LRP5 and LRP6 Are Not Required for Protective Antigen–Mediated Internalization or Lethality of Anthrax Lethal Toxin

Anthrax toxin (AnTx) plays a key role in the pathogenesis of anthrax. AnTx is composed of three proteins: protective antigen (PA), edema factor, and lethal factor (LF). PA is not toxic but serves to bind cells and translocate the toxic edema factor or LF moieties to the cytosol. Recently, the low-density lipoprotein receptor–related protein LRP6 has been reported to mediate internalization and lethality of AnTx. Based on its similarity to LRP6, we hypothesized that LRP5 may also play a role in cellular uptake of AnTx. We assayed PA-dependent uptake of anthrax LF or a cytotoxic LF fusion protein (FP59) in cells and mice harboring targeted deletions of Lrp5 or Lrp6. Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression. Moreover, we observed efficient PA-mediated uptake into anthrax toxin receptor (ANTXR)–deficient Chinese hamster ovary cells (PR230) that had been stably engineered to express either human ANTXR1 or human ANTXR2 in the presence or absence of siRNA specific for LRP5 or LRP6. Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin

Perturbation of Mouse Retinal Vascular Morphogenesis by Anthrax Lethal Toxin

Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK). In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis

Partial Deletion of the Sulfate Transporter <em>SLC13A1</em> Is Associated with an Osteochondrodysplasia in the Miniature Poodle Breed

<div><p>A crippling dwarfism was first described in the Miniature Poodle in Great Britain in 1956. Here, we resolve the genetic basis of this recessively inherited disorder. A case-control analysis (8∶8) of genotype data from 173 k SNPs revealed a single associated locus on <em>CFA14</em> (P_raw <10^–8). All affected dogs were homozygous for an ancestral haplotype consistent with a founder effect and an identical-by-descent mutation. Systematic failure of nine, nearly contiguous SNPs, was observed solely in affected dogs, suggesting a deletion was the causal mutation. A 130-kb deletion was confirmed both by fluorescence <em>in situ</em> hybridization (FISH) analysis and by cloning the physical breakpoints. The mutation was perfectly associated in all cases and obligate heterozygotes. The deletion ablated all but the first exon of <em>SLC13A1</em>, a sodium/sulfate symporter responsible for regulating serum levels of inorganic sulfate. Our results corroborate earlier findings from an <em>Slc13a1</em> mouse knockout, which resulted in hyposulfatemia and syndromic defects. Interestingly, the metabolic disorder in Miniature Poodles appears to share more clinical signs with a spectrum of human disorders caused by <em>SLC26A2</em> than with the mouse <em>Slc13a1</em> model. <em>SLC26A2</em> is the primary sodium-independent sulfate transporter in cartilage and bone and is important for the sulfation of proteoglycans such as aggregan. We propose that disruption of <em>SLC13A1</em> in the dog similarly causes undersulfation of proteoglycans in the extracellular matrix (ECM), which impacts the conversion of cartilage to bone. A co-dominant DNA test of the deletion was developed to enable breeders to avoid producing affected dogs and to selectively eliminate the mutation from the gene pool.</p> </div

Genetic mapping of the osteochondrodysplasia locus.

<p>(A) Manhattan plot of genome-wide results displaying a statistically significant peak of association on the terminal end of <i>CFA14</i>. Plotted <i>p</i>-values were calculated by Fisher’s Exact Test. The p-values for the six most strongly associated SNPs (overlapping on the graph), adjusted by permutation, were p = 0.0006. (B) Genotypic pattern of SNPs across the interval of interest on <i>CFA14</i> (62.5–64.0 Mb). Red, major allele in cases; orange, heterozygous SNP genotypes; yellow, homozygous minor allele in cases. Genotype data are shown horizontally for each of seven controls and eight cases. The arrow notes an historical crossover that bounds the ancestral haplotype shared among cases, upon which the founder mutation is predicted to have occurred. Nine SNPs systematically failed in the case samples (white block with orange), suggesting a large deletion. (C) Raw fluorescence intensity readings from the SNP array for loci across the region of interest (62.5–64.0 Mb). Although several SNPs were initially scored heterozygous (orange, in B), the raw intensity data shows systematic failure in cases for all 12 SNPs in this interval (red bracket), relative to controls. (D) The potential deletion was bounded by two SNPs that were scored in cases. The putative deletion includes a single gene, <i>SLC13A1</i>, drawn to scale, with exon structure in red. The location of physical breakpoints that were subsequently cloned are shown as opaque boxes.</p

Cytogenetic confirmation of a deletion on chromosome 14 by <i>FISH</i>.

<p>Dual-color FISH probe results are shown for representative metaphase spreads obtained from (A) wildtype, (B) obligate carrier, and (C) affected Miniature Poodle dogs, respectively. The green LR-PCR 14–63.6 g FISH signal is within the deleted region and shows some centromeric background staining. The red BAC probe 14–59.1or signal distinguishes <i>CFA14</i> within the canine metaphase spread. Wildtype shows co-localization of red (control) and green (experimental) probes. This co-localization is absent in the affected dog (homozygous for deletion), and is heterozygous in the carrier dog.</p

DNA test of large scale deletion.

<p>(A) A three-primer, dual reporter design exploits known breakpoints to create differentially dye-labeled and differentially-sized PCR products for a co-dominant DNA test readout. (B) The assay converted to a high-throughput format by analysis with a semi-automated DNA sequencing instrument for fluorescence-based fragment analysis. The electropherogram shows standard fragments for sizing (red peaks), product corresponding to the deletion-specific allele (blue peak), and product corresponding to the wildtype reference allele (green peak). Test outcomes are shown for each of the three genotypes possible. There is a size difference in the products corresponding to the wildtype allele in the clear dog and the carrier dog. This is attributable to a small insertion/deletion in the interval that segregates in the Miniature Poodle population.</p

Gross presentation of Miniature Poodle osteochondrodysplasia.

<p>(A) An affected dog (foreground) shows dwarfism relative to an age-matched normal (wildtype) dog (background). (B) The misshapen paw of an affected dog (Aff) relative to that of a wildtype (WT) dog. (C) Close-up of the affected foot, which is similar to talipes varus, or clubfoot, a distinguishing feature of human osteochondrodsyplasias.</p

Serum sulfate levels by <i>SLC13A1</i> genotypic class.

<p>A scatter plot for sulfate concentration measurements from serum obtained from several dogs of each genotypic class: <i>SLC13A1</i> homozygotes (n = 5); heterozygotes (n = 4); and Δ<i>slc13a1</i> homozygotes (affected dogs; n = 3). The minimum sensitivity threshold for the assay is 0.2 mM. Differences between genotypes were assessed with the Kruskal Wallis test. Although sample sizes were relatively small, the mean sulfate level of affected dogs was significantly lower than the means of the other genotypic classes (<i>p</i> = 0.02).</p

Phosphorylation of TXNIP by AKT Mediates Acute Influx of Glucose in Response to Insulin

Growth factors, such as insulin, can induce both acute and long-term glucose uptake into cells. Apart from the rapid, insulin-induced fusion of glucose transporter (GLUT)4 storage vesicles with the cell surface that occurs in muscle and adipose tissues, the mechanism behind acute induction has been unclear in other systems. Thioredoxin interacting protein (TXNIP) has been shown to be a negative regulator of cellular glucose uptake. TXNIP is transcriptionally induced by glucose and reduces glucose influx by promoting GLUT1 endocytosis. Here, we report that TXNIP is a direct substrate of protein kinase B (AKT) and is responsible for mediating AKT-dependent acute glucose influx after growth factor stimulation. Furthermore, TXNIP functions as an adaptor for the basal endocytosis of GLUT4 in vivo, its absence allows excess glucose uptake in muscle and adipose tissues, causing hypoglycemia during fasting. Altogether, TXNIP serves as a key node of signal regulation and response for modulating glucose influx through GLUT1 and GLUT4