Growth factor withdrawal from primary human erythroid progenitors induces apoptosis through a pathway involving glycogen synthase kinase-3 and Bax.
The prevention of apoptosis is a key function of growth factors in the regulation of erythropoiesis. This study examined the role of the constitutively active serine/threonine kinase glycogen synthase kinase-3 (GSK3), a target of the phosphoinositide-3-kinase (PI3K)/Akt pathway, in the regulation of apoptosis in primary human erythroid progenitors. GSK3 phosphorylation at its key regulatory residues S21 (alpha isoform) and S9 (beta isoform) was high in steady-state culture, disappeared on growth factor withdrawal, and returned in response to treatment of cells with either erythropoietin or stem cell factor. Phosphorylation correlated with a PI3K-dependent reduction of 25% to 30% in measured GSK3 activity. LY294002, a specific inhibitor of PI3K, induced apoptosis in growth factor-replete erythroid cells to a degree similar to growth factor deprivation, whereas the Mek1 inhibitor U0126 had no effect, implicating PI3K and not mitogen-activated protein kinase in survival signaling. Growth factor-deprived erythroblasts, which undergo apoptosis rapidly, were protected from apoptosis by both lithium chloride, a GSK3 selective inhibitor, and inhibition of caspase activity. However, the clonogenic potential of single cells, which more accurately reflects cell survival, was maintained by lithium chloride, but not by caspase inhibition. Furthermore, lithium chloride, but not caspase inhibition, prevented the appearance of the conformational form of Bax associated with apoptosis induction. In summary, GSK3 activity is suppressed by erythropoietin and stem cell factor in human erythroid progenitor cells, and increased GSK3 activity, brought about by growth factor withdrawal, may regulate commitment to cell death through a caspase-independent pathway that results in a conformational change in Bax.
Somervaille
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TC
Department of Hematology, Royal Free and University College Medical School, London, United Kingdom. t.somervaille@ucl.ac.uk
Linch
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DC
Khwaja
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A
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D053148:Caspase 3; D061945:Caspase Inhibitors; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D002867:Chromones / Q000494:pharmacology; D003114:Colony-Forming Units Assay; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D004921:Erythropoietin / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology; D018021:Lithium Chloride / Q000494:pharmacology; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008856:Microscopy, Fluorescence; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D015398:Signal Transduction / Q000187:drug effects*; D019089:Stem Cell Factor / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein
D000590:Amino Acid Chloromethyl Ketones; C494826:BAX protein, human; D061945:Caspase Inhibitors; D002867:Chromones; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007527:Isoenzymes; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D019089:Stem Cell Factor; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D004921:Erythropoietin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D038341:Glycogen Synthase Kinases; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D018021:Lithium Chloride; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one