The Effects And Mechanism Of GPI-PLD On The Adhesion And Migration Of Hematopoietic Cells

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD)is an enzyme which is highly specific for the GPIanchor(glycan-phosphatidylinositol linkages) and does not cleave anyother phospholipids. GPI-PLD could regulate the expression ofGPI-anchored proteins and modulated their correspondent functions byreleasing GPI-anchored proteins from cell membrane. A group ofcell-surface proteins attached to the outer leaflet of the plasma membraneby GPI anchors are named GPI-anchored proteins, some of which arecellular adhesion molecules. These GPI-anchored proteins have beenproved to participate in many important cellular functions, including theregulation of adhesion and migration of leukocytes and tumor cells.Hematopoietic cells are proved to be able to produce GPI-PLD as well asexpress GPI-anchored proteins, some of which are also adhesionmolecules.The processes governing intravenous hematopoietic stem/progenitorcells(HS/PC) homing to the bone marrow also regulate leukemic cellsmigration to extramedullary tissues, which include cellular interactions ofrecognition, adhesion, migration, and so on. Recent studies suggested thatthe mechanisms of the malignancy and prognosis of certain tumors werecorrelated with GPI-PLD. GPI-PLD also played an important role in theprocess of metastasis of these tumor cells. HS/PC homing and leukemiccells extramedullary infiltratation was similar to these solid tumorinvasion and metastasis in some processes of crossing the physicalendothelium/extracellular matrix(ECM) barrier, which make us curiouswhether GPI-PLD contribute to the homing of HS/PC and leukemic cellsextramedullary infiltratation. In this work, we detected the expression ofcellular GPI-PLD and related GPI-anchored proteins in CD34⁺ cells fromumbilical cord blood(UCB), bone marrow(BM), mobilized peripheralblood(MPB) as well as various leukemic cell lines(K562, HL-60, THP-1),then the effects on the abilities of adhesion and migration in thesehematopoietic cells were observed in vitro by modifying GPI-PLDactivity. And we try to explored the initial mechanisms in these processes.Firstly, the CD34⁺ cells derived from UCB, BM and MPB wereisolated by MACS, then Semi-quantitive RT-PCR was used to detectGPI-PLD expression in these CD34⁺ cells. Additionally, their differencesin adhesion, migration of the three different CD34⁺ cells untreated ortreated with 0.5mmol/L 1,10-phenanthroline were assayed, respectively.The expression of GPI-anchored proteins(CDg8,CD90) and cell cyclestates of these CD34⁺ cells were analyzed by flow cytometry. The resultsshowed that GPI-PLD mRNA was undetectable in MPB, however, therewere no obvious difference in adhesiveness between UCB and BMCD34⁺ cells. While, spontaneous and SDF-1 induced migration of UCBand MPB were decreased after treatment with 1,10-phenanthroline.Nevertheless, little difference in the expression of GPI-anchored CD48and CD90 as well as cell cycle states were found between these untreatedand pretreated CD34⁺ cells.Moreover, the mRNA and protein expressive levels of GPI-PLD anduPAR(CD87) in the K562, HL-60 and THP-1 leukemic cells weredetected using semi-quantitive RT-PCR and western blot assay.Simultaneously, the expression of membrane GPI-anchored uPAR inthese leukemic cells were analyzed by flow cytometry andimmunohistochemistry, respectively. The abilities of adhesion andinvasion of the three myeloid leukemic cell lines were assayed,respectively. The results showed that the mRNA and protein expressionof GPI-PLD and uPAR in K562 and HL-60 cells were lower thanTHP-1 cells. The capability of adhesion to Fn in THP-1 cell line washigher than K562 and HL-60 cells, and the invasion capacity of THP-1and HL-60 leukemic cells in vitro were also higher than K562 cells.To further study the effects of GPI-PLD on the cleavage ofmembrane-bound uPAR and elucidate the implication for adhesive andinvasive potential in myeloid leukemic cells. The three myeloid leukemiccells were treated with 0.5mmol/L 1,10-phenanthroline orinsulin(10^-7mol/L) plus glucose (16.7mmol/L) which is an inhibitor oractivators of GPI-PLD. The changes of adhesiveness and invasiveness ofthree leukemic cells after treatment were detected. And the expression of GPI-anchored uPAR on the membrane of these cells were measured byflow cytometry. After incubated for 4 hours with 0.5mmol/L1,10-phenanthroline, the up-regulations of adhesion rate and membraneGPI-anchored uPAR expression were only observed in THP-1 cells,compared with untreated cells. Semi-quantitive RT-PCR and western blotresults showed that no obvious changes of GPI-PLD expression observedseen in these cells. Conversely, the down-regulations of the adhesiveness,invasiveness, and expression of uPAR and GPI-PLD were detected inTHP-1 cells after treatment with insulin (10^-7 mol/L) plus glucose (16.7mmol/L) for 24 hours.In conclusion, the results suggested that GPI-PLD probably made nocontribution to the processes of adhesion and migration of HS/PC, whichmay due to its low or no expression in UCB, BM, and MPB CD34⁺ cells.The expression of cellular GPI-PLD and membrane GPI-anchored uPARin THP-1 cells were higher than that in K562 and HL-60 cells, which maycorrelate with its superior adhesiveness and invasiveness. It alsosuggested that GPI-PLD play an important role in the adhesion andinvasion of THP-1 cells. It may be the potential mechanism in theprocesses that the GPI-PLD influence the adhesiveness and invasivenessof THP-1 cells by regulating the expression of membrane GPI-anchoreduPAR. The work may made a new experimental foundation for furtherstudying of HS/PC homing, enhancing hematopoietic stem celltransplantation success as well as the development of therapies toleukemia extramedullary infiltratation for clinical purposes.