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

The endoplasmic reticulum (ER) plays a key role in Ca2+ signalling through Ca2+ release via inositol 1,4,5-trisphosphate receptors (InsP(3)-Rs) and Ca 2+ uptake by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs). Here, we in vestigated the organization of platelet ER and its biogenesis during megaka ryocytopoiesis. First, erythro/megakaryoblastic MEG 01, UT7, M-O7e and CHRF 288-11 cell lines, platelets and thrombopoietin-induced 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. Se cond, these studies were extended to the newly described alternatively spli ced SERCA3a-c RNAs and InsP(3)-Rs using the in vitro model of PMA-induced d ifferentiation of MEG 01 cells. Time-course and dose-response studies showe d a maximal approx. 4-fold up-regulation of SERCA3 proteins using 10(-8) M PMA for 3 days, which paralleled induction of GPIIIa expression. SERCA3 ind uction was found to occur at the level of mRNA. The modulation of the diffe rent SERCA3 species (i.e. 3a, 3b and 3c) was isoform-specific: while SERCA3 a was slightly increased, an approx. 3-fold induction of SERCA3b, and a 4-f old induction of SERCA3c, was observed after 24 h of PMA treatment. Isoform -specific Western blotting and/or reverse transcriptase PCR studies showed that InsP(3)-R types I, II and III are expressed in MEG 01 cells, as well a s in platelets. Study of the expression of these InsP(3)-R types in PMA-ind uced MEG 01 cells revealed that: (i) InsP(3)-RI protein and mRNA showed no changes; (ii) InsP(3)-RII mRNA was upregulated and peaked at hour 48 and (i ii) InsP(3)-RIII mRNA and protein showed a transitory maximal 3- and 2.3-fo ld increase at hours 6 and 30, respectively. Upon PMA treatment of CHRF 288 -11 cells, in which GPIIIa is not induced upon treatment, a similar pattern of regulation of InsP(3)-R types II and III was seen, but a distinct patte rn of SERCA3 regulation was observed. These results suggest a profound reor ganization of ER-protein patterns during megakaryocytopoiesis and underline the role of SERCA3 gene regulation in the control of Ca2+-dependent platel et functions.