Abstract
Enhancer of zeste homolog 2 (EZH2) regulates stem cells renewal, maintenance, and differentiation into different cell lineages including neuron. Changes in intracellular Ca(2+) concentration play a critical role in the differentiation of neurons. However, whether EZH2 modulates intracellular Ca(2+) signaling in regulating neuronal differentiation from human mesenchymal stem cells (hMSCs) still remains unclear. When hMSCs were treated with a Ca(2+) chelator or a PLC inhibitor to block IP(3)-mediated Ca(2+) signaling, neuronal differentiation was disrupted. EZH2 bound to the promoter region of PIP5K1C to suppress its transcription in proliferating hMSCs. Interestingly, knockdown of EZH2 enhanced the expression of PIP5K1C, which in turn increased the amount of PI(4,5)P(2), a precursor of IP(3), and resulted in increasing the intracellular Ca(2+) level, suggesting that EZH2 negatively regulates intracellular Ca(2+) through suppression of PIP5K1C. Knockdown of EZH2 also enhanced hMSCs differentiation into functional neuron both in vitro and in vivo. In contrast, knockdown of PIP5K1C significantly reduced PI(4,5)P(2) contents and intracellular Ca(2+) release in EZH2-silenced cells and resulted in the disruption of neuronal differentiation from hMSCs. Here, we provide the first evidence to demonstrate that after induction to neuronal differentiation, decreased EZH2 activates the expression of PIP5K1C to evoke intracellular Ca(2+) signaling, which leads hMSCs to differentiate into functional neuron lineage. Activation of intracellular Ca(2+) signaling by repressing or knocking down EZH2 might be a potential strategy to promote neuronal differentiation from hMSCs for application to neurological dysfunction diseases.
Highlights
Human mesenchymal stem cells4 derived from bone marrow are obtained [1], safely expanded in vitro and are not susceptible to malignant transformation; hMSCs are suitable for therapeutic applications [2]. hMSCs can be induced to differentiate into multiple lineages, including bone, fat, cartilage, as well as neuron in vitro [1, 3,4,5]
The involvement of Ca2ϩ in neuronal differentiation has been reported from 1990s [42], it remains unclear whether intracellular Ca2ϩ transient is required for differentiation from hMSCs into neuronal lineage
We demonstrated that intracellular Ca2ϩ transient peaks at day 1 post-induction (Fig. 2A) and is required for neuronal differentiation from hMSCs (Fig. 2, B and C)
Summary
Materials—Chemicals used in induction of neuronal differentiation, dexamethasone, ascorbic acid-2-phosphate, indomethacin, insulin, and 3-isobutyl-1-methyl-xanthine, an intracellular Ca2ϩ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM), polybrene, and puromycin were purchased from Sigma. The q-PCR was examined by incubating the cDNA in a reaction mixture containing, 0.5 M of each primer, 0.1 M UPL probe, and 1-fold concentration of Probes Master reagent (Roche). The protein/DNA crosslinks of the immunoprecipitated complexes were reversed by incubation in 0.2 M NaCl at 65 °C for 4 –5 h, and the DNA was purified and applied to PCR as described above to determine the binding ability of EZH2 to its potential target gene, PIP5K1C. After removing the reaction mixture and washing three times with PBST, the peroxidase-linked secondary detection reagent was added into the wells, and the plate was incubated at room temperature for 1 h to detect the PI[4,5]P2 detector protein bound to the plate. The three-dimensional images were observed under a Carl Zeiss LSM510 laserscanning confocal microscope
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