Promegapoietin, a family of chimeric growth factors, supports megakaryocyte development through activation of IL-3 and c-Mpl ligand signaling pathways.
OBJECTIVE
The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated.


METHODS
The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses.


RESULTS
Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist.


CONCLUSIONS
PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.
Doshi
P D
PD
Pharmacia Discovery Research, Chesterfield, Mo. 63198, USA. parul.doshi@pharmacia.com
Giri
J G
JG
Abegg
A L
AL
Favara
J P
JP
Huynh
M S
MS
Kahn
L E
LE
Minnerly
J C
JC
Pegg
L E
LE
Villani-Price
D
D
Siegel
N R
NR
Staten
N R
NR
Thomas
J W
JW
McKearn
J P
JP
Smith
W G
WG
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D001854:Bone Marrow Cells / Q000166:cytology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D006133:Growth Substances / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D007377:Interleukin-3; D053614:Janus Kinase 2; D008253:Macaca mulatta; D008533:Megakaryocytes / Q000187:drug effects / Q000276:immunology*; D008894:Milk Proteins; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000502:physiology; D018121:Receptors, Cytokine / Q000378:metabolism; D016185:Receptors, Interleukin-3 / Q000502:physiology; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D013926:Thrombopoietin / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection
D004268:DNA-Binding Proteins; D006133:Growth Substances; D007377:Interleukin-3; D008894:Milk Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D016185:Receptors, Interleukin-3; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; C494095:STAT5B protein, human; D015534:Trans-Activators; C082218:MPL protein, human; D019000:Phosphotyrosine; D013926:Thrombopoietin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2