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Phosphatidylinositol 3-Kinase Is Necessary but Not Sufficient for Thrombopoietin-induced Proliferation in Engineered Mpl-bearing Cell Lines as Well as in Primary Megakaryocytic Progenitors*

Open AccessPublished:September 14, 2001DOI:https://doi.org/10.1074/jbc.M105178200
      Thrombopoietin and its receptor (Mpl) support survival and proliferation in megakaryocyte progenitors and in BaF3 cells engineered to stably express Mpl (BaF3/Mpl). The binding of thrombopoietin to Mpl activates multiple kinase pathways, including the Jak/STAT, Ras/Raf/MAPK, and phosphatidylinositol 3-kinase pathways, but it is not clear how these kinases promote cell cycling. Here, we show that thrombopoietin induces phosphatidylinositol 3-kinase and that phosphatidylinositol 3-kinase is required for thrombopoietin-induced cell cycling in BaF3/Mpl cells and in primary megakaryocyte progenitors. Treatment of BaF3/Mpl cells and megakaryocytes with the phosphatidylinositol 3-kinase inhibitor LY294002 inhibited mitotic and endomitotic cell cycl-ing. BaF3/Mpl cells treated with thrombopoietin and LY294002 were blocked in G1, whereas megakaryocyte progenitors treated with thrombopoietin and LY294002 showed both a G1 and a G2 cell cycle block. Expression of constitutively active Akt in BaF3/Mpl cells restored the ability of thrombopoietin to promote cell cycling in the presence of LY294002. Constitutively active Akt was not sufficient to drive proliferation of BaF3/Mpl cells in the absence of thrombopoietin. We conclude that in BaF3/Mpl cells and megakaryocyte progenitors, thrombopoietin-induced phosphatidylinositol 3-kinase activity is necessary but not sufficient for thrombopoietin-induced cell cycle progression. Phosphatidylinositol 3-kinase activity is likely to be involved in regulating the G1/S transition.
      TPO
      thrombopoietin
      BrdUrd
      bromodeoxyuridine
      BSA
      bovine serum albumin
      IL
      interleukin
      IMDM
      Iscove's modified Dulbecco's medium
      MK
      megakaryocyte
      PI3K
      phosphatidylinositol 3-kinase
      PI
      propidium iodide
      rhTPO
      recombinant human TPO
      TBST
      Tris-buffered saline (pH 8.0) containing 0.05% Tween-20
      Mature megakaryocytes are large, polyploid bone marrow cells that give rise to circulating platelets (
      • Kaushansky K.
      ). Thrombopoietin (TPO)1 has been characterized as the primary hematopoietic cytokine regulating normal megakaryocyte (MK) development. The receptor for TPO is encoded by the c-mpl proto-oncogene, a member of the hematopoietic growth factor receptor family, which in altered form causes a myeloproliferative syndrome in mice (
      • Wendling F.
      • Varlet P.
      • Charon M.
      • Tambourin P.
      ). In addition to its effects on MK development, TPO has been found to play a significant role in promoting stem cell survival, and in conjunction with other cytokines, it can support stem cell expansion (
      • Sitnicka E.
      • Lin N.
      • Priestley G.V.
      • Fox N.
      • Broudy V.C.
      • Wolf N.S.
      • Kaushansky K.
      ,
      • Yagi M.
      • Ritchie K.A.
      • Sitnicka E.
      • Storey C.
      • Roth G.J.
      • Bartelmez S.
      ,
      • Alexander W.S.
      • Roberts A.W.
      • Nicola N.A.
      • Li R.
      • Metcalf D.
      ). Characterization of the pathways by which TPO signals to promote survival and proliferation in stem cells and developing MKs is critical to a better understanding of the physiologic, pathologic, and potentially therapeutic roles of the cytokine in stem cell expansion, myeloproliferative disorders, and states of bone marrow failure.
      Many of the effects of TPO on cell survival and proliferation have been ascribed to activation of the Jak/STAT and Ras/Raf/MAPK pathways (reviewed in Ref.
      • Drachman J.G.
      • Rojnuckarin P.
      • Kaushansky K.
      ). Activation of Jak2 leads to tyrosine phosphorylation of Mpl as well as STAT3 and STAT5 (
      • Bacon C.M.
      • Tortolani P.J.
      • Shimosaka A.
      • Rees R.C.
      • Longo D.L.
      • O'Shea J.J.
      ,
      • Drachman J.G.
      • Griffin J.D.
      • Kaushansky K.
      ), and the phosphorylated STATs dimerize and translocate to the nucleus, where they stimulate transcription. In addition, phosphorylation of distal tyrosine residues on Mpl creates docking sites for SHC, which undergoes phosphorylation and then recruits Grb2/SOS, thus activating the Ras/Raf/MAPK pathway (
      • Drachman J.G.
      • Griffin J.D.
      • Kaushansky K.
      ,
      • Yamada M.
      • Komatsu N.
      • Okada K.
      • Kato T.
      • Miyazaki H.
      • Miura Y.
      ,
      • Alexander W.S.
      • Maurer A.B.
      • Novak U.
      • Harrison-Smith M.
      ). Activation of the Ras/Raf/MAPK pathway can also occur by an alternative, as yet undetermined mechanism, independent of the distal receptor phosphotyrosine residues (
      • Rojnuckarin P.
      • Drachman J.G.
      • Kaushansky K.
      ).
      Although these studies and others have shown that activation of the Jak/STAT and Ras/Raf/MAPK pathways is important for signaling by Mpl, other pathways likely contribute to the cellular response to TPO. One such pathway that may contribute to Mpl signaling is phosphatidylinositol 3-kinase (PI3K). PI3K has been shown to be activated by many growth factors involved in hematopoiesis, including stem cell factor, platelet-derived growth factor, erythropoietin, interleukin 3 (IL-3), IL-2, granulocyte-macrophage colony-stimulating factor, granulocyte colony stimulating factor, insulin-like growth factor 1, and TPO (
      • Corey S.
      • Eguinoa A.
      • Puyana-Theall K.
      • Bolen J.B.
      • Cantley L.
      • Mollinedo F.
      • Jackson T.R.
      • Hawkins P.T.
      • Stephens L.R.
      ,
      • Sattler M.
      • Salgia R.
      • Durstin M.A.
      • Prasad K.V.
      • Griffin J.D.
      ,
      • Zhang X.
      • Vik T.A.
      ,
      • Timokhina I.
      • Kissel H.
      • Stella G.
      • Besmer P.
      ,
      • Dong F.
      • Larner A.C.
      ). It has been well established that activation of PI3K plays an important role in promoting cell survival (
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ,
      • Songyang Z.
      • Baltimore D.
      • Cantley L.C.
      • Kaplan D.R.
      • Franke T.F.
      ,
      • Downward J.
      ). However, although there are ample data supporting a requirement for PI3K in proliferation of nonhematopoietic cells (
      • Coughlin S.R.
      • Escobedo J.A.
      • Williams L.T.
      ,
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ,
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ), data showing specific requirements for PI3K in the cytokine-dependent proliferation of hematopoietic cells are inconclusive. Studies in cell lines with IL-2, IL-3, and erythropoietin have supported a role for PI3K in the proliferative response (
      • Ahmed N.N.
      • Grimes H.L.
      • Bellacosa A.
      • Chan T.O.
      • Tsichlis P.N.
      ,
      • Jaster R.
      • Bittorf T.
      • Brock J.
      ,
      • Shigematsu H.
      • Iwasaki H.
      • Otsuka T.
      • Ohno Y.
      • Arima F.
      • Niho Y.
      ,
      • Craddock B.L.
      • Orchiston E.A.
      • Hinton H.J.
      • Welham M.J.
      ); however, studies with Flt-3L have not (
      • Beslu N.
      • LaRose J.
      • Casteran N.
      • Birnbaum D.
      • Lecocq E.
      • Dubreuil P.
      • Rottapel R.
      ). Importantly, the conclusions from studies performed in primary cells often differ from those performed in cell lines. For example, data from cell lines support the role of PI3K in erythropoietin-induced proliferation (
      • Jaster R.
      • Bittorf T.
      • Brock J.
      ,
      • Shigematsu H.
      • Iwasaki H.
      • Otsuka T.
      • Ohno Y.
      • Arima F.
      • Niho Y.
      ), whereas a study done in primary erythroid progenitors concluded that the specific role of PI3K was to promote erythropoietin-induced survival (
      • Haseyama Y.
      • Sawada K.
      • Oda A.
      • Koizumi K.
      • Takano H.
      • Tarumi T.
      • Nishio M.
      • Handa M.
      • Ikeda Y.
      • Koike T.
      ). These studies demonstrate that the role of PI3K in cell proliferation may be cytokine- and cell type-specific. Therefore, in this, work we examined TPO signaling both in a TPO-responsive cell line (BaF3/Mpl) and in primary murine MK progenitors, and we show that an intact PI3K pathway is necessary but not sufficient for cell cycling.

      DISCUSSION

      As research into cytokine signaling progresses, it is becoming apparent that a complex array of signals is directed by cytokines, which cooperate to amplify or modulate the cellular response. Cell survival signals in particular synergize with proliferative signals to provide maximal cell growth. TPO is known to stimulate cell growth through activation of the Jak/STAT and Ras/Raf/MAPK pathways, although the specific contributions of these pathways to cell survival and cell cycling are not yet fully understood. In this work, we show that PI3K transduces a signal that promotes cell cycling in response to TPO both in a Mpl-bearing cell line and in primary MK progenitors and that that this signal can be distinguished from its effects on cell survival.
      TPO is among many growth factors that have been shown to stimulate PI3K; however, the mechanism by which TPO activates PI3K is not understood. Mpl does not contain the YXXM or YVAC motifs, which are classically required to bind the SH2 domains of p85 (
      • Damen J.E.
      • Cutler R.L.
      • Jiao H.
      • Yi T.
      • Krystal G.
      ). In addition, attempts to immunoprecipitate p85 with Mpl have been unsuccessful (
      • Miyakawa Y.
      • Rojnuckarin P.
      • Habib T.
      • Kaushansky K.
      ), suggesting that the interaction is indirect or of low affinity. It has been demonstrated that a complex forms between Gab-2 and SHP-2 that may mediate the activation of PI3K by TPO in BaF3/Mpl cells (
      • Rojnuckarin P.
      • Drachman J.G.
      • Kaushansky K.
      ).
      Because our conclusions rely largely on the use LY294002, it is important to consider its specificity as an inhibitor of PI3K. LY294002 works by reversibly inhibiting the ability of PI3K to catalyze the transfer of phosphate from ATP to its substrate (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ). LY294002 and the other commonly used PI3K inhibitor wortmannin have been shown to inhibit PI3K-like kinases, such as DNA-PK and ATM, as well as casein kinase (
      • Izzard R.A.
      • Jackson S.P.
      • Smith G.C.
      ,
      • Rosenzweig K.E.
      • Youmell M.B.
      • Palayoor S.T.
      • Price B.D.
      ,
      • Davies S.P.
      • Reddy H.
      • Caivano M.
      • Cohen P.
      ); although we have not looked directly for an effect on these other kinases, we have attempted to perform our assays at the lowest effective LY294002 doses. It is notable that the cell cycle block attributed to the inhibition of DNA-PK and ATM by LY294002 was reported to be in G2/M, due to the presence of unrepaired DNA breaks occurring after radiation (
      • Rosenzweig K.E.
      • Youmell M.B.
      • Palayoor S.T.
      • Price B.D.
      ). Inhibitors of PI3K have also been noted to inhibit the distantly related kinase FRAP/TOR, which is an upstream regulator of p70S6K and has been implicated in cell cycle regulation (
      • Grewe M.
      • Gansauge F.
      • Schmid R.M.
      • Adler G.
      • Seufferlein T.
      ). We did not note, however, any effect on cell survival or cell cycling when treating cells with rapamycin (data not shown), which effectively inhibits TOR (
      • Brown E.J.
      • Albers M.W.
      • Shin T.B.
      • Ichikawa K.
      • Keith C.T.
      • Lane W.S.
      • Schreiber S.L.
      ), and expression of constitutively active Akt is sufficient to rescue MyrAktMpl cells from LY294002-induced cell cycle arrest and apoptosis. Finally, several reports indicate that inhibition of PI3K can result in reduced activation of MAPK (
      • Cross D.A.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ,
      • Ferby I.M.
      • Waga I.
      • Hoshino M.
      • Kume K.
      • Shimizu T.
      ), whereas other reports indicate that inhibition of PI3K has no effect on activation of MAPK (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ,
      • Scheid M.P.
      • Duronio V.
      ). In BaF3/Mpl cells, the concentrations of LY294002 that we employed did not result in significant inhibition of the MAPK pathway as measured by ERK phosphorylation; furthermore, specific inhibition of the MAPK pathway using PD98059 did not significantly reduce TPO-induced cell survival or cell cycling (data not shown). The fact that expression of a constitutively active Akt completely rescued TPO-treated BaF3/Mpl cells from the effects of LY294002, allowing them to cycle normally, provides a strong argument for both the specificity of LY294002 in PI3K signaling and the role of Akt in PI3K-dependent cell cycling and survival in response to TPO. Active Akt, however, was not sufficient to promote significant cell cycling and survival by itself, and a second TPO-dependent signal was required. Therefore, our results differ somewhat from those of others, who reported that constitutively active Akt could provide growth factor independence from IL-3 (
      • Songyang Z.
      • Baltimore D.
      • Cantley L.C.
      • Kaplan D.R.
      • Franke T.F.
      ) or IL-4 (
      • Cerezo A.
      • Martinez A.C.
      • Lanzarot D.
      • Fischer S.
      • Franke T.F.
      • Rebollo A.
      ).
      The role of PI3K in promoting cell survival is well documented (
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ,
      • Songyang Z.
      • Baltimore D.
      • Cantley L.C.
      • Kaplan D.R.
      • Franke T.F.
      ,
      • Downward J.
      ,
      • Yao R.
      • Cooper G.M.
      ,
      • Minshall C.
      • Arkins S.
      • Freund G.G.
      • Kelley K.W.
      ,
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ,
      • Blume-Jensen P.
      • Janknecht R.
      • Hunter T.
      ). In this work, we observed that PI3K is also required for TPO-induced cell cycling. We did observe increased apoptosis in response to the inhibition of PI3K, but the effects of LY294002 on cell cycling were evident as much as 16 h before significant apoptosis was measured by Annexin and Apotag assays. The mechanisms by which PI3K might promote cell proliferation are not known. Nevertheless, our data would suggest that in TPO signaling, PI3K activity is important for the cells to transit from G1 to S phase in both mitotically (BaF3/Mpl cells and 2N MKs) and endomitotically (4N MKs) cycling cells. Endomitosis is a defining feature of maturing megakaryocytes that is thought to involve a specific defect in late anaphase, with the resulting failure of telophase and the development of large polyploid cells (
      • Nagata Y.
      • Muro Y.
      • Todokoro K.
      ,
      • Vitrat N.
      • Cohen-Solal K.
      • Pique C.
      • Le Couedic J.P.
      • Norol F.
      • Larsen A.K.
      • Katz A.
      • Vainchenker W.
      • Debili N.
      ,
      • Roy L.
      • Coullin P.
      • Vitrat N.
      • Hellio R.
      • Debili N.
      • Weinstein J.
      • Bernheim A.
      • Vainchenker W.
      ). Cells that become polyploid are theorized to have a functional advantage from increased size. The differences between G1 of the mitotic cell cycle and the endomitotic cell cycle are not understood, but G1 in endomitosis is significantly shorter and thus is likely to be regulated differently. In addition, the D-type cyclin in MKs is primarily cyclin D3, whereas much of the data in the literature pertains to cyclin D1 (
      • Ando K.
      • Ajchenbaum-Cymbalista F.
      • Griffin J.D.
      ,
      • Wang Z.
      • Zhang Y.
      • Kamen D.
      • Lees E.
      • Ravid K.
      ,
      • Zimmet J.M.
      • Ladd D.
      • Jackson C.W.
      • Stenberg P.E.
      • Ravid K.
      ,
      • Bacqueville D.
      • Casagrande F.
      • Perret B.
      • Chap H.
      • Darbon J.M.
      • Breton-Douillon M.
      ). G1arrest has been previously described in response to inhibition of PI3K in several nonhematopoietic cell types, and in these systems, it has been attributed to decreased cdk2 and cdk4 activities, decreased cyclin D1, and increased p27kip (
      • Bacqueville D.
      • Casagrande F.
      • Perret B.
      • Chap H.
      • Darbon J.M.
      • Breton-Douillon M.
      ,
      • Casagrande F.
      • Bacqueville D.
      • Pillaire M.J.
      • Malecaze F.
      • Manenti S.
      • Breton-Douillon M.
      • Darbon J.M.
      ,
      • Takuwa N.
      • Fukui Y.
      • Takuwa Y.
      ). Activated Akt has been reported to increase the posttranslational stability of cyclin D1 through its inhibition of GSK-3β (
      • Takuwa N.
      • Fukui Y.
      • Takuwa Y.
      ,
      • Diehl J.A.
      • Cheng M.
      • Roussel M.F.
      • Sherr C.J.
      ). In hematopoietic cells, PI3K signals were shown to be necessary and sufficient for stimulation of E2F activity by IL-2 in a T cell line (
      • Brennan P.
      • Babbage J.W.
      • Burgering B.M.
      • Groner B.
      • Reif K.
      • Cantrell D.A.
      ). Active Akt was shown to increase cyclin D1 and decrease p27kip in this system; however, PI3K signals were not sufficient for cell cycling.
      In summary, we have shown that TPO induces PI3K activity and that this activity is required for optimal cell survival and cell cycling in response to TPO, both in a factor-dependent leukemic cell line and in primary MK progenitors. The absence of PI3K activity results in a block in the cell cycle, which occurs in G1 in BaF3/Mpl cells and results in absence of endomitosis in MK progenitors. Constitutively active Akt can replace the activity of PI3K in promoting cell cycling and cell survival, although without another non-PI3K TPO signal, it is not itself sufficient for cell growth, implying a requirement for a second signal from another pathway such as Jak/STAT or MAPK.

      Acknowledgments

      We thank Kathy Allen and David Coder for their expert assistance in cell sorting and cell cycle analysis.

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