Human Erythropoietin Blood Vascular Cells To Produce The Hormone On Endothelial Cell Proliferation And Its Mechanism

Objectives:Hemangiopoietin(HAPO) is a novel growth factor,originally identified and purified from the urine of patients with aplastic anemia.Previously,we have demonstrated that human recombinant HAPO(rhHAPO),which has been produced based on the N-terminal amino acid sequence of purified native HAPO,supports survival and proliferation of primitive cells of both hematopoietic and endothelial cell lineages,and efficiently protects mice from radiation damage.Recently,we have reported that rhHAPO protects MO7e cells from apoptosis by inactivating caspase 3 and blocking the PARP degradation through the PI-3K/Akt pathway.However,the underlying mechanisms by which HAPO stimulates cell proliferation remain unclear. Thus,we studied the signal transduction pathway through which rhHAPO promotes cell proliferation in human umbilical vein endothelial cells(HUVECs).Methods:We carried out two assays;MTT colorimetric assay and BrdU incorporation assay to determine the effect of HAPO on cell proliferation.We performed the cell cycle analysis on HAPO-treated cells.We examined the activation of different signal transduction pathways and the expression levels of cell-cycle regulatory proteins using Western blot analysis.Results:We demonstrated that rhHAPO induced HUVEC proliferation in a dose-dependent manner,with the maximum effect observed at a concentration of 500 ng/ml.HAPO activated PI-3K/Akt pathway in HUVEC,and PI-3K/Akt pathway was involved in HAPO-induced HUVEC proliferation,rhHAPO markedly increased the expression of cyclin D1 in a time-course manner.We finally found that HAPO enhances VEGF-,but not bFGF-induced endothelial cell proliferation in vitro.Conclusions:We have demonstrated that HAPO,a novel growth factor,exerts a direct effect on HUVEC proliferation.Akt signaling was activated and the expression of cyclin D1 was also affected by HAPO.Pretreatment of LY294002 inhibited the activation of Akt and blocked HAPO-induced HUVEC proliferation.Furthermore, HAPO significantly enhanced VEGF-induced endothelial cell proliferation.These results indicate that HAPO promotes HUVEC proliferation through PI-3K/Akt pathway. Objectives:ABCG2,also known as breast cancer resistance protein(BCRP)/placental ABC protein(ABCP)/mitoxantrone resistance protein(MXR),belongs to the ATP-binding cassette(ABC) transporter family.To date,more than 50 ABC transporters have been described,classified,localized and functionally evaluated in diverse organisms from microbes to humans.The majority of these proteins actively transport an array of biological compounds,including peptides,steroids,ions,and phospholipids,contributing to physiology,pathology,toxicology,and pharmacology. The ATP-binding cassette efflux transporter,ABCG2,is widely expressed in a variety of normal tissues,stem cells as well as cancer cells.Existing data suggest that ABCG2 plays important roles in the maintenance of the stem cell phenotype and multidrug resistance of cancer cells.However,the potential role of ABCG2 in other cellular processes remains speculative and poorly understood.In the present study,we explored the possible role of ABCG2 in cell proliferation.Methods:To better elucidate the molecular mechanisms involved in ABCG2-mediated cell proliferation,MCF-7/MX and A549 were used as two independent cell models. MCF-7/MX is a mitoxantrone-selected,multidrug-resistant breast cancer cell line.This cell line overexpresses ABCG2 compared with parental MCF-7 cells,and is regarded as the source of induced ABCG2 expression.A549 is a lung carcinoma cell line with moderate expression of ABCG2.This cell line exhibits a strong SP phenotype and is considered as the source of endogenous ABCG2 expression.To study the cellular role of ABCG2,we performed gene silencing by gene-targeted siRNA method.We carried out two assays,MTT colorimetric assay and BrdU incorporation assy to determine the effect of ABCG2 knockdown on cell proliferation.We performed the cell cycle analysis on cells transfected with siRNA against ABCG2 to determine if ABCG2 is involved in cell cycle progression.To confirm that knockdown of ABCG2 reduced the S-phase entry,BrdU incorporation assay was performed.We examined the expression levels of cell-cycle regulatory proteins using Western blot analysis.Results:ABCG2 expression in both MCF-7/MX and A549 cells was strongly reduced by ABCG2 gene specific siRNA.Both expression and function of ABCG2 are markedly suppressed by gene-specific siRNA.Down-regulation of ABCG2 by transient RNAi inhibited the proliferation of MCF-7/MX and A549 cells.However,it had no effect on the proliferation of DU145 cell line.Down-regulation of ABCG2 by transient RNAi blocked cell cycle progression in G0/G1 phase,and reduced the S-phase entry.Western blot analysis demonstrated that silencing of ABCG2 gene resulted in pronounced increase in p21 Cip1 levels,and down-regulation of cyclin D3 in MCF-7/MX and A549 cells.In contrast,the levels of cyclin D3 and p21 in DU145 cell line were not affected by ABCG2 siRNA treatment.Furthermore,blocking of ABCG2 function by chemical inhibitor,fumitremorgin C,also inhibited cell proliferation via the prolonged G0/G1 interval.Our data suggest that the FTC-induced cytotoxic effect with sucha high concentration of the compound may be mediated by other mechanisms than induction of p21 Cip1 and suppression of cyclin D3.Conclusions:The present study suggests that suppression of ABCG2,using gene-specific siRNA or chemical inhibitor,decreased cell proliferation and caused a G0/G1 phase arrest in the cell cycle progression.These findings define a novel functional role of ABCG2 in cell proliferation.However,more studies are needed to determine the potential physiological substrates for this transporter,and to demonstrate the interactions between ABCG2 and pathways that regulate the proliferation of stem cells and cancer cells.