Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin.

Thrombopoietin (TPO) has recently been cloned and shown to regulate megakaryocyte and platelet production by activating the cytokine receptor c-mpl. To determine whether TPO is the only ligand for c-mpl and the major regulator of megakaryocytopoiesis, TPO deficient mice were generated by gene targeting. TPO-/- mice have a >80% decrease in their platelets and megakaryocytes but have normal levels of all the other hematopoietic cell types. A gene dosage effect observed in heterozygous mice suggests that the TPO gene is constitutively expressed and that the circulating TPO level is directly regulated by the platelet mass. Bone marrow from TPO-/- mice have decreased numbers of megakaryocyte-committed progenitors as well as lower ploidy in the megakaryocytes that are present. These results demonstrate that TPO alone is the major physiological regulator of both proliferation and differentiation of hematopoietic progenitor cells into mature megakaryocytes but that TPO is not critical to the final step of platelet production.

Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo.

The cytokine thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells. TPO exerts its effect through activation of the c-Mpl receptor and of multiple downstream signal transduction pathways. While the membrane-proximal half of the cytoplasmic domain appears to be required for the activation of signaling molecules that drive proliferation, the distal half and activation of the mitogen-activated protein kinase pathway have been implicated in mediating megakaryocyte maturation in vitro. To investigate the contribution of these two regions of c-Mpl and the signaling pathways they direct in mediating the function of TPO in vivo, we used a knock-in (KI) approach to delete the carboxy-terminal 60 amino acids of the c-Mpl receptor intracellular domain. Mice lacking the C-terminal 60 amino acids of c-Mpl (Delta60 mice) have normal platelet and megakaryocyte counts compared to wild-type mice. Furthermore, platelets in the KI mice are functionally normal, indicating that activation of signaling pathways connected to the C-terminal half of the receptor is not required for megakaryocyte differentiation or platelet production. However, Delta60 mice have an impaired response to exogenous TPO stimulation and display slower recovery from myelosuppressive treatment, suggesting that combinatorial signaling by both ends of the receptor intracellular domain is necessary for an appropriate acute response to TPO.

Isolation of cDNA clones encoding the mouse protein V-1.

The amino acid (aa) sequence of rat V-1, a developmentally regulated brain protein, was used to isolate clones encoding mouse V-1, a protein of 118 aa, from an embryoid body cDNA library. The aa sequences of rat and mouse V-1 are identical. V-1 shares several properties (including a 23 of 24 aa match of a tryptic peptide) with myotrophin, a protein that induces cardiac myocyte hypertrophy. Attempts to show that V-1 produced in Escherichia coli could induce the cardiac myocyte hypertrophy ascribed to myotrophin were unsuccessful.

Cloning of FRK, a novel human intracellular SRC-like tyrosine kinase-encoding gene.

We report the cloning of a novel tyrosine kinase (TyK)-encoding gene (TYK) from the human hepatoma cell line Hep3B. Using the polymerase chain reaction (PCR) and oligodeoxyribonucleotide primers based on conserved TYK motifs, a 180-bp fragment was cloned and used to obtain full-length cDNA clones of 2.9 kb, with an open reading frame of 505 amino acids (aa). Restricted expression was detected by Northern blotting or reverse-transcribed PCR in a broad range of cell lines. The predicted aa sequence contains characteristic TyK motifs without a transmembrane region, suggesting an intracellular localization. There was 49% aa sequence identity with human FYN product and 47% with human SRC product; however, several structural differences distinguish this clone from other SRC subfamily members. This clone, FYN-related kinase or FRK, is a novel member of the intracellular TYK gene family.

Cloning and characterization of a human leptin receptor using a biologically active leptin immunoadhesin.

Leptin, the product of the ob gene, is a hormone secreted by fat cells which is primarily involved in the regulation of body weight. We have generated a leptin immunoadhesin (leptin-IgG) which was more potent than leptin alone at reducing body weight and food intake when injected into ob/ob mice. This molecule was used to identify high affinity binding sites on human embryonic 293 kidney cells and subsequently to isolate a cDNA encoding the leptin receptor from this cell line by expression cloning. This receptor corresponds to the short form of the recently isolated leptin receptor. Analysis of the expression pattern of the two forms of receptor by Northern blot, in situ hybridization and quantitative PCR showed that the receptor is expressed in most tissues but that the long form is prevalent in the hypothalamus.

Hemagglutinin mutations related to antigenic variation in H1 swine influenza viruses.

The hemagglutinin (HA) of a recent swine influenza virus, A/Sw/IN/1726/88 (H1N1), was shown previously to have four antigenic sites, as determined from analysis of monoclonal antibody (MAb)-selected escape mutants. To define the HA mutations related to these antigenic sites, we cloned and sequenced the HA genes amplified by polymerase chain reaction of parent virus and MAb-selected escape mutants. The genetic data indicated the presence of four amino acid changes. After alignment with the three-dimensional structure of H3 HA, three changes were located on the distal tip of the HA, and the fourth was located within the loop on the HA. We then compared our antigenic sites, as defined by the changed amino acids, with the well-defined sites on the H1 HA of A/PR/8/34. The four amino acid residues corresponded with three antigenic sites on the HA of A/PR/8/34. This finding, in conjunction with our previous antigenic data, indicated that two of the four antigenic sites were overlapping. In addition, our previous studies indicated that one MAb-selected mutant and a recent, naturally occurring swine isolate reacted similarly with the MAb panel. However, their amino acid changes were different and also distant on the primary sequence but close topographically. This finding indicates that changes outside the antigenic site may also affect the site. A comparison of the HA amino acid sequences of early and recent swine isolates showed striking conservation of genetic sequences as well as of the antigenic sites. Thus, swine influenza viruses evolve more slowly than human viruses, possibly because they are not subjected to the same degree of immune selection.

The bglX gene located at 47.8 min on the Escherichia coli chromosome encodes a periplasmic beta-glucosidase.

A new Escherichia coli gene, bgIX, encoding a beta-D-glucosidase (EC 3.2.1.21) has been characterized. The bgIX gene is located adjacent to the dld gene at 47.8 min or 2225 kb on the E. coli chromosome. The sequence of a 2.6 kb DNA fragment from this region revealed a large open reading frame encoding a protein of 765 amino acids. The BgIX protein was purified from the periplasm, and amino-terminal sequencing suggests that the mature protein is derived from cleavage of a 20 residue signal peptide. A search of the sequence databases revealed that BgIX is a member of a large family of beta-glucosidases from a variety of bacteria and fungi, and the plant Arabidopsis thaliana. It differs from the other four E. coli phospho-beta-glucosidases in sequence and in its periplasmic rather than cytoplasmic location. The BgIX enzyme has a Km of 18 +/- 1 mM and a Vmax of 3 +/- 0.7 mumol min-1 for the colorimetric substrate o-nitrophenyl beta-D-glucopyranoside.

Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4.

The tyrosine kinases Flt4, Flt1, and Flk1 (or KDR) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain. Flt1 and Flk1 have been shown to play key roles in vascular development; these two receptors bind and are activated by vascular endothelial growth factor (VEGF). No ligand has been identified for Flt4, whose expression becomes restricted during development to the lymphatic endothelium. We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32% amino acid identity to VEGF. This protein, designated VEGF-related protein (VRP), specifically binds to the extracellular domain of Flt4, stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells, and promotes the mitogenesis of human lung endothelial cells. VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1. The protein contains a C-terminal, cysteine-rich region of about 180 amino acids that is not found in VEGF. A 2.4-kb VRP mRNA is found in several human tissues including adult heart, placenta, ovary, and small intestine and in fetal lung and kidney.

Mutations of the growth hormone receptor in children with idiopathic short stature. The Growth Hormone Insensitivity Study Group.

BACKGROUND: Short stature in children who are not deficient in growth hormone (GH) is probably caused by a variety of defects. Some children with idiopathic short stature have low serum concentrations of GH-binding protein, which is derived from the GH receptor. The possibility that low serum concentrations of GH-binding protein might indicate partial insensitivity to GH led us to investigate possible defects in the gene for the GH receptor in children with idiopathic short stature and low serum concentrations of GH-binding protein. METHODS: We studied 14 children with idiopathic short stature who were selected on the basis of normal GH secretion and low serum concentrations of GH-binding protein. Analysis of single-strand conformation polymorphisms and DNA sequencing were both used to identify mutations in the GH-receptor gene. RESULTS: Mutations in the region of the GH-receptor gene that codes for the extracellular domain of the receptor were found in 4 of the 14 children, but in none of 24 normal subjects. One of the four children with mutations was a compound heterozygote, with one mutation that reduced the affinity of the receptor for GH and a second mutation that may affect a function other than ligand binding. The remaining three children had single mutations in one allele of the gene. One mutation introduced a premature termination codon, and two caused substitutions of single amino acids in a structurally conserved domain of the receptor. CONCLUSIONS: Some children with idiopathic short stature may have partial insensitivity to GH due to mutations in the GH-receptor gene.

Low levels of erythroid and myeloid progenitors in thrombopoietin-and c-mpl-deficient mice.

Thrombopoietin (TPO), the ligand for the c-mpl receptor, has been shown to be the major regulator of platelet production. Mice deficient in either c-mpl or TPO generated by homologous recombination show a dramatic decrease in platelet counts, but other blood cell counts are normal. Because TPO treatment of myelosuppressed mice not only enhances the recovery of platelets but also accelerates erythroid recovery, we investigated the levels of myeloid and erythroid progenitor cells in TPO-or c-mpl-deficient mice. Our results show that the number of megakaryocyte, granulocyte-macrophage, erythroid, and multilineage progenitors are significantly reduced in the bone marrow, spleen, and peripheral blood of either TPO-or c-mpl-deficient mice. Administration of recombinant murine TPO to TPO-deficient mice and control littermate mice significantly increased the absolute number of myeloid, erythroid, and mixed progenitors in bone marrow and spleen. This increase was especially apparent in TPO-deficient mice where numbers were increased to a level greater than in diluent-treated control mice and approached or equaled that in the TPO-treated control mice. Moreover, TPO-administration greatly increased the number of circulating progenitors as well as platelets in both TPO-deficient and control mice. Furthermore, the megakaryocytopoietic activity of other cytokines in the absence of a functional TPO or c-mpl gene was shown both in vitro and in vivo.

Expression cloning of cardiotrophin 1, a cytokine that induces cardiac myocyte hypertrophy.

Heart failure continues to be a leading cause of mortality worldwide. A hallmark of this disease is dilated cardiac hypertrophy, which is accompanied by a reactivation of genes expressed in fetal heart development. Reasoning that fetal or embryonic growth factors may mediate the onset of cardiac hypertrophy, we have coupled expression cloning with an embryonic stem cell-based model of cardiogenesis to isolate a 21.5-kDa protein, cardiotrophin 1, that potently induces cardiac myocyte hypertrophy in vitro. Amino acid similarity data indicate that cardiotrophin 1 is a member of the leukemia inhibitory factor/ciliary neurotrophic factor/oncostatin M/interleukin 6/interleukin 11 family of cytokines. Several members of this family that are known to signal through the transmembrane protein gp130 stimulate cardiac myocyte hypertrophy, like cardiotrophin 1, suggesting that the gp130 signaling pathway may play a role in cardiac hypertrophy. A 1.4-kb cardiotrophin 1 mRNA is expressed in the heart and several other mouse tissues.

Activating Smoothened mutations in sporadic basal-cell carcinoma.

Basal-cell carcinomas (BCCs) are the commonest human cancer. Insight into their genesis came from identification of mutations in the PATCHED gene (PTCH) in patients with the basal-cell nevus syndrome, a hereditary disease characterized by multiple BCCs and by developmental abnormalities. The binding of Sonic hedgehog (SHH) to its receptor, PTCH, is thought to prevent normal inhibition by PTCH of Smoothened (SMO), a seven-span transmembrane protein. According to this model, the inhibition of SMO signalling is relieved following mutational inactivation of PTCH in basal-cell nevus syndrome. We report here the identification of activating somatic missense mutations in the SMO gene itself in sporadic BCCs from three patients. Mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblast cells in culture. Furthermore, skin abnormalities similar to BCCs developed in transgenic murine skin overexpressing mutant SMO. These findings support the role of SMO as a signalling component of the SHH-receptor complex and provide direct evidence that mutated SMO can function as an oncogene in BCCs.