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. 2004 Sep 6;200(5):569-80.
doi: 10.1084/jem.20040762. Epub 2004 Aug 30.

Lnk inhibits Tpo-mpl signaling and Tpo-mediated megakaryocytopoiesis

Wei Tong  1 , Harvey F Lodish
Affiliations

V体育安卓版 - Lnk inhibits Tpo-mpl signaling and Tpo-mediated megakaryocytopoiesis

Wei Tong et al. J Exp Med. .

Abstract

Thrombopoietin (Tpo) is the primary cytokine regulating megakaryocyte development and platelet production. Tpo signaling through its receptor, c-mpl, activates multiple pathways including signal transducer and activator of transcription (STAT)3, STAT5, phosphoinositide 3-kinase-Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor and immunoreceptor signaling. Here, we show that Lnk overexpression negatively regulates Tpo-mediated cell proliferation and endomitosis in hematopoietic cell lines and primary hematopoietic cells. Lnk attenuates Tpo-induced S-phase progression in 32D cells expressing mpl, and Lnk decreases Tpo-dependent megakaryocyte growth in bone marrow (BM)-derived megakaryocyte culture. Consistent with this result, we found that in both BM and spleen, Lnk-deficient mice exhibited increased numbers of megakaryocytes with increased ploidy compared with wild-type mice VSports手机版. In addition, Lnk-deficient megakaryocytes derived from BM and spleen showed enhanced sensitivity to Tpo during culture. The absence of Lnk caused enhanced and prolonged Tpo induction of STAT3, STAT5, Akt, and MAPK signaling pathways in CD41+ megakaryocytes. Furthermore, the Src homology 2 domain of Lnk is essential for Lnk's inhibitory function. In contrast, the conserved tyrosine near the COOH terminus is dispensable and the pleckstrin homology domain of Lnk contributes to, but is not essential for, inhibiting Tpo-dependent 32D cell growth or megakaryocyte development. Thus, Lnk negatively modulates mpl signaling pathways and is important for Tpo-mediated megakaryocytopoiesis in vivo. .

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Figures

Figure 1.
Figure 1.
Lnk inhibits 32D cell growth via blocking S-phase progression, and the SH2 domain of Lnk is essential for its inhibitory function. (a) Establishment of mpl-expressing 32D cells. Parental 32D cells and cells stably expressing mpl were cultured in IL-3 or different concentrations of Tpo. Live cell numbers in the presence of Tpo relative to that in IL-3 after 3 d of culture were determined by MTT absorbance. (b) Overexpression of Lnk in 32D/mpl cells inhibits cell growth in response to Tpo. We introduced either vector alone or WT Lnk into 32D/mpl cells and determined the proportion of infected cells as those that express GFP 2 d later. We measured the GFP+ fraction every 3 d, and the percentage of GFP+ cells relative to the infection rate 2 d after infection was plotted. Results are representative of more than five independent experiments. (c) 2 d after the infection, total cell numbers were counted and GFP+ percentages were determined daily. The numbers of vector- or Lnk-expressing GFP+ cells were calculated and plotted (mean ± SD). (d) Cells infected with either vector alone or Lnk were subjected to cell cycle analysis after overnight culture in the presence of Tpo; only GFP+ cells were gated in both plots. Results are representative of three independent experiments. (e) We introduced the WT or the mutant Lnk cDNA constructs into 32D/mpl cells and measured the GFP+ fraction every 3 d as described in b. The cells were maintained in the presence of 1 ng/ml Tpo, and the percentage of GFP+ cells relative to the infection rate 2 d after infection was plotted. Results are representative of more than three independent experiments.
Figure 2.
Figure 2.
The SH2 domain of Lnk is critical for inhibiting sustained STAT5 and p42/44 MAPK phosphorylation induced by Tpo. 32D/mpl cells infected with either vector alone or the WT or the mutant forms of Lnk were purified, stimulated with Tpo, and lysed at indicated intervals followed by Western blotting analysis. (a) STAT5 phosphorylation and protein levels after Tpo administration. The triangles indicate the 120-min time points. (b) P42/44 MAPK phosphorylation and protein levels after Tpo administration. White lines indicate that intervening lanes have been spliced out. Representatives of three independent experiments are shown.
Figure 3.
Figure 3.
The SH2 domain of Lnk is required to reduce the size and ploidy of megakaryocytes. Lin− BM cells were transduced with either vector alone, or with WT or mutant forms of Lnk, and GFP+ cells were purified and cultured in serum-free media containing only Tpo (see Materials and Methods). After 4 d of growth and differentiation, the diameters of the megakaryocytes were measured and the median diameters (±SD) are shown in a. Student's t tests were performed on each form of Lnk compared to the vector control, and p-values are shown on the top of each bar. (b) Differential interference contrast images of megakaryocytes derived from vector-transduced Lin− progenitor cells (left) and megakaryocytes expressing Lnk (right). White arrows, long thin arrows, and short wide arrows indicate megakaryocytes at different developmental stages (see Results). Double-lined arrows point to dead cells that do not respond to Tpo. Bar, 50 μm. (c–g) Ploidy analysis of the megakaryocytes derived from vector-transduced (c), Lnk-transduced (d), Lnk (W191A)-transduced (e), Lnk (R364E)-transduced (f), and Lnk (Y536F)-transduced (g) Lin− BM cells. GFP+ cells were gated, and cells with 8N and greater ploidy were analyzed.
Figure 4.
Figure 4.
Increased megakaryocyte ploidy in Lnk nullizygous mice. CD41+ megakaryocytes from WT BM (a) and Lnk-deficient BM (b) were analyzed for DNA content. Only mature megakaryocytes with 8N and greater ploidy are shown. (c) Quantification of the percentages of individual ploidy classes (≥8N) of BM megakaryocytes (mean ± SD). Mean ploidy was calculated as stated in Materials and Methods. Student's t test was performed. P < 0.001.
Figure 5.
Figure 5.
Enhanced megakaryocyte growth and ploidy in response to Tpo during in vitro megakaryocyte culture. Total BM (a) or spleen (b) cells from WT mice or Lnk nullizygous mice were cultured in different concentrations of Tpo for 4 d, the numbers of CD41+ megakaryocytes with 8N and greater ploidy were enumerated (mean ± SD). (c) Mean ploidies (±SD) of BM-derived megakaryocytes were plotted. A Student's t test was performed, and p-values are shown on top of each pair of points.
Figure 6.
Figure 6.
Enhanced Tpo-induced signaling pathways in Lnk-deficient megakaryocytes. (a–c) CD41+ BM megakaryocytes from WT mice or Lnk nullizygous mice were stimulated with 0, 1, or 10 ng/ml of Tpo for 10 min. Protein lysates from equal numbers of cells were subjected to Western blotting analysis. Phosphorylation and total protein levels of p42/44MAPK and Akt (a), STAT5 (b), and STAT3 (c) are shown. (d and e) Purified CD41+ BM megakaryocytes from WT mice or Lnk nullizygous mice were stimulated with 10 ng/ml of Tpo, and lysed at indicated intervals followed by Western blotting analysis. Phosphorylation and total protein levels of Akt (d) and p42/44MAPK (e) are shown.
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References

    1. Kaushansky, K. 1995. Thrombopoietin: the primary regulator of platelet production. Blood. 86:419–431. - PubMed
    1. Kaushansky, K. 2003. Thrombopoietin: a tool for understanding thrombopoiesis. J. Thromb. Haemost. 1:1587–1592. - "VSports最新版本" PubMed
    1. Gurney, A.L., K. Carver-Moore, F.J. de Sauvage, and M.W. Moore. 1994. Thrombocytopenia in c-mpl-deficient mice. Science. 265:1445–1447. - PubMed
    1. de Sauvage, F.J., K. Carver-Moore, S.M. Luoh, A. Ryan, M. Dowd, D.L. Eaton, and M.W. Moore. 1996. Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin. J. Exp. Med. 183:651–656. - PMC - PubMed
    1. Alexander, W.S., A.W. Roberts, N.A. Nicola, R. Li, and D. Metcalf. 1996. Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl. Blood. 87:2162–2170. - PubMed

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