Phosphorylation status of transcription factor C/EBPα determines cell-surface poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis

The cell-surface straight and branched repeats of N-acetyllactosamine (LacNAc) units, called poly-LacNAc chains, characterize the histo-blood group i and I antigens, respectively. The transition of straight to branched poly-LacNAc chain (i to I) is determined by the I locus, which expresses 3 IGnT transcripts, IGnTA, IGnTB, and IGnTC. Our previous investigation demonstrated that the i-to-I transition in erythroid differentiation is regulated by the transcription factor CCAAT/enhancer binding protein α (C/EBPα). In the present investigation, the K-562 cell line was used as a model to show that the i-to-I transition is determined by the phosphorylation status of the C/EBPα Ser-21 residue, with dephosphorylated C/EBPα Ser-21 stimulating the transcription of the IGnTC gene, consequently resulting in I branching. Results from studies using adult erythropoietic and granulopoietic progenitor cells agreed with those derived using the K-562 cell model, with lentiviral expression of C/EBPα in CD34+ hematopoietic cells demonstrating that the dephosphorylated form of C/EBPα Ser-21 induced the expression of I antigen, granulocytic CD15, and also erythroid CD71 antigens. Taken together, these results demonstrate that the regulation of poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis share a common mechanism, with dephosphorylation of the Ser-21 residue on C/EBPα playing the critical role.

A Secreted Proform of Neutrophil Proteinase 3 Regulates the Proliferation of Granulopoietic Progenitor Cells

Myeloid leukemia cells, the human promyelocytic cell line HL-60, and a subpopulation of normal marrow cells produce a leukemia-associated inhibitor (LAI) that reversibly downmodulates DNA synthesis of normal granulopoietic progenitor cells colony-forming unit granulocyte-macrophage (CFU-GM). We isolated an active 125-kD component of LAI from HL-60 conditioned medium (CM), subjected it to cyanogen bromide cleavage and show by amino acid sequencing of the resulting peptides that it consists of a complex of the serine proteinase inhibitor 1-antitrypsin and a 31-kD fragment that retained the S-phase inhibitory activity, but resisted sequencing. This finding suggested that the 31-kD fragment originated from one of the neutrophil serine proteases (ie, elastase, proteinase 3, or cathepsin G) produced by normal promyelocytes, as well as HL-60 cells, for storage in primary granules and partly secreted during synthesis as enzymatically inactive proforms. Immunoblot analysis showed that the 125-kD complex contained proteinase 3 (PR3), and immunoprecipitation of PR3 from HL-60 CM abrogated the S-phase inhibitory activity, whereas immunoprecipitation of cathepsin G or elastase did not. Immunoprecipitation of PR3 from CM of a subpopulation of normal marrow cells also abrogated the S-phase inhibitory effect. Furthermore, CM from rat RBL and murine 32D cell lines transfected with human PR3 both reduced the fraction of CFU-GM in S-phase with 30% to 80% at 1 to 35 ng/mL PR3, whereas CM of the same cells transfected with cathepsin G or elastase did not. Also, an enzymatically silent mutant of PR3 exerted full activity, showing that the S-phase modulatory effect is not dependent on proteolytic activity. Amino acid sequencing of biosynthetically radiolabeled PR3 showed that PR3 from transfected cells is secreted after synthesis as proforms retaining amino terminal propeptides. In contrast, mature PR3 extracted from mature neutrophils has only minor activity. The inhibitory effect of secreted PR3 is reversible and abrogated by granulocyte (G)- or granulocyte-macrophage colony-stimulating factor (GM-CSF). Experiments with highly purified CD34+ bone marrow cells suggested that PR3 acts directly on the granulopoietic progenitor cells. These observations suggest a role for PR3 in regulation of granulopoiesis, and possibly in suppression of normal granulopoiesis in leukemia.

A Secreted Proform of Neutrophil Proteinase 3 Regulates the Proliferation of Granulopoietic Progenitor Cells

Myeloid leukemia cells, the human promyelocytic cell line HL-60, and a subpopulation of normal marrow cells produce a leukemia-associated inhibitor (LAI) that reversibly downmodulates DNA synthesis of normal granulopoietic progenitor cells colony-forming unit granulocyte-macrophage (CFU-GM). We isolated an active 125-kD component of LAI from HL-60 conditioned medium (CM), subjected it to cyanogen bromide cleavage and show by amino acid sequencing of the resulting peptides that it consists of a complex of the serine proteinase inhibitor 1-antitrypsin and a 31-kD fragment that retained the S-phase inhibitory activity, but resisted sequencing. This finding suggested that the 31-kD fragment originated from one of the neutrophil serine proteases (ie, elastase, proteinase 3, or cathepsin G) produced by normal promyelocytes, as well as HL-60 cells, for storage in primary granules and partly secreted during synthesis as enzymatically inactive proforms. Immunoblot analysis showed that the 125-kD complex contained proteinase 3 (PR3), and immunoprecipitation of PR3 from HL-60 CM abrogated the S-phase inhibitory activity, whereas immunoprecipitation of cathepsin G or elastase did not. Immunoprecipitation of PR3 from CM of a subpopulation of normal marrow cells also abrogated the S-phase inhibitory effect. Furthermore, CM from rat RBL and murine 32D cell lines transfected with human PR3 both reduced the fraction of CFU-GM in S-phase with 30% to 80% at 1 to 35 ng/mL PR3, whereas CM of the same cells transfected with cathepsin G or elastase did not. Also, an enzymatically silent mutant of PR3 exerted full activity, showing that the S-phase modulatory effect is not dependent on proteolytic activity. Amino acid sequencing of biosynthetically radiolabeled PR3 showed that PR3 from transfected cells is secreted after synthesis as proforms retaining amino terminal propeptides. In contrast, mature PR3 extracted from mature neutrophils has only minor activity. The inhibitory effect of secreted PR3 is reversible and abrogated by granulocyte (G)- or granulocyte-macrophage colony-stimulating factor (GM-CSF). Experiments with highly purified CD34+ bone marrow cells suggested that PR3 acts directly on the granulopoietic progenitor cells. These observations suggest a role for PR3 in regulation of granulopoiesis, and possibly in suppression of normal granulopoiesis in leukemia.

A comparison of granulopoiesis in culture from blood and marrow cells of nonleukemic individuals and patients with acute leukemia

The objective of this study was to compare the concentration of committed granulopoietic progenitor cells (CFU-C) in marrow and blood. For individuals without leukemia, a highly significant correlation was observed between the concentration of CFU-C obtained from the two sites. However, CFU-C in blood had a slower sedimentation velocity than that reported for marrow and were found not to be in the DNA synthetic phase of the cycle using the tritiated thymidine suicide tehcnique. In patients with acute leukemia, no correlation was observed between concentrations of CFU-C in marrow and peripheral blood, regardless of whether the patients were newly diagnosed, in remission, or in relapse. We concluded that studies of the peripheral blood do not yield the same information in respect to granulopoietic progenitor cells as do studies of the marrow.

A comparison of granulopoiesis in culture from blood and marrow cells of nonleukemic individuals and patients with acute leukemia

The objective of this study was to compare the concentration of committed granulopoietic progenitor cells (CFU-C) in marrow and blood. For individuals without leukemia, a highly significant correlation was observed between the concentration of CFU-C obtained from the two sites. However, CFU-C in blood had a slower sedimentation velocity than that reported for marrow and were found not to be in the DNA synthetic phase of the cycle using the tritiated thymidine suicide tehcnique. In patients with acute leukemia, no correlation was observed between concentrations of CFU-C in marrow and peripheral blood, regardless of whether the patients were newly diagnosed, in remission, or in relapse. We concluded that studies of the peripheral blood do not yield the same information in respect to granulopoietic progenitor cells as do studies of the marrow.

Granulopoietic progenitors in suspension culture: a comparison of stimulatory cells and conditioned media

Kinetic studies have been carried out to investigate the functional heterogeneity previously observed in populations of human marrow or peripheral blood cells separated by velocity sedimentation. The results obtained confirm the earlier results, in that slowly-sedimenting cells were found to stimulate both colony formation by granulopoietic progenitors and an increase in numbers of granulopoietic progenitors in suspension culture, while rapidly-sedimenting cells stimulated only colony formation and not increased progenitors in suspension cultures. Investigations of the properties of media conditioned by these two subpopulations of cells revealed no clear differences between them; both stimulated suspension cultures as well as colony formation, and both lost the former activity, but not the latter, after dialysis. The results contribute to the evidence that more than one process is regulated in cultures of granulopoietic progenitor cells.