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First published online 31 January 2006
doi: 10.1242/jcs.02784

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Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes

¹ Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy
² Department of Cellular Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy
³ Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy
⁴ T. Jefferson University, Kimmel Cancer Institute, 233 South 10th St, Philadelphia, PA 19107-5541, USA

View larger version (26K): [in a new window]   Fig. 1. The effect of MAPK and PI3K inhibitors on MK proliferation. Proliferation of HPCs along the megakaryocytic lineage in the presence of 100 ng/ml TPO alone (Control), or in combination with 10 µM PD98059 or 10 µM U0126 or 10 µM LY294002 or 50 nM rapamycin. All the inhibitors were dissolved in DMSO, and an equivalent volume of DMSO was added to the control cultures. Data are expressed as the mean ± s.e.m. of six separate experiments.

View larger version (35K): [in a new window]   Fig. 2. The effect of PD98059 and rapamycin on megakaryocytic morphology. (A) Cell morphology of MKs, derived from peripheral blood HPCs grown in serum-free liquid culture with 100 ng/mL TPO supplemented or not with 10 µM PD98059 or 50 nM rapamycin. Representative results from cells at day 12 of culture are shown (May-Grünwald staining; original magnification x400). (B) Effects of PD98059 or rapamycin treatment on polyploidization. The percentage of HPC-derived MKs with 1, 2, 4 or more than 8 lobes was evaluated by morphological analysis. The data relative to day 12 of culture are presented as the mean ± s.e.m. of six independent experiments. *P<0.05, **P<0.01, ***P<0.001 when compared with the control by Student's t-test.

View larger version (17K): [in a new window]   Fig. 3. Analysis of the DNA content of PD98059- or rapamycin-treated megakaryocytic cells. Polyploidy (2N, 4N, etc) was evaluated by flow cytometry analysis at day 10 of culture on MKs grown either in the presence of 100 ng/mL TPO alone (Control) or in combination with 10 µM PD98059 or 50 nM rapamycin. MKs were stained with PI as described in the Materials and Methods. A representative experiment from three separate experiments is shown.

View larger version (19K): [in a new window]   Fig. 4. Membrane phenotype of megakaryocytic cells. HPCs grown in serum-free medium in the presence of 100 ng/ml TPO alone (Control), or in combination with 10 µM PD98059 or 50 nM rapamycin were analyzed throughout the culture (from day 0 up to day 12) by flow cytometry using PE- or FITC-conjugated mAbs. The percentages of CD34+, CD41+, CD61+, CD42b+ and CD62+ cells are shown as the mean ± s.e.m. of three independent experiments.

View larger version (40K): [in a new window]   Fig. 5. Effects of MAPK and PI3K inhibitors on the TPO-induced signaling pathway. (A) 2x105 CD34+ cells were incubated overnight in serum-free medium starvation conditions, and then pre-treated for a brief period (20 minutes) with specific MAPK or PI3K pathway inhibitors followed by TPO incubation (40 minutes). The cells were then lyzed and the protein separated by SDS PAGE. The blots were probed with specific antibodies against phospho-ERK 1/2 (P-ERK 1/2), ERK 1/2, P-AKT, AKT, P-p70S6K (Thr389), P-p70S6K (Thr421/Ser424) or p70S6K. Representative immunoblots are shown. (B) The densitometric analysis of phosphorylated protein blots was performed with the chemidoc program Biorad, and expressed as fold increase of unstimulated cells, after normalization of total ERK, AKT and p706SK protein of each loading, and is presented as a bar graph. Results are expressed as the mean ± s.d. from three independent experiments.

View larger version (81K): [in a new window]   Fig. 6. Cellular localization of cyclin D1 and cyclin D3. Differentiating MKs grown for 3, 6 and 10 days either in the absence (Control) or in presence of PD98059 or rapamycin were cytocentrifuged, fixed and labeled with anti-cyclin D1 (A) or anti-cyclin D3 (B) and then with FITC-conjugated anti-mouse IgGs. Immunofluorescent cells were detected using an optical microscope equipped for immunofluorescence. Original magnifications: A, 630x; B, 400x.

View larger version (19K): [in a new window]   Fig. 7. Schematic model of the TPO-induced signaling pathway in megakaryocytopoiesis. (A) The sites of inhibition in the MAPK and PI3K pathways are shown with particular emphasis on the proposed mechanism, through PI3K-AKT-mTOR-p70S6K-cyclin D3, mediating MK polyploidization (green). (B) The phospho-ERK (p-ERK) and phospho-AKT (p-AKT) expression are inversely correlated in promoting MK proliferation and endomitosis, respectively.

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