Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study

Abstract

The endoplasmic reticulum (ER) plays a key role in Ca# + signalling through Ca# + release via inositol 1,4,5-trisphosphate receptors (InsP $ -Rs) and Ca# + uptake by sarco\endoplasmic reticulum Ca# + -ATPases (SERCAs). Here, we investigated the organization of platelet ER and its biogenesis during megakaryocytopoiesis. First, erythro\megakaryoblastic MEG 01, UT7, M-O7e and CHRF 288-11 cell lines, platelets and thrombopoietininduced UT7-Mpl cells were selected for the study of SERCA2b and SERCA3 proteins by Western blotting using the antibodies IID8 and PL\IM430, respectively. As judged by platelet glycoprotein IIIa (GPIIIa) expression, an increase in SERCA3 proteins was observed while that of SERCA2b remained unchanged throughout maturation. Second, these studies were extended to the newly described alternatively spliced SERCA3a-c RNAs and InsP $ -Rs using the in itro model of PMA-induced differentiation of MEG 01 cells. Time-course and dose-response studies showed a maximal approx. 4-fold up-regulation of SERCA3 proteins using 10 −) M PMA for 3 days, which paralleled induction of GPIIIa expression. SERCA3 induction was found to occur at the level of mRNA. The modulation of the different SERCA3 species (i.e. 3a, 3b and 3c) was isoform-specific : while

Human platelet Ca2+-ATPases: New markers of cell differentiation as illustrated in idiopathic scoliosis

“…The combination of these findings with those previously obtained [12,21], and studies performed on human megakaryocytopoiesis (P. Nurden, N. Debili, W. Vainchenker, R. Bobe, R. Bredoux, E. Corvazier, R. Combrié, E. Fressinaud, D. Meyer, A. Nurden and J. Enouf, Blood on line) establishes that platelet maturation involves a concerted down-regulation of PMCA4b protein and PARP, coupled to an up-regulation of SERCA3a protein. Table III summarizes the overall data on the expression of PMCA4b and SERCA3a proteins along with platelet maturation and tentatively locates the apparent differentiation stages of platelets of patients with AIS, according to their expressions of these Ca 2þ -ATPases.…”

“…The expression of actin was used as internal control. Figure 4 shows the results of the expression of Ca 2þ -ATPases obtained in MEG 01 cells treated for up to 3 days with 10 À8 M PMA [21]. Experiments were also performed for 2 days by using 10 À7 M, which gave similar results (data not shown).…”

“…This hypothesis was based on previous findings obtained using different experimental models pointing to a down-modulation of the expression of PMCA4b protein [12] and an up-regulation of total SERCA3 proteins visualized by PL/IM430 antibody [21] upon platelet maturation. Here, we investigated the expression of PMCA4b and SERCA3a isoforms in the same model of PMA-treated MEG 01 cells and looked for an additional role of caspase-3 activity in the down-regulation of PMCA4b isoform.…”

“…Platelets were isolated as previously described [21]. Total RNA and protein were prepared using TRIzol solution.…”

N‐methyl‐d‐aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines

“…Normal megakaryocytic differentiation requires fine regulation of SOCE 18. TPO increases expression of molecules that facilitate SOCE, including those that release and refill intracellular Ca 2+ stores 19, 20, 21, 22. Emerging data indicate that SOCE peaks during proplatelet formation and contributes to megakaryocyte adhesion and motility 23…”