METHODS: Immortalized IFRS1 SCs and human primary SCs generated two distinct subtypes of SCs: undifferentiated and differentiated SCs. Quantitative PCR was employed to evaluate the expression of differentiation markers and mechanosensitive channels, including TRP channels (TRPV4, TRPM7 and TRPA1) and Piezo channels (Piezo1 and Piezo2). To validate the functionality of specific mechanosensitive channels, Ca2+ imaging and electronic cell sizing experiments were conducted under hypotonic conditions, and inhibitors and siRNAs were used. Protein expression was assessed by Western blotting and immunostaining. Additionally, secretome analysis was performed to evaluate the release of neurotrophic factors in response to hypotonic stimulation, with BDNF, a representative trophic factor, quantified using ELISA.
RESULTS: Induction of differentiation increased Piezo2 mRNA expression levels both in IFRS1 and in human primary SCs. Both cell types were responsive to hypotonic solutions, with differentiated SCs displaying a more pronounced response. Gd3+ and FM1-43 effectively inhibited hypotonicity-induced Ca2+ transients in differentiated SCs, implicating Piezo2 channels. Conversely, inhibitors of Piezo1 and TRPM7 (Dooku1 and NS8593, respectively) had no discernible impact. Moreover, Piezo2 in differentiated SCs appeared to participate in regulatory volume decreases (RVD) after cell swelling induced by hypotonic stimulation. A Piezo2 deficiency correlated with reduced RVD and prolonged cell swelling, leading to heightened release of the neurotrophic factor BDNF by upregulating the function of endogenously expressed Ca2+-permeable TRPV4.
CONCLUSIONS: Our study unveils the mechanosensitivity of SCs and implicates Piezo2 channels in the release of neurotrophic factors from SCs. These results suggest that Piezo2 may contribute to RVD, thereby maintaining cellular homeostasis, and may also serve as a negative regulator of neurotrophic factor release. These findings underscore the need for further investigation into the role of Piezo2 in SC function and neurotrophic regulation.
方法:永生化IFRS1SCs和人原代SCs产生两种不同的SCs亚型:未分化和分化的SCs。定量PCR用于评估分化标记和机械敏感通道的表达,包括TRP通道(TRPV4、TRPM7和TRPA1)和压电通道(Piezo1和Piezo2)。为了验证特定机械敏感通道的功能,在低渗条件下进行了Ca2+成像和电子细胞大小实验,使用抑制剂和siRNA。通过蛋白质印迹和免疫染色评估蛋白质表达。此外,进行了分泌组分析,以评估响应低张性刺激的神经营养因子的释放,BDNF,代表性的营养因子,使用ELISA定量。
结果:诱导分化增加了IFRS1和人原代SCs中Piezo2mRNA的表达水平。两种细胞类型都对低渗溶液有反应,分化的SCs表现出更明显的反应。Gd3+和FM1-43有效抑制低渗性诱导的分化SCs中的Ca2+瞬变,涉及Piezo2通道。相反,Piezo1和TRPM7(分别为Dooku1和NS8593)的抑制剂没有明显的影响。此外,分化的SCs中的Piezo2似乎参与了低渗刺激诱导的细胞肿胀后的调节体积减少(RVD)。Piezo2缺乏与降低的RVD和延长的细胞肿胀相关,通过上调内源性表达的Ca2通透性TRPV4的功能,导致神经营养因子BDNF的释放增加。
结论:我们的研究揭示了SCs的机械敏感性,并暗示Piezo2通道参与SCs释放神经营养因子。这些结果表明Piezo2可能有助于RVD,从而维持细胞内稳态,也可以作为神经营养因子释放的负调节因子。这些发现强调需要进一步研究Piezo2在SC功能和神经营养调节中的作用。