

Animals↗