The ABCs of flower development: mutational analysis of AP1/FUL‐like genes in rice provides evidence for a homeotic (A)‐function in grasses

The well‐known ABC model describes the combinatorial interaction of homeotic genes in specifying floral organ identities. While the B‐ and C‐functions are highly conserved throughout flowering plants and even in gymnosperms, the A‐function, which specifies the identity of perianth organs (sepals and petals in eudicots), remains controversial. One reason for this is that in most plants that have been investigated thus far, with Arabidopsis being a remarkable exception, one does not find recessive mutants in which the identity of both types of perianth organs is affected. Here we report a comprehensive mutational analysis of all four members of the AP1/FUL‐like subfamily of MADS‐box genes in rice (Oryza sativa). We demonstrate that OsMADS14 and OsMADS15, in addition to their function of specifying meristem identity, are also required to specify palea and lodicule identities. Because these two grass‐specific organs are very likely homologous to sepals and petals of eudicots, respectively, we conclude that there is a floral homeotic (A)‐function in rice as defined previously. Together with other recent findings, our data suggest that AP1/FUL‐like genes were independently recruited to fulfil the (A)‐function in grasses and some eudicots, even though other scenarios cannot be excluded and are discussed.     

SIRT1‐mediated epigenetic downregulation of plasminogen activator inhibitor‐1 prevents vascular endothelial replicative senescence

The inactivation of plasminogen activator inhibitor‐1 (PAI‐1) has been shown to exert beneficial effects in age‐related vascular diseases. Limited information is available on the molecular mechanisms regarding the negatively regulated expression of PAI‐1 in the vascular system. In this study, we observed an inverse correlation between SIRT1, a class III histone deacetylase, and PAI‐1 expression in human atherosclerotic plaques and the aortas of old mice, suggesting that internal negative regulation exists between SIRT1 and PAI‐1. SIRT1 overexpression reversed the increased PAI‐1 expression in senescent human umbilical vein endothelial cells (HUVECs) and aortas of old mice, accompanied by decreased SA‐β‐gal activity in vitro and improved endothelial function and reduced arterial stiffness in vivo. Moreover, the SIRT1‐mediated inhibition of PAI‐1 expression exerted an antisenescence effect in HUVECs. Furthermore, we demonstrated that SIRT1 is able to bind to the PAI‐1 promoter, resulting in a decrease in the acetylation of histone H4 lysine 16 (H4K16) on the PAI‐1 promoter region. Thus, our findings suggest that the SIRT1‐mediated epigenetic inhibition of PAI‐1 expression exerts a protective effect in vascular endothelial senescence.     

Dwarf and deformed flower 1, encoding an F‐box protein,is critical for vegetative and floral development in rice (Oryza sativa L.)

Recent studies have shown that F‐box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1‐1 (ddf1‐1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1‐1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume‐like organs and pistil‐like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F‐box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B‐class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F‐box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.     

Functional specialization of duplicated AP3‐like genes in Medicago truncatula

The B–class of MADS box genes has been studied in a wide range of plant species, but has remained largely uncharacterized in legumes. Here we investigate the evolutionary fate of the duplicated AP3‐like genes of a legume species. To obtain insight into the extent to which B‐class MADS box gene functions are conserved or have diversified in legumes, we isolated and characterized the two members of the AP3 lineage in Medicago truncatula: MtNMH7 and MtTM6 (euAP3 and paleoAP3 genes, respectively). A non‐overlapping and complementary expression pattern of both genes was observed in petals and stamens. MtTM6 was expressed predominantly in the outer cell layers of both floral organs, and MtNMH7 in the inner cell layers of petals and stamens. Functional analyses by reverse genetics approaches (RNAi and Tnt1 mutagenesis) showed that the contribution of MtNMH7 to petal identity is more important than that of MtTM6, whereas MtTM6 plays a more important role in stamen identity than its paralog MtNMH7. Our results suggest that the M. truncatula AP3‐like genes have undergone a functional specialization process associated with complete partitioning of gene expression patterns of the ancestral gene lineage. We provide information regarding the similarities and differences in petal and stamen development among core eudicots.     

Crystal structure of human WBSCR16, an RCC1‐like protein in mitochondria

WBSCR16 (Williams‐Beuren Syndrome Chromosomal Region 16) gene is located in a large deletion region of Williams‐Beuren syndrome (WBS), which is a neurodevelopmental disorder. Although the relationship between WBSCR16 and WBS remains unclear, it has been reported that WBSCR16 is a member of a functional module that regulates mitochondrial 16S rRNA abundance and intra‐mitochondrial translation. WBSCR16 has RCC1 (Regulator of Chromosome Condensation 1)‐like amino acid sequence repeats but the function of WBSCR16 appears to be different from that of other RCC1 superfamily members. Here, we demonstrate that WBSCR16 localizes to mitochondria in HeLa cells, and report the crystal structure of WBSCR16 determined to 2.0 Å resolution using multi‐wavelength anomalous diffraction. WBSCR16 adopts the seven‐bladed β‐propeller fold characteristic of RCC1‐like proteins. A comparison of the WBSCR16 structure with that of RCC1 and other RCC1‐like proteins reveals that, although many of the residues buried in the core of the β‐propeller are highly conserved, the surface residues are poorly conserved and conformationally divergent.     

Long noncoding RNA FAM83H‐AS1 exerts an oncogenic role in glioma through epigenetically silencing CDKN1A (p21)

Gliomas are the commonest and most aggressive primary malignant tumor in the central nervous system. Long noncoding RNAs (lncRNAs) have been identified to act as crucial regulators in multiple biological processes, including tumorigenesis. FAM83H antisense RNA1 (FAM83H‐AS1) has been uncovered to be dysregulated in several cancers. However, the biological role of FAM83H‐AS1 in glioma still needs to be investigated. Currently, our findings indicated that FAM83H‐AS1 was upregulated in glioma tissues and cell lines and high level of FAM83H‐AS1 was associated with poor prognosis of glioma. Loss‐of‐function assays demonstrated that silenced FAM83H‐AS1 obviously suppressed cell proliferation via regulating the cell‐cycle distribution and cell apoptosis rate, and mechanistic experiments revealed that FAM83H‐AS1 could epidemically silence CDKN1A expression through recruiting EZH2 to the promoter of CDKN1A, thereby influencing the cell cycle and proliferation. Collectively, our findings suggested that FAM83H‐AS1 participated in the progression of glioma and might act as a potential therapeutic target and prognosis biomarker for human glioma.     

Regulation of insulin resistance by targeting the insulin‐like growth factor 1 receptor with microRNA‐122‐5p in hepatic cells

Insulin resistance (IR) is a common etiology of type 2 diabetes (T2D) defined by a state of decreased reactivity to insulin in multiple organs, such as the liver. This study aims to investigate how microRNA‐122‐5p (miR‐122) regulates the hepatic IR in vitro. We first found that the miR‐122 level was upregulated in the liver of rats fed with a high‐fat diet and injected with streptozotocin (T2D rats), while the expression level of insulin‐like growth factor 1 receptor (IGF‐1R), a potential target of miR‐122, was downregulated in the diabetic liver. In vitro, glucosamine‐induced IR was introduced in HepG2 hepatic cells, and the levels of miR‐122 and IGF‐1R were further assessed. An increase of miR‐122 level and a decrease of IGF‐IR level were observed in IR hepatic cells, which was the same as that in the diabetic liver. Results of the luciferase reporter assay validated IGF‐1R as a direct target of miR‐122. Moreover, in IR HepG2 cells, antagonizing miR‐122 with its specific inhibitor enhanced glucose uptake and suppressed the expression of glucose 6‐phosphatase and phosphoenolpyruvate carboxykinase, two key enzymes in regulating gluconeogenesis. Such alterations induced by the miR‐122 inhibitor in IR hepatic cells were impaired when IGF‐1R was simultaneously knocked down. In addition, the PI3K/Akt pathway was deactivated in IR cells, and then reactivated with miR‐122 inhibitor transfection. In conclusion, our study demonstrates that miR‐122 is able to regulate IR in hepatic cells by targeting IGF‐1R.     

Tumour necrosis factor‐α‐induced protein 8‐like 2 is a novel regulator of proliferation,migration, and invasion in human rectal adenocarcinoma cells

Tumour necrosis factor‐α‐induced protein 8‐like 2 (TIPE2) is a tumour suppressor in many types of cancer. However, the mechanism of action of TIPE2 on the growth of rectal adenocarcinoma is unknown. Our results showed that the expression levels of TIPE2 in human rectal adenocarcinoma tissues were higher than those in adjacent non‐tumour tissues. Overexpression of TIPE2 reduced the proliferation, migration, and invasion of human rectal adenocarcinoma cells and down‐regulation of TIPE2 showed reverse effects. TIPE2 overexpression increased apoptosis through down‐regulating the expression levels of Wnt3a, phospho (p)‐β‐Catenin, and p‐glycogen synthase kinase‐3β in rectal adenocarcinoma cells, however, TIPE2 knockdown exhibited reverse trends. TIPE2 overexpression decreased autophagy by reducing the expression levels of p‐Smad2, p‐Smad3, and transforming growth factor‐beta (TGF‐β) in rectal adenocarcinoma cells, however, TIPE2 knockdown showed opposite effects. Furthermore, TIPE2 overexpression reduced the growth of xenografted human rectal adenocarcinoma, whereas TIPE2 knockdown promoted the growth of rectal adenocarcinoma tumours by modulating angiogenesis. In conclusion, TIPE2 could regulate the proliferation, migration, and invasion of human rectal adenocarcinoma cells through Wnt/β‐Catenin and TGF‐β/Smad2/3 signalling pathways. TIPE2 is a potential therapeutic target for the treatment of rectal adenocarcinoma.     

Protective efficacy of a DNA vaccine encoding capsid protein of porcine circovirus‐like virus P1 against porcine circovirus 2 in mice

The capsid protein is the major immunogenic protein of porcine circovirus 2 (PCV2). The nucleotide sequence of porcine circovirus‐like virus P1 shares high homology with open reading frame (ORF) 2 of PCV2, and ORF1 of P1 encodes its structural protein. Mice were vaccinated twice intramuscularly with a plasmid expressing the P1 ORF1 protein (pcDNA3.1(+)‐ORF1) at 2‐week intervals. All animals vaccinated with pcDNA3.1(+)‐ORF1 developed higher specific anti‐P1 antibody levels, and had less PCV2 viremia and milder histopathological changes than PCV2‐challenged mice in the control group. Our results show that the P1 DNA vaccine elicited immune responses against PCV2 infection in a mouse model.

     

Dickkopf‐1‐promoted vasculogenic mimicry in non‐small cell lung cancer is associated with EMT and development of a cancer stem‐like cell phenotype

To characterize the contributions of Dickkopf‐1 (DKK1) towards the induction of vasculogenic mimicry (VM) in non‐small cell lung cancer (NSCLC), we evaluated cohorts of primary tumours, performed in vitro functional studies and generated xenograft mouse models. Vasculogenic mimicry was observed in 28 of 205 NSCLC tumours, while DKK1 was detected in 133 cases. Notably, DKK1 was positively associated with VM. Statistical analysis showed that VM and DKK1 were both related to aggressive clinical course and thus were indicators of a poor prognosis. Moreover, expression of epithelial‐mesenchymal transition (EMT)‐related proteins (vimentin, Slug, and Twist), cancer stem‐like cell (CSC)‐related proteins (nestin and CD44), VM‐related proteins (MMP2, MMP9, and vascular endothelial‐cadherin), and β‐catenin‐nu were all elevated in VM‐positive and DKK1‐positive tumours, whereas the epithelial marker (E‐cadherin) was reduced in the VM‐positive and DKK1‐positive groups. Non‐small cell lung cancer cell lines with overexpressed or silenced DKK1 highlighted its role in the restoration of mesenchymal phenotypes and development of CSC characteristics. Moreover, DKK1 significantly promotes NSCLC tumour cells to migrate, invade and proliferate. In vivo animal studies demonstrated that DKK1 enhances the growth of transplanted human tumours cells, as well as increased VM formation, mesenthymal phenotypes and CSC properties. Our results suggest that DKK1 can promote VM formation via induction of the expression of EMT and CSC‐related proteins. As such, we feel that DKK1 may represent a novel target of NSCLC therapy.     

The role of albumin and PPAR‐α in differentiation‐dependent change of fatty acid profile during differentiation of mesenchymal stem cells to hepatocyte‐like cells

Differentiation of mesenchymal stem cells (MSCs) to hepatocyte‐like cells is associated with morphological and biological changes. In this study, the effect of hepatogenic differentiation on fatty acid profile and the expression of proliferator‐activated receptors‐α (PPAR‐α) have been studied. For this purpose, MSCs isolated from human umbilical cord were differentiated into hepatocyte‐like cells on selective culture media. The morphological and biochemical changes, PPAR‐α expression and reactive oxygen species (ROS) levels were studied during the differentiation process. Besides, the cells were processed to determine changes in fatty acid profile using gas chromatography analysis. The results showed that hepatic differentiation of the MSCs is associated with a decrease in major polyunsaturated fatty acids in mature hepatocytes, whereas there was an increase in the saturated fatty acid (SFA) levels during hepatocyte maturation. The differentiation‐dependent shift in the ratio of SFA/USFA was associated with changes in albumin and PPAR‐α expression, whereas changes in fatty acid profile were independent of ROS production and lipid peroxidation in differentiating cells. In conclusion, these data may suggest that hepatocyte formation during the stem cell differentiation is associated with a shift in the fatty acid profile that is probably a normal phenomenon in hepatogenic differentiation of the MSCs. Copyright © 2014 John Wiley & Sons, Ltd.     

Follistatin‐like protein 1 contributes to dendritic cell and T‐lymphocyte activation in nasopharyngeal carcinoma patients by altering nuclear factor κb and Jun N‐terminal kinase expression

Follistatin‐like protein 1 (FSTL1) is a newly characterized protein that can regulate the immune response in various ways. Dendritic cells (DCs) are central to immune regulation. In this study, we explored the impact of FSTL1 on DC activity in nasopharyngeal carcinoma (NPC) patients. The surface expression of CD40, CD86, and HLA‐DR on DCs was analyzed and showed significantly elevated expression levels, indicating DC maturity. After FSTL1 was added to DCs collected from NPC patients (n = 50), controls (n = 47), and healthy donors (n = 10), interferon γ secretion and T‐cell receptor expression in cytotoxic T lymphocytes were also investigated. In the experimental groups, the expression of the critical immune protein nuclear factor (NF)‐κb was upregulated, whereas Jun N‐terminal kinase (JNK) was downregulated. Our findings demonstrate that FSTL1 plays a critical role in immune regulation, enhancing the antigen presentation ability of DCs by up‐regulating NF‐κb expression and down‐regulating JNK expression.     

The essentiality of α‐2‐macroglobulin in human salivary innate immunity against new H1N1 swine origin influenza A virus

A novel strain of influenza A H1N1 emerged in the spring of 2009 and has spread rapidly throughout the world. Although vaccines have recently been developed that are expected to be protective, their availability was delayed until well into the influenza season. Although anti‐influenza drugs such as neuraminidase inhibitors can be effective, resistance to these drugs has already been reported. Although human saliva was known to inhibit viral infection and may thus prevent viral transmission, the components responsible for this activity on influenza virus, in particular, influenza A swine origin influenza A virus (S‐OIV), have not yet been defined. By using a proteomic approach in conjunction with beads that bind α‐2,6‐sialylated glycoprotein, we determined that an α‐2‐macroglobulin (A2M) and an A2M‐like protein are essential components in salivary innate immunity against hemagglutination mediated by a clinical isolate of S‐OIV (San Diego/01/09 S‐OIV). A model of an A2M‐based “double‐edged sword” on competition of α‐2,6‐sialylated glycoprotein receptors and inactivation of host proteases is proposed. We emphasize that endogenous A2M in human innate immunity functions as a natural inhibitor against S‐OIV.     

MiR‐29 mediates TGFβ1‐induced extracellular matrix synthesis through activation of PI3K‐AKT pathway in human lung fibroblasts

TGFβ1 is very important in the synthesis and degradation of extracellular matrix, and also in the mediation of human lung fibroblasts proliferation, and miR‐29 plays an important role in this process. To explore the interactions of miR‐29 family members and TGFβ1, the effects of transforming growth factor TGFβ1 on the expression of miR‐29 and whether miR‐29 is involved in pro‐survival signaling pathways mediated by TGFβ1 were examined in human lung fibroblasts. Treatment of the human embryonic lung fibroblast cell line IMR90 with TGFβ1 caused a decrease in expression of miR‐29a/b/c by real‐time PCR analysis. TGFβ1 stimulation increased cell proliferation, colony formation and up‐regulated expression of COL1A1; transfecting with miR‐29a/b/c mimics reverse TGFβ1‐induced phenotype changes in IMR90 cells. Western blot analyses showed that TGFβ1 treatment unchanged total protein expression levels of PI3K or AKT, but the expression levels of p‐PI3K, p‐AKT, and COL1A1 were increased; and miR‐19a/b/c mimics interfering blocked phosphorylation of PI3K or AKT and decreased expression of COL1A1 after TGFβ1 treatment. The results indicate that TGFβ1 beta uses the PI3k‐Akt pathway in these embryonic fibroblasts and miR29 blocks this activation pathway. It indicates a novel biological function of the PI3K‐Akt pathway in IMR90. Elevated expression of miR‐29 may play an important role in the pathogenesis of diseases related to fibrogenic reactions in human lung fibroblasts. J. Cell. Biochem. 114: 1336–1342, 2013. © 2013 Wiley Periodicals, Inc.