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G-CSF and GM-CSF play important roles in regulating neutrophil production, survival, differentiation, and function. However, we have shown previously that G-CSF/GM-CSF double-deficient [knockout (KO)] mice still develop a profound neutrophilia in bone marrow and blood after infection with Candida albicans. This finding suggests the existence of other systems, which can regulate emergency neutrophil production. We have now developed an “in vitro” technique to detect and characterize a neutrophil-promoting activity (NPA) in the media conditioned by mouse embryonic fibroblasts (MEFs) derived from G-CSF−/−/GM-CSF−/− mice. NPA is produced in vitro by the MEFs after stimulation with LPS or heat-inactivated C. albicans. Although M-CSF added directly to bone marrow cultures does not sustain granulocyte production, our studies indicate that production of NPA requires activation of the M-CSF receptor (c-fms). First, G-CSF−/−/GM-CSF−/− MEFs produce high levels of NPA after stimulation with LPS or C. albicans, and G-CSF/GM-CSF/M-CSF triple-KO MEFs do not. Second, the production of NPA by the G-CSF−/−/GM-CSF−/− MEFs is reduced significantly upon incubation with neutralizing antibodies to M-CSF or c-fms. Third, NPA production by G-CSF−/−/GM-CSF−/−/M-CSF−/− fibroblasts is enhanced by supplementing culture medium with M-CSF. Thus, stimulation of c-fms by M-CSF is a prerequisite for the production of NPA.

Downstream of kinase (Dok)–related protein (DokR, also known as p56dok/FRIP/Dok-R) is implicated in cytokine and immunoreceptor signaling in myeloid and T cells. Tyrosine phosphorylation induces DokR to bind the signal relay molecules, RasGTPase-activating protein (RasGAP) and Nck. Here, we have examined the function of DokR during hematopoietic development and the requirement for RasGAP and Nck binding sites in its biological function. Retroviral-mediated expression of DokR in bone marrow cells dramatically inhibited their capacity to form colonies in vitro in response to the cytokines macrophage colony–stimulating factor and stem cell factor, whereas responses to interleukin-3 and granulocyte macrophage colony–stimulating factor were only weakly affected. When introduced into lethally irradiated mice, hematopoietic cells expressing DokR showed a drastically reduced capacity to repopulate lymphoid tissues. Most notably, DokR dramatically reduced repopulation of the thymus, in part by reducing the number of T cell precursors seeding in the thymus, but equally, through inhibiting the transition of CD4−CD8− to CD4+CD8+ T cells. Consequently, the number of mature peripheral T cells was markedly reduced. In contrast, a minimal effect on B cell and myeloid lineage development was observed. Importantly, functional RasGAP and Nck binding sites were found to be essential for the biological effects of DokR in vitro and in vivo.

Autophosphorylation is a key event in the activation of protein kinases. In this study, we demonstrate that autophosphorylation of the recombinant Src family kinase Hck leads to a 20-fold increase in its specific enzymatic activity. Hck was found to autophosphorylate readily to a stoichiometry of 1.3 mol of phosphate per mol of enzyme, indicating that the kinase autophosphorylated at more than one site. Solid phase sequencing and two-dimensional mapping of the phosphopeptide fragments derived from the autophosphorylated enzyme revealed that the kinase can undergo autophosphorylation at the following two sites: (i) Tyr-388, which is located to the consensus autophosphorylation site commonly found in the activation loop of many protein kinases, and (ii) Tyr-29, which is located in the unique domain of Hck. Hck purified from mouse bone marrow-derived macrophages could also autophosphorylate in vitro at both Tyr-388 and Tyr-29, indicating that naturally occurring Hck can also autophosphorylate at Tyr-29. Furthermore, Hck transiently expressed in human embryonic kidney 293T cells was found to be phosphorylated at Tyr-29 and Tyr-388, proving that Hck can also undergo autophosphorylation at both sites in vivo. The recombinant enzyme carrying the mutation of Tyr-388 to Phe was also able to autophosphorylate at Tyr-29, albeit at a significantly slower rate. A 2-fold increase in the specific enzymatic activity was seen with this mutant despite the stoichiometry of autophosphorylation only approaching 0.2 mol of phosphate per mol of enzyme. This indicates that autophosphorylation of Tyr-29 contributes significantly to the activation of Hck. Regulation of the catalytic activity by phosphorylation of Tyr-29 in the unique domain may represent a new mechanism of regulation of Src family tyrosine kinases.

Genetic studies have previously revealed that Cdc37p is required for the catalytic competence of v-Src in yeast. We have reasoned that temperature-sensitive mutants of Src family kinases might be more sensitive to the cellular level of p50(Cdc37), the mammalian homolog of Cdc37p, than their wild-type counterpart, thus potentially providing a unique opportunity to elucidate the involvement of p50(Cdc37) in the folding and stabilization of Src family kinases. A temperature-sensitive mutant of a constitutively active form of Hck (i.e., tsHck499F) was created by mutating two amino acids within the kinase domain of Hck499F. Significantly, overexpression of p50(Cdc37) rescues the catalytic activity of tsHck499F at 33 degrees C, while partially buffering it against inactivation at higher temperatures (e.g., 37 and 39 degrees C). Hsp90 function is required for tsHck499F activity and its stabilization by p50(Cdc37), but overexpression of Hsp90 is not sufficient to stabilize tsHck499F. Overexpression of p50(Cdc37) promotes the association of tsHck499F with Hsp90, suggesting that the cellular level of p50(Cdc37) might be the rate-limiting step in the association of tsHck499F with Hsp90. A truncation mutant of p50(Cdc37) that cannot bind Hsp90 still has a limited capacity to rescue the catalytic activity of tsHck499F and promote its association with Hsp90. This is a particularly important observation, since it argues that rather than solely acting as a passive adapter protein to tether tsHck499F to Hsp90, p50(Cdc37) may also act allosterically to enhance the association of tsHck499F with Hsp90. The findings presented here might also have implications for our understanding of the evolution of protein kinases and tumor development.

A murine embryonic cDNA library was screened for potential substrates of the Src family kinase, Lyn, using a phosphorylation-screening strategy. One cDNA that we identified encodes Dok-related protein (DokR), a protein with homology to p62(dok) (Dok), and members of the insulin receptor substrate-1 family of proteins. Analysis of murine tissue extracts with DokR-specific antisera revealed that DokR protein is expressed at highest levels in lymphoid tissues. Co-expression of a FLAG epitope-tagged form of DokR (FLAG-DokR) with Lyn in embryonic kidney 293T cells resulted in constitutive phosphorylation of FLAG-DokR on tyrosine residues and consequential physical association with RasGTPase-activating protein (GAP) and the Nck adaptor protein. Stimulation of BaF/3 hematopoietic cells co-expressing the epidermal growth factor (EGF) receptor tyrosine kinase and FLAG-DokR with EGF also induced phosphorylation of FLAG-DokR and promoted its association with GAP. Immunoprecipitation experiments using DokR-specific antibodies revealed an interaction between endogenous DokR and a 150-kDa protein that is tyrosine-phosphorylated in EGF-stimulated BaF/3 cells. The molecular basis of the interactions involving DokR with GAP and Nck was investigated using a novel glutathione S-transferase fusion protein binding assay and/or site-directed mutagenesis. Tandem SH2-binding sites containing Tyr-276 and Tyr-304 were shown to mediate binding of DokR to GAP, whereas Tyr-351 mediated the binding of DokR to Nck. These results suggest that DokR participates in numerous signaling pathways.

Despite evidence that implicates transforming growth factor-alpha (TGF-alpha) in the pathogenesis of acute lung injury, the contribution of TGF-alpha to the fibroproliferative response is unknown. To determine whether the development of pulmonary fibrosis depends on TGF-alpha, we induced lung injury with bleomycin in TGF-alpha null-mutation transgenic mice and wild-type mice. Lung hydroxyproline content was 1.3, 1.2, and 1.6 times greater in wild-genotype mice than in TGF-alpha-deficient animals at Days 10, 21, and 28, respectively, after a single intratracheal injection of bleomycin. At Days 7 and 10 after bleomycin treatment, lung total RNA content was 1.5 times greater in wild-genotype mice than in TGF-alpha-deficient animals. There was no significant difference between mice of the two genotypes in lung total DNA content or nuclear labeling indices after bleomycin administration. Wild-genotype mice had significantly higher lung fibrosis scores at Days 7 and 14 after bleomycin treatment than did TGF-alpha-deficient animals. There was no significant difference between TGF-alpha-deficient mice and wild-genotype mice in lung inflammation scores after bleomycin administration. To determine whether expression of other members of the epidermal growth factor (EGF) family is increased after bleomycin-induced injury, we measured lung EGF and heparin-binding- epidermal growth factor (HB-EGF) mRNA levels. Steady-state HB-EGF mRNA levels were 321% and 478% of control values in bleomycin-treated lungs at Days 7 and 10, respectively, but were not significantly different in TGF-alpha-deficient and in wild-genotype mice. EGF mRNA was not detected in normal or bleomycin-treated lungs of mice of either genotype. These results show that TGF-alpha contributes significantly to the pathogenesis of pulmonary fibrosis after bleomycin-induced injury, and that compensatory increases in other EGF family members do not occur in TGF-alpha-deficient mice.

Cell type-specific responses to the leukemia inhibitory factor (LIF)/interleukin 6 cytokine family are mediated by dimerization of the LIF receptor alpha-chain (LIFRalpha) with the signal transducer gp130 or of two gp130 molecules followed by activation of the JAK/STAT and Ras/mitogen-activated protein kinase cascades. In order to dissect the contribution of gp130 and LIFRalpha individually, chimeric molecules consisting of the extracellular domain of the granulocyte colony stimulating factor receptor (GCSF-R) and various mutant forms of the cytoplasmic domains of gp130 or LIFRalpha were expressed in embryonic stem (ES) cells to test for suppression of differentiation, or in a factor-dependent plasma cytoma cell line to assess for induction of proliferation. Carboxyl-terminal domains downstream of the phosphatase (SHP2)-binding sites were dispensable for mitogen-activated protein kinase activation and the transduction of proliferative signals. Moreover, carboxyl-terminal truncation mutants which lacked intact Box 3 homology domains showed decreased STAT3 activation, failed to induce Hck kinase activity and suppress ES cell differentiation. Moreover, STAT3 antisense oligonucleotides impaired LIF-dependent inhibition of differentiation. Substitution of the tyrosine residue within the Box 3 region of the GSCF-R abolished receptor-mediated suppression of differentiation without affecting the transduction of proliferative signals. Thus, distinct cytoplasmic domains within the LIFRalpha, gp130, and GCSF-R transduce proliferative and differentiation suppressing signals.

Hck and Src are members of the Src family of protein- tyrosine kinases that carry out distinct and overlapping functions in vivo (Lowell, C. A., Niwa, M., Soriano, P., and Varmus, H. E. (1996) Blood 87, 1780-1792). In an attempt to understand how Hck and Src can function both independently and in concert, we have compared 1) their in vitro substrate specificity and 2) the accessibility of their Src homology 2 (SH2) domain. Using several synthetic peptides, we have demonstrated that Hck and Src recognize similar structural features in the substrate peptides, suggesting that both kinases have the intrinsic ability to carry out overlapping cellular functions by phosphorylating similar cellular proteins in vivo. Using a phosphotyrosine-containing peptide that has previously been shown to bind the SH2 domain of Src family kinases with high affinity, we found that although Src could bind to the phosphopeptide, Hck showed no interaction. The inability of Hck to bind the phosphopeptide was not a result of a stable intramolecular interaction between its SH2 domain and C-terminal regulatory phosphotyrosine residue (Tyr-520), as most Hck molecules in the purified Hck preparation were not tyrosine-phosphorylated. In contrast to intact Hck, a recombinant truncation analog of Hck was able to bind the phosphopeptide with an affinity similar to that of the Src SH2 domain, suggesting that conformational constraints are imposed on intact Hck that limit accessibility of its SH2 domain to the phosphopeptide. Furthermore, the difference in SH2 domain accessibility is a potential mechanism that enables Src and Hck to perform their respective unique functions by 1) targeting them to different subcellular compartments, whereupon they phosphorylate different cellular proteins, and/or 2) facilitating direct binding to their cellular substrates.

Leukemia inhibitory factor (LIF) is a mammalian cytokine that has a wide range of physiological activities, including the inhibition of differentiation of embryonic stem (ES) cells. We have used insertional mutagenesis in an attempt to isolate molecules that participate in LIF signal transduction via the LIF receptor. Using a robust screen for undifferentiated cells, we have isolated one ES cell line, Poly 27, that does not require exogenous LIF to remain undifferentiated in vitro. We present evidence that Poly 27 is not irreversibly committed to an undifferentiated phenotype, but can differentiate in vitro if cultured in the presence of chemical differentiating agents, while in syngeneic mice Poly 27 cells form tumours which are composed largely of undifferentiated cells. We have characterized the mechanism of factor independence in Poly 27, and shown it to be a result of autocrine LIF production. This LIF production is potentially the result of a mutation in a gene critically involved in regulating LIF production in ES cells.

To ascertain whether the development of dendritic cells (DC) in mouse lymphoid organs is dependent on granulocyte/macrophage colony-stimulating factor (GM-CSF), we determined the number of DC in the thymus, spleen and lymph nodes of normal mice, of mice with the genes coding for GM-CSF or its receptor inactivated, and of transgenic mice with excessive levels of GM-CSE DC were extracted from the tissues and enriched prior to flow cytometric analysis. The total DC level and the incidence of DC expressing lymphoid-related markers (CD8(hi) CD11b(lo)) and myeloid-related markers (CD8(lo) CD11b(hi)) were monitored. Both in GM-CSF null mice, and GM-CSF receptor null mice, DC of all surface phenotypes were present in all lymphoid organs; only small decreases in DC levels were recorded, except for the lymph nodes of GM-CSF receptor null mice which showed a more pronounced (threefold) decrease in DC numbers. Since the GM-CSF receptor null mice lacked the beta chain common to the GM-CSF, interleukin (IL)-3 and IL-5 receptors, the development of DC in the absence of GM-CSF was not due to common beta chain mediated developmental signals elicited by IL-3 or IL-5. In GM-CSF transgenic mice, there was only a 50 % increase in DC numbers in thymus and spleen, paralleling an increase in overall cellularity, but a more pronounced (threefold) increase in DC numbers in lymph nodes. There was no evidence that GM-CSF had a selective effect on any particular DC subpopulation defined by CD8 or CD11b expression. We conclude that the development of most lymphoid tissue DC can proceed in the absence of GM-CSF, although this cytokine can produce some elevation of DC levels. It is not clear whether the enhancing effect of GM-CSF is direct or an indirect effect mediated by other cytokines.

Lyn is a member of the Src family of protein-tyrosine kinases that can readily undergo autophosphorylation in vitro. The site of autophosphorylation is Tyr397 which corresponds to the consensus autophosphorylation site of other Src family tyrosine kinases. The rate of autophosphorylation is concentration-dependent, indicating that the reaction follows an intermolecular mechanism. Autophosphorylation results in a 17-fold increase in protein-tyrosine kinase activity. Kinetic analysis demonstrates that phosphorylation of a substrate peptide by Lyn following autophosphorylation occurs with a 63-fold decrease in Km but no significant change in Vmax, suggesting that autophosphorylation relieves the conformational constraint that prevents binding of the substrate peptide to the active site of the kinase. Using a phosphotyrosine-containing peptide (pYEEI) that has previously been shown to bind to the Src homology 2 (SH2) domain of Src family tyrosine kinases with high affinity, we found that autophosphorylation results in a significant decrease in accessibility of the Lyn SH2 domain, indicating that conformational changes in the protein kinase domain induced by autophosphorylation can be propagated to the SH2 domain. Our study suggests that autophosphorylation plays an important role in regulating Lyn by modulating both its kinase activity and its interaction with other phosphotyrosine-containing molecules.

The role played by the Src-related tyrosine kinase, Hck, in embryonic stem (ES) cell differentiation was investigated by replacing a conserved C-terminally located tyrosine with phenylalanine by gene targeting. Targeted ES cells display a 7- to 9-fold elevation in constitutive Hck kinase activity and require approximately 15 times less leukaemia inhibitory factor (LIF) than parental ES cells to maintain their stem cell character in vitro. We also demonstrate a rapid and transient increase in Hck tyrosine kinase activity in parental ES cells stimulated by LIF and, finally, show that Hck is physically associated with gp130, an affinity converter and signal transducing component of the LIF receptor. Thus, these results provide biological and biochemical evidence that Hck participates in signal transduction from the LIF receptor.

In this study a rapid, simple and inexpensive procedure is described which allows potential germ-line male mice to be identified with confidence. Spermatozoa recovered by uterine washing following mating with normal female mice was analysed in two ways. First, the patterns of expression of the different isoforms of glucose phosphate isomerase were determined. Since the glucose phosphate isomerase isoforms expressed in embryo stem (ES) cell lines are frequently different from those associated with the host blastocyst, it is possible to determine the proportion of spermatozoa produced by an individual animal that was of ES cell or host-blastocyst origin. Second, DNA of spermatozoa was subjected to polymerase chain reaction (PCR) analysis using primers with specificity for the targeted mutation in the ES cells. The PCR analysis was particularly valuable in identifying germ cell chimaeras in which the contribution of ES-derived spermatozoa was significantly less than that specified by the host blastocyst.

The aim of this study was to investigate the metabolic and structural consequences of a decrease in glucose transporter-4 (GLUT4) levels on the heart. The CreLoxP system was utilised to delete GLUT4 in muscle tIssue including heart. The presence of the PGK-neoR cassette in the GLUT4-Lox mice resulted in reduced expression in all tIssues to levels 15-30% of wild-type control mice. In mice expressing Cre recombinase, there was a further reduction of GLUT4 in cardiac tIssue to almost undetectable levels. Cardiac glucose uptake was measured basally and during a euglycaemic/hyperinsulinaemic clamp using 2-deoxy-[1-(14)C]glucose. Insulin-stimulated glucose uptake was normal in hearts expressing 15% of normal GLUT4 levels but markedly reduced in mice with more profound reduction in GLUT4. Cardiac enlargement occurred only when GLUT4 levels were less than 5% of normal values. In heart there is a threshold level of GLUT4 above which insulin-stimulated glucose uptake is maintained. As little as 5% of normal GLUT4 levels expressed in heart is sufficient to prevent the development of cardiac hypertrophy.

Objective Studies on mice lacking the common receptor subunit gp130 reveal that activation of gp130-dependent signaling pathways is essential for normal fetal and adult hematopoiesis. However, the extent to which hematopoiesis is dependent upon activation of a particular gp130 signaling pathway, namely STAT1/3 or SHP2/MAPK, is unknown. This study examined the specific contribution of gp130-mediated STAT1/3 signaling to the regulation of hematopoiesis. Materials and Methods Hematopoiesis was examined at various developmental stages in mice homozygous for a targeted carboxy-terminal truncation mutation in gp130 (gp130 Δ /Δ ) that deletes all STAT1/3 binding sites, thereby abolishing gp130-mediated STAT1/3 activation. Results Adult gp130 Δ / Δ mice have increased numbers of immature colony-forming unit spleen progenitor cells in the bone marrow and spleen, elevated numbers of committed myeloid progenitor cells in the spleen and peripheral blood, and leukocytosis. Increased progenitor cell production was observed in gp130 Δ /Δ fetal livers from 14 days of gestation onward. In contrast, the circulating platelet count was reduced by 30% in gp130 Δ / Δ mice, without any corresponding decrease in the number of bone marrow and splenic megakaryocytes. In liquid cultures, megakaryocytes from gp130 Δ/ Δ mice are smaller than those from wild-type mice and do not increase in size upon stimulation with interleukin-6 or interleukin-11. Administration of either interleukin-6 or interleukin-11 to gp130 Δ / Δ mice failed to increase platelet numbers, despite an increase in the production of megakaryocytes. Conclusions Collectively, these results reveal that gp130-mediated STAT1/3 activation is required to maintain the normal balance of hematopoietic progenitors during fetal and adult hematopoiesis. Furthermore, they suggest two distinct roles for gp130-mediated STAT1/3 activation in hematopoiesis, one restricting the production of immature hematopoietic progenitor cells and the other promoting the functional maturation of megakaryocytes to produce platelets.

TGF alpha is a mitogenic polypeptide found in the conditioned media of transformed cell lines as well as in various solid tumors. Although its physiological role in normal tissues is uncertain, the autocrine action of TGF alpha on the EGF receptor is postulated to play a role in tumorigenesis. To explore the possibility that pre-existing mouse mutants might have concordance with the mouse TGF alpha locus (Tgfa) we sought to establish the chromosomal localization of the murine TGF alpha gene. Using Southern analysis we have detected NcoI and PvuII RFLPs in the TGF alpha gene of progenitor RI mouse strains. These RFLPs have been used to analyze four different RI sets of DNA and to assign Tgfa to the 35-cM region of chromosome 6. Linkage has been established and the data suggest that the distance between Igk and wa-1 anchor loci may be less than 8 cM and that the gene order for the proximal to mid region of mouse chromosome 6 may be: Ggc-Xmmv27-[Brp-1, Lvp-1, Ms6-4]-[Igk, Ly2, Ly3 Odc-rs5, Rn7s-6, Fabp1]-[Tgfa/wa-1]-IL5-R. Homology of synteny has been further defined between the proximal region of mouse chromosome 6 and with the 2p13-p11 region of human chromosome 2 encompassing TGFA, IGK, CD8A, and FABP1.

Hck, a member of the Src-family of protein tyrosine kinases, is expressed primarily in hematopoietic cells of the myeloid and B-lymphocyte lineages. Hybridoma cell lines were established that secrete monoclonal antibodies (MAbs) to Hck. Three of the MAbs were extensively characterized and designated H7, H34, and H42. The MAbs H7 and H34 recognized an epitope within the SH3 domain of Hck, while the epitope recognized by the H42 MAb resides within the Unique domain. All three MAbs specifically recognized the p59 and p56 isoforms of Hck in transiently transfected 293T cells and in a murine macrophage cell line. Notably, the antibodies did not cross-react with other Src-family kinases tested. Under native conditions, the MAbs H34 and H42 efficiently immunoprecipitated Hck from transfected cells. Both MAbs were also successfully used for the immunofluorescent staining of Hck in intact cells. Thus, the MAbs described herein should be useful in studies of Hck function and expression.

Src family tyrosine kinases have previously been proposed to mediate some of the biological effects of lipopolysaccharide on macrophages. Accordingly, we have sought to identify substrates of Src family kinases in lipopolysaccharide-stimulated macrophages. Stimulation of Bac1.2F5 macrophage cells with lipopolysaccharide was found to induce gradual and persistent tyrosine phosphorylation of Cbl in an Src family kinase-dependent manner. Immunoprecipitation experiments revealed that Cbl associates with Hck in Bac1.2F5 cells, while expression of an activated form of Hck in Bac1.2F5 cells induces tyrosine phosphorylation of Cbl in the absence of lipopolysaccharide stimulation. The Src homology 3 domain of Hck can directly bind Cbl, and this interaction is important for phosphorylation of Cbl. Association of the p85 subunit of phosphatidylinositol (PI) 3-kinase with Cbl is enhanced following lipopolysaccharide stimulation of Bac1.2F5 cells, and transient expression experiments indicate that phosphorylation of Cbl by Hck can facilitate the association of p85 with Cbl. Lipopolysaccharide treatment also stimulates the partial translocation of Hck to the cytoskeleton of Bac1.2F5 cells. Notably, lipopolysaccharide enhances the adherence of Bac1.2F5 cells, an effect that is dependent on the activity of Src family kinases and PI 3-kinase. Thus, we postulate that Hck enhances the adherence of lipopolysaccharide-stimulated macrophages, at least in part, via Cbl and PI 3-kinase.

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF −/− mice. To investigate the responses of CD8+and CD4+T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8+T cells with specificity for ovalbumin peptide could not be induced in GM-CSF −/− mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF −/− mice. Purified CD4+T cells from GM-CSF −/− mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-γ (IFN-γ) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4+T cells from GM-CSF −/− mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-γ and IL-4. To analyze the rescue effect of DC on CD4+T cells, supernatants from (i) CD4+T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4+T cells and KLH-pulsed spleen cells from GM-CSF −/− mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-γ production of CD4+T cells from GM-CSF −/− mice.

The leukemia inhibitory factor/interleukin 6 (LIF/IL6) family of cytokines promotes cell type-specific pleiotropic effects by engaging multimeric receptor complexes that share the common affinity converter/signal transducing subunit gp130. While the maintenance of embryonic stem (ES) cell self-renewal is an activity unique to this family of cytokines, the intracellular signaling events mediated by gp130 remain largely unknown. Here we show a rapid and transient increase in the specific activity of the Src- related kinase Hck as well as of the Janus kinases Jak1, Jak2, and Tyk2 following treatment of ES cells with LIF or a combination of IL6 plus a soluble form of the IL6 receptor. Within 2 min of stimulation, we also observed increased tyrosine phosphorylation of SHC, activation of the guanidine nucleotide exchange activity on p21(ras), and an electrophoretic mobility shift of MAP kinase. Functional involvement of Hck and p21(ras) activation in gp130-mediated signaling is supported by the finding that the introduction of constitutively activated Hck or v-Ha-ras partially alleviates the requirement of ES cells for LIF to remain undifferentiated. In contrast, suppression of Jak1 in ES cells by antisense technology increased the amount of LIF required to retain their pluripotentiality. These results are consistent with the notion that gp130-mediated suppression of ES cell differentiation depends on signaling through at least two cascades, namely a p21(ras)-dependent pathway that possibly involves Hck, as well as a Jak kinase-dependent pathway.

We have determined the nucleotide sequence of a cDNA encoding a protein kinase that is closely related to the enzyme encoded by the Drosophila melanogaster mutant polo and that we have designated Plk (polo-like kinase). Plk is also related to the products of the Saccharomyces cerevisiae cell cycle gene MSD2 (CDC5) and the recently described early growth response gene Snk. Together, Plk, polo, Snk, and MSD2 define a subfamily of serine/threonine protein kinases. Plk is expressed at high levels in a number of fetal and newborn mouse tissues but is not expressed in the corresponding adult organs. With the exception of adult hemopoietic tissues, the only adult tissues in which we could detect Plk expression were ovaries and testes. Taken together, the patterns of Plk expression suggest an association with proliferating cells. Since polo is required for mitosis in Drosophila it is possible that Plk is involved in some aspect of cell cycle regulation in mammalian cells.

Mice homozygous for a disrupted transforming growth factor alpha (TGF alpha) gene are healthy and fertile, although some older mice show evidence of corneal inflammation. In contrast with TGF alpha +/- and +/+ animals, TGF alpha -/- mice have a pronounced waviness of the coat. Histological examination of the skin from TGF alpha -/- mice reveals a dramatic derangement of hair follicles. Mice with a disrupted TGF alpha gene also have curly whiskers, first evident on the day of birth. The phenotype of TGF alpha -/- mice is remarkably similar to that of the mouse mutant waved-1 (wa-1). Offspring resulting from crosses between TGF alpha -/- and wa-1 mice display the curly whisker-coat phenotype, indicating that the basis of the wa-1 phenotype is a mutation in the TGF alpha gene. These observations suggest that TGF alpha plays a pivotal role in determining skin architecture and in regulating hair development.

Transgenic mice aberrantly expressing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene develop an unusual syndrome of blindness, tissue damage and wasting which is associated with accumulations of hemopoietic cells. In order to further characterize this disease state, we have used messenger RNA detection techniques to show that the genes for tumor necrosis factor (TNF alpha), interleukin-1 alpha (IL-1 alpha) and basic fibroblast growth factor (bFGF) are expressed at abnormally high levels in both macrophages and granulocytes in transgenic mice. Furthermore, since these cell types also express the GM-CSF transgene, it is likely that they are autocrine stimulated by GM-CSF. These observations raise the possibilities that, first, the expression of tumor necrosis factor alpha, interleukin-1 alpha and basic fibroblast growth factor in hemopoietic cells is a direct consequence of their autostimulation by GM-CSF, and second, that these cytokines may be responsible for some aspects of the transgenic mouse disease.

In normal steady-state hematopoiesis, G-CSF not only regulates the production of neutrophils, it is also involved in release of neutrophil from bone marrow into the circulation. In the present study we have evaluated the physiological role of G-CSF in the release of granulocytes from marrow into circulation. The thymidine analogue, 5′-bromo-2′-deoxyuridine (BrdU), was used to label dividing bone marrow cells and follow the release of granulocytes into circulation in G-CSF-deficient and wild type mice. The bone marrow and blood cells were labeled with anti-BrdU antibody conjugated to FITC and anti-Gr-1 antibody conjugated to PE (marker for granulocytes) and analysed by FACScan. Interestingly, the labeling index and the amount of BrdU incorporated by bone marrow cells was greater in G-CSF-deficient mice compared to wild type mice. This phenomenon was most striking in the blast cell population clearly indicating that this population of cells is cycling faster in G-CSF-deficient mice compared to wild type mice. However, a greater proportion of early granulocytic cells in the bone marrow was undergoing apoptosis in G-CSF-deficient mice compared to wild type mice. The kinetics of release of granulocytes from marrow into circulation was similar in both G-CSF and wild type mice. Similarly, the transit time of granulocytes through the mitotic and post-mitotic pool in the marrow was unaffected in absence of G-CSF. Furthermore, the peripheral half-lives of granulocytes of G-CSF-deficient and wild-type mice were comparable. These findings demonstrate that while G-CSF is important for survival of granulocytic cells, under physiological conditions, G-CSF is dispensable for mobilisation of granulocytes from marrow into circulation.

The structure and function of the promoter region and exon 1 of the murine hck gene have been characterized in detail. RNase protection analysis has established that hck transcripts initiate from heterogeneous start sites located within the hck gene. Fusion gene constructs containing hck 5'-flanking sequences and the bacterial Neor gene have been introduced into the hematopoietic cell lines FDC-P1 and WEHI-265 by using a self-inactivating retroviral vector. The transcriptional start sites of the fusion gene are essentially identical to those of the endogenous hck gene. Analysis of infected WEHI-265 cell lines treated with bacterial lipopolysaccharide (LPS) reveals a 3- to 5-fold elevation in the levels of endogenous hck mRNA and a 1.4- to 2.6-fold increase in the level of Neor fusion gene transcripts, indicating that hck 5'-flanking sequences are capable of conferring LPS responsiveness on the Neor gene. The 5'-flanking region of the hck gene contains sequences similar to an element which is thought to be involved in the LPS responsiveness of the class II major histocompatibility gene A alpha k. A subset of these sequences are also found in the 5'-flanking regions of other LPS-responsive genes. Moreover, this motif is related to the consensus binding sequence of NF-kappa B, a transcription factor which is known to be regulated by LPS.

A method has been devised which permits mapping of transcripts by a two-step hybridization procedure (sandwich hybridization). RNA extracted from cells infected with an adenovirus-SV40 hybrid (Ad2+ND1) was hybridized to restriction endonuclease fragments of adenovirus type 2 (Ad2) DNA immobilized on nitrocellulose filters. RNAs containing both Ad2 and SV40 sequences formed duplexes through their Ad2 sequences, leaving their SV40 sequences as protruding tails. Annealing with 32P-labeled SV40 DNA caused these tails to become labeled, permitting autoradiographic identification of the sequences of Ad2 DNA which are homologous to the RNA. The high sensitivity of this technique, achieved through the use of 32P-labeled RNA of high specific activity, has led to the observation that hybridization of Ad2+ND1 RNA occurs at several locations on the Ad2 genome, in addition to the expected sites of hybridization proximal to the SV40 insertion.

Adenovirus type 2 (Ad2) causes respiratory infections in humans, grows productively on human cell lines such as HeLa and KB, and can transform rat primary cell lines (Tooze 1973). The Ad2 virion consists of a linear duplex DNA chromosome 35,000 base pairs (bp) long (23 × 106 daltons) contained within an icosahedral capsid composed of at least ten different proteins. After penetration of the cell membrane, the viral DNA and several core proteins associated with it are transported to the nucleus, where early RNA transcription begins within 2 hours. About 8 hours after infection, viral DNA replication commences, reaching a maximum rate several hours later. Late transcription to produce messenger RNA (mRNA) for capsid and other virus-specific proteins begins after the onset of DNA replication and continues for about 2 days, when the infected cells die.

cDNA clones specifying the murine granulocyte–macrophage colony stimulating factor have been isolated. This haematopoietic growth factor is encoded by a unique gene specifying a messenger RNA of 1,200 nucleotides and a polypeptide of 118 amino acids. It bears no structural similarity to the functionally related factor, interleukin-3, described recently.

We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb.

To study the control mechanism(s) that govern the transcription of c-myb, genomic clones corresponding to the 5' region of the murine c-myb gene have been isolated and characterized structurally and functionally. Primer extension and nuclease protection analysis have revealed the presence of multiple transcriptional initiation sites, that are utilized in several hemopoietic cell lines (WEHI3B(D+). FDC-P1 and RB22.2). Some of the sites are used in all cell lines but others are unique; all are located in a region of the c-myb gene that is G-C rich, contains a number of potential Sp1 binding sites and lacks classical promoter consensus sequences. Experiments in which well characterized promoters controlling expression of a reporter gene have been replaced by fragments of c-myb DNA (including the observed cap sites) were performed in an attempt to demonstrate promoter activity in various cell types. It was shown that a region of the c-myb gene (approximately 1.0 kbp upstream from the splice donor site of the first exon) contains a weak promoter that has a low level of transcriptional activity in hemopoietic as well as in fibroblastic cells. These results support the suggestion that c-myb expression is not regulated primarily at the level of initiation of transcription.

During the course of lytic infection with the adenovirus 2-SV40 hybrid Ad2 + ND1 dp2, a viral mRNA is synthesized whose 5′ end includes the start of the adenovirus 2 fiber gene and whose 3′ end lies in SV40 sequences. This hybrid mRNA codes for a protein(s) of 23–24K daltons (G. Fey, J. B. Lewis, T. Grodzicker and A. Bothwell, manuscript submitted). Biochemical and electron microscope heteroduplex analyses reveal that two forms of this mRNA exist in the cytoplasm of cells at late times in infection. Both forms contain the conventional tripartite leader present on "late" adenovirus 2 mRNAs, but they differ in the presence or absence of a fourth leader sequence consisting of 180 nucleotides of adenovirus 2 sequences located 2,000 nucleotides upstream from the gene. By fractionation of total poly(A)-containing Ad2 + ND1 dp2 "late" cytoplasmic RNA in methyl mercuric hydroxide-agarose gels, we have isolated both forms of the hybrid mRNA and established that the presence or absence of the fourth leader sequence has little or no influence on the ability of the messenger to direct the synthesis of the 23–24K dalton protein(s) in a cell-free translation system derived from rabbit reticulocytes.

A cDNA containing a virtually complete copy of the mRNA for the haemopoietic growth regulator, granulocyte-macrophage colony stimulating factor (GM-CSF), has been isolated from a murine T lymphocyte cDNA library. When a eukaryotic expression vector with this cDNA coupled to the SV40 late promoter was introduced into simian COS cells, significant quantities of GM-CSF were secreted. Since all of the biological activities previously ascribed to highly purified GM-CSF were exhibited in the COS cell-derived GM-CSF, all of these activities are intrinsic to the product of a single gene. There are two potential translational initiation codons in the GM-CSF mRNA; the first is buried in the stem and the second located in the loop of a very stable hairpin structure. Expression studies using deletion derivatives of the cDNA indicated that the second AUG is able to initiate the translation and secretion of GM-CSF. The amino acid sequence of the leader peptide is rather atypical for a secreted protein and we speculate that molecules which initiate at the first AUG might exist as integral membrane proteins whereas those initiating at the second are secreted.

Fiber mRNA of adenovirus 2 has been used as a template for RNA-dependent DNA polymerase. The resulting cDNA/RNA hybrids have been inserted at the Pst I site of the plasmid vector pBR322 after A:T tailing. One recombinant plasmid, pJAW 43, has been characterized in detail and shown to contain sequences from the main body of fiber mRNA, the three leaders common to most late adenoviral mRNAs and a fourth leader found in some species of fiber mRNA. The complete DNA sequence of the leader region has been determined and does not contain the initiation codon AUG, although this codon does occur immediately downstream from the junction between the fourth leader and the main body of the fiber mRNA. The first leader (map coordinate 16.6) is 41 nucleotides long, the second (from 19.6) is 71 nucleotides, the third (from 26.6) is 88 nucleotides and the fourth (from 78.5) is 181 nucleotides. The location of junctions between viral leaders and intervening sequences has been determined by reference, where possible, to sequences of the adenovirus 2 genome. Although the presence of short repeated sequences at the boundaries of intervening sequences and leaders makes it impossible to locate the splice point unambiguously, all of the leader-intervening sequence junctions can be arranged to stress a common feature--the presence of the dinucleotides GT and AG at the 5' and 3' ends, respectively, of the intervening sequences. This prototype sequence, which has also been recognized at or near the splice points in other eucaryotic systems, is possibly part of a larger unit which serves as a recognition site for specific excision-ligation events that ultimately lead to the production of mature mRNAs.

Publisher Summary This chapter describes the technique for mapping viral mRNAs by sandwich hybridization. The technique is of application and can be used to analyze the transcription products of any segment of DNA whose restriction maps are known. The sequences contained within the tail can be determined by a second round of hybridization using 32P-labeled fragments of viral DNA as probes. Hybridization to two adjacent fragments means that a particular mRNA contains sequences that are contiguous within the genome and span the restriction endonuclease cleavage site. Several factors contribute to the overall sensitivity of the sandwich hybridization technique. Of primary importance is the integrity of the unpaired RNA tails during and after the first stage hybridization. An attractive application of the sandwich hybridization technique takes advantage of the fact that the polyriboadenylic acid residues contained at the 3' end of mRNAs are not encoded within the structural gene.

It has become apparent from studies in vitro that the survival, proliferation and differentiation of progenitor cells and functional activation of the various mature cell types of the haemopoietic system are controlled by a group of glycoprotein regulators, notably erythropoietin, T cell growth factor (IL2) and the colony-stimulating factors (CSFs) [1, 2]. Four CSFs with distinet biochemical and biological properties have been purified: M-CSF is a selective proliferative stimulus for macrophages [3]; G-CSF for granulocytes [4]; GM-CSF for both granulocytes and macrophages [5]; while multi-CSF (also known as interleukin- 3 [6], burst-promoting activity [7], P cell-stimulating factor [8], mast cell growth factor [9] and haemopoietic cell growth factor [10]) stimulates the proliferation not only of neutrophilic granulocytes and macrophages, but also eosinophils, megakaryocytes, erythroid and mast cells.

Transgenic mice carrying the murine granulocyte-macrophage colony stimulating factor (GM-CSF) gene expressed from a retroviral promoter exhibit elevated levels of GM-CSF in the serum, urine, peritoneal cavity, and eye. The eyes of transgenic mice are opaque, contain accumulations of macrophages, and develop retinal damage. Similarly, lesions containing macrophages develop in striated muscle. The mice also display an accumulation of large, often multinucleate, activated macrophages in the peritoneal and pleural cavities. The transgene is transcribed in peritoneal cells, as well as in eyes and infiltrated striated muscle. A high proportion of transgenic mice die with muscle wasting when aged 2-4 months, possibly because of macrophage activation resulting from the high levels of GM-CSF.

A growth factor-dependent hemopoietic cell-line, FDC-P1, was treated with a chemical mutagen, EMS, and a number of growth factor-independent variants isolated. Six lines have been extensively analyzed with respect to their growth kinetics, morphology, karyotype, tumorigenicity, and hemopoietic growth factor production. Four lines produced at least one growth factor, subsequently demonstrated to be GM-CSF, while two lines showed no evidence of hemopoietic growth factor production. The observation that the autonomous proliferation of those EMS-derived cell lines that produced GM-CSF can be inhibited by incubation in growth media containing 10-50 JM anti-sense GM-CSF oligonucleotides demonstrated directly that the autonomous behavior of these cells is based on an autocrine mechanism. The induction of the expression of the GM-CSF gene represents a rare class of EMS-induced mutants, and is strongly suggestive of repressor inactivation rather than promoter activation.

Two overlapping genomic clones containing the murine granulocyte-macrophage colony stimulating factor (GM-CSF) gene have been isolated. On the basis of transfection experiments, we have established that a 9-kb BamHI fragment from one of these recombinants encodes biologically active GM-CSF. As deduced from nucleotide sequence analysis, the GM-CSF gene comprises four exons encompassing 2.5 kb of genomic DNA. Primer extension analysis of GM-CSF mRNA identifies a transcriptional initiation site 35 bp upstream of a single translational initiation codon in-frame with the GM-CSF coding sequences and 28 bp downstream of a TATA promoter consensus sequence. Pre-GM-CSF molecules encoded by mRNAs originating from this promoter would include a hydrophobic leader sequence typical for a secreted protein. Intriguingly, sequences present at the 5' end of a GM-CSF cDNA clone previously isolated in our laboratory are not contained within either of the genomic clones and must therefore be transcribed from a promoter located at least 10 kb 5' of the main body of the gene. mRNAs transcribed from this alternative upstream promoter possess an additional initiating codon and potentially encode a pre-GM-CSF polypeptide with an atypical NH2-terminal leader peptide. Comparison of the nucleotide sequence of the GM-CSF gene with that of other haemopoietic growth factor genes has revealed a common decanucleotide (5'-GPuGPuTTPyCAPy-3') within their respective 5'-flanking regions which may be involved in their co-ordinate regulation.

We have isolated a murine cDNA that shares extensive homology with genes encoding the src (Rous sarcoma virus oncogene)-related family of protein-tyrosine kinases. The cDNA includes an open reading frame of 1509 base pairs, and conceptual translation predicts a protein of 56 kDa. Blot-hybridization analysis indicates that this src-related gene is expressed in normal macrophages and in cell lines representing both the myeloid and lymphoid B-cell lineages and, accordingly, is designated "bmk" (B cell/myeloid kinase). In addition, bmk mRNA levels increase following the induced differentiation of the murine myelomonocytic leukemic cell line WEHI-3B.

Two lyn proteins of 56 and 53 kDa have been observed in immunoprecipitates from a variety of murine and human cell lines and tissues. We report the cloning and nucleotide sequence of two distinct murine lyn cDNAs isolated from an FDC-P1 cDNA library. One of the cDNAs, designated lyn11, encodes a protein of 56 kDa which shares 96% similarity with human lyn. The other cDNA, designated lyn12, encodes a protein of 53 kDa. The proteins differ in the presence or absence of a 21-amino-acid sequence located 24 amino acids C terminal of the translational initiation codon. Using RNase protection analysis, we have identified mRNAs corresponding to both cDNAs in murine cell lines and tissues. Sequence analysis of murine genomic clones suggests that the distinct mRNAs are alternatively spliced transcripts derived from a single gene. Expression of both cDNAs in COS cells leads to the production of lyn proteins with the same molecular weight as the two forms of lyn proteins immunoprecipitated from extracts of FDC-P1 cells and mouse spleen. Subcellular fractionation studies and Western immunoblotting analysis suggest that both isoforms of lyn are membrane associated. The association of both lyn isoforms with the membrane fraction supports the notion that lyn, like other src-related kinases, may interact with the intracellular domain of cell surface receptors.

The structure and function of the promoter region and exon 1 of the murine hck gene have been characterized in detail. RNase protection analysis has established that hck transcripts initiate from heterogeneous start sites located within the hck gene. Fusion gene constructs containing hck 5'-flanking sequences and the bacterial Neor gene have been introduced into the hematopoietic cell lines FDC-P1 and WEHI-265 by using a self-inactivating retroviral vector. The transcriptional start sites of the fusion gene are essentially identical to those of the endogenous hck gene. Analysis of infected WEHI-265 cell lines treated with bacterial lipopolysaccharide (LPS) reveals a 3- to 5-fold elevation in the levels of endogenous hck mRNA and a 1.4- to 2.6-fold increase in the level of Neor fusion gene transcripts, indicating that hck 5'-flanking sequences are capable of conferring LPS responsiveness on the Neor gene. The 5'-flanking region of the hck gene contains sequences similar to an element which is thought to be involved in the LPS responsiveness of the class II major histocompatibility gene A alpha k. A subset of these sequences are also found in the 5'-flanking regions of other LPS-responsive genes. Moreover, this motif is related to the consensus binding sequence of NF-kappa B, a transcription factor which is known to be regulated by LPS.

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.