Novel point mutation in the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor in a case of severe congenital neutropenia hyporesponsive to G-CSF treatment.

Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand-receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness.

Convergence of congenic mapping and allele-specific alterations in tumors for the resolution of the Skts1 skin tumor susceptibility locus.

Although several familial cancer genes with high-penetrance mutations have been identified, the major genetic component of susceptibility to sporadic cancers is attributable to low-penetrance alleles. These 'weak' tumor susceptibility genes do not segregate as single Mendelian traits and are therefore difficult to find in studies of human populations. Previously, we have proposed that a combination of germline mapping and analysis of allele-specific imbalance in tumors may be used to refine the locations of susceptibility genes using mouse models of cancer. Here, we have used linkage analysis and congenic mouse strains to map the major skin tumor susceptibility locus Skts1 within a genetic interval of 0.9 cM on proximal chromosome 7. This interval lies in an apparent recombination cold spot, and corresponds to a physical distance of about 15 Mb. We therefore, used patterns of allele-specific imbalances in tumors from backcross and congenic mice to refine the location of Skts1. We demonstrate that this single tumor modifier locus has a dramatic effect on the allelic preference for imbalance on chromosome 7, with at least 90% of tumors from the congenics showing preferential gain of markers on the chromosome carrying the susceptibility variant. Importantly, these alterations enabled us to refine the location of Skts1 at higher resolution than that attained using the congenic mice. We conclude that low-penetrance susceptibility genes can have strong effects on patterns of allele-specific somatic genetic changes in tumors, and that analysis of the directionality of these somatic events provides an important and rapid route to identification of germline genetic variants that confer increased cancer risk.

Epistatic interactions between skin tumor modifier loci in interspecific (spretus/musculus) backcross mice.

The development of cancer is influenced both by exposure to environmental carcinogens and by the host genetic background. Epistatic interactions between genes are important in determining phenotype in plant and animal systems and are likely to be major contributors to cancer susceptibility in humans. Several tumor modifier loci have been identified from studies of mouse models of human cancer, and genetic interactions between modifier loci have been detected by genome scanning using recombinant congenic strains of mice (R. Fijneman et al., Nat. Genet., 14: 465-467, 1996; T. van Wezel et al., Nat. Genet., 14: 468-470, 1996; W. N. Frankel et al., Nat. Genet., 14, 371-373, 1996). We demonstrate here that strong genetic interactions between skin tumor modifier loci can be detected by hierarchical whole genome scanning of a complete interspecific backcross [outbred Mus spretus X Mus musculus (NIH/Ola)]. A locus on chromosome 7 (Skts1) showed a highly significant interaction with Skts5 on chromosome 12 (P < 10(-16)), whereas additional significant interactions were detected between loci on chromosomes 4 and 5, and 16 and 15. Some of these quantitative trait loci and their interactions, in particular the Skts1-Skts5 interaction, were confirmed in two completely independent backcrosses using inbred spretus strains (SEG/Pas and SPRET/Ei) and NIH/Ola. These results, therefore, illustrate the general use of interspecific crosses between Mus musculus and Mus spretus for the detection of strong genetic interactions between tumor modifier genes.

STAT3-mediated differentiation and survival and of myeloid cells in response to granulocyte colony-stimulating factor: role for the cyclin-dependent kinase inhibitor p27(Kip1).

The signal transducer and activator of transcription (STAT) proteins have been implicated in cytokine-regulated proliferation, differentiation and cell survival. Granulocyte colony-stimulating factor (G-CSF), a regulator of granulocytic differentiation, induces a robust and sustained activation of STAT3. Here, we show that introduction of dominant negative (DN) forms of STAT3 interferes with G-CSF-induced differentiation and survival in murine 32D cells. G-CSF induces expression of the cyclin-dependent kinase (cdk) inhibitor p27(KiP1) (but not p21(CiP1)), which is completely blocked by DN-STAT3. The ability of tyrosine-to-phenylalanine substitution mutants of the G-CSF receptor to activate STAT3 strongly correlated with their capacity to induce p27 expression and their ability to mediate differentiation and survival, suggesting a causal relationship between STAT3 activation, p27 expression and the observed cellular responses. We identified a putative STAT binding site in the promoter region of p27 that showed both STAT3 binding in electrophoretic mobility shift assays and functional activity in luciferase reporter assays. Finally, we studied G-CSF-induced responses in primary bone marrow and spleen cells of p27-deficient mice. Compared with wild-type, myeloid progenitors from p27-deficient mice showed significantly increased proliferation and reduced differentiation in response to G-CSF. These findings indicate that STAT3 controls myeloid differentiation, at least partly, via upregulation of p27(Kip1).

Signaling mechanisms of cytokine receptors and their perturbances in disease.

Cytokines regulate the proliferation and differentiation of cells through their interaction with specific receptors on the surface of target cells which are coupled to intracellular signal transduction pathways. The cytokine receptor class I superfamily, characterized by structural homology in the extracellular domain, includes receptors for many interleukins and hematopoietic growth factors, but also those of growth hormone, leptin, ciliary neurotrophic factor (CNTF), oncostatin M (OSM), leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1). The receptors for interferons are structurally distinct and have therefore been categorized separately (class II cytokine receptors). The discovery of the JAK/STAT pathway in the early 1990s has been an important step forward in deciphering cytokine mediated signaling. This pathway connects activation of the receptor complexes directly to transcription of genes. Studies of humans and mice, deficient for one of the JAKs or STATs, have revealed crucial roles of these molecules in embryonic development, blood cell formation and immune responses. In addition, recent studies have revealed some of the mechanisms that control the activation of the JAKs and STATs, which contribute to signal intensity and specificity. In this review we will summarize these recent insights and discuss their implications for a variety of pathological conditions.

STAT3beta does not interfere with granulocyte colony-stimulating factor-induced neutrophilic differentiation.

INTRODUCTION: Activation of the signal transducer and activator of transcription protein STAT3 is a crucial step in granulocyte colony-stimulating factor (G-CSF)-mediated cell cycle exit and subsequent neutrophilic differentiation of myeloid precursor cells. We have recently demonstrated that this is mediated, at least in part, by upregulation of the cyclin-dependent kinase inhibitor p27(Kip1). The splice variant STAT3beta, that lacks a C-terminal serine residue implicated in the transcriptional activity of STAT3, has been shown to inhibit STAT3-mediated transcription in certain situations. STAT3beta is known to be expressed in hematopoietic cells, but its role in controlling the balance between proliferation and differentiation has not been established. MATERIALS AND METHODS: We ectopically introduced STAT3beta in differentiation-competent 32D cell transfectants expressing human wild type (WT) G-CSF receptors and studied the consequences for G-CSF-mediated responses. RESULTS: Overexpression of STAT3beta did not alter the kinetics of G-CSF-mediated neutrophilic differentiation or p27 induction in 32D/G-CSF-R WT cells. In addition, we found that p27(Kip1) promoter activity was not inhibited by STAT3beta, while inhibition of p27 transactivation by a dominant-negative STAT3 mutant could in fact be alleviated by coexpression of the beta form. CONCLUSION: These findings argue against a role of STAT3beta as a negative regulator of G-CSF-induced expression of p27 and myeloid differentiation.

Effect of an external resistance to airflow on the inspiratory flow curve.

Inhalation is a convenient way to deliver drugs to the respiratory tract in the treatment of respiratory diseases. For dry powder inhalers (DPI's), the principle of operation is to use the patient-generated inspiratory flow as energy source for emptying of the dose system and the delivery of fine drug particles into the respiratory tract. Resistance to airflow of the inhaler device is a major determinant for the inspiratory flow profile through the dry powder inhaler that can be generated by the patient. Therefore, resistance to airflow is one of the design parameters for DPI's, that could be used to control the inspiratory flow profile, and is one of the parameters to optimise particle deposition in the airways. In this study the effect of resistance to airflow on different parameters of the inspiratory flow curves as generated by healthy subjects, asthmatics and COPD patients was determined. As a result of increased resistance to airflow, the peak inspiratory flow (PIF), the flow increase rate (FIR) and the inhaled volume to reach PIF is decreased. On the other hand, the total inhalation time as well as the 80% dwell time is increased. In general, tuning of the resistance to airflow in the design of a dry powder inhaler may improve the drug deposition in the respiratory tract.

Advances in understanding the biology and function of the G-CSF receptor.

G-CSF is the major growth factor involved in the production of neutrophilic granulocytes. G-CSF exerts its function via the activation of a membrane receptor that belongs to the super-family of hematopoietin receptors, also referred to as class I cytokine receptors. In this review we summarize the current knowledge of the function of the G-CSF receptor in normal granulopoiesis, as well as in some patients with severe congenital neutropenia and acute myeloblastic leukemia, diseases characterized by disturbed myeloid maturation.

Identification of functional domains of interleukin-3 by construction of primate interspecies chimera.

Interleukin-3 (IL-3) is involved in regulation of proliferation and differentiation of multipotent hemopoietic cells and stimulates the production of most blood cell types. The observed functional specificity across species concurs with an extreme rate of IL-3 amino acid substitutions during mammalian evolution. Tamarin IL-3 exhibited 70.5% sequence identity with human IL-3 and was severely impaired in supporting proliferation of human IL-3-dependent cells. In contrast, chimpanzee IL-3 displayed high amino acid sequence homology (98.5%) and could substitute for human IL-3. A panel of interspecies chimera between the chimpanzee and tamarin IL-3 genes has been constructed and expressed in Escherichia coli and eukaryotic cells to investigate the role of substitutions in different protein domains on the functional species specificity. Our analyses show that substitutions at residues encoded by the first two exons appear crucial in the functional species specificity, whereas C-terminal alterations show only moderate effects.

Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G-CSF-mediated activation of signaling complexes of the p21ras route.

Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G-CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G-CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R.

Stimulation of Stat5 by granulocyte colony-stimulating factor (G-CSF) is modulated by two distinct cytoplasmic regions of the G-CSF receptor.

In a manner similar to many other cytokines, treatment of cells with granulocyte CSF (G-CSF) has been shown to induce the tyrosine phosphorylation of the STAT proteins. Activation of Stat1 and Stat5 by G-CSF requires the membrane-proximal cytoplasmic domain of the receptor, including box1 and box2, while G-CSF-stimulated tyrosine phosphorylation of Stat3 also requires a region distal to box 2. In this study, we show that although the membrane-proximal 55 amino acids of the G-CSF receptor are sufficient for activation of Stat5, the maximal rate of Stat5 activation requires an additional 30 amino acids of the cytoplasmic domain. In contrast, the distal carboxyl-terminal region of the receptor appears to down-regulate Stat5 activation in that deletion of this carboxyl terminus results in increased amplitude and prolonged duration of Stat5 activation by G-CSF. Significantly, expression of a truncated dominant-negative Stat5 protein in hemopoietic cells not only inhibits G-CSF-dependent cell proliferation, but also suppresses cell survival upon G-CSF withdrawal. We further show that a potential protein tyrosine phosphatase may play a critical role in the down-regulation of G-CSF-stimulated Stat5 activation. These results demonstrate that two distinct cytoplasmic regions of the G-CSF receptor are involved in the regulation of the intensity and duration of Stat5 activation, and that Stat5 may be an important player in G-CSF-mediated cell proliferation and survival.

Multiple signals mediate proliferation, differentiation, and survival from the granulocyte colony-stimulating factor receptor in myeloid 32D cells.

Granulocyte colony-stimulating factor (G-CSF) regulates neutrophil production through activation of its cognate receptor, the G-CSF-R. Previous studies with deletion mutants have shown that the membrane-proximal cytoplasmic domain of the receptor is sufficient for mitogenic signaling, whereas the membrane-distal domain is required for differentiation signaling. However, the function of the four cytoplasmic tyrosines of the G-CSF-R in the control of proliferation, differentiation, and survival has remained unclear. Here we investigated the role of these tyrosines by expressing a tyrosine "null" mutant and single tyrosine "add back" mutants in maturation-competent myeloid 32D cells. Clones expressing the null mutant showed only minimal proliferation and differentiation, with survival also reduced at low G-CSF concentrations. Analysis of clones expressing the add-back mutants revealed that multiple tyrosines contribute to proliferation, differentiation, and survival signals from the G-CSF-R. Analysis of signaling pathways downstream of these tyrosines suggested a positive role for STAT3 activation in both differentiation and survival signaling, whereas SHP-2, Grb2 and Shc appear important for proliferation signaling. In addition, we show that a tyrosine-independent "differentiation domain" in the membrane-distal region of the G-CSF-R appears necessary but not sufficient for mediating neutrophilic differentiation in these cells.

Specific involvement of tyrosine 764 of human granulocyte colony-stimulating factor receptor in signal transduction mediated by p145/Shc/GRB2 or p90/GRB2 complexes.

Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) occurs via multiple pathways, one of which involves activation of p21Ras and mitogen-activated protein kinase. The SH2 domain-containing proteins Shc and GRB2 have been implicated in this latter signaling route. We studied the role of these proteins in signal transduction from wild type (WT) G-CSF-R, C-terminal deletion mutants, and tyrosine-to-phenylalanine substitution mutants in transfectants of the mouse pro-B cell line, BAF3. G-CSF stimulation of BAF3 cells expressing WT G-CSF-R induced tyrosine phosphorylation of Shc. Anti-Shc antibodies co-immunoprecipitated tyrosine-phosphorylated 145-kD proteins (p145), whereas GRB2 immunoprecipitates contained phosphorylated Shc, Syp, and proteins of 145 and 90 kD (p90). Neither of these complexes were detected after activation of a C-terminal deletion mutant of G-CSF-R that lacked all four conserved cytoplasmic tyrosine residues. G-CSF induced formation of Syp/GRB2 complexes in all the tyrosine-substitution mutants, suggesting that this association did not depend on the presence of single specific tyrosine residues in G-CSF-R. In contrast, tyrosine 764 of G-CSF-R appeared to be exclusively required for tyrosine phosphorylation of Shc and its association with p145 and GRB2. In addition, tyrosine 764 also specifically mediated binding of GRB2 to p90 without the involvement of Shc. These findings indicate that tyrosine 764 of G-CSF-R has a prominent role in G-CSF signal transduction.

Proliferation signaling and activation of Shc, p21Ras, and Myc via tyrosine 764 of human granulocyte colony-stimulating factor receptor.

The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF-mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF-containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF-mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF-driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.

The membrane-distal cytoplasmic region of human granulocyte colony-stimulating factor receptor is required for STAT3 but not STAT1 homodimer formation.

Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) involves the activation of the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway. G-CSF induces tyrosine phosphorylation of Jak1, Jak2, STAT1, and STAT3. The membrane-proximal region of G-CSF-R is sufficient for activation of Jaks. It is still unclear how STAT proteins are activated by G-CSF-R. We investigated the possible involvement of the C-terminal region of G-CSF-R in the recruitment of STAT proteins using BAF3 cell transfectants expressing wild type (WT) G-CSF-R, C-terminal deletion mutants and tyrosine-to-phenylalanine substitution mutants. Electrophoretic mobility shift assays with STAT-binding oligonucleotides (m67) showed that activation of WT G-CSF-R induces three distinct STAT complexes, namely STAT3 homodimers, STAT1-STAT3 heterodimers, and STAT1 homodimers. However, STAT1 homodimers and STAT1-STAT3 heterodimers were predominantly formed after activation of a C-terminal deletion mutant d685, which lacks all four conserved cytoplasmic tyrosine residues, located at positions 704, 729, 744, and 764. Antiphosphotyrosine immunoblots of STAT3 immunoprecipitates showed that activation of WT G-CSF-R induced phosphorylation of STAT3. In contrast, no phosphorylation of STAT3 was observed after activation of deletion mutant d685. These findings establish that the C-terminal region of G-CSF-R plays a major role in the activation of STAT3. By using tyrosine-to-phenylalanine substitution mutants of G-CSF-R, we further show that tyrosine 704, present in a YXXQ consensus sequence shown to be essential for STAT3 binding to gp130, is not exclusively involved in the activation of STAT3 by G-CSF-R.