Mitigating cadmium accumulation in rice without compromising growth via modifying the regulatory region of OsNRAMP5.

Cadmium (Cd) intake poses a significant health risk to humans, and the contamination of rice grains with Cd is a major concern in regions where rice is a staple food. Although the knockout of OsNRAMP5, which encodes a key transporter responsible for Cd and manganese (Mn) uptake, can significantly reduce Cd accumulation in rice grains, recent studies have revealed that this knockout adversely affects plant growth, grain yield, and increases vulnerability to abiotic and biotic stresses due to reduced Mn accumulation. In this study, we employed CRISPR/Cas9 technology to modify the regulatory region of OsNRAMP5 with the aim of reducing Cd accumulation in rice grains. Our findings demonstrate that mutations in the regulatory region of OsNRAMP5 do not impact its expression pattern but result in a reduction in translation. The decreased translation of OsNRAMP5 effectively decreases grain Cd accumulation while leaving Mn accumulation and important agronomic traits, including yield, unaffected. Thus, our study presents a practical and viable strategy for reducing Cd accumulation in rice grains without compromising Mn accumulation or overall rice production.

Wnt/ß-Catenin Promotes the Osteoblastic Potential of BMP9 Through Down-Regulating Cyp26b1 in Mesenchymal Stem Cells.

BACKGROUND: All-trans retinoic acid (ATRA) promotes the osteogenic differentiation induced by bone morphogenetic protein 9 (BMP9), but the intrinsic relationship between BMP9 and ATRA keeps unknown. Herein, we investigated the effect of Cyp26b1, a critical enzyme of ATRA degradation, on the BMP9-induced osteogenic differentiation in mesenchymal stem cells (MSCs), and unveiled possible mechanism through which BMP9 regulates the expression of Cyp26b1. METHODS: ATRA content was detected with ELISA and HPLC-MS/MS. PCR, Western blot, and histochemical staining were used to assay the osteogenic markers. Fetal limbs culture, cranial defect repair model, and micro-computed tomographic were used to evaluate the quality of bone formation. IP and ChIP assay were used to explore possible mechanism. RESULTS: We found that the protein level of Cyp26b1 was increased with age, whereas the ATRA content decreased. The osteogenic markers induced by BMP9 were increased by inhibiting or silencing Cyp26b1 but reduced by exogenous Cyp26b1. The BMP9-induced bone formation was enhanced by inhibiting Cyp26b1. The cranial defect repair was promoted by BMP9, which was strengthened by silencing Cyp26b1 and reduced by exogenous Cyp26b1. Mechanically, Cyp26b1 was reduced by BMP9, which was enhanced by activating Wnt/ß-catenin, and reduced by inhibiting this pathway. ß-catenin interacts with Smad1/5/9, and both were recruited at the promoter of Cyp26b1. CONCLUSIONS: Our findings suggested the BMP9-induced osteoblastic differentiation was mediated by activating retinoic acid signalling, viadown-regulating Cyp26b1. Meanwhile, Cyp26b1 may be a novel potential therapeutic target for the treatment of bone-related diseases or accelerating bone-tissue engineering.

Dysfunction of the 4-coumarate:coenzyme A ligase 4CL4 impacts aluminum resistance and lignin accumulation in rice.

The root cell wall is the first and primary target of aluminum (Al) toxicity. Monocots such as rice (Oryza sativa) can accumulate appreciable levels of hydroxycinnamic acids (HCAs) to modify and cross-link hemicellulose and/or lignin of the cell wall. Nevertheless, it is unclear whether this HCA-mediated modification of the cell wall is important for Al accumulation and resistance. We previously isolated and characterized a rice ral1 (resistance to aluminum 1) mutant that shows enhanced Al resistance. In this study, we cloned RAL1 and found that it encodes the 4-coumarate:coenzyme A ligase 4CL4, an enzyme putatively involved in lignin biosynthesis. Mutation of RAL1/4CL4 reduces lignin content and increases the accumulation of its substrates 4-coumaric acid (PA) and ferulic acid (FA). We demonstrate that altered lignin accumulation is not required for the enhanced Al resistance in ral1/4cl4 mutants. We found that the increased accumulation of PA and FA can reduce Al binding to hemicellulose and consequently enhance Al resistance in ral1/4cl4 mutants. Al stress is able to trigger PA and FA accumulation, which is likely caused by the repression of the expression of RAL1/4CL4 and its homologous genes. Our results thus reveal that Al-induced PA and FA accumulation is actively and positively involved in Al resistance in rice through the modification of the cell wall and thereby the reduced Al binding to the cell wall.

Caseinate-gelatin and caseinate-hydrolyzed gelatin composites formed via transglutaminase: chemical and functional properties.

BACKGROUND: Treatment of food proteins by enzymatic crosslinking and other reactions can confer modified properties on the treated proteins. Bovine gelatin and hydrolyzed bovine gelatin were used to generate two caseinate-based composites via transglutaminase, and potential useful properties to food processing were investigated for both composites. RESULTS: Caseinate-gelatin and caseinate-hydrolyzed gelatin composites contained 33.4 and 10.3 g kg(-1) protein of 4-hydroxyproline, respectively. Caseinate conjugation with gelatin and hydrolyzed gelatin resulted in two composites with stronger absorption at five wavenumbers during Fourier transform-infrared analysis, demonstrating that they were rich in hydroxyl and carboxyl groups. Both composites exhibited higher viscosity values in aqueous dispersions, lower thermal stability (i.e. higher mass loss) during thermogravimetric analysis and worse emulsifying properties than original caseinate, owing to conjugation and crosslinking via transglutaminase. However, confocal laser scanning microscopy (CLSM) analysis revealed that both composites actually had better emulsion stability after 2 weeks of storage. CONCLUSION: The composites generated were different in chemical characteristics and better in viscosity and emulsion stability than original caseinate. They might have potential as protein thickeners and emulsifiers. CLSM is a better technique to assess emulsion stability of food proteins than the classic turbidity method.

MicroRNA-200C and -150 play an important role in endothelial cell differentiation and vasculogenesis by targeting transcription repressor ZEB1.

To investigate the role of miRNA in controlling human embryonic stem (hES) cell differentiation toward the endothelial lineage and chick embryonic blood vessel formation, undifferentiated hES cells were first cultured on Matrigel-coated flasks and in endothelial cell growth medium-2 (EGM-2) to initiate endothelial cell (EC) differentiation. CD146(+) cells were isolated from differentiating hES cells and expanded in vitro. The in vitro expanded CD146(+) cells were positive for EC markers, capable of Ac-LDL uptake, lectin binding, and the formation of vascular structures in vitro and in vivo. miRNA gain/loss-of-function analyses revealed that miR-150 and miR-200c were crucial in EC differentiation. Transcriptional repressor zinc finger E-box-binding homeobox 1 (ZEB1) was identified as the communal target gene of miRNA-200C and -150, and inhibition of ZEB1 was required for miRNA-200C or -150 mediated EC gene expressions. Moreover, we demonstrated that ZEB1 could transcriptionally repress EC gene expression through direct binding to promoters of EC genes. Finally, we also demonstrated that miRNA-200c and -150 played an important role in chick embryonic blood vessel formation by in vivo inhibition of miRNA-200C or -150 in developing chick embryos, and blocking ZEB1 signaling in CD146-positive cells could rescue the inhibitory effects of miR-200c inhibiton in in vivo vasculogenesis. Our findings revealed that miR-150 and miR-200c play an important role in human endothelial lineage specification and chick embryonic vasculogenesis by targeting ZEB1.

Nrf3-Pla2g7 interaction plays an essential role in smooth muscle differentiation from stem cells.

OBJECTIVE: Phospholipase A2, group 7 (Pla2g7) is an important mediator in cardiovascular development and diseases because of its divergent physiological and pathological functions in inflammation and oxidative stress. However, little is known about the functional role of Pla2g7 in smooth muscle cell (SMC) differentiation from stem cells. METHODS AND RESULTS: In the present study, embryonic stem cells were cultivated on collagen IV-coated plates to allow SMC differentiation. Pla2g7 gene expression and activity were upregulated significantly following 4 to 14 days of cell differentiation and colocalized with SMC differentiation markers in the differentiated SMCs. Knockdown of Pla2g7 resulted in downregulation of smooth muscle-specific markers in vitro and impairment of SMC differentiation in vivo, whereas enforced expression of Pla2g7 enhanced SMC differentiation and increased reactive oxygen species generation. Importantly, enforced expression of Pla2g7 significantly increased the binding of serum response factor to SMC differentiation gene promoters, resulting in SMC differentiation, which was abolished by free radical scavenger and flavoprotein inhibitor of NADPH oxidase but not hydrogen peroxide inhibitor. Moreover, we demonstrated that nuclear factor erythroid 2-related factor 3 (Nrf3) regulates Pla2g7 gene expression through direct binding to the promoter regions of Pla2g7 gene. CONCLUSION: Our findings demonstrated that Pla2g7 plays a crucial physiological role in SMC differentiation from stem cells, and the fine interactions between Nrf3 and Pla2g7 are essential for SMC differentiation.

Functional impact of heterogeneous nuclear ribonucleoprotein A2/B1 in smooth muscle differentiation from stem cells and embryonic arteriogenesis.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) play various roles in transcriptional and post-transcriptional modulation of gene expression. However, it remains unclear if hnRNPs are associated with smooth muscle cell (SMC) differentiation from stem cells and embryonic arteriogenesis. In this study, mouse embryonic stem (ES) cells were cultivated on collagen IV-coated plates and smooth muscle differentiation medium. We found that hnRNPA2/B1 gene and protein expression was significantly up-regulated following 3-7 days of cell differentiation. hnRNPA2/B1 knockdown resulted in down-regulation of specific smooth muscle markers and transcription factors, whereas enforced expression of hnRNPA2/B1 enhanced the expression of these genes. Moreover, we demonstrated by using luciferase and chromatin immunoprecipitation assays that hnRNPA2/B1 could transcriptionally regulate SMC gene expression through direct binding to promoters of Smαa and Sm22α genes. We further demonstrated that chromobox protein homolog gene 3, a previously identified SMC differentiation regulatory nuclear protein, is required for hnRNPA2/B1-mediated SMC differentiation gene expression. Importantly, specifically designed Hnrnpa2/b1 morpholinos for in vivo knockdown could inhibit the migration and differentiation of neural crest cells into SMCs in chick embryos. This resulted in the maldevelopment of branchial arch arteries and increased embryo lethality at a later developmental stage. Our findings demonstrated that hnRNPA2/B1 plays a functional role in SMC differentiation from stem cells in vitro and embryonic branchial arch artery development. This indicates that hnRNPA2/B1 is a potential modulating target for deriving SMCs from stem cells and cardiovascular regenerative medicine.

Chromobox protein homolog 3 is essential for stem cell differentiation to smooth muscles in vitro and in embryonic arteriogenesis.

OBJECTIVE: Smooth muscle cell (SMC) differentiation is a critical process during cardiovascular formation and development, but the underlying molecular mechanism remains unclear. METHODS AND RESULTS: Here we demonstrated that chromobox protein homolog 3 (Cbx3) is crucial for SMC differentiation from stem cells and that the chromodomain and chromoshadow domain of Cbx3 are responsible for Cbx3-induced SMC differentiation. Moreover, we identified that 4 amino acids (165 to 168) within the chromoshadow domain of Cbx3 are key elements for Cbx3 interaction with Dia-1- and Cbx3-induced SMC differentiation. Mechanistically, we found that Cbx3 mediates SMC differentiation through modulating serum response factor (SRF) recruitment to the promoters of SMC genes, in which the interaction between Cbx3 and Dia-1/SRF plays a crucial role in this process. Moreover, our in vivo study demonstrated that the misexpression of Cbx3 within neural crest cells of chick embryos resulted in the death of chick embryos at early stages because of the maldevelopment of branchial arch arteries. CONCLUSIONS: Our findings suggest that the interaction between Cbx3 and Dia-1/SRF is essential for SMC differentiation from stem cells and for the development of functional cardiovascular system.

Signalling pathways that regulate endothelial differentiation from stem cells.

Endothelial cells play a vital role in the human vascular system. Injury to this layer of cells can lead to devastating consequences and eventually mortality. As demonstrated by recent accumulating evidences, the injured endothelial layer can be rescued by endothelial cell-based therapy. However, the limited source of functional endothelial cells which can be used in clinical surgery, is hugely hampered. The discovery of pluripotent embryonic stem cells, nevertheless has raised hope for generating endothelial cells in the regenerative medicine field. It was demonstrated that the concerted and coordinated series of specific signaling pathways involving different molecules, guide the differentiation of these embryonic stem cells into functional endothelial cells. Moreover, it is believed that understanding the molecular mechanisms of endothelial development and signal pathways leading to endothelial differentiation from stem cells, will be essential for potential cell therapy for vascular disease. This review therefore, will summarize and discuss recent insights into endothelial development and the signaling pathways regulating embryonic stem cell differentiation towards the endothelial lineage.

Pluripotent stem cell differentiation into vascular cells: a novel technology with promises for vascular re(generation).

Several types of stem and progenitor cells are currently under investigation for their potential to accomplish vascular regeneration. This review focuses on embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We will discuss the technologies allowing for their derivation, culture expansion and maintenance in a pluripotent status. Moreover, both ESCs and iPSCs can be differentiated in endothelial cells (ECs) and mural cell, including vascular smooth muscle cells (VSMCs). Here, we will describe the involvements of growth factors (vascular endothelial growth factors-VEGFs-, platet-derived growth factors-PDGFs-), Wnt and Notch signal pathways, reactive oxygen species (ROS), histone deacetylases (HDACs), and microRNAs (miRNAs) in vascular cell differentiation from pluripotent stem cells. We will additionally describe the therapeutic potential of stem cells for vascular medicine.

Sp1-dependent activation of HDAC7 is required for platelet-derived growth factor-BB-induced smooth muscle cell differentiation from stem cells.

We have previously demonstrated that histone deacetylase 7 (HDAC7) expression and splicing play an important role in smooth muscle cell (SMC) differentiation from embryonic stem (ES) cells, but the molecular mechanisms of increased HDAC7 expression during SMC differentiation are currently unknown. In this study, we found that platelet-derived growth factor-BB (PDGF-BB) induced a 3-fold increase in the transcripts of HDAC7 in differentiating ES cells. Importantly, our data also revealed that PDGF-BB regulated HDAC7 expression not through phosphorylation of HDAC7 but through transcriptional activation. By dissecting its promoters with progressive deletion analysis, we identified the sequence between -343 and -292 bp in the 5'-flanking region of the Hdac7 gene promoter as the minimal PDGF-BB-responsive element, which contains one binding site for the transcription factor, specificity protein 1 (Sp1). Mutation of the Sp1 site within this PDGF-BB-responsive element abolished PDGF-BB-induced HDAC7 activity. PDGF-BB treatment enhanced Sp1 binding to the Hdac7 promoter in differentiated SMCs in vivo as demonstrated by the chromatin immunoprecipitation assay. Moreover, we also demonstrated that knockdown of Sp1 abrogated PDGF-BB-induced HDAC7 up-regulation and SMC differentiation gene expression in differentiating ES cells, although enforced expression of Sp1 alone was sufficient to increase the activity of the Hdac7 promoter and expression levels of SMC differentiation genes. Importantly, we further demonstrated that HDAC7 was required for Sp1-induced SMC differentiation of gene expression. Our data suggest that Sp1 plays an important role in the regulation of Hdac7 gene expression in SMC differentiation from ES cells. These findings provide novel molecular insights into the regulation of HDAC7 and enhance our knowledge in SMC differentiation and vessel formation during embryonic development.

The mechanism of stem cell differentiation into smooth muscle cells.

Stem cells represent one of the most promising areas in biological and medical research. All stem cells are defined as having two basic properties: unlimited self-renewal and the broad potential to differentiate in vitro, via "progenitor cells", into somatic cells of many tissue types, in which smooth muscle cell (SMC) differentiation is a complicated and not well defined process. It is known that serum response factors (SRF) and co-activator myocardin are essential transcription factors in SMC differentiation. Upstream activators or regulators for the transcription factors have been recently identified, such as reactive oxygen species, histone deacetylases, microRNAs and extracellular matrix (ECM) proteins and integrins. In this review we, therefore, aim to briefly summarise recent progress in the mechanism of stem cell differentiation into SMCs to highlight the potential targets for promoting/inhibiting SMC differentiation useful for vessel-tissue engineering and treatment of vascular disease.

Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.

NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-beta(1) activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.