Thrombopoietin regulates Bcl-xL gene expression through Stat5 and phosphatidylinositol 3-kinase activation pathways.
Thrombopoietin (TPO), an essential factor for megakaryopoiesis and thrombopoiesis, works as a survival factor for megakaryocytic lineage cells. However, little is known about the molecular mechanism in detail. We show here that TPO supports the survival of TPO-dependent leukemia cell line UT-7/TPO and normal megakaryocytic progenitors via the induction of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. We further analyzed the signal transduction pathways required for TPO-induced Bcl-xL gene expression. A reporter assay with various lengths of Bcl-x gene promoter revealed that both Stat- and nuclear factor kappa B-binding sites are prerequisites for TPO-induced promoter activity. Consistent with these results, TPO induced the binding of Stat5 and subunits of nuclear factor kappa B, p50, and c-Rel to the Bcl-x gene promoter. AG490, a specific inhibitor for Jak2, and LY294002, a specific inhibitor for phosphatidylinositol (PI) 3-kinase, reduced the protein level of Bcl-xL in UT-7/TPO cells, accompanied by an increase in the ratio of apoptotic cells. Interestingly, LY294002 enhanced the TPO-induced DNA binding activity of Stat5 without affecting the Jak2 activation and tyrosine phosphorylation of Stat5. Concomitantly, confocal microscopy revealed that LY294002 clearly inhibited the nuclear export of Stat5, suggesting that PI 3-kinase regulates the subcellular localization of Stat5. Taken together, our results suggest that both Jak-Stat and PI 3-kinase activation pathways regulate the TPO-induced survival of megakaryocytic cells via Bcl-xL gene expression. In addition, our data suggest possible cross-talk between these two signaling pathways.
Kirito
Keita
K
Division of Hematology, Department of Medicine, Jichi Medical School, Tochigi 329-04, Japan.
Watanabe
Tomoko
T
Sawada
Ken-ichi
K
Endo
Hitoshi
H
Ozawa
Keiya
K
Komatsu
Norio
N
D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D019070:Cell Lineage; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002867:Chromones / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005799:Genes, Dominant; D006801:Humans; D053614:Janus Kinase 2; D008156:Luciferases / Q000378:metabolism; D018613:Microscopy, Confocal; D008894:Milk Proteins; D009025:Morpholines / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000378:metabolism*; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D013926:Thrombopoietin / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D002867:Chromones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008894:Milk Proteins; D009025:Morpholines; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D051020:bcl-X Protein; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D013926:Thrombopoietin; D008156:Luciferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2