Abstract 1956: Nox4 is Crucial for Stem Cell Differentiation into Smooth Muscle Cells
Background: Our previous study has demonstrated that embryonic stem cells can differentiate into smooth muscle cells (SMCs) in which matrix protein collagen IV and growth factors play a role in the process. However, little is known about the molecular mechanisms of SMC differentiation in such a process. Since NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that is important for cell behavior, it would be interesting to explore the potential impact of Nox4 in stem cell differentiation.
Methods and Results: Mouse ES cells were plated on collagen IV-coated dishes/flasks, and total RNA were harvested and subjected to microarray analysis at various time points. A panel of SMC-specific genes was significantly and consistently upregulated during ES cell differentiation, in which Nox4 expression was markedly correlated with SMC marker gene induction during ES cell differentiation. The result was confirmed by real-time PCR, immunofluorescence and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, while knockdown of Nox4 showed a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin, but not other cell lineage markers, were activated by Nox4 gene expression induced by transfection. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of ROS, especial hydrogen peroxide (H2O2) production. Confocal microscope analysis indicates that SRF was translocated into the nucleus during SMC differentiation, in which SRF was phosphorylated. Additionally, Nox4 overexpression resulted in TGF-β1 secretion that positively enhanced SMC differentiation. Interestingly, a cell line generated from stem cells by Nox4 transfection and selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function.
Conclusions: We demonstrate for the first time that Nox4 is crucial for SMC differentiation from embryonic stem cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation under physiological and pathological conditions.