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Biomedical subjects

E F Wagner

Publications and source records attributed to E F Wagner.

At least 55 records · Page 3Linked to original sources
PubMed1999

Fra-1 potentiates osteoclastic differentiation in osteoclast-macrophage precursor cell lines.

c-Fos, a component of the dimeric transcription factor AP-1, is necessary for osteoclast formation. To determine whether c-Fos can substitute for any or all of the stimuli needed for osteoclast induction, we infected osteoclast precursors with retroviral vectors expressing c-Fos or the Fos-related protein, Fra-1. The infected cells were incubated with or without osteoclast-inductive stimuli. Osteoclast formation from retroviral-infected precursors remained completely dependent on osteoclast-inductive stromal cells. Unexpectedly, infection of bipotential osteoclast-macrophage precursor cell lines with retroviruses expressing Fra-1 but not c-Fos caused a 10-100-fold increase in the number of precursors that developed calcitonin receptors associated with an increase in bone resorption. These observations suggest that, in the precursor cell lines, Fra-1 is a limiting factor for full responsiveness to the osteoclast-inductive environment. Fra-1 is therefore likely to play a role in osteoclast differentiation which is distinct from that of c-Fos.

Autoradiography↗
PubMed1999

A putative role for c-Fos in the pathophysiology of Paget's disease.

The molecular mechanisms underlying Paget's disease and subsequent osteosarcoma formation are not well understood. In this study, we aim to delineate the function of the c-Fos oncogene in Paget's disease using transgenic mice, based on previous findings that c-Fos is highly expressed in Pagetic osteoclasts and that c-Fos is an essential gene for osteoclast differentiation and skeletal neoplasia. We have generated transgenic mice in which c-Fos is overexpressed specifically in osteoclasts using the tartrate-resistant acid phosphatase (TRAP) promoter, and five founder mice have been identified. All transgene-expressing animals developed severe bone remodeling lesions, some of which progressed to large bone tumors. Histopathologic analysis indicated that the lesions contained a marked increase in the number of osteoclasts that contained a large number of nuclei. Osteoclasts were identified by histochemical staining for TRAP and by in situ hybridization for matrix metalloproteinase-9 (MMP-9) expression. Moreover, transgenic osteoclasts, and in some cases, osteoblasts and chondrocytes, expressed high levels of c-Fos protein as judged by immunocytochemistry. This phenotype of increased osteoclast number and activity, together with an apparently high rate of bone turnover, resembles some characteristics of Paget's disease. These data therefore support an important function for c-Fos in the Pagetic phenotype, and further support the notion that this gene is important in osteoclastogenesis and in bone remodeling disorders.

Acid Phosphatase↗
PubMed1999

Defective neural tube morphogenesis and altered apoptosis in the absence of both JNK1 and JNK2.

Mice lacking both c-Jun-NH(2)-terminal kinases (JNK1 and JNK2) were generated to define their roles in development. Jnk1/jnk2 double mutant fetuses die around embryonic day 11 (E11) and were found to display an open neural tube (exencephaly) at the hindbrain level with reduced apoptosis in the hindbrain neuroepithelium at E9.25. In contrast, a dramatic increase in cell death was observed one day later at E10.5 in both the hindbrain and forebrain regions. Moreover, about 25% of jnk1-/-jnk2+/- fetuses display exencephaly probably due to reduced levels of JNK proteins, whereas jnk1+/-jnk2-/- mice are viable. These results assign both pro- and anti-apoptotic functions for JNK1 and JNK2 in the development of the fetal brain.

Animals↗
PubMed1999

Mice lacking the poly(ADP-ribose) polymerase gene are resistant to pancreatic beta-cell destruction and diabetes development induced by streptozocin.

Human type 1 diabetes results from the selective destruction of insulin-producing pancreatic beta cells during islet inflammation. Cytokines and reactive radicals released during this process contribute to beta-cell death. Here we show that mice with a disrupted gene coding for poly (ADP-ribose) polymerase (PARP-/- mice) are completely resistant to the development of diabetes induced by the beta-cell toxin streptozocin. The mice remained normoglycemic and maintained normal levels of total pancreatic insulin content and normal islet ultrastructure. Cultivated PARP-/- islet cells resisted streptozocin-induced lysis and maintained intracellular NAD+ levels. Our results identify NAD+ depletion caused by PARP activation as the dominant metabolic event in islet-cell destruction, and provide information for the development of strategies to prevent the progression or manifestation of the disease in individuals at risk of developing type 1 diabetes.

Animals↗
PubMed1999

Amino-terminal phosphorylation of c-Jun regulates stress-induced apoptosis and cellular proliferation.

c-Jun is a major component of the heterodimeric transcription factor AP-1 and is essential for embryonic development, as fetuses lacking Jun die at mid-gestation with impaired hepatogenesis and primary Jun-/- fibroblasts have a severe proliferation defect and undergo premature senescence in vitro. c-Jun and AP-1 activities are regulated by c-Jun N-terminal phosphorylation (JNP) at serines 63 and 73 through Jun N-terminal kinases(JNKs). JNP is thought to be required for the anti-apoptotic function of c-Jun during hepatogenesis, as mice lacking the JNK kinase SEK1 exhibit liver defects similar to those seen in Jun-/- fetuses. To investigate the physiological relevance of JNP, we replaced endogenous Jun by a mutant Jun allele with serines 63 and 73 mutated to alanines (Jun(tm1wag); hereafter referred to as JunAA). Here we show that primary JunAA fibroblasts have proliferation- and stress-induced apoptotic defects, accompanied by reduced AP-1 activity. JunAA mice are viable and fertile, smaller than controls and resistant to epileptic seizures and neuronal apoptosis induced by the excitatory amino acid kainate. Primary mutant neurons are also protected from apoptosis and exhibit unaltered JNK activity. Our results provide evidence that JNP is dispensable for mouse development, and identify c-Jun as the essential substrate of JNK signalling during kainate-induced neuronal apoptosis.

Animals↗
PubMed1999

Cross-validation of the Temptation Coping Questionnaire: adolescent coping with temptations to use alcohol and illicit drugs.

OBJECTIVE: The present study had two primary objectives: (1) to examine differences in adolescent coping with temptations to use alcohol versus temptations for marijuana and/or other drug use and (2) to replicate and extend previous findings regarding the factor structure and psychometric validity of the Temptation Coping Questionnaire (TCQ). METHOD: This was a cross-sectional study of 1,273 high school students, 52% female and, on average, 16.6 years old. The TCQ was completed as part of a school-wide survey of cigarette, alcohol and other drug involvement. Responses to the TCQ were elicited in response to two hypothetical temptation situations, one involving offers of alcohol only and the other involving offers of marijuana and/or other drugs. RESULTS: The factor structure of the TCQ coping scale was confirmed for the original 11 items, both within and across situations representing temptations to use alcohol or marijuana and/or other drugs. In addition, the mean levels of perceived importance of abstaining and extent of coping efforts endorsed were higher for a marijuana and other drug than for an alcohol temptation situation. Finally, construct and criterion validity of the TCQ was replicated in the present sample. CONCLUSIONS: Present results suggest that similar coping strategies are employed regardless of the particular substance encountered in a temptation situation. However, situation appraisal and extent of coping efforts appeared to differ for distinct substance types. These findings corroborate previous evidence for the utility of the TCQ as a brief, reliable measure of adolescent temptation coping and provide convergent support for the temptation-coping construct.

Adaptation, Psychological↗
PubMed1999

A preliminary evaluation of the effectiveness of a standardized Student Assistance Program.

We examined the effectiveness of Student Assistance Program group counseling. One hundred and forty-four (144) substance using high school seniors completed a questionnaire in 1995 concerning their substance use before and after counseling, their experience in counseling, and the impact of counseling on their substance use. Participants reported significant decreases in substance use and attributed these changes to counseling. Most participants liked counseling, and higher frequency drinkers did not differ from lower frequency drinkers in liking counseling and reporting decreased substance use. These results offer support for the effectiveness of such interventions and highlight the need for additional research in the area.

Adolescent↗
PubMed1999

Innovations in adolescent substance abuse intervention.

Adolescent alcohol and other drug abuse is an important public health concern, and the past two decades has seen a dramatic increase in the demand for interventions to address substance use problems among teenagers. This demand has led to the development of multiple primary, secondary, and tertiary substance abuse prevention programs, some of which have little theoretical basis and most of which currently operate in the absence of data supporting their effectiveness. Very recently, there has been increased emphasis on the goal of developing and testing theoretically based and empirically supported intervention approaches for adolescent substance abuse. We describe five ongoing research programs devoted to meeting this goal. The background and rationale for each research program are discussed, and preliminary efficacy data concerning the specific interventions are presented.

Adolescent↗
PubMed1999

[Loss of junB expression causes a myeloproliferative syndrome resembling chronic myeloid leukemia].

JunB, a member of the AP-1 family of transcription factors, has been implicated in the control of proliferation and differentiation of various cell types including myeloid cells. We found that absence of junB expression in the myeloid lineage of mice lead to a myeloproliferative syndrome resembling human chronic myeloid leukaemia. The disease was due to a cell autonomous increase in the proliferation of immature myeloid cells. These results suggest that junB is a negative regulator of myelopoiesis.

Animals↗
PubMed1998

A pivotal role of cyclin D3 and cyclin-dependent kinase inhibitor p27 in the regulation of IL-2-, IL-4-, or IL-10-mediated human B cell proliferation.

The functional differences between IgDhighCD38- naive and IgD-CD38- memory (M) or IgDlowCD38+ germinal center (GC) B cells may stem from their variable response to signals that regulate activation, proliferation, and differentiation. In this report, we provide evidence for differential induction of cell cycle regulators in tonsillar human B cell subpopulations that were activated with anti-IgM and anti-CD40 in the presence or absence of IL-2, IL-4, or IL-10. Naive (IgDhigh) B cells exhibited a significant proliferative response to IL-4, but not to IL-2 or IL-10, whereas these cytokines triggered variable levels of growth in the combined GC/M subpopulation (referred to as IgDlow), as measured by [3H]thymidine incorporation. Induction of growth by cytokines in B cell subpopulations strictly correlated with the increased levels of cyclin D3 and cyclin-dependent protein kinase (cdk) 6. Moreover, only cyclin D3/cdk6 complexes were functional as observed in both naive and GC/M B cells stimulated in the presence of IL-4. In addition, active growth was associated with cytokine-mediated elimination of the cell cycle inhibitor p27. The significance of p27 in human B cell cycle was further demonstrated by rapamycin-mediated growth inhibition of IL-4-dependent proliferation, which resulted in strikingly increased p27 levels. Taken together, our findings suggest that cyclin D3, cdk6, and p27 play key roles in IL-2-, IL-4-, and IL-10-mediated human B cell proliferation. Furthermore, these results may provide a molecular basis for different cycling characteristics of naive and GC/M B cell subpopulations.

Antibodies, Anti-Idiotypic↗
PubMed1998

A strain-independent postnatal neurodegeneration in mice lacking the EGF receptor.

Mice lacking the epidermal growth factor receptor (EGFR) exhibit strain-dependent phenotypes ranging from placental to postnatal skin, lung and brain defects. After birth, all mutant mice develop a progressive neurodegeneration in the frontal cortex, olfactory bulb and thalamus, characterized by massive apoptosis and upregulation of c-fos. These defects occur in a strain-independent manner, since neither rescue of the placental phenotype by aggregation of diploid 129/Sv EGFR mutant and tetraploid wild-type embryos, nor promotion of lung maturation by transplacental dexamethasone administration alters the course of neurodegeneration. VEGF is not induced during the degenerative process, excluding hypoxia and ischemia as causes of cell death. A migratory disorder is detected in the hippocampus with nests of ectopic neurons, which are also apoptotic. Cerebral cortices from EGFR mutants contain lower numbers of GFAP positive astrocytes, which display reduced proliferation in vitro. Since EGFR is expressed in the affected cell-types, these results define a specific function for EGFR in the proliferation and/or differentiation of astrocytes and in the survival of postmitotic neurons.

Animals↗
PubMed1998

recessive spotting: a linked locus that interacts with W/Kit but is not allelic.

BACKGROUND: The murine coat-colour mutation recessive spotting (rs) maps very closely to the W/Kit locus, encoding the proto-oncoprotein Kit, the protein tyrosine kinase receptor for stem cell factor. Kit is important in the development of melanocytes, germ cells, interstitial cells of Cajal (ICC) and haemopoietic lineages, including mast cells. rs has never been genetically separated from Kit, and interacts with Kit mutations, suggesting that it is a recessive allele of Kit. Here we have tested this possibility. We have shown previously that diploid rs/rs melanocytes proliferated more slowly than did +/+ melanocytes, as did an immortal line of rs/rs melanocytes, melan-rs. RESULTS: The Kit mRNA level in rs/rs melanocytes was indistinguishable from that of other melanocyte lines. The Kit cDNA sequence from rs/rs melanocytes and the kinase activity of Kit in rs/rs mast cells appeared to be normal. No deficiency of mast cells or ICC was observed in rs/rs mice. Moreover, following the overexpression of a normal Kit cDNA, proliferation of rs/rs melanocytes was retarded further, but that of +/+ melanocytes was increased, indicating an intracellular interaction between rs and Kit. Of other closely linked tyrosine kinase genes, melanocytes and melanoblasts did not express mRNA for Pdgfra, Flk-1 or Txk, but both expressed Tec, encoding a nonreceptor kinase that interacts with Kit. CONCLUSIONS: rs is not a mutation in Kit, although we have confirmed that rs interacts with Kit. It seems unlikely that rs affects Pdgfra, Flk-1 or Txk, but Tec remains a candidate for rs.

Alleles↗
PubMed1998

Embryonic overexpression of the c-Fos protooncogene. A murine stem cell chimera applicable to the study of fibrodysplasia ossificans progressiva in humans.

Murine embryonic overexpression of the c-fos protooncogene leads to early postnatal heterotopic chondrogenesis and osteogenesis with phenotypic features similar to those seen in children who have the disabling heritable disease fibrodysplasia ossificans progressiva. The overexpression of Fos in embryonic stem cell chimeras leads to heterotopic endochondral osteogenesis at least in part through a bone morphogenetic protein 4 mediated signal transduction pathway. In contrast, early fibrodysplasia ossificans progressiva lesions express abundant bone morphogenetic protein 4, without abundant expression of c-Fos, suggesting that the primary molecular defect in fibrodysplasia ossificans progressiva may be independent of the sustained Fos effects on chondrogenesis and osteogenesis. Comparisons of the clinical, molecular, and pathogenetic features of the c-Fos embryonic stem cell chimeras with those of fibrodysplasia ossificans progressiva provide insight into the earliest events in the molecular pathogenesis of genetically induced heterotopic chondrogenesis and osteogenesis. The relevance of the c-Fos embryonic stem cell chimera to the study of the currently untreatable human disease fibrodysplasia ossificans progressiva demonstrates the power of using embryonic stem cell technology for generating gain of function mutations in the study of human bone disease.

Animals↗
PubMed1997

Imprinted expression of the Igf2r gene depends on an intronic CpG island.

Gametic imprinting is a developmental process that induces parental-specific expression or repression of autosomal and X-chromosome-linked genes. The mouse Igf2r gene (encoding the receptor for insulin- like growth factor type-2) is imprinted and is expressed from the maternal allele after embryonic implantation. We previously proposed that methylation of region 2, a region rich in cytosine-guanine doublets (a 'CpG island') in the second intron of Igf2r, is the imprinting signal that maintains expression of the maternal allele. Here we use mouse transgenes to test the role of region 2 and the influence of chromosome location on Igf2r imprinting. Yeast artificial chromosome transgenes successfully reproduced the imprinted methylation and expression pattern of the endogenous Igf2r gene; deletion of region 2 from these transgenes caused a loss of imprinting and restored biallelic Igf2r expression. These results define a primary role for region 2 and a negligible role for chromosomal location in Igf2r imprinting; they also show that methylation imprints can maintain allelic expression. Short transgenes containing only region 2 and yeast artificial chromosome transgenes with an inactive Igf2r promoter do not attract parental-specific methylation. All transgenes showing paternal-specific repression of Igf2r produced an antisense RNA whose transcription was dependent on region 2. The production of an antisense RNA by the repressed parental allele is reminiscent of the imprinting of the Igf2/H19 gene pair and may indicate that expression competition could play a general role in imprinting.

Animals↗
PubMed1997

Regulation of ES cell differentiation by functional and conformational modulation of p53.

Embryonic stem (ES) cell lines were used to examine the role of p53 during in vitro differentiation. Undifferentiated ES cells express high levels of p53 exclusively in the wild-type conformation, immunoprecipitable by monoclonal antibody PAb246, and p53 was found to be functionally active as determined by its ability to bind DNA specifically and to activate transcription of target genes. Differentiation in vitro resulted in a decrease in the levels of p53 and in a shift in its conformational status to the mutant form, detectable by monoclonal antibody PAb240, with a concomitant loss of functional activity. The presence of functional p53 in the undifferentiated ES cells renders them hypersensitive to UV irradiation, whereas the differentiated cells were resistant to UV treatment. ES cells lacking p53 exhibit enhanced proliferation in both the undifferentiated and differentiated state, and apoptosis accompanying differentiation was found to be reduced. Furthermore, wild-type ES cells undergoing apoptosis expressed functional p53. Expression of the temperature-sensitive p53val135 mutant in wild-type ES cells resulted in a reduction of apoptosis accompanying differentiation when it adopted a mutant conformation at 39 degrees C. These data demonstrate that functional inactivation of p53 allows differentiating cells to escape from apoptosis, and suggest that the conformational switch could regulate the inactivation process.

Animals↗
PubMed1997

PARP is important for genomic stability but dispensable in apoptosis.

Mice lacking the gene encoding poly(ADP-ribosyl) transferase (PARP or ADPRT) display no phenotypic abnormalities, although aged mice are susceptible to epidermal hyperplasia and obesity in a mixed genetic background. Whereas embryonic fibroblasts lacking PARP exhibit normal DNA excision repair, they grow more slowly in vitro. Here we investigated the putative roles of PARP in cell proliferation, cell death, radiosensitivity, and DNA recombination, as well as chromosomal stability. We show that the proliferation deficiency in vitro and in vivo is most likely caused by a hypersensitive response to environmental stress. Although PARP is specifically cleaved during apoptosis, cells lacking this molecule apoptosed normally in response to treatment with anti-Fas, tumor neurosis factor alpha, gamma-irradiation, and dexamethasone, indicating that PARP is dispensable in apoptosis and that PARP-/- thymocytes are not hypersensitive to ionizing radiation. Furthermore, the capacity of mutant cells to carry out immunoglobulin class switching and V(D)J recombination is normal. Finally, primary PARP mutant fibroblasts and splenocytes exhibited an elevated frequency of spontaneous sister chromatid exchanges and elevated micronuclei formation after treatment with genotoxic agents, establishing an important role for PARP in the maintenance of genomic integrity.

Animals↗
PubMed1997

Structure and chromosomal assignment of the mouse fra-1 gene, and its exclusion as a candidate gene for oc (osteosclerosis).

We have determined the genomic structure of the mouse fra-1 gene, which consists of four exons and three introns at positions also found in the other members of the fos gene family. Fra-1 is expressed rather highly in the brain and testes of adult mice, and at low levels in most other tissues. Absence of c-Fos leads to significantly reduced serum stimulation of fra-1 expression in gene targeted mouse fibroblasts, demonstrating that mitogen induction of fra-1 is partially mediated by c-Fos/AP-1. A polymorphic (CA)n microsatellite marker was found in intron 2 of fra-1 and used to map the gene to the centromeric region of mouse chromosome 19. Since fra-1 maps to the same genomic region as oc (osteosclerosis), an autosomal recessive disorder leading to the bone remodelling disease osteopetrosis, we tested it as a candidate gene for oc. The segregation of fra-1 in two different crosses of mice carrying oc and an allelism test between oc and a targeted disruption of fra-1 demonstrate that fra-1 and oc are two distinct genes rather than oc being a mutant allele of fra-1.

Amino Acid Sequence↗