Tescalcin is an essential factor in megakaryocytic differentiation associated with Ets family gene expression
Konstantin Levay, Vladlen Z. Slepak
Konstantin Levay, Vladlen Z. Slepak
Published September 4, 2007
Citation Information: J Clin Invest. 2007;117(9):2672-2683. https://doi.org/10.1172/JCI27465.
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Research Article Hematology

Tescalcin is an essential factor in megakaryocytic differentiation associated with Ets family gene expression

Abstract

We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation, such as expression of specific cell surface antigens, inhibition of cell proliferation, and polyploidization. Conversely, knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin, the expression of Fli-1, Ets-1, and Ets-2 transcription factors, but not GATA-1 or MafB, is blocked. Thus, tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation.

Authors

Konstantin Levay, Vladlen Z. Slepak

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Figure 2

Overexpression of tescalcin in K562 cells induces events of early megakaryocytic differentiation.

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Overexpression of tescalcin in K562 cells induces events of early megaka...
(A) Overexpression of tescalcin leads to an increased level of surface GPIIb. Control [K562-Ctrl(+)] and tescalcin overexpressing [K562-Tsc(+)] cells were stained with FITC-conjugated CD41-specific antibody. Samples were analyzed using BD FACScan, with a minimum of 10,000 events acquired per sample. Expression profile of surface CD41 in K562-Ctrl(+) and K562-Tsc(+) cells is shown as filled histogram. FITC-conjugated isotype mouse IgG was used as negative control (open histogram). (B) Overexpression of tescalcin promotes polyploidy in K562 cells. K562-Ctrl(+) and K562-Tsc(+) cells were fixed and stained with propidium iodide and their DNA content was analyzed by FACS. PI log, propidium iodide, logarithmic scale. (C) The onset of PMA-induced polyploidy occurs faster in tescalcin-overexpressing cells. K562-Ctrl(+) and K562-Tsc(+) cells were cultured in the presence of PMA for indicated times and analyzed as described in B. (D) Cyclin D3 accumulation in tescalcin-overexpressing cells is increased. Wild-type [K562-WT], K562-Ctrl(+), and K562-Tsc(+) cell lysates were probed with cyclin D3– and tescalcin-specific antibodies. β-Actin was used as a loading control. (E) Reduction of proliferation rate in tescalcin-overexpressing cells. Absorbance at 490 nm of K562-Ctrl(+) and K562-Tsc(+) cell lines were compared in the MTS-based cell proliferation assay, as described in Methods. Data represent 3 independent experiments (mean ± SD).