Interleukin 9: A candidate gene for asthma

Asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of atopy and bronchial hyperresponsiveness. Recent studies localized a major gene for asthma to chromosome 5q31-q33 in humans. Thus, this segment of the genome represents a candidate region for genes that determine susceptibility to bronchial hyperresponsiveness and atopy in animal models. Homologs of candidate genes on human chromosome 5q31-q33 are found in four regions in the mouse genome, two on chromosome 18, and one each on chromosomes 11 and 13. We assessed bronchial responsiveness as a quantitative trait in mice and found it linked to chromosome 13. Interleukin 9 (IL-9) is located in the linked region and was analyzed as a gene candidate. The expression of IL-9 was markedly reduced in bronchial hyporesponsive mice, and the level of expression was determined by sequences within the qualitative trait locus (QTL). These data suggest a role for IL-9 in the complex pathogenesis of bronchial hyperresponsiveness as a risk factor for asthma.

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse)

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Thiazolidinediones and insulin resistance: Peroxisome proliferatoractivated receptor γ activation stimulates expression of the CAP gene

c-Cbl-associated protein (CAP) is a signaling protein that interacts with both c-Cbl and the insulin receptor that may be involved in the specific insulin-stimulated tyrosine phosphorylation of c-Cbl. The restricted expression of CAP in cells metabolically sensitive to insulin suggests an important potential role in insulin action. The expression of CAP mRNA and proteins are increased in 3T3-L1 adipocytes by the insulin sensitizing thiazolidinedione drugs, which are activators of the peroxisome proliferator-activated receptor γ (PPARγ). The stimulation of CAP expression by PPARγ activators results from increased transcription. This increased expression of CAP was accompanied by a potentiation of insulin-stimulated c-Cbl tyrosine phosphorylation. Administration of the thiazolidinedione troglitazone to Zucker (fa/fa) rats markedly increased the expression of the major CAP isoform in adipose tissue. This effect was sustained for up to 12 weeks of treatment and accompanied the ability of troglitazone to prevent the onset of diabetes and its complications. Thus, CAP is the first PPARγ-sensitive gene identified that participates in insulin signaling and may play a role in thiazolidinedione-induced insulin sensitization.

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (Eδ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by Eδ and the TCR α enhancer, and that it prevents Eδ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.

MRG1, the product of a melanocyte-specific gene related gene, is a cytokine-inducible transcription factor with transformation activity

Identification of cytokine-inducible genes is imperative for determining the mechanisms of cytokine action. A cytokine-inducible gene, mrg1 [melanocyte-specific gene (msg1) related gene], was identified through mRNA differential display of interleukin (IL) 9-stimulated and unstimulated mouse helper T cells. In addition to IL-9, mrg1 can be induced by other cytokines and biological stimuli, including IL-1α, -2, -4, -6, and -11, granulocyte/macrophage colony-stimulating factor, interferon γ, platelet-derived growth factor, insulin, serum, and lipopolysaccharide in diverse cell types. The induction of mrg1 by these stimuli appears to be transient, with induction kinetics similar to other primary response genes, implicating its role in diverse biological processes. Deletion or point mutations of either the Box1 motif (binds Janus kinase 1) or the signal transducer and activator of transcription 3 binding site-containing region within the intracellular domain of the IL-9 receptor ligand binding subunit abolished or greatly reduced mrg1 induction by IL-9, suggesting that the Janus kinase/signal transducer and activator of transcription signaling pathway is required for mrg1 induction, at least in response to IL-9. Transfection of mrg1 cDNA into TS1, an IL-9-dependent mouse T cell line, converted these cells to IL-9-independent growth through a nonautocrine mechanism. Overexpression of mrg1 in Rat1 cells resulted in loss of cell contact inhibition, anchorage-independent growth in soft agar, and tumor formation in nude mice, demonstrating that mrg1 is a transforming gene. MRG1 is a transcriptional activator and may represent a founding member of an additional family of transcription factors.

Regulation of dopaminergic pathways by retinoids: Activation of the D2 receptor promoter by members of the retinoic acid receptor–retinoid X receptor family

Dopamine is a neuromodulator involved in the control of key physiological functions. Dopamine-dependent signal transduction is activated through the interaction with membrane receptors of the seven-transmembrane domain G protein-coupled family. Among them, dopamine D2 receptor is highly expressed in the striatum and the pituitary gland as well as by mesencephalic dopaminergic neurons. Lack of D2 receptors in mice leads to a locomotor parkinsonian-like phenotype and to pituitary tumors. The D2 receptor promoter has characteristics of a housekeeping gene. However, the restricted expression of this gene to particular neurons and cells points to a strict regulation of its expression by cell-specific transcription factors. We demonstrate here that the D2 receptor promoter contains a functional retinoic acid response element. Furthermore, analysis of retinoic acid receptor-null mice supports our finding and shows that in these animals D2 receptor expression is reduced. This finding assigns to retinoids an important role in the control of gene expression in the central nervous system.

A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by ≈31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.

Genomic and cDNA sequence analysis of the cell matrix adhesion regulator gene

The cell matrix adhesion regulator (CMAR) gene has been suggested to be a signal transduction molecule influencing cell adhesion to collagen and, through this, possibly involved in tumor suppression. The originally reported CMAR cDNA was 464 bp long with a tyrosine phosphorylation site at the extreme 3′ end, which mutagenesis studies had shown to be central to the function of this gene. Since the discovery of a 4-bp insertion polymorphism within the originally reported coding region, further sequence information has been obtained. The cDNA has been extended 5′ by ≈2 kb revealing a 559-bp region showing strong homology to the proposed 5′ untranslated sequence of a murine protein kinase receptor family member, variant in kinase (vik). CMAR genomic sequencing has shown the presence of an intron, the intron/exon boundary lying within this region of homology. An RNA transcript for CMAR of ≈2.5 kb has also been identified. The data suggest complex mechanisms for control of expression of two closely associated genes, CMAR and the vik- associated sequence.

Identification of functional domains on human interleukin 10

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein–Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152–160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1β-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-α production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-γ-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A

Natriuretic peptides, produced in the heart, bind to the natriuretic peptide receptor A (NPRA) and cause vasodilation and natriuresis important in the regulation of blood pressure. We here report that mice lacking a functional Npr1 gene coding for NPRA have elevated blood pressures and hearts exhibiting marked hypertrophy with interstitial fibrosis resembling that seen in human hypertensive heart disease. Echocardiographic evaluation of the mice demonstrated a compensated state of systemic hypertension in which cardiac hypertrophy and dilatation are evident but with no reduction in ventricular performance. Nevertheless, sudden death, with morphologic evidence indicative in some animals of congestive heart failure and in others of aortic dissection, occurred in all 15 male mice lacking Npr1 before 6 months of age, and in one of 16 females in our study. Thus complete absence of NPRA causes hypertension in mice and leads to cardiac hypertrophy and, particularly in males, lethal vascular events similar to those seen in untreated human hypertensive patients.

Targeted deletion of all isoforms of the trkC gene suggests the use of alternate receptors by its ligand neurotrophin-3 in neuronal development and implicates trkC in normal cardiogenesis

We have generated null mutant mice that lack expression of all isoforms encoded by the trkC locus. These mice display a behavioral phenotype characterized by a loss of proprioceptive neurons. Neuronal counts of sensory ganglia in the trkC mutant mice reveal less severe losses than those in NT-3 null mutant mice, strongly suggesting that NT-3, in vivo, may signal through receptors other than trkC. Mice lacking either NT-3 or all trkC receptor isoforms die in the early postnatal period. Histological examination of trkC-deficient mice reveals severe cardiac defects such as atrial and ventricular septal defects, and valvular defects including pulmonic stenosis. Formation of these structures during development is dependent on cardiac neural crest function. The similarities in cardiac defects observed in the trkC and NT-3 null mutant mice indicate that the trkC receptor mediates most NT-3 effects on the cardiac neural crest.

Aberrant B cell receptor signaling from B29 (Igβ, CD79b) gene mutations of chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia (CLL) B cells characteristically exhibit low or undetectable surface B cell receptor (BCR) and diminished responses to BCR-mediated signaling. These features suggest that CLL cells may have sustained mutations affecting one or more of the BCR proteins required for receptor surface assembly and signal transduction. Loss of expression and mutations in the critical BCR protein B29 (Igβ, CD79b), are prevalent in CLL and could produce the hallmark features of these leukemic B cells. Because patient CLL cells are intractable to manipulation, we developed a model system to analyze B29 mutations. Jurkat T cells stably expressing μ, κ, and mb1 efficiently assembled a functional BCR when infected with recombinant vaccinia virus bearing wild-type B29. In contrast, a B29 CLL mutant protein truncated in the transmembrane domain did not associate with μ or mb1 at the cell surface. Another B29 CLL mutant lacking the C-terminal immunoreceptor tyrosine activation motif tyrosine and distal residues brought the receptor to the surface as well as wild-type B29 but showed significant impairment in anti-IgM-stimulated signaling events including mitogen-activated protein kinase activation. These findings demonstrate that B29 mutations previously identified in CLL patients can affect BCR-dependent signaling and may contribute to the unresponsive B cell phenotype in CLL. Finally, the features of the B29 mutations in CLL predict that they may be generated by somatic hypermutation.

Lipophosphoglycan from Leishmania suppresses agonist-induced interleukin 1β gene expression in human monocytes via a unique promoter sequence

Leishmania are parasites that survive within macrophages by mechanism(s) not entirely known. Depression of cellular immunity and diminished production of interleukin 1β (IL-1β) and tumor necrosis factor α are potential ways by which the parasite survives within macrophages. We examined the mechanism(s) by which lipophosphoglycan (LPG), a major glycolipid of Leishmania, perturbs cytokine gene expression. LPG treatment of THP-1 monocytes suppressed endotoxin induction of IL-1β steady-state mRNA by greater than 90%, while having no effect on the expression of a control gene. The addition of LPG 2 h before or 2 h after endotoxin challenge significantly suppressed steady-state IL-1β mRNA by 90% and 70%, respectively. LPG also inhibited tumor necrosis factor α and Staphylococcus induction of IL-1β gene expression. The inhibitory effect of LPG is agonist-specific because LPG did not suppress the induction of IL-1β mRNA by phorbol 12-myristate 13-acetate. A unique DNA sequence located within the −310 to −57 nucleotide region of the IL-1β promoter was found to mediate LPG’s inhibitory activity. The requirement for the −310 to −57 promoter gene sequence for LPG’s effect is demonstrated by the abrogation of LPG’s inhibitory activity by truncation or deletion of the −310 to −57 promoter gene sequence. Furthermore, the minimal IL-1β promoter (positions −310 to +15) mediated LPG’s inhibitory activity with dose and kinetic profiles that were similar to LPG’s suppression of steady-state IL-1β mRNA. These findings delineated a promoter gene sequence that responds to LPG to act as a “gene silencer,” a function, to our knowledge, not previously described. LPG’s inhibitory activity for several mediators of inflammation and the persistence of significant inhibitory activity 2 h after endotoxin challenge suggest that LPG has therapeutic potential and may be exploited for therapy of sepsis, acute respiratory distress syndrome, and autoimmune diseases.

Isolation and characterization of the chicken m2 acetylcholine receptor promoter region: Induction of gene transcription by leukemia inhibitory factor and ciliary neurotrophic factor

We have isolated the promoter region and determined the start sites of transcription for the gene encoding the chicken m2 (cm2) muscarinic acetylcholine receptor. Transfection experiments, using cm2-luciferase reporter gene constructs, demonstrated that a 789-bp genomic fragment was sufficient to drive high level expression in chicken heart primary cultures, while an additional 1.2-kb region was required for maximal expression in mouse septal/neuroblastoma (SN56) cells. Treatment of SN56 cells with the cytokines ciliary neurotrophic factor and leukemia inhibitory factor increases expression of endogenous muscarinic acetylcholine receptors and results in a 4- to 6-fold induction of cm2 promoter driven luciferase expression. We have mapped a region of the cm2 promoter that is necessary for induction by cytokines.

Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1β are differentially regulated in human blood mononuclear cells and mouse spleen cells

Interleukin (IL)-18, formerly called interferon γ (IFN-γ)-inducing factor, is biologically and structurally related to IL-1β. A comparison of gene expression, synthesis, and processing of IL-18 with that of IL-1β was made in human peripheral blood mononuclear cells (PBMCs) and in human whole blood. Similar to IL-1β, the precursor for IL-18 requires processing by caspase 1. In PBMCs, mature but not precursor IL-18 induces IFN-γ; in whole human blood stimulated with endotoxin, inhibition of caspase 1 reduces IFN-γ production by an IL-1β-independent mechanism. Unlike the precursor for IL-1β, precursor for IL-18 was expressed constitutively in PBMCs and in fresh whole blood from healthy human donors. Western blotting of endotoxin-stimulated PBMCs revealed processed IL-1β in the supernatants via an caspase 1-dependent pathway. However, in the same supernatants, only unprocessed precursor IL-18 was found. Unexpectedly, precursor IL-18 was found in freshly obtained PBMCs and constitutive IL-18 gene expression was present in whole blood of healthy donors, whereas constitutive IL-1β gene expression is absent. Similar to human PBMCs, mouse spleen cells also constitutively contained the preformed precursor for IL-18 and expressed steady-state IL-18 mRNA, but there was no IL-1β protein and no spontaneous gene expression for IL-1β in these same preparations. We conclude that although IL-18 and IL-1β are likely members of the same family, constitutive gene expression, synthesis, and processing are different for the two cytokines.