Abstract
Store-operated Ca2+ entry (SOCE) channels are the main pathway of Ca2+ entry in non-excitable cells such as neural progenitor cells (NPCs). However, the role of SOCE channels has not been defined in the neuronal differentiation from NPCs. Here, we show that canonical transient receptor potential channel (TRPC) as SOCE channel influences the induction of the neuronal differentiation of A2B5+ NPCs isolated from postnatal-12-day rat cerebrums. The amplitudes of SOCE were significantly higher in neural cells differentiated from proliferating A2B5+ NPCs and applications of SOCE blockers, 2-aminoethoxy-diphenylborane (2-APB), and ruthenium red (RR), inhibited their rise of SOCE. Among TRPC subtypes (TRPC1-7), marked expression of TRPC5 and TRPC6 with turned-off TRPC1 expression was observed in neuronal cells differentiated from proliferating A2B5+ NPCs. TRPC5 small interfering RNA (siRNA) blocked the neuronal differentiation from A2B5+ NPCs and reduced the rise of SOCE. In contrast, TRPC6 siRNA had no significant effect on the neuronal differentiation from A2B5+ NPCs. These results indicate that calcium regulation by TRPC5 would play a key role as a switch between proliferation and neuronal differentiation from NPCs.
Highlights
Cytosolic Ca2+ is a ubiquitous second messenger to control a great number of cellular functions ranging from short-term responses such as contraction and secretion to long-term regulation of transcription, growth and cell division as well as development of embryonic cells [1,2,3]
Immunofluorescence was performed on the A2B5+ Neural progenitor cells (NPCs) after magnetic activated cell sorting (MACS) sorting; A2B5+ NPCs expressed both A2B5 and Nestin, a neural stem cell marker (Figure 1F)
Immunocytochemistry was performed at 1 week after MACS, and most of the neurospheres were positive for A2B5 and the majority of them co-expressed Nestin (Figure 2A), GFAP, a glial cell or a subventricular zone stem cell marker (Figure 2C); Tuj1, an early neural cell marker (Figure 2D)
Summary
Cytosolic Ca2+ is a ubiquitous second messenger to control a great number of cellular functions ranging from short-term responses such as contraction and secretion to long-term regulation of transcription, growth and cell division as well as development of embryonic cells [1,2,3]. Neural progenitor cells (NPCs) have few VOCCs, indicating that non-VOCCs may regulate the differentiation of NPCs [5], [6], and canonical transient receptor potential channel (TRPC) is one of the non-VOCCs [7]. We focused on the physiological function of the TRPCs as store-operated Ca2+ entry (SOCE) in the neural differentiation of NPCs. Based on sequence similarity and function, seven TRPC homologs (TRPC1-7) can be subdivided into four groups: TRPC 4/5 (group 1), TRPC 1 (group 2), TRPC 3/6/7 (group 3), and TRPC 2 (group 4) [8], [9]. TRPCs operate as receptor-operated Ca2+ entry (ROCE) channels which are activated by agonist of receptors or SOCE channels which are activated by emptying of
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