Crucial Role of Nrf3 in Smooth Muscle Cell Differentiation From Stem Cells

Abstract

Rationale: Nuclear factor erythroid 2-related factor (Nrf)3, a member of the cap 'N' collar family of transcription factors that bind to the DNA-antioxidant responsive elements, is involved in reactive oxygen species balancing and in muscle precursor migration during early embryo development. Objective: To investigate the functional role of Nrf3 in smooth muscle cell (SMC) differentiation in vitro and in vivo. Methods and Results: Nrf3 was upregulated significantly following 1 to 8 days of SMC differentiation. Knockdown of Nrf3 resulted in downregulation of smooth muscle specific markers expression, whereas enforced expression of Nrf3 enhanced SMC differentiation in a dose-dependent manner. SMC-specific transcription factor myocardin, but not serum response factor, was significantly upregulated by Nrf3 overexpression. Strikingly, the binding of SRF and myocardin to the promoter of smooth muscle differentiation genes was dramatically increased by Nrf3 overexpression, and Nrf3 can directly bind to the promoters of SMC differentiation genes as demonstrated by chromatin immunoprecipitation assay. Moreover, NADPH-derived reactive oxygen species production during SMC differentiation was further enhanced by Nrf3 overexpression through upregulation of NADPH oxidase and inhibition of antioxidant signaling pathway. In addition, Nrf3 was involved in the endoplasmic reticulum stressor induced SMC differentiation. (SMCs). [1][2][3] Our previous studies showed that growth factor stimulation, 2 mechanical force or extracellular matrix 4 can be used to drive ESC differentiation toward the vascular lineage. We have established an in vitro model for ESC differentiation into SMCs, 3 which involves the choice of extracellular matrix protein collagen IV, 3,5,6 to investigate SMC differentiation more closely. In the adult organism, SMCs have the main physiological function of contracting and controlling the blood vessel tone, diameter, blood pressure and blood flow. In response to vascular injury, SMCs dramatically increase their rate of proliferation, migration and synthetic capacity. 7,8 Atherosclerosis and postangioplasty restenosis, for example, are characterized by the development of an enlarged neointima. 9 The cells responsible for the neointima formation have been identified as not only SMC migrating from the tunica media, 10 but also vascular progenitors that originate from the circulation and the perivascular adventitia, which differentiate into vascular SMCs in the lesion. 11-14 However, the mechanisms involved in SMC differentiation in vitro and in vivo is still poorly understood.In a microarray screen for factors involved in vascular progenitor differentiation, we identified nuclear factor erythroid 2-related factor (Nrf)3 expression to be constantly modulated. Nrf3 is the most recently identified member of the Nrf family and contains a domain homologous to the cap 'N' collar (CNC) transcription factors. 15 The CNC domain is a unique, conserved region, located at the amino-terminus of the bZIP domain. 16 Nrf3 heterodim...

Upregulated sirtuin 1 by miRNA-34a is required for smooth muscle cell differentiation from pluripotent stem cells

“…Murine ESCs were induced to differentiate towards SMCs as described previously (Supplementary Figure S1). [11][12][13][14] miRNA microarrays analysis was conducted to identify potential miRNA candidates for SMC differentiation, and the data revealed that miR-290 family members, the reported ESC-specific miRNA cluster, 15 were dramatically downregulated upon differentiation (Supplementary Table S1). Conversely, muscle differentiation-related miRNAs (miR-143/145/133) were increased in our SMC differentiation model, while SMC proliferation-related miR-21 was undetectable at early stage but dramatically increased at late stage of SMC differentiation, indicating that some miRNAs may initiate SMC differentiation, whereas others may have important roles in the late stage of SMC differentiation.…”

“…Detailed protocols for mouse ESCs (mESCs) (ES-D3 cell line, CRL-1934; ATCC, Manassas, VA, USA) culture and SMC differentiation were described in our previous studies. [11][12][13][14]17,22,34,43,44 The Shef-1, 2, 3, 6, and 7 human ESC lines were obtained from the United Kingdom Stem Cell Bank (UKSCB, Hertfordshire, UK) and maintained in our Laboratory as described in our previous study. 18 Briefly, undifferentiated ESCs were dissociated into single cells and seeded onto collagen I/IV (5 μg/ml)-coated flasks or plates in differentiation medium (DM, MEM alpha medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin) for 0-9 days prior to further treatment.…”

Functional Impact of Heterogeneous Nuclear Ribonucleoprotein A2/B1 in Smooth Muscle Differentiation from Stem Cells and Embryonic Arteriogenesis

“…Cell Culture and SMC Differentiation-Detailed protocols for mouse ES cell (ES-D3 cell line, CRL-1934; ATCC, Manassas, VA) culture and SMC differentiation were described in our previous studies (2)(3)(4)(5)(6)(7)(8). For SMC differentiation, undifferentiated ES cells were seeded on mouse collagen IV (5 g/ml)-coated flasks or plates in differentiation medium (DM), ␣-minimal essential medium (Invitrogen) supplemented with 10% FBS, 0.05 mM ␤-mercaptoethanol, 100 units/ml penicillin, and 100 g/ml streptomycin for 3-7 days prior to further treatments.…”

“…Other Procedures-Experiments of nucleofection, siRNA experiments, indirect immunofluorescent staining for cells, and real time quantitative PCRs (RT-qPCR) were performed according to the standard procedures as described previously (2)(3)(4)(5)(6)(7)(8). Whole mount immunohistochemistry and in situ hybridization were carried out as described previously (7).…”

“…Understanding the signaling pathways and molecular mechanisms in regulating stem/progenitor cell differentiation into cardiomyocytes and endothelial and smooth muscle cells (SMCs) 5 is crucial. This is especially so for cardiovascular regenerative medicine that eventually benefits patients in clinical applications.…”