[Activation of signal transducers and activators of transcription induced by vascular endothelial growth factor in CD34+ hematopoietic progenitor cells in vitro].
OBJECTIVE
To investigate the activation pattern of signal transducers and activators of transcription (STAT) induced by vascular endothelial growth factor (VEGF) in CD34+ hematopoietic progenitor cells, and gain an insight into the molecular mechanism and signal transduction pathway of VEGF that has an effect on CD34+ hematopoietic progenitor cells.


METHODS
After isolated from umbilical cord blood by using a high-gradient magnetically activated cell sorting system (MACS), CD34+ cells were stimulated by VEGF (50 ng/ml) for different time (0, 15, 30, 45, 60, 90 min) to detect the tyrosine phosphorylation and nuclear translocation of STAT-3 and STAT-5 with Western blot and immunocytochemistry methods. The expression of VEGF receptor-2 (VEGFR2) on the membrane of CD34+ progenitor cells was examined by immunocytochemistry. ATWLPPR, an effective peptide screened from phage epitope library by affinity for membrane-expressed VEGFR2 and blocking the binding of VEGF to VEGFR2, was used to determine whether the activation of STAT pathway induced by VEGF was blocked.


RESULTS
Tyrosine phosphorylation of STAT-3 and STAT-5 was undetectable in unstimulated CD34+ cells, but was evident at 15 min in response to VEGF stimulation. VEGF resulted in a rapid and transient tyrosine phosphorylation of STAT-3 and STAT-5. The maximal tyrosine phosphorylation was catched at 30 and 45 min, respectively (P = 0.0001), and returned to basal levels at 90 min. Immunocytochemistry confirmed that increased phosphorylated STAT-3 was translocated into the nuclei at 30 min (P = 0.0001), and mainly in cytoplasms again at 90 min after stimulation with VEGF. However, compared with unstimulated CD34+ cells, there was only increased phosphorylation of STAT-5 appeared mainly in cytoplasms, but no significant nuclear translocation was found after stimulation with VEGF (P > 0.05). The presence of VEGFR2 was confirmed using anti-VEGFR2 antibody staining by immunocytochemistry, moreover, the phosphorylation of STAT-3 and STAT-5 failed to be activated by the co-culture with ATWLPPR and VEGF, suggesting that activation of the STAT pathway be specifically mediated by VEGFR2 in CD34+ progenitor cells.


CONCLUSIONS
STAT signaling pathway participates in the signal transduction of VEGF via VEGFR2 in CD34+ hemopoietic progenitor cells.
Ye
Feng
F
Department of Gynecologic Oncology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China.
Ye
Da-feng
DF
Xie
Xing
X
Chen
Huai-zeng
HZ
Lü
Wei-guo
WG
D000328:Adult; D018952:Antigens, CD34 / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005312:Fetal Blood / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008894:Milk Proteins; D010766:Phosphorylation; D011247:Pregnancy; D040262:Receptors, Vascular Endothelial Growth Factor / Q000378:metabolism; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014443:Tyrosine / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
D018952:Antigens, CD34; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D042461:Vascular Endothelial Growth Factor A; D014443:Tyrosine; D040262:Receptors, Vascular Endothelial Growth Factor