Expression of transient receptor potential mRNA isoforms and Ca2+ influx in differentiating human stem cells and platelets

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Abstract

Store-regulated Ca2+ entry (SOCE) is an important mechanism of elevating cytosolic [Ca2+]i in platelets, though the Ca2+ influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34+) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61+/CD42blow) and mature (CD61+/CD42bhigh) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14+/CD61/CD42b) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca2+ after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE.

Keywords

Calcium channel
Megakaryocyte
Monocyte
Stem cell
Trp
Platelet

Abbreviations

[Ca2+]i, cytosolic free calcium concentration
FITC, fluorescein isothiocyanate
InsP3, inositol 1,4,5-trisphosphate
PCR, polymerase chain reaction
SOCE, store-operated Ca2+ entry
Trp, transient receptor potential

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These authors contributed equally to this paper.