Overexpression of the alfalfa (Medicago sativa) gene, MsKMS1, negatively regulates seed germination in transgenic tobacco (Nicotiana tabacum).

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-associated proteins are a class of transmembrane proteins involved in intracellular trafficking pathways. However, the functions of many SNARE domain-containing proteins remain unclear. We have previously identified a SNARE-associated gene in alfalfa (Medicago sativa ) KILLING ME SLOWLY1 (MsKMS1 ), which is involved in various abiotic stresses. In this study, we investigated the function of MsKMS1 in the seed germination of transgenic tobacco (Nicotiana tabacum ). Phylogenetic analysis showed that MsKMS1 was homologous to the SNARE-associated or MAPR component-related proteins of other plants. Germination assays revealed that MsKMS1 negatively regulated seed germination under normal, D-mannitol and abscisic acid-induced stress conditions, yet MsKMS1 -overexpression could confer enhanced heat tolerance in transgenic tobacco. The suppressive effect on germination in MsKMS1 -overexpression lines was associated with higher abscisic acid and salicylic acid contents in seeds. This was accompanied by the upregulation of abscisic acid biosynthetic genes (ZEP and NCED ) and the downregulation of gibberellin biosynthetic genes (GA20ox2 and GA20ox3 ). Taken together, these results suggested that MsKMS1 negatively regulated seed germination by increasing abscisic acid and salicylic acid contents through the expression of genes related to abscisic acid and gibberellin biosynthesis. In addition, MsKMS1 could improve heat tolerance during the germination of transgenic tobacco seeds.

A 3-Ketoacyl-CoA Synthase 10 (KCS10) Homologue from Alfalfa Enhances Drought Tolerance by Regulating Cuticular Wax Biosynthesis.

Cuticular wax, forming the first line of defense against adverse environmental stresses, comprises very long-chain fatty acids (VLCFAs) and their derivatives. 3-Ketoacyl-CoA synthase (KCS) is a rate-limiting enzyme for VLCFA biosynthesis. In this study, we isolated KCS10, a KCS gene from alfalfa, and analyzed the effect of gene expression on wax production and drought stress in transgenic plants. MsKCS10 overexpression increased compact platelet-like crystal deposition and promoted primary alcohol biosynthesis through acyl reduction pathways in alfalfa leaves. Overexpression of MsKCS10 induced the formation of coiled-rodlet-like crystals and increased n-alkane content through decarbonylation pathways in tobacco and tomato fruits. Overexpression of MsKCS10 enhanced drought tolerance by limiting nonstomatal water loss, improving photosynthesis, and maintaining osmotic potential under drought stress in transgenic tobacco. In summary, MsKCS10 plays an important role in wax biosynthesis, wax crystal morphology, and drought tolerance, although the mechanisms are different among the plant species. MsKCS10 can be targeted in future breeding programs to improve drought tolerance in plants.

PARP14 promotes the growth and glycolysis of acute myeloid leukemia cells by regulating HIF-1α expression.

OBJECTIVE: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a poor prognosis. This study aimed to investigate the action of PARP14 in the growth and glycolysis of AML. METHODS: The clinical samples of AML patients were collected, and the expression of PARP14 was detected. AML cells were transfected with PARP14, HIF-1α or treated with NF-KB inhibitor (BAY11-7082) or PARP14 inhibitor (RBN012759). Cell proliferation was detected by CCK-8 and colony formation assays, apoptosis by flow cytometry, glucose consumption and lactate production by glucose and lactate kits, ECAR and OCR by XF96 bioenergy analyzer, and related protein levels by Western blot. A mouse xenograft tumor model was established to evaluate the effect of PARP14 on tumor formation. RESULTS: Significant upregulation of PARP14 expression was observed in AML. PARP14 promoted AML cell proliferation and glycolysis and inhibited apoptosis, while PARP14 deficiency had the opposite effect. PARP14 promoted HIF-1α expression by activating NF-κB. HIF-1α silencing reversed the cancer-promoting effect of PARP14. In vivo results suggested that PARP14 promoted tumor formation. CONCLUSION: PARP14 induces AML cell growth and glycolysis by activating NF-κB and promoting HIF-1α expression, which may suggest new insights into the pathogenesis of AML.

ICAM-1-related noncoding RNA accelerates atherosclerosis by amplifying NF-κB signaling.

Long noncoding RNAs (lncRNAs) are critical regulators of inflammation with great potential as new therapeutic targets. However, the role of lncRNAs in early atherosclerosis remains poorly characterized. This study aimed to identify the key lncRNA players in activated endothelial cells (ECs). The lncRNAs in response to pro-inflammatory factors in ECs were screened through RNA sequencing. ICAM-1-related non-coding RNA (ICR) was identified as the most potential candidate for early atherosclerosis. ICR is essential for intercellular adhesion molecule-1 (ICAM1) expression, EC adhesion and migration. In a high fat diet-induced atherosclerosis model in mice, ICR is upregulated in the development of atherosclerosis. After intravenous injection of adenovirus carrying shRNA for mouse ICR, the atherosclerotic plaque area was markedly reduced with the declined expression of ICR and ICAM1. Mechanistically, ICR stabilized the mRNA of ICAM1 in quiescent ECs; while under inflammatory stress, ICR upregulated ICAM1 in a nuclear factor kappa B (NF-κB) dependent manner. RNA-seq analysis showed pro-inflammatory targets of NF-κB were regulated by ICR. Furthermore, the chromatin immunoprecipitation assays showed that p65 binds to ICR promoter and facilitates its transcription. Interestingly, ICR, in turn, promotes p65 accumulation and activity, forming a positive feedback loop to amplify NF-κB signaling. Preventing the degradation of p65 using proteasome inhibitors rescued the expression of NF-κB targets suppressed by ICR. Taken together, ICR acts as an accelerator to amplify NF-κB signaling in activated ECs and suppressing ICR is a promising early intervention for atherosclerosis through ICR/p65 loop blockade.

Hyperglycemia Promotes Endothelial Cell Senescence through AQR/PLAU Signaling Axis.

Hyperglycemia is reported to accelerate endothelial cell senescence that contributes to diabetic complications. The underlying mechanism, however, remains elusive. We previously demonstrated AQR as a susceptibility gene for type 2 diabetes mellitus (T2DM) and showed that it was increased in multiple tissues in models with T2DM or metabolic syndrome. This study aimed to investigate the role of AQR in hyperglycemia-induced senescence and its underlying mechanism. Here, we retrieved several datasets of the aging models and found the expression of AQR was increased by high glucose and by aging across species, including C. elegans (whole-body), rat (cardiac tissues), and monkey (blood). we validated the increased AQR expression in senescent human umbilical vein endothelial cells (HUVECs). When overexpressed, AQR promoted the endothelial cell senescence, confirmed by an increased number of cells stained with senescence-associated beta-galactosidase and upregulation of CDKN1A (P21) as well as the prohibited cellular colony formation and G2/M phase arrest. To explore the mechanism by which AQR regulated the cellular senescence, transcriptomic analyses of HUVECs with the overexpression and knockdown of the AQR were performed. We identified 52 co-expressed genes that were enriched, in the terms of plasminogen activation, innate immunity, immunity, and antiviral defense. Among co-expressed genes, PLAU was selected to evaluate its contribution to senescence for its highest strength in the enrichment of the biological process. We demonstrated that the knockdown of PLAU rescued senescence-related phenotypes, endothelial cell activation, and inflammation in models induced by AQR or TNF-α. These findings, for the first time, indicate that AQR/PLAU is a critical signaling axis in the modulation of endothelial cell senescence, revealing a novel link between hyperglycemia and vascular dysfunction. The study may have implications in the prevention of premature vascular aging associated with T2DM.

Allelic distribution of ABO gene in Chinese centenarians.

OBJECTIVE: Human ABO blood groups are determined by the alleles A, B, and O (O01 and O02) of the ABO gene and have been linked to the risks for cardiovascular diseases and cancers that affect lifespan.We examined the genetic associations of the ABO gene and blood groups with longevity. METHODS: We inspected the frequencies of the A, B, O, and O02 alleles in a large Chinese centenarian population (n = 2201) and in middle-aged controls (n = 2330). The single nucleotide polymorphisms were selected as allele A (rs507666), B (rs8176743, rs8176746, and rs8176749), O (rs687289), and O02 (rs688976, rs549446, and rs512770). RESULTS: Supported by allelic and genotypic association studies, the frequencies of blood types A, B, O, and AB in centenarian versus control participants were not statistically different: 0.2821 versus 0.2781 (χ2 = 0.09, P = 0.76), 0.2867 versus 0.3060 (χ2 = 2.03, P = 0.15), 0.3380 versus 0.3159 (χ2 = 2.52, P = 0.11), and 0.0859 versus 0.0910 (χ2 = 0.37, P = 0.54), respectively. Sex had little effect on these distributions. CONCLUSION: Integrated with other previous reports, we conclude from this large Chinese cohort that genetic variants of the ABO gene and blood groups are not associated with longevity.

AQR is a novel type 2 diabetes-associated gene that regulates signaling pathways critical for glucose metabolism.

Type 2 diabetes mellitus (T2DM) is a common metabolic disease influenced by both genetic and environmental factors. In this study, we performed an in-house genotyping and meta-analysis study using three independent GWAS datasets of T2DM and found that rs3743121, located 1 kb downstream of AQR, was a novel susceptibility SNP associated with T2DM. The risk allele C of rs3743121 was correlated with the increased expression of AQR in white blood cells, similar to that observed in T2DM models. The knockdown of AQR in HepG2 facilitated the glucose uptake, decreased the expression level of PCK2, increased the phosphorylation of GSK-3ß, and restored the insulin sensitivity. Furthermore, the suppression of AQR inhibited the mTOR pathway and the protein ubiquitination process. Our study suggests that AQR is a novel type 2 diabetes-associated gene that regulates signaling pathways critical for glucose metabolism.

Ctenopharyngodon idella p53 mediates between NF-κB and PKR at the transcriptional level.

p53, NF-κB and PKR are well-known to be involved in antiviral response. Although p53 has been reported in fish, its role in the regulation of NF-κB and PKR is not well understood. Here, we cloned and characterized the full length of cDNA sequence of grass carp (Ctenopharyngodon idella) p53 (Cip53) and its promoter sequence. The full length cDNA of Cip53 was 1879 bp with an ORF of 1116 bp encoding a polypeptide of 371 amino acids. Phylogenetic tree analysis revealed that Cip53 shares high homology with Dario rerio p53 (Drp53). Similar to those of Cip65 and CiPKR, the expression of Cip53 in CIK cells was significantly up-regulated after stimulation with poly I:C. To further understand the roles of fish p53 in the transcriptional control of NF-κB and PKR, Cip53 and Cip65 were expressed in E. coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, gel mobility shift assays demonstrated that the high affinity interaction between Cip65 and Cip53 promoter. Similarly, Cip53 bound to CiPKR promoter with high affinity. Dual-luciferase reporter assays showed that Cip65 activated Cip53 promoter and Cip53 activated CiPKR promoter, respectively. In addition, the role of p53 in p65-p53-PKR transcription pathway was explored. When Cip53 was knockdown in CIK cells, the mRNA levels of Cip65 and CiPKR were decreased. Taken together, p53 may play pivotal roles in transcription pathway of NF-κB and PKR in fish.

A mechanism underlies fish GRP78 protection against Pb2+ toxicity.

Heavy metal exposure impacts basic cellular processes and results in serious toxicological effects. Pb2+ can activate the response to endoplasmic reticulum (ER) stress by protein denaturation, changing intracellular calcium homeostasis, and inducing cell death. As an ER retention protein, 78-kDa glucose-regulated protein (GRP78) can relieve the Pb2+-induced ER stress and enhance cell viability. We previously showed that heavy metal ions such as Pb2+ etc. are harmful to fish cell lines in a time- and dose-dependent manner. The phenomenon is accompanied by the increasing accumulation of grass carp GRP78 (CiGRP78), which can protect the cells from heavy metal ion cytotoxicity. Here, we investigated the mechanism in which CiGRP78 exerted its protective function. Using metal ions affinity elution method and fluorescent spectral analysis, we showed that CiGRP78 could respectively form a complex with Calcium, Lead and Cadmium ions, especially with Lead ion in vitro. However, another ER retention protein CiGRP94 could not bind to Pb2+, highlighting the functional differentiation might exist in CiGRP78 and CiGRP94 in regulating heavy metal cytotoxicity. Our results suggested that CiGRP78 might increase cellular tolerance to Pb2+ via the direct interaction with it.

Ctenopharyngodon idella IRF2 and ATF4 down-regulate the transcriptional level of PRKRA.

PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a protective protein which regulates the adaptation of cells to ER stress and virus-stimulated signaling pathways by activating PKR. In the present study, a grass carp (Ctenopharyngodon idella) PRKRA full-length cDNA (named CiPRKRA, KT891991) was cloned and identified. The full-length cDNA is comprised of a 5' UTR (36 bp), a 3' UTR (350 bp) and the longest ORF (882 bp) encoding a polypeptide of 293 amino acids. The deduced amino acid sequence of CiPRKRA contains three typical dsRNA binding motifs (dsRBM). Phylogenetic tree analysis revealed a closer evolutionary relationship of CiPRKRA with other fish PRKRA, especially with Danio rerio PRKRA. qRT-PCR showed that CiPRKRA was significantly up-regulated after stimulation with tunicamycin (Tm) and Poly I:C in C. idella kidney (CIK) cells. To further study its transcriptional regulation, the partial promoter sequence of CiPRKRA (1463 bp) containing one ISRE and one CARE was cloned by Tail-PCR. Subsequently, grass carp IRF2 (CiIRF2) and ATF4 (CiATF4) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. In vitro, both CiIRF2 and CiATF4 bound to CiPRKRA promoter with high affinity by gel mobility shift assays, revealing that IRF2 and ATF4 might be potential transcriptional regulatory factors for CiPRKRA. Dual-luciferase reporter assays were applied to further investigate the transcriptional regulation of CiPRKRA in vivo. Recombinant plasmid of pGL3-PRKRAPro was constructed and transiently co-transfected into CIK cells with pcDNA3.1-CiIRF2 and pcDNA3.1-CiATF4, respectively. The results showed that both CiIRF2 and CiATF4 significantly decreased the luciferase activity of pGL3-PRKRAPro, suggesting that they play a negative role in CiPRKRA transcription.

Identification of grass carp (Ctenopharyngodon idella) XBP1S as a primary member in ER stress.

X-box binding protein 1 (XBP1), a vital basic leucine zipper transcription factor for the related gene transcription in endoplasmic reticulum (ER) stress, belongs to the CREB/ATF family. In mammals, XBP1S is the activated one of XBP1 isoform. In order to study the role of fish XBP1S, we cloned and identified the XBP1S (KU509247) from grass carp (Ctenopharyngodon idella) (named CiXBP1S) by homologous cloning and RACE technique. The full length of CiXBP1S is 1694 bp along with 124 bp of 5' UTR, 418 bp of 3' UTR and the longest open reading frame (1152 bp) encoding a polypeptide of 383 amino acids with a well conserved DNA binding domain (BRLZ domain). CiXBP1S shares significant homology to zebrafish XBP1S (∼90%) at amino acid level. RT-PCR showed that the expression of CiXBP1S was ubiquitous in all tested grass carp tissues and was significantly up-regulated under the stimulation with tunicamycin (Tm) in CIK (C. idellus kidney) cells. To study the molecular mechanism of transcriptional regulation for XBP1 signaling pathway in fish, we cloned grass carp XBP1 promoter sequence. Its promoter is 1036 bp in length and divided into two distinct regions in which an ER stress response element (ERSE) exists in the proximal region. Meanwhile, grass carp ATF6 (CiATF6N) and CiXBP1S were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. Gel mobility shift assay showed that CiATF6N and CiXBP1S had the high affinity with CiXBP1 promoter sequence in vitro. Co-transfection of pcDNA3.1-CiATF6 (or pcDNA3.1-CiXBP1S respectively) with pGL3-CiXBP1P2 (or pGL3-CiXBP1P1 respectively) into epithelioma papulosum cyprini (EPC) cells showed that CiATF6 and CiXBP1S played a positive role in CiXBP1S transcription. CiXBP1S also had high affinity with CiGRP78 and CiGRP94 promoter sequences. In addition, recombinant plasmids of pGL3-CiGRP78P and pGL3-CiGRP94P were constructed and transiently co-transfected with pcDNA3.1-CiXBP1S (pcDN3.1-CiXBP1S-nBRLZ, respectively) into EPC cells. The result showed that CiXBP1S can activate CiGRP78 and CiGRP94 promoters.