Expression of recombinant human plasminogen in mammalian cells is augmented by suppression of plasmin activity.

We present evidence that over-expression of human plasminogen, the precursor to the serine protease plasmin, can be cytotoxic to mammalian cells. When an expression vector containing plasminogen cDNA is transfected into baby hamster kidney cells, the number of drug-resistant colonies as well as the levels of plasminogen secreted by those colonies is lower than observed in similar transfections of other protease precursor genes. The recombinant plasminogen accumulates intracellularly as degraded NH2-terminal fragments. In contrast, a mutant of plasminogen that produces inactive plasmin (active site Ser740 changed to Ala) is synthesized by these cells as a full-length plasminogen molecule, and the colony numbers and expression levels are normal. Thus, the generation of plasmin activity is responsible for the cytotoxic phenomena and the degradation associated with plasminogen expression. In addition, experiments using a plasminogen mutant that cannot be activated to plasmin (activation cleavage site Arg560 to Gly) or using coexpression of antisense urokinase RNA indicate that an endogenous plasminogen activator is responsible for converting newly synthesized plasminogen to plasmin. Finally, coexpression of plasminogen with alpha 2-plasmin inhibitor, a serpin which is the physiologic inhibitor of plasmin, prevents the toxic effects of intracellular plasmin activity and allows the synthesis and secretion of native human plasminogen.

Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.

The major regulator of circulating platelet levels is believed to be a cytokine termed thrombopoietin. It is thought to be a lineage-specific cytokine affecting the proliferation and maturation of committed cells resulting in the production of megakaryocytes and platelets. Despite considerable efforts by a number of laboratories, the unequivocal identification of thrombopoietin has proven elusive. Here we report the functional cloning of a murine complementary DNA encoding a ligand for the receptor encoded by the c-mpl proto-oncogene (c-Mpl). The encoded polypeptide has a predicted molecular mass of 35,000 (M(r) 35K). The protein has a novel two-domain structure with an amino-terminal domain homologous with erythropoietin and a carboxy-terminal domain rich in serine, threonine and proline residues and containing seven potential N-linked glycosylation sites. Intraperitoneal injections of mice with recombinant protein increase circulating platelet levels by greater than fourfold after 7 days. These results along with those presented in the accompanying report strongly suggest that the ligand for c-Mpl is thrombopoietin.

Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization.

Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO). Southern blot analysis of human genomic DNA reveals a hybridization pattern consistent with a single gene locus. The locus was mapped by in situ hybridization of metaphase chromosome preparations to chromosome 3q26-27, a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. A human TPO cDNA was isolated by PCR from kidney mRNA. The cDNA encodes a protein with 80% identity to previously described murine TPO and is capable of initiating a proliferative signal to murine interleukin 3-dependent BaF3 cells expressing the murine or human TPO receptor.

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.