Survey of 800+ data sets from human tissue and body fluid reveals xenomiRs are likely artifacts.

miRNAs are small 22-nucleotide RNAs that can post-transcriptionally regulate gene expression. It has been proposed that dietary plant miRNAs can enter the human bloodstream and regulate host transcripts; however, these findings have been widely disputed. We here conduct the first comprehensive meta-study in the field, surveying the presence and abundances of cross-species miRNAs (xenomiRs) in 824 sequencing data sets from various human tissues and body fluids. We find that xenomiRs are commonly present in tissues (17%) and body fluids (69%); however, the abundances are low, comprising 0.001% of host human miRNA counts. Further, we do not detect a significant enrichment of xenomiRs in sequencing data originating from tissues and body fluids that are exposed to dietary intake (such as liver). Likewise, there is no significant depletion of xenomiRs in tissues and body fluids that are relatively separated from the main bloodstream (such as brain and cerebro-spinal fluids). Interestingly, the majority (81%) of body fluid xenomiRs stem from rodents, which are a rare human dietary contribution but common laboratory animals. Body fluid samples from the same studies tend to group together when clustered by xenomiR compositions, suggesting technical batch effects. Last, we performed carefully designed and controlled animal feeding studies, in which we detected no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood. In summary, our comprehensive computational and experimental results indicate that xenomiRs originate from technical artifacts rather than dietary intake.

Evidence for the biogenesis of more than 1,000 novel human microRNAs.

BACKGROUND: MicroRNAs (miRNAs) are established regulators of development, cell identity and disease. Although nearly two thousand human miRNA genes are known and new ones are continuously discovered, no attempt has been made to gauge the total miRNA content of the human genome. RESULTS: Employing an innovative computational method on massively pooled small RNA sequencing data, we report 2,469 novel human miRNA candidates of which 1,098 are validated by in-house and published experiments. Almost 300 candidates are robustly expressed in a neuronal cell system and are regulated during differentiation or when biogenesis factors Dicer, Drosha, DGCR8 or Ago2 are silenced. To improve expression profiling, we devised a quantitative miRNA capture system. In a kidney cell system, 400 candidates interact with DGCR8 at transcript positions that suggest miRNA hairpin recognition, and 1,000 of the new miRNA candidates interact with Ago1 or Ago2, indicating that they are directly bound by miRNA effector proteins. From kidney cell CLASH experiments, in which miRNA-target pairs are ligated and sequenced, we observe hundreds of interactions between novel miRNAs and mRNA targets. The novel miRNA candidates are specifically but lowly expressed, raising the possibility that not all may be functional. Interestingly, the majority are evolutionarily young and overrepresented in the human brain. CONCLUSIONS: In summary, we present evidence that the complement of human miRNA genes is substantially larger than anticipated, and that more are likely to be discovered in the future as more tissues and experimental conditions are sequenced to greater depth.

Autotaxin in embryonic development.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the multifunctional lipid mediator lysophosphatidic acid (LPA). LPA signals through six distinct G protein-coupled receptors, acting alone or in concert to activate multiple effector pathways. The ATX-LPA signaling axis is implicated in a remarkably wide variety of physiological and pathological processes and plays a vital role in embryonic development. Disruption of the ATX-encoding gene (Enpp2) in mice results in intrauterine death due to vascular defects in the extra-embryonic yolk sac and embryo proper. In addition, Enpp2 (-/-) embryos show impaired neural development. The observed angiogenic defects are attributable, at least in part, to loss of LPA signaling through the Gα(12/13)-linked RhoA-ROCK-actin remodeling pathway. Studies in zebrafish also have uncovered a dual role for ATX in both vascular and neural development; furthermore, they point to a key role for ATX-LPA signaling in the regulation of left-right asymmetry. Here we discuss our present understanding of the role of ATX-LPA signaling in vertebrate development. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

The polybasic insertion in autotaxin α confers specific binding to heparin and cell surface heparan sulfate proteoglycans.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), playing a key role in diverse physiological and pathological processes. ATX exists in distinct splice variants, but isoform-specific functions remain elusive. Here we characterize the ATXα isoform, which differs from the canonical form (ATXß) in having a 52-residue polybasic insertion of unknown function in the catalytic domain. We find that the ATXα insertion is susceptible to cleavage by extracellular furin-like endoproteases, but cleaved ATXα remains structurally and functionally intact due to strong interactions within the catalytic domain. Through ELISA and surface plasmon resonance assays, we show that ATXα binds specifically to heparin with high affinity (K(d) ~10(-8) M), whereas ATXß does not; furthermore, heparin moderately enhanced the lysophospholipase D activity of ATXα. We further show that ATXα, but not ATXß, binds abundantly to SKOV3 carcinoma cells. ATXα binding was abolished after treating the cells with heparinase III, but not after chondroitinase treatment. Thus, the ATXα insertion constitutes a cleavable heparin-binding domain that mediates interaction with heparan sulfate proteoglycans, thereby targeting LPA production to the plasma membrane.

Autotaxin/Lpar3 signaling regulates Kupffer's vesicle formation and left-right asymmetry in zebrafish.

Left-right (L-R) patterning is essential for proper organ morphogenesis and function. Calcium fluxes in dorsal forerunner cells (DFCs) are known to regulate the formation of Kupffer's vesicle (KV), a central organ for establishing L-R asymmetry in zebrafish. Here, we identify the lipid mediator lysophosphatidic acid (LPA) as a regulator of L-R asymmetry in zebrafish embryos. LPA is produced by Autotaxin (Atx), a secreted lysophospholipase D, and triggers various cellular responses through activation of specific G protein-coupled receptors (Lpar1-6). Knockdown of Atx or LPA receptor 3 (Lpar3) by morpholino oligonucleotides perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ L-R asymmetries, whereas overexpression of lpar3 partially rescued those defects in both atx and lpar3 morphants. Similar defects were observed in embryos treated with the Atx inhibitor HA130 and the Lpar1-3 inhibitor Ki16425. Knockdown of either Atx or Lpar3 impaired calcium fluxes in DFCs during mid-epiboly stage and compromised DFC cohesive migration, KV formation and ciliogenesis. Application of LPA to DFCs rescued the calcium signal and laterality defects in atx morphants. This LPA-dependent L-R asymmetry is mediated via Wnt signaling, as shown by the accumulation of ß-catenin in nuclei at the dorsal side of both atx and lpar3 morphants. Our results suggest a major role for the Atx/Lpar3 signaling axis in regulating KV formation, ciliogenesis and L-R asymmetry via a Wnt-dependent pathway.

Autotaxin and LPA receptor signaling in cancer.

Lysophosphatidic acid (LPA; monoacyl-glycerol-3-phosphate) is a lipid mediator that functions as a mitogen and motility factor for many cell types. LPA signals through six specific G protein-coupled receptors, named LPA(1-6), which trigger both overlapping and distinct signaling pathways. LPA is produced from extracellular lysophosphatidylcholine by a secreted lysophospholipase D, named autotaxin (ATX), originally identified as an "autocrine motility factor" for tumor cells. ATX-LPA signaling is vital for embryonic development and promotes tumor formation, angiogenesis, and experimental metastasis in mice. Elevated expression of ATX and/or aberrant expression of LPA receptors are found in several human malignancies, while loss of LPA(6) function has been implicated in bladder cancer. In this review, we summarize our present understanding of ATX and LPA receptor signaling in cancer.

Structural basis of substrate discrimination and integrin binding by autotaxin.

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

Development of an activity-based probe for autotaxin.

Autotaxin (ATX), or ecto-nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted lysophospholipase D that hydrolyses lysophosphatidylcholine into the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX has been implicated in tumour progression and inflammation, and might serve as a biomarker. Here we describe the development of a fluorescent activity-based probe that covalently binds to the active site of ATX. The probe consists of a lysophospholipid-based backbone linked to a trapping moiety that becomes reactive after phosphate ester hydrolysis, and a Cy5 fluorescent dye to allow visualisation of active ATX. The probe reacts specifically with the three known isoforms of ATX, it competes with small-molecule inhibitors for binding to ATX and allows ATX activity in plasma to be determined. Our activity-based reporter will be useful for monitoring ATX activity in biological fluids and for inhibitor screening.

HIF-1 alpha expression is associated with an atheromatous inflammatory plaque phenotype and upregulated in activated macrophages.

OBJECTIVE: Angiogenesis and inflammation are important features in atherosclerotic plaque destabilization. The transcription factor hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a key regulator of angiogenesis and is also involved in inflammatory reactions. We studied HIF-1 alpha expression in different atherosclerotic plaque phenotypes. METHODS AND RESULTS: HIF-1 alpha expression was observed in 18/37 (49%) carotid and in 9/15 (60%) femoral endarterectomy specimens. Expression of HIF-1 alpha was associated with the presence of a large extracellular lipid core (P=0.03) and macrophages (P=0.02). HIF-1 alpha co-localized with vascular endothelial growth factor (VEGF), an important downstream target of HIF-1 alpha. In addition, a strong association was observed between expression levels of HIF-1 alpha and VEGF (P=0.001). The average number of plaque microvessels was higher in plaques with no or minor HIF-1 alpha staining than in plaques with moderate or heavy HIF-1 alpha staining (P=0.03). In human macrophages, lipopolysaccharide activation induced HIF-1 alpha expression. In embryonic fibroblasts derived from wild-type mice, lipopolysaccharide activation induced an increase in HIF-1 alpha mRNA, whereas in Toll-like receptor 4 defective embryonic fibroblasts no effect was observed after lipopolysaccharide stimulation. CONCLUSIONS: In atherosclerotic plaque, the transcription factor HIF-1 alpha is associated with an atheromatous inflammatory plaque phenotype and with VEGF expression. HIF-1 alpha expression is upregulated in activated macrophages under normoxic conditions.

Identification of HLA-A*0111N: a synonymous substitution, introducing an alternative splice site in exon 3, silenced the expression of an HLA-A allele.

A new variant of the HLA-A*010101 allele designated as HLA-A*0111N, previously known as HLA-A*010101var, was identified in a patient requiring a stem-cell transplantation. The patient was typed by serologic methods as HLA-A2 homozygous and by sequence-based typing (SBT) as A*010101,020601. Flow-cytometric (FCM) analysis with 11 human monoclonal antibodies (mAbs) for the A1 molecule confirmed lack of any cell membrane expression of the A*0111N allele. One-dimensional isoelectric focusing (1D-IEF) of total cell lysate from the patient's cells revealed no cell surface and cytoplasmic A1 protein expression, whereas the HLA-A2 molecule was identified by both FCM analysis and 1D-IEF. DNA sequence analysis showed the presence of a synonymous substitution from G to T at position 597 in codon 175. RNA SBT revealed a deletion of 24 bp in exon 3, position 596 through 619, encoding codons 175 through 182 of the HLA-A*0111N allele. The synonymous substitution introduced a new splice site, resulting in an efficient splicing, because no classical A1 protein could be detected in the patient. This alternative splicing prevented the translation into a correct and stable class I molecule expression on the cell surface.