Panax notoginseng saponins induced up-regulation, phosphorylation and binding activity of MEK, ERK, AKT, PI-3K protein kinases and GATA transcription factors in hematopoietic cells.
OBJECTIVE
To investigate the effects of panax notoginseng saponins (PNS) on expression, regulation and phosphorylation of multiple protein kinases in mitogen activated protein kinase (MAPK) intracellular signal pathway and GATA transcription factors in hematopoietic cells, so as to explore its mechanism of proliferation and differentiation activity on hematopoiesis.


METHODS
The human granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF-288 and Meg-01 cell lines were treated by PNS, the positive control of K562, CHRF-288 cells treated by recombination human erythropoietin (Epo) and thrombopoietin (Tpo) respectively. The total cell lysate and nuclei protein were extracted after being treated by PNS, subsequently, analyzed by both Western blot and immune-precipitation. Meanwhile, the nuclei extract was performed for electrophoretic mobility shift assay (EMSA) by using (32)P radio labeled double-stranded GATA consensus oligonucleotide.


RESULTS
The expression levels of kinase MEK-1, MEK-2, ERK-1, ERK-2, AKT-1, AKT-2 and PI-3K were increased by PNS treatment to different extent in four cell lines, depending on cellular heterogeneity and sensitivity to PNS, also phosphorylation of MEK-1, ERK-1 was differentially promoted by PNS respectively P<0.05, 0.01, 0.001). The expression levels of transcription factors GATA-1 and GATA-2 were increased, moreover, their DNA binding activities were raised dramatically in PNS treated K562, CHRF-288 and Meg-01 cells compared with the controls respectively (P<0.05, 0.01, 0.001). The positive control of K562, CHRF-288 cells treated by Epo or Tpo respectively also displayed up-regulation of protein kinases and GATA transcription factors respectively (P<0.05, 0.01, 0.001).


CONCLUSIONS
The results indicated that intracellular signal pathway initiated by PNS was involved in MAPK pathway and transcription factors of GATA family in hematopoietic cells. PNS displayed the role to promote proliferation and differentiation, by means of increasing expression level and phosphorylation status of multiple protein kinases, also inducing synthesis of GATA transcription factors and upregulation its DNA binding activity.
Department of Oncology, Zhejiang Provincial People's Hospital, Hangzhou, China.
Xin
X
Sun
Rui-Lan
RL
Gao
Xiao-Jie
XJ
Lin
Wei-Hong
WH
Xu
Xiao-Hong
XH
Chen
D015153:Blotting, Western; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D050980:GATA Transcription Factors / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D047468:Immunoprecipitation; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D053723:Panax notoginseng / Q000737:chemistry*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects*
D050980:GATA Transcription Factors; D012503:Saponins; D004247:DNA; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases