Metatranscriptomic analysis of ectomycorrhizal roots reveals genes associated with Piloderma-Pinus symbiosis: improved methodologies for assessing gene expression in situ.

Ectomycorrhizal (EM) fungi form symbiotic associations with plant roots that regulate nutrient exchange between forest plants and soil. Environmental metagenomics approaches that employ next-generation sequencing show great promise for studying EM symbioses; however, metatranscriptomic studies have been constrained by the inherent difficulties associated with isolation and sequencing of RNA from mycorrhizae. Here we apply an optimized method for combined DNA/RNA extraction using field-collected EM fungal-pine root clusters, together with protocols for taxonomic identification of expressed ribosomal RNA, and inference of EM function based on plant and fungal metatranscriptomics. We used transcribed portions of ribosomal RNA genes to identify several transcriptionally dominant fungal taxa associated with loblolly pine including Amphinema, Russula and Piloderma spp. One taxon, Piloderma croceum, has a publically available genome that allowed us to identify patterns of gene content and transcript abundance. Over 1500 abundantly expressed Piloderma genes were detected from mycorrhizal roots, including genes for protein metabolism, cell signalling, electron transport, terpene synthesis and other extracellular activities. In contrast, Piloderma gene encoding an ammonia transporter showed highest transcript abundance in soil samples. Our methodology highlights the potential of metatranscriptomics to identify genes associated with symbiosis and ecosystem function using field-collected samples.

Cholesterol enrichment upregulates intercellular adhesion molecule-1 in human vascular endothelial cells.

Hypercholesterolemia is a major risk factor for atherosclerosis, but the mechanism by which cholesterol activates the endothelium remains undocumented. The present investigation was undertaken to investigate the role of cholesterol, one of the bioactive moieties of the low-density lipoprotein (LDL) particle, in initiating of intracellular signaling in endothelial cells (ECs) and culminating in increased abundance of the intercellular adhesion molecule-1 (ICAM-1). Cholesterol was delivered to human umbilical vein ECs (HUVECs) via cholesterol-enriched liposomes. In HUVECs, the cellular cholesterol:phospholipid ratio increased after 1 h of exposure to cholesterol. The level of ICAM-1 increased in both mRNA and protein after 24 h of cholesterol exposure. ICAM-1 mRNA half-life was not affected by cholesterol exposure. Promoter studies showed greater than two-fold activation of the ICAM-1 gene expression after cholesterol exposure. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) activity substantially increased after 2 h of exposure to cholesterol. In contrast, cholesterol did not affect nuclear factor-kappaB (NF-kappaB) activity. Results of trans-reporting assay revealed 2.5-fold increased expression of the AP-1-dependent reporter gene after cholesterol exposure whereas NF-kappaB-dependent expression was not affected. The AP-1/Ets (-891 to -908) site, one of the three AP-1-like sites in the ICAM-1 promoter, was most responsive to cholesterol. These data demonstrate for the first time that cholesterol enrichment phenotypically modulates ECs by transcriptionally upregulating ICAM-1 expression.

Lipoprotein promotes caveolin-1 and Ras translocation to caveolae: role of cholesterol in endothelial signaling.

To explore the role of LDL in caveolin-Ras regulation in human endothelial cells (ECs), we incubated confluent human umbilical vein endothelial cells (HUVECs) with LDL. This resulted in a high steady-state caveolin-1 (Cav-1) expression at both the mRNA and protein levels. LDL exposure appeared not to regulate the abundance of Cav-1. Immunofluorescence staining showed that Cav-1 protein migrated from the cytoplasm to the cell membrane after LDL exposure. Cav-1 protein and cholesterol partitioned mainly into the caveola fractions, and LDL increased both Cav-1 and cholesterol in these fractions. Ras protein in caveola fractions was also increased by LDL. Increased Ras was detected in Cav-1 immunoprecipitated samples, and conversely, increased Cav-1 was found in Ras-immunoprecipitated samples. We also demonstrated LDL-increased Ras activity in HUVECs by measuring the GTP/GTP+GDP ratio of Ras with [(32)P]orthophosphate labeling in the cells. Finally, we determined the binding of [(3)H]-labeled free cholesterol and recombinant H-Ras to Cav-1 fusion proteins in vitro. Both cholesterol and Ras bound to full-length GST-Cav-1, scaffolding domain (61-101), and C-terminal (135-178) Cav-1 fusion peptides. Addition of cholesterol enhanced Ras binding to the full-length and scaffolding domain of Cav-1 but not to the C-terminal Cav-1. These findings strongly suggest a role for Cav-1 in cholesterol trafficking and cholesterol-mediated intracellular signaling, which may mediate EC activation by LDL.

Selective activation of endothelial cells by the antioxidant pyrrolidine dithiocarbamate: involvement of C-jun N-terminal kinase and AP-1 activation.

The antioxidant agent pyrrolidine dithiocarbamate (PDTC) has been shown to protect endothelial cells (EC) from pro-inflammatory-induced and pro-oxidant-induced NF-kappaB activation. It also perturbs EC by altering activator protein-1 (AP-1) status and inducing ICAM-1. Experiments were performed to investigate the upstream mechanism by which PDTC produces these effects. We have demonstrated that PDTC not only induced AP-1 binding and ICAM-1 expression by itself, but it also augmented AP-1 activation and ICAM-1 induction in low-dose IL-1alpha treated cells. To dissect the mechanism of these effects, we measured c-Jun and c-Fos expression, and the activity of c-Jun NH2-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) in human umbilical vein endothelial cells (HUVEC). We detected an increase in JNK activity in PDTC-treated HUVEC. Following cotransfection with JNK[K-M], a kinase-deficient JNK1, the PDTC-increased AP-1-driven-luciferase activity was attenuated. Utilizing a specific trans-reporting system we confirmed c-Jun activation by upstream signaling mechanisms. The results show that c-Jun activity was increased 9-fold after PDTC treatment. In addition, PDTC promoted more transient activation in ERK-c-fos. In contrast, PDTC produced sustained JNK-c-Jun activation, which translated into long-lasting ICAM-1 production. These results suggest that an antioxidant may contribute to chronic vascular endothelial activation.

Low-density lipoprotein augments interleukin-1-induced vascular adhesion molecule expression in human endothelial cells.

In this study, the effect of low density lipoproteins (LDL) on the ability of the vascular endothelium to respond to vascular cell adhesion molecule 1 (VCAM-1) activation by a cytokine was investigated. After a 4-day pre-exposure to 240 mg/dl of LDL, human umbilical vein endothelial cells (HUVECs) were hyperresponsive to minute amounts of interleukin 1 alpha (IL-1 alpha) as demonstrated by an augmentation of VCAM-1 gene expression. Furthermore, in response to LDL exposure, endothelial recruitment of monocytes induced by minute amounts of IL-1 alpha was increased. This enhancing effect was blocked by an anti-VCAM antibody. The increased response appears not to be due to changes in IL-1 binding affinity or induction of endogenous IL-1 alpha. Transient transfection of HUVECs with a reporter driven by the VCAM promoter showed that LDL increased cellular response to IL-1 alpha by 46%. LDL itself does not increase NF-kappa B binding in endothelial cells (ECs). However, after a 2-day LDL incubation, NF-kappa B binding could be induced by over 63% with a very low dose of IL-1 alpha. IL-1 alpha at this dose (which activates NF-kappa B, but not AP-1) also enhanced LDL-activated AP-1 binding. This cross-enhanced effect may be an important intracellular signaling mechanism for EC activation. The results from this study provide new clues to understanding the mechanisms governing combined risk factors for atherosclerosis.

Low-density lipoprotein activates Jun N-terminal kinase (JNK) in human endothelial cells.

We have reported previously that native low-density lipoprotein (LDL) activates c-Jun and transcription factor AP-1 in human umbilical vein endothelial cells (HUVEC). The aim of this study was to elucidate the upstream signaling mechanisms mediating LDL activation of c-Jun/AP-1. Using a c-Jun NH2-terminal kinase (JNK) activity assay, we have detected an increase in JNK activity in LDL-exposed HUVEC, which started at 15 min and reached maximum activity after 1-2 h. This JNK activity, increased by LDL, occurred in a dose-dependent fashion starting at a concentration of 80 mg/dl of LDL and reaching maximum activation at a concentration of 160-240 mg/dl. Following cotransfection, the increase of AP-1 driven luciferase activity by LDL was attenuated 54% by a kinase-deficient JNK1. Furthermore, a specific trans-reporting system was utilized to confirm c-Jun activation by upstream signal mechanisms. The results show c-Jun activity increased by 3-fold after LDL exposure when compared with respective controls. In contrast, LDL exposure did not affect the activation of extracellular signal regulated kinase 1 and 2 (ERK1/2), even though phorbol 12-myristate 13-acetate treatment remarkably increased the activity of these kinases. Thus, this study demonstrates, for the first time, that JNK mediates LDL-induced endothelial cell activation.

LDL induces transcription factor activator protein-1 in human endothelial cells.

Low density lipoprotein (LDL) has been shown to perturb endothelial cells, with manifestations ranging from alterations in free radicals and arachidonate metabolism to stress fiber formation and monocyte recruitment. Some of these changes are regulated by LDL at the transcriptional level. Using mobility shift assays with consensus sequences for various transcription factors, we have detected an increase in activator protein 1 (AP-1), but not nuclear factor-kappaB (NF-kappaB), binding in human umbilical vein endothelial cells exposed to LDL. Following transfection, AP-1-driven chloramphenicol acetyltransferase and AP-1-driven-luciferase are upregulated by LDL. In contrast, there is no effect on NF-kappaB-driven chloramphenicol acetyltransferase. AP-1 increases in a biphasic fashion, with the first peak occurring 6 hours after and the second 48 hours after exposure to LDL. This AP-1 binding increase involves c-Jun, but not c-Fos, as shown by gel supershift, Northern hybridization, and Western blotting analyses. c-Jun mRNA levels are elevated by 9 hours after and remain so until at least 24 hours after exposure to LDL. c-Jun protein levels increase at 12 hours and continue to rise for 24 hours after exposure to LDL. Moreover, this LDL-increased AP-1 binding is suppressed by several protein kinase (PK) inhibitors: the PKC inhibitor calphostin C, the cAMP-dependent PK inhibitor H89, and the tyrosine PK inhibitors genistein and lavendustin A. This study demonstrates that (1) LDL is an endothelial agonist distinct from other cell stimulators, such as cytokines, endotoxin, and phorbol 12-myristate 13-acetate, because LDL appears to activate human umbilical vein endothelial cells predominantly through the transcription factor AP-1 and not NF-kappaB; and (2) LDL increases AP-1 via mechanisms involving multiple kinase activities and c-Jun transcription.

Activation of ICAM-1 promoter by lysophosphatidylcholine: possible involvement of protein tyrosine kinases.

Lysophosphatidylcholine (lyso-PC) selectively upregulates the mRNA level of intercellular adhesion molecule-1 (ICAM-1) but not that of vascular cell adhesion molecule-1 (VCAM-1) in cultured human umbilical vein endothelial cells. Transfection studies show that lyso-PC activates the ICAM-1 promoter but not the VCAM-1 promoter. Gel mobility shift assays document an increase in NF-kappa B binding in cells treated with lyso-PC. The increases of ICAM-1 mRNA and NF-kappa B binding were inhibited by the protein tyrosine kinase inhibitors, genistein and lavendustin A, but not by inhibitors for cyclic AMP-dependent protein kinases or protein kinase C. Our results suggest that lyso-PC induces ICAM-1 expression most likely by activating NF-kappa B, and that the effect appears to be protein tyrosine kinase-dependent.

Induction of vascular cell adhesion molecule-1 by low-density lipoprotein.

Low-density lipoprotein (LDL) is a well-established risk factor for atherosclerosis. When endothelial cells are incubated with this lipoprotein in pathophysiologic amounts, the cells are activated. Among the documented cellular responses to LDL is increased recruitment of monocytes, which are believed to play a major role in promoting intimal plaque formation. The findings presented here link an atheogenic lipoprotein, LDL, with the induction of an adhesion molecule important in atherogenesis Human LDL induces the vascular cell adhesion molecule-1 (VCAM-1) transcriptionally with an increase in mRNA levels through activation of the VCAM promoter. This effect is blocked by anti-VCAM antibodies. After a 2-day incubation in LDL, the binding of NF-kappa B, which is believed to be a key oxidative-stress sensor for VCAM regulation, remains at basal level. In contrast, the binding activities of AP-1 and GATA, on the other hand, are increased by LDL. Thus, a component of LDL-enhanced endothelial recruitment of monocytes is attributed to VCAM-1 expression, which appears to be mediated through AP-1 and GATA. These data identify LDL as a VCAM-inducer possibly distinct from cytokines and endotoxin.