Biomedical subjects

K Nocka

Publications and source records attributed to K Nocka.

9 recordsLinked to original sources

PubMed1996

Targeted disruption of guanosine diphosphate-dissociation inhibitor for Rho-related proteins, GDID4: normal hematopoietic differentiation but subtle defect in superoxide production by macrophages derived from in vitro embryonal stem cell differentiation.

The Rho subfamily of small guanosine triphosphate (GTP)-binding proteins, through their role in cytoskeletal organization, is involved in diverse cellular functions, including cell motility and morphologic changes during differentiation. Rac also has a special role in the production of superoxide, a key component in phagocytic antimicrobial function. Guanosine diphosphate (GDP)-dissociation inhibitors (GDIs) belong to one of three classes of proteins that regulate the critical cycling of GTP-binding proteins between the inactive and active states. Two homologous GDIs for the Rho subfamily have been identified. GDID4 is preferentially expressed in hematopoietic cells, while RhoGDI is ubiquitously expressed. Whether different physiologic functions are subserved by the two GDIs is unknown. We have derived embryonal stem (ES) cells with targeted disruption of both alleles of the GDID4 gene and examined hematopoiesis and phagocytic functions of macrophages derived from in vitro ES-cell differentiation. GDID4-/- ES cells develop like wild-type cells into colonies that contain heterogeneous populations of progenitor cells and differentiated erythromyeloid cells. GDID4-/- cells express no GDID4 protein, but have normal levels of RhoGDI. GDID4-/- macrophages phagocytose yeasts and antibody-opsonized erythrocytes as effectively as wild-type macrophages. However, a slight but consistent reduction in their capacity to generate superoxide was observed, which suggests new insight into the cellular role of GDID4. The minimal phenotypic effect of a loss of function of GDID4 also indicates a significant redundancy of function between GDID4 and RhoGDI. Their functional repertoire may be better revealed by a disruption of both genes. The use of hematopoietic cells derived in vitro from genotypically altered ES cells avoids the difficulties inherent in generating knockout animals and is a useful complementary approach for evaluating the gene function.

Animals↗

PubMed1996

Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen.

The phenotypes of mice that harbor a defect in the genes encoding either stem cell factor (SCF) or its receptor, c-kit, indicate that this ligand/receptor pair is necessary for maintenance of normal hematopoiesis in the adult. Our objective was to determine whether SCF, like erythropoietin, is necessary for acute erythroid expansion during recovery from hemolytic anemia. Monoclonal antibody ACK2, which recognizes the murine c-kit receptor, was used to selectively block the hematopoietic growth-promoting effects of SCF. Mice were treated with phenylhydrazine on day 0 and day 1 to induce hemolytic anemia and also received no antibody, control IgG, or ACK2 on day 0. The mice were killed on day 3 and the hematocrit (Hct), reticulocyte count, and numbers of erythroid and myeloid hematopoietic progenitor cells (colony-forming unit-erythroid [CFU-E], burst-forming unit [BFU]-E, and CFU-granulocyte-macrophage [GM]) were quantitated in the femoral marrow and spleen using hematopoietic colony-forming assays. Induction of hemolytic anemia with phenylhydrazine resulted in a drop in the Hct from approximately 50% to 30%, and an approximate 8- to 10-fold increase in the reticulocyte count. The numbers of CFU-E increased modestly in the femur, and approximately 25- to 50-fold in the spleen, in comparison with normal mice. BFU-E and CFU-GM values did not increase in the femur but expanded 6- to 10-fold in the spleen, in comparison with normal mice. This confirms that much of the erythroid expansion in response to hemolytic anemia occurs in the murine spleen. Neutralizing quantities of the ACK2 antibody reduced femoral CFU-E, BFU-E, and CFU-GM content to less than half that found in phenylhydrazine-treated control mice and nearly totally ablated splenic hematopoiesis. These results suggest that c-kit receptor function may be required for optimal response to acute erythropoietic demand and that erythropoiesis in the splenic microenvironment is more dependent on SCF/c-kit receptor interaction than is erythropoiesis in the marrow microenvironment. Because expansion of late erythropoiesis in the spleen was preferentially blocked, we tested the hypothesis that homing of more primitive hematopoietic cells to the spleen was dependent on c-kit receptor function. Lethally irradiated mice were injected with marrow cells obtained from mice that had received phenylhydrazine plus control IgG or with marrow cells obtained from mice that had received phenylhydrazine plus ACK2. In parallel experiments, normal murine marrow cells were treated in vitro with control IgG or with ACK2 and were injected into lethally irradiated mice. The fraction of BFU-E and CFU-GM retrieved from the marrow and spleen of the recipient mice 4 hours later was reduced by approximately 75% when progenitor cells had been exposed to ACK2, in comparison with control IgG. These data suggest that interaction of SCF with the c-kit receptor affects the homing behavior of hematopoietic progenitor cells in the adult animal.

Anemia, Hemolytic↗

PubMed1990

The hematopoietic growth factor KL is encoded by the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus.

Mutations at the steel locus (Sl) of the mouse affect the same cellular targets as mutations at the white spotting locus (W), which is allelic with the c-kit proto-oncogene. We show that KL, a hematopoietic growth factor obtained from conditioned medium of BALB/c 3T3 fibroblasts that stimulates the proliferation of mast cells and early erythroid progenitors, specifically binds to the c-kit receptor. The predicted amino acid sequence of isolated KL-specific cDNA clones suggests that KL is synthesized as an integral transmembrane protein. Linkage analysis maps the KL gene to the Sl locus on mouse chromosome 10, and KL sequences are deleted in the genome of the Sl mouse. These results indicate that the Sl locus encodes the ligand of the c-kit receptor, KL.

Amino Acid Sequence↗

PubMed1990

The dominant W42 spotting phenotype results from a missense mutation in the c-kit receptor kinase.

The murine white spotting locus (W) is allelic with the proto-oncogene c-kit, which encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand. Mutations at the W locus affect various aspects of hematopoiesis and the proliferation and migration of primordial germ cells and melanoblasts during development to varying degrees of severity. The W42 mutation has a particularly severe effect in both the homozygous and the heterozygous states. The molecular basis of the W42 mutation was determined. The c-kit protein products in homozygous mutant mast cells were expressed normally but displayed a defective tyrosine kinase activity in vitro. Nucleotide sequence analysis of mutant complementary DNAs revealed a missense mutation that replaces aspartic acid with asparagine at position 790 in the c-kit protein product. Aspartic acid-790 is a conserved residue in all protein kinases. These results provide an explanation for the dominant nature of the W42 mutation and provide insight into the mechanism of c-kit-mediated signal transduction.

Amino Acid Sequence↗

PubMed1990

Candidate ligand for the c-kit transmembrane kinase receptor: KL, a fibroblast derived growth factor stimulates mast cells and erythroid progenitors.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor for an unidentified ligand and is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis and hematopoiesis during development and in adult life. Cellular targets of W mutations in hematopoiesis include distinct cell populations in the erythroid and mast cell lineages as well as stem cells. In the absence of interleukin-3 (IL-3) mast cells derived from normal mice but not from W mutant mice can be maintained by co-culture with 3T3 fibroblasts. Based on the defective proliferative response of W mast cells in the 3T3 fibroblast co-culture system it had been proposed that fibroblasts produce the c-kit ligand. We have used a mast cell proliferation assay to purify a 30 kd protein, designated KL, from conditioned medium of Balb/3T3 fibroblasts to apparent homogeneity. KL stimulates the proliferation of normal bone marrow derived mast cells but not mast cells from W mice, although both normal and mutant mast cells respond similarly to IL-3. Connective tissue-type mast cells derived from the peritoneal cavity of normal mice were found to express a high level of c-kit protein on their surface and to proliferate in response to KL. The effect of KL on erythroid progenitor cells was investigated as well. In combination with erythropoietin, KL was found to stimulate early erythroid progenitors (BFU-E) from fetal liver and spleen cells but not from bone marrow cells of adult mice and from fetal liver cells of W/W mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed1990

Molecular bases of dominant negative and loss of function mutations at the murine c-kit/white spotting locus: W37, Wv, W41 and W.

The proto-oncogene c-kit encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand and is allelic with the murine white-spotting locus (W). Mutations at the W locus affect various aspects of hematopoiesis, the proliferation and migration of primordial germ cells and melanoblasts during development. The original W mutation and W37 are severe lethal mutations when homozygous. In the heterozygous state the W mutation has a weak phenotype while W37 has dominant characteristics. Wv and W41 are weak W mutations with dominant characteristics. We have characterized the molecular basis of these four W mutations and determined their effects on mast cell differentiation by using a fibroblast/mast cell co-culture assay. We show that W37, Wv and W41 are the result of missense mutations in the kinase domain of the c-kit coding sequence (W37 E----K at position 582; Wv T----M position 660 and W41 V----M position 831), which affect the c-kit associated tyrosine kinase to varying degrees. The c-kit protein products in homozygous mutant mast cells are expressed normally, although the 160 kd cell membrane form of the c-kitW37 protein displays accelerated turnover characteristics. The W mutation is the result of a 78 amino acid deletion which includes the transmembrane domain of the c-kit protein. A 125 kd c-kit protein was detected in homozygous W/W mast cells which lacks kinase activity and is not expressed on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed1990

Gonadal expression of c-kit encoded at the W locus of the mouse.

Recently, it has been shown that the c-kit proto-oncogene is encoded at the white spotting (W) locus in mice. Mutations of this gene cause depletion of germ cells, some hematopoietic cells and melanocytes. In order to define further the role of c-kit in gametogenesis, we have examined its expression in late fetal and postnatal ovaries and in postnatal testis. By RNA blot analysis, c-kit transcripts were not detected in late fetal ovaries but appeared at birth. The relative amount reached a maximum in ovaries of juvenile mice, and decreased in adult ovaries. c-kit transcripts were present in increasing amounts in isolated primordial, growing and full-grown oocytes, as well as in ovulated eggs. Little was detected in early 2-cell embryos and none in blastocysts. In situ hybridization revealed c-kit transcripts in a few oocytes of late fetal ovaries and in all oocytes (from primordial to full-grown) in ovaries from juvenile and adult mice. Expression was also observed in ovarian interstitial tissue from 14 days of age onward. Using indirect immunofluorescence, the c-kit protein was detected on the surface of primordial, growing and full-grown oocytes, as well as on embryos at the 1- and 2-cell stages; little remained in blastocysts. In situ hybridization analysis of testes from mice of different ages demonstrated expression in spermatogonia from 6 days of age onward. Using information provided by determining the stage of the cycle of the seminiferous epithelium for a given tubule and by following the age dependence of labeling, it was concluded that the period of expression of c-kit extends from at least as early as type A2 spermatogonia through type B spermatogonia and into preleptotene spermatocytes. Leydig cells were labelled at all ages examined. The expression pattern in oocytes correlates most strongly with oocyte growth and in male germ cells with gonial proliferation.

Animals↗

PubMed1990

The mouse W/c-kit locus.

The mature cells in the haemopoietic system arise as the result of the extensive developmental and proliferative capacity of pluripotential stem cells. In order to understand the molecular basis for these developmental processes, it will be necessary to identify and characterize the cellular genes that control early steps in haemopoiesis. Mutations at the mouse W locus on chromosome 5 lead to pleiotropic developmental defects, including sterility, coat colour abnormalities, severe macrocytic anaemia and mast cell deficiency. The defects in all these lineages are cell autonomous and intrinsic, suggesting that the W locus encodes a gene product required directly for cellular differentiation. In an attempt to understand this classical mouse developmental mutation, we have demonstrated that the c-kit proto-oncogene, which encodes a transmembrane receptor tyrosine kinase, is very closely linked to W. Several further observations are consistent with the idea that W and c-kit are allelic: first, c-kit is expressed in those cell populations affected by W mutations; second, the expression of c-kit transcripts can be affected by mutations at the W locus; third, the tyrosine kinase activity associated with the protein encoded by c-kit is functionally impaired in mast cells derived from mutant W/Wv mice; and fourth, rearrangements within the c-kit gene have been reported in two W mutant alleles. These observations suggest that the dominant phenotype associated with W mutations results from loss-of-function alterations that affect the receptor tyrosine kinase encoded by c-kit. The demonstration that the W locus encodes a transmembrane growth factor receptor provides a molecular basis for understanding the intrinsic haemopoietic defect in W mutant mice and the role that this cellular proto-oncogene plays in haemopoiesis and other developmental processes.

Animals↗

PubMed1989

Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice--evidence for an impaired c-kit kinase in mutant mice.

The proto-oncogene c-kit, a transmembrane tyrosine protein kinase receptor for an unknown ligand, was shown recently to map to the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis, as well as the proliferation and/or migration of primordial germ cells and melanoblasts during development. Here, we show that c-kit is expressed in tissues known to be affected by W mutations in fetal and adult erythropoietic tissues, mast cells, and neural-crest-derived melanocytes. We demonstrate that the c-kit associated tyrosine-specific protein kinase is functionally impaired in W/WV mast cells, thus providing a molecular basis for understanding the developmental defects that result from these mutations.

Alleles↗