Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death.

The low-affinity receptor for leukemia inhibitory factor (LIFR) interacts with gp130 to induce an intracellular signal cascade. The LIFR-gp130 heterodimer is implicated in the function of diverse systems. Normal placentation is disrupted in LIFR mutant animals, which leads to poor intrauterine nutrition but allows fetuses to continue to term. Fetal bone volume is reduced greater than three-fold and the number of osteoclasts is increased six-fold, resulting in severe osteopenia of perinatal bone. Astrocyte numbers are reduced in the spinal cord and brain stem. Late gestation fetal livers contain relatively high stores of glycogen, indicating a metabolic disorder. Hematologic and primordial germ cell compartments appear normal. Pleiotropic defects in the mutant animals preclude survival beyond the day of birth.

Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice.

Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.

Role of the PU.1 transcription factor in controlling differentiation of Friend erythroleukemia cells.

Both viral and cellular genes have been directly implicated in pathogenesis of Friend viral erythroleukemia. The virus-encoded gp55 glycoprotein binds to erythropoietin receptors to cause mitogenesis and differentiation of erythroblasts. However, if the provirus integrates adjacent to the gene for the PU.1 transcription factor, the cell loses its commitment to terminally differentiate and becomes immortal, as indicated by its transplantability and by its potential for indefinite growth in culture (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 63:4434-4437, 1989; R. Paul, S. Schuetze, S. L. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test the implications of these results, we produced polyclonal antiserum to bacterially synthesized PU.1, and we used it to analyze PU.1 expression throughout leukemic progression and during chemically induced differentiation of Friend erythroleukemia (F-MEL) cell lines. This antiserum identified three electrophoretically distinct PU.1 components in extracts of F-MEL cells and demonstrated their nuclear localization. Although PU.1 proteins are abundant in F-MEL cells, they are absent or present in only trace amounts in normal erythroblasts or in differentiating erythroblasts from the preleukemic stage of Friend disease. Furthermore, chemicals (dimethyl sulfoxide or N,N'-hexamethylenebisacetamide) that overcome the blocked differentiation of F-MEL cells induce rapid declines of PU.1 mRNA and PU.1 proteins. The elimination of PU.1 proteins coincides with recommitment to the program of erythroid differentiation and with loss of immortality. These results support the hypothesis that PU.1 interferes with the commitment of erythroblasts to differentiate and that chemicals that reduce PU.1 expression reinstate the erythropoietic program.

A GM-colony-stimulating factor (CSF) activated ribonuclease system transregulates M-CSF receptor expression in the murine FDC-P1/MAC myeloid cell line.

The murine myeloid precursor cell line FDC-P1/MAC simultaneously expresses receptors for multi-colony-stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF. Growth of FDC-P1/MAC cells in either multi-CSF or GM-CSF results in the posttranscriptional suppression of M-CSF receptor (c-fms proto-oncogene) expression. We use the term transregulation to describe this control of receptor expression and have further characterized this regulatory process. The removal of FDC-P1/MAC cells from GM-CSF stimulation resulted in the re-expression of c-fms mRNA independent of M-CSF stimulation and new protein synthesis. Switching FDC-P1/MAC cells from growth in M-CSF to GM-CSF caused the selective degradation of c-fms mRNA within 6 h after factor switching. Blocking protein synthesis or gene transcription with metabolic inhibitors effectively prevented GM-CSF stimulated degradation of c-fms mRNA. These results suggest that the transregulation of c-fms transcripts by GM-CSF requires the transcriptional activation of a selective mRNA degradation factor. In vitro analysis, the use of cytoplasmic cell extracts, provided evidence that a ribonuclease is preferentially active in GM-CSF stimulated cells, although the specificity for mRNA degradation in vitro is broader than seen in vivo. Together, these data suggest that GM-CSF can dominantly transregulate the level of c-fms transcript through the transcriptional activation of a ribonuclease degradation system.

Disulfide bonding controls the processing of retroviral envelope glycoproteins.

The mitogenic membrane glycoprotein (gp55) encoded by Friend erythroleukemia virus is inefficiently processed from the rough endoplasmic reticulum (RER) and only 3-5% reaches plasma membranes. Because this processed component (gp55P) contains larger and more complex oligosaccharides, it can be separated from RER gp55. In nonreducing conditions, gp55P is a unique disulfide-bonded dimer, whereas RER gp55 consists of monomers and dimers with diverse intrachain and interchain disulfide bonds. This suggests that gp55 folds heterogeneously and that only one homodimer is competent for export from the RER. Pulse-chase analyses of gp55 components labeled with radioactive amino acids indicated that formation of diverse disulfide-bonded components occurred within minutes of polypeptide synthesis and that malfolded components did not later isomerize to generate dimers competent for export from the RER. Chemical studies suggested that all 12 cysteines of gp55 were oxidized within 5 min after synthesis of the protein. In contrast, the envelope glycoprotein precursor (gPr90) encoded by a replication-competent murine leukemia virus folds more homogeneously, and it is then processed and cleaved to form an extracellular glycoprotein gp70 plus a transmembrane protein p15E. The fully processed glycoprotein contains an unoxidized cysteine sulfhydryl that isomerizes reversibly with a disulfide bond that links gp70 to p15E. Consequently, only a proportion of gp70 and p15E is disulfide-bonded, and dissociation occurs when the environment becomes even slightly reducing. The gp55 glycoprotein appears to be an extreme example of protein malfolding associated with imprecise and irreversible disulfide bonding. We discuss evidence that folding inefficiencies are common for retroviral proteins that have newly evolving pathogenic functions.

Expression of the M-CSF receptor is controlled posttranscriptionally by the dominant actions of GM-CSF or multi-CSF.

We have isolated a murine myeloid precursor cell line (FDC-P1/MAC) that simultaneously expresses receptors for multi-CSF, GM-CSF, and M-CSF (c-fms protooncogene). FDC-P1/MAC cells express high levels of c-fms mRNA and protein when grown in M-CSF, whereas growth in multi-CSF or GM-CSF caused a dramatic reduction of c-fms glycoprotein and mRNA. Nuclear run-off assays demonstrated that c-fms transcription was not growth factor dependent and the regulation occurred posttranscriptionally. Factor switching experiments have shown that both multi-CSF and GM-CSF act dominantly and in a factor concentration dependent manner to suppress c-fms expression. In vitro agar assays of bone marrow cells grown in the presence of GM-CSF and M-CSF, individually and in combination, support the concept that GM-CSF can act dominantly to prevent monocyte/macrophage development. These results suggest that GM-CSF and multi-CSF can suppress development along the monocyte/macrophage lineage and offer a simple stochastic mechanism governing myeloid lineage restriction.

Splenic accumulation of interleukin-3-dependent hematopoietic cells in Friend erythroleukemia.

Interleukin-3-dependent hematopoietic stem cells commonly accumulate in spleens of mice infected with leukemia viruses. To study their origins, a molecularly tagged helper-free Friend spleen focus-forming virus was used to produce erythroleukemias. Uninfected interleukin-3-dependent basophil-mast cell progenitors coproliferated amidst the spleen focus-forming virus-infected leukemic cells. Splenic proliferation of normal stem cells is apparently a host response to leukemogenesis, and we propose that it may contribute to certain retroviral diseases.

Leukemogenic membrane glycoprotein encoded by Friend spleen focus-forming virus: transport to cell surfaces and shedding are controlled by disulfide-bonded dimerization and by cleavage of a hydrophobic membrane anchor.

The leukemogenic glycoprotein (gp55) encoded by Friend spleen focus-forming virus is predominantly retained in the rough endoplasmic reticulum (RER). However, a small proportion (ca. 5%) is processed to form a derivative that occurs on plasma membranes and causes mitosis of infected erythroblasts. We have now found that gp55 folds heterogeneously in the RER to form components with different disulfide bonds and that this difference may determine their processing fates. RER gp55 consists predominantly of monomers with intrachain disulfide bonds. In contrast, the processed molecules are disulfide-bonded dimers. These dimers are extensively modified in transit to cell surfaces by conversion of four N-linked high-mannose oligosaccharides to complex derivatives and by attachment of a sialylated O-linked oligosaccharide. The plasma membrane dimers are then slowly shed into the medium by a mechanism that involves proteolytic cleavage of approximately 25 membrane-anchoring hydrophobic amino acids from the carboxyl termini of the glycoproteins. Consequently, shed molecules have shorter polypeptide chains than cell-associated gp55. We conclude that gp55 folds into different disulfide-bonded components that do not substantially isomerize, and that only one specific dimer is competent for export from the RER. Mitogenic activity of gp55 could be caused by the cell surface dimers, by the shed derivative, or by the carboxyl-terminal hydrophobic anchors that remain in the membranes after the shedding reaction.

Topological correlation between the cell-recognition protein, R-cognin and α-bungarotoxin receptor in retinal plasma membrane.

The amount of the neural retina cell recognition protein, R-cognin, in the plasma membrane of chick embryo neural retina cells declined 43% between 10 and 17 days of embryonic development. Over this period there was a 27% increase in the plasma membrane content of the α-bungarotoxin receptor. Plasma membranes of both these ages were sonicated into vesicles and these vesicles partitioned on α-bungarotoxin agarose beads into those which contained detectable α-bungarotoxin receptor and those which did not. At 10 days, approximately 6% of the plasma membrane vesicles contained receptor. At 17 days, <2% did. At 10 days, 60% of the R-cognin was found in the α-bungarotoxin receptor-containing vesicles, at 17 days 86%. At 17 days, 6% of the retina membrane with a high concentration of both α-bungarotoxin receptor and R-cognin was of a density indicative of it being of synaptic origin. These results suggested that R-cognin and α-bungarotoxin receptor occurred close together in the plasma membrane of retina cells. However, the lack of competition between R-cognin gamma globulin and specific α-bungarotoxin binding indicated that the α-bungarotoxin receptor and R-cognin were not the same protein. Thus, R-cognin and the α-bungarotoxin receptor appear to be separate proteins which occur in close proximity on the retina plasma membrane.