Maturation of Megakaryoblastic Cells Is Accompanied by Upregulation of G_Sα-L Subtype and Increased cAMP Accumulation

Summary

In platelets and megakaryoblasts G_s, the trimeric G-protein that stimulates adenylyl cyclase, is present in a short, 45 kDa, and a long, 52 kDa isoform termed G_sα-S and G_sα-L, respectively. To assess the relative contribution of these isoforms in the cellular synthesis of cAMP, the ratio G_sα-S/G_sα-L was changed in the megakaryoblastic cell line DAMI by inducing cell maturation with recombinant human thrombopoietin (TPO) or the phorbol ester PMA. Flow cytometric analysis confirmed that this treatment induced a moderate (TPO) and extensive (PMA) increase in nuclear ploidy and expression of the glycoproteins-IIIa and -Ib. Northern blot analysis revealed down-regulation of total G_S-mRNA after treatment of DAMI-cells with TPO and PMA. Western blot analysis showed significant (P <0.05) upregulation of G_s-L with respective amounts of 27 ± 4% of total G s in untreated cells, 35 ± 1% in TPO- and 41 ± 3% in PMA-treated DAMI cells (n = 3-4). DAMI cells contained 6 ± 1 pmol cAMP/10⁶ cells, which was not changed by treatment with TPO or PMA. In untreated cells this level increased to 70 ± 9 pmol cAMP/10⁶ cells after 10 min stimulation with 1 μmol/l of the stable prostacyclin analog iloprost. The same stimulation with iloprost resulted in 165 ± 32 pmol cAMP/10⁶ in TPO-treated cells and in 588 ± 100 pmol cAMP/10⁶ in cells treated with PMA. Thus, a shift from G_sα-S to G_sα-L during megakaryoblast maturation strongly potentiates the production of cAMP. A similar shift may occur during normal megakaryocyte maturation and may explain the extreme sensitivity to prostacyclin of platelets, which contain G_sα-S and G_sα-L in approximately equal amounts.

• References

• 1 van Willigen G, Donath J, Lapetina EG, Akkerman JWN. Identification of α-subunits of trimeric GTP-binding proteins in human platelets by RT-PCR. Biochem Biophys Res Commun 1995; 214: 254-62.
• 2 van der Vuurst H, van Willigen G, van Spronsen A, Hendriks M, Donath J, Akkerman JWN. Signal transduction through trimeric G-proteins in megakaryoblastic cell lines. Arterioscler Thromb Vasc Biol 1997; 17: 1830-6.
• 3 Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, Methia N, Breton-Gorius J, Cosman D, Vainchenker W. c-Mpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 1994; 369: 571-4.
• 4 Metcalf D. Blood: Thrombopoietin – At last. Nature 1994; 369: 519-20.
• 5 Alexander WS, Maurer AM, Novak U, Harrison-Smith M. Tyrosine-599 of the c-Mpl receptor is required for Shc phosphorylation and the introduction of cellular differentiation. EMBO J 1996; 15: 6531-40.
• 6 Methia N, Louache F, Vainchenker W, Wendling F. Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis. Blood 1993; 82: 1395-401.
• 7 Debili N, Wendling F, Cosman D, Titeux M, Florindo C, Dusanter-Fourt I, Schooley K, Methia N, Charon M, Nador R, Bettaieb A, Vainchenker W. The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets. Blood 1995; 85: 391-401.
• 8 Wilhide CC, Van Dang C, Dipersio J, Kenedy AA, Bray PF. Overexpression of cyclin D1 in the Dami megakaryocytic cell line causes growth arrest. Blood 1995; 86: 294-304.
• 9 Greenberg AM, Rosenthal DS, Greeley TA, Tantravahi R, Handin RL. Characterization of a new megakaryocytic cell line: the Dami cell. Blood 1988; 72: 1968-77.
• 10 Davis LG, Dibner MD, Battey JF. Basic Methods in Molecular Biology. New York: Elsevier Science Publishing Co., Inc.; 1986. pp 129-56.
• 11 Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 1980; 77: 5201-5.
• 12 Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983; 132: 6-13.
• 13 Feinberg AP, Vogelstein B. Addendum, A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1984; 137: 266-7.
• 14 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescent properties. J Biol Chem 1985; 260: 3440-50.
• 15 Gillis JM, Gailly P. Measurements of [Ca²⁺]_i with the diffusable Fura-2 AM: Can some potential pitfalls be evaluated. Biophys J 1994; 67: 476-7.
• 16 van der Vuurst H, Hendriks M, van Willigen G, Akkerman JWN. Maturation of megakaryoblastic cells by TPO or phorbolester is accompanied by a shift in expression of G_sα subtypes and a change in cAMP accumulation. Thromb Haemost 1997; 78: 175 (abstract).
• 17 Brass LF, Woolkalis MJ. Dual regulation of cyclic AMP formation by thrombin in HEL cells, a leukaemic cell line with megakaryocytic properties. Biochem J 1992; 281: 73-80.
• 18 Kozasa T, Itoh H, Tsukamoto T, Kaziro Y. Isolation and characterization of the human G_sα gene. Proc Natl Acad Sci USA 1988; 85: 2081-5.
• 19 Nanoff C, Boehm S, Hohenegger M, Schutz W, Freissmuth M. 2’,3’-dialdehyde GTP as an irreversible G protein antagonist. J Biol Chem 1994; 269: 31999-2007.
• 20 Taussig R, Gilman AG. Mammalian membrane-bound adenylyl cyclases. J Biol Chem 1995; 270: 1-4.
• 21 Cooper DMF, Mons N, Karpen JW. Adenylyl cyclases and the interaction between calcium and cAMP signaling. Nature 1995; 374: 421-4.
• 22 Sheth SB, Colman RW. Regulatory and catalytic domains of platelet cAMP phosphodiesterases: Targets for drug design. Semin Hematol 1995; 32: 110-9.
• 23 Kaushansky K, Broudy VC, Grossmann A, Humes J, Lin N, Ren HP, Bailey MC, Papayannopoulou T, Forstrom JW, Sprugel KH. Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy. J Clin Invest 1995; 96: 1683-7.
• 24 Kaushansky K, Lok S, Holly RD, Broudy VC, Lin N, Bailey MC, Forstrom JW, Buddle MM, Oort PJ, Hagen FS, Roth GJ, Papayannopoulou T, Foster DC. Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature 1994; 369: 568-71.
• 25 Matsumura I, Kanakura Y, Kato T, Ikeda H, Horikawa V, Ishikawa J, Kitayama H, Nishiura T, Tomiyama Y, Miyazaki H, Matsuzawa Y. The biologic properties of recombinant human thrombopoietin in the proliferation and megakaryocytic differentiation of acute myeloblastic leukemia cells. Blood 1996; 88: 3074-82.
• 26 Morgan DA, Soslau G, Brodsky I. Thrombopoietin (TPO) induces bi-potential HU-3 cell line to lineage commitment with enhancement of megakaryocyte antigen expression and loss of erythroid potential. (Abstract) Blood 1995; 86 (supplement) 3604.
• 27 Bacon CM, Tortolani PJ, Shimosaka A, Rees RC, Longo DL, O’Shea JJ. Thrombopoietin (TPO) induces tyrosine phosphorylation and activation of STAT5 and STAT3. FEBS Lett 1995; 370: 63-8.
• 28 Nagata Y, Todokoro K. Thrombopoietin induces activation of at least two distinct signaling pathways. FEBS Lett 1995; 377: 497-501.
• 29 Ezumi Y, Takayama H, Okuma M. Thrombopoietin, c-Mpl ligand, induces tyrosine phosphorylation of Tyk2, JAK2, and STAT3, and enhances agonists-induced aggregation in platelets in vitro. FEBS Lett 1995; 374: 48-52.
• 30 Yamada M, Komatsu N, Okada K, Kato T, Miyazaki H, Miura Y. Thrombopoietin induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in a human thrombopoietin-dependent cell line. Biochem Biophys Res Commun 1995; 217: 230-7.
• 31 Moroi M, Ohnuma M, Komatsu N, Miura Y, Sato T, Jung SM. Phorbol ester treatment of two megakaryoblastic cell lines, CMK and UT-7, induces specific protein composition changes with non-coincident time courses. Biochim Biophys Acta Mol Cell Res 1993; 1177: 139-46.
• 32 Ogura M, Morishima Y, Okumura M, Hotta T, Takamoto S, Ohno R, Hirabayashi N, Nagura H, Saito H. Functional and morphological differentiation induction of a human megakaryoblastic leukemia cell line (MEG-01s) by phorbol diesters. Blood 1988; 72: 49-60.
• 33 Adachi M, Ryo R, Yoshida A, Sugano W, Yasunaga M, Saigo K, Yamaguchi N, Sato K, Kaibuchi K, Takai Y. Induction of smg p21/rap1Ap21/krev-1 p21 gene expression during phorbol ester-induced differentiation of a human megakaryocytic leukemia cell line. Oncogene 1992; 7: 323-9.
• 34 Namciu S, Lieberman MA, Stavnezer E. Induction of the c-ski protooncogene by phorbol ester correlates with induction of megakaryocyte differentiation. Oncogene 1994; 9: 1407-16.
• 35 Dorn GW II, Davis MG, D’Angelo DD. Gene expression during phorbol ester-induced differentiation of cultured human megakaryoblastic cells. Am J Physiol 1994; 266: C1231-9.
• 36 Bennett BD, Cowley S, Jiang S, London R, Deng B, Grabarek J, Groopman JE, Goeddel DV, Avraham H. Identification and characterization of a novel tyrosine kinase from megakaryocytes. J Biol Chem 1994; 269: 1068-74.
• 37 Konkle BA, Schick PK, He X, Liu RJ, Mazur EM. Plasminogen activator inhibitor-1 mRNA is expressed in platelets and megakaryocytes and the megakaryoblastic cell line CHRF- 288. Arterioscler Thromb 1993; 13: 669-74.
• 38 Hayward CP, Bainton DF, Smith JW, Horsewood P, Stead RH, Podor TJ, Warkentin TE, Kelton JG. Multimerin is found in the alpha-granules of resting platelets and is synthesized by a megakaryocytic cell line. J Clin Invest 1993; 91: 2630-9.
• 39 Nagata K, Okano Y, Nozawa Y. Identification of heterotrimeric GTP-binding proteins in human megakaryoblastic leukemia cell line, MEG-01, and their alteration during cellular differentiation. Life Sci 1995; 57: 1675-81.