Activation of phosphatidylinositol 3-kinase is important for erythropoietin-induced erythropoiesis from CD34(+) hematopoietic progenitor cells.
OBJECTIVE
Several transducing molecules, including JAK2, STAT5, MAP kinases, phosphatidylinositol 3-kinase (PI3K), phospholipase C-gamma1, and PKC are activated by interaction between erythropoietin (EPO) and the EPO receptor. The aim of this was to examine the relative involvement of PI3K in the development of glycophorin A (GPA)(+) erythroid cells from normal hematopoietic progenitor cells.


METHODS
CD34(+) hematopoietic progenitor cells or subpopulations obtained by FACS sorting were cultured in serum-free medium containing EPO with or without inhibitors for PI3K, p38, MEK, or PKC for various time periods before phenotypic analysis or detection of apoptosis by flow cytometry, cell cycle analysis, high-resolution tracking of cell division, Western blot analysis, or Akt kinase assay were performed.


RESULTS
The PI3K inhibitor LY294002 completely counteracted the EPO-induced proliferation of CD34(+) progenitor cells and CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 also highly suppressed the expanded erythropoiesis induced by the combined action of EPO and stem cell factor. The profound inhibitory effect of LY294002 on proliferation was caused by its induction of cell cycle arrest in the G(0)/G(1) phase of the cell cycle. Some cells acquired GPA expression before they went through cell division. This was completely blocked by LY294002, implying an inhibitory effect on maturation. In addition, LY294002 completely blocked the viability-enhancing effect of EPO in CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 and various inhibitors of PKC completely suppressed the EPO-induced increase in the activity of Akt kinase, a direct downstream target of PI3K.


CONCLUSIONS
Our results point to an important role for PI3K in mediating EPO-induced survival, proliferation, and possibly maturation of early erythroid progenitors.
Myklebust
June Helen
JH
Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway. junehm@ulrik.uio.no
Blomhoff
Heidi Kiil
HK
Rusten
Leiv Sindre
LS
Stokke
Trond
T
Smeland
Erlend B
EB
D000328:Adult; D018952:Antigens, CD34 / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D002070:Butadienes / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects*; D004920:Erythropoiesis / Q000187:drug effects / Q000502:physiology*; D004921:Erythropoietin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology; D006021:Glycophorins / Q000032:analysis; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology; D009025:Morpholines / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D019089:Stem Cell Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology
D018952:Antigens, CD34; D000943:Antigens, Differentiation; D002070:Butadienes; D002227:Carbazoles; D002867:Chromones; D004791:Enzyme Inhibitors; D004921:Erythropoietin; D005419:Flavonoids; D006021:Glycophorins; D007211:Indoles; D007527:Isoenzymes; D009025:Morpholines; D009281:Naphthalenes; D009570:Nitriles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D019311:Staurosporine; D019089:Stem Cell Factor; C113580:U 0126; C081021:Go 6976; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494918:AKT1 protein, human; C496628:PRKCA protein, human; C058819:calphostin C; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one