Limits...
LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin.

Young JJ, Bromberg-White JL, Zylstra C, Church JT, Boguslawski E, Resau JH, Williams BO, Duesbery NS - PLoS Pathog. (2007)

Bottom Line: Recently, the low-density lipoprotein receptor-related protein LRP6 has been reported to mediate internalization and lethality of AnTx.Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression.Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer and Developmental Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, United States of America.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

In Vitro Treatment of Embryonic Fibroblasts from LRP5- and LRP6-Deficient Mice with PA Plus FP59 or LF(A) To assess the effects of LRP5 or LRP6 deficiency upon LeTx-sensitivity in vitro, embryonic fibroblasts from LRP5 and LRP6 parental (+/+), heterozygous (ko/+), and izygous (ko/ko) mice were treated with PA (1 ug/ml) and FP59 (20 pg/ml–2 ug/ml). Cell viability (oordinate) at the end of 24 h of incubation is plotted versus the concentration of FP59 (abcissa). Error bars indicate standard deviation between three replicates in a single experiment.(B) Alternatively, embryonic fibroblasts from the same mice were treated with FP59 (1 ng/ml) and PA (1 pg/ml–10 ug/ml). Cell viability (oordinate) at the end of 24 h of incubation is plotted versus the concentration of PA (abcissa). Error bars indicate standard deviation between three replicates in a single experiment. This plot is representative of three independent experiments.(C) As an independent indicator of PA entry into embryonic fibroblasts derived from mice with targeted deletions of LRP5 (upper panel) and LRP6 (lower panel), cleavage of MEK1 was assessed by immunoblotting with antibodies that are specific for the NH2-terminus of MEK1. UT, untreated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1808072&req=5

ppat-0030027-g002: In Vitro Treatment of Embryonic Fibroblasts from LRP5- and LRP6-Deficient Mice with PA Plus FP59 or LF(A) To assess the effects of LRP5 or LRP6 deficiency upon LeTx-sensitivity in vitro, embryonic fibroblasts from LRP5 and LRP6 parental (+/+), heterozygous (ko/+), and izygous (ko/ko) mice were treated with PA (1 ug/ml) and FP59 (20 pg/ml–2 ug/ml). Cell viability (oordinate) at the end of 24 h of incubation is plotted versus the concentration of FP59 (abcissa). Error bars indicate standard deviation between three replicates in a single experiment.(B) Alternatively, embryonic fibroblasts from the same mice were treated with FP59 (1 ng/ml) and PA (1 pg/ml–10 ug/ml). Cell viability (oordinate) at the end of 24 h of incubation is plotted versus the concentration of PA (abcissa). Error bars indicate standard deviation between three replicates in a single experiment. This plot is representative of three independent experiments.(C) As an independent indicator of PA entry into embryonic fibroblasts derived from mice with targeted deletions of LRP5 (upper panel) and LRP6 (lower panel), cleavage of MEK1 was assessed by immunoblotting with antibodies that are specific for the NH2-terminus of MEK1. UT, untreated.

Mentions: Wei et al. [22] observed that antisense expression of an EST corresponding to an intronic sequence between exons 21 and 22 of the LRP6 gene could 1) reduce expression of LRP6, and 2) protect M2182 prostate carcinoma cells from PA-mediated uptake of FP59, a cytotoxic fusion protein consisting of the N-terminus of LF genetically fused with the ADP-ribosylating domain of Pseudomonas exotoxin A [43]. These observations formed the basis for their conclusion that LRP6 was essential for PA-dependent uptake into cells. To test whether Lrp5 was essential for PA-mediated internalization, we isolated murine embryonic fibroblasts (MEFs) from Lrp5+/+ parental, Lrp5+/− heterozygous, and Lrp5−/− izygous mice, and treated them with PA plus FP59. Lrp5+/+ parental, Lrp5+/− heterozygous, and Lrp5−/− izygous MEFs demonstrated equal sensitivity to treatment with a constant amount of PA plus varying concentrations of FP59 (Figure 2A), or with a varying amount of PA plus constant concentrations of FP59 (Figure 2B). These results indicate that loss of Lrp5 expression alters neither MEF sensitivity to FP59 nor the ability of PA to translocate FP59 into cells. As an independent measure of PA-mediated entry, MEFs were treated with PA plus LF, and lysates were immunoblotted for N-terminal proteolysis of mitogen activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1. Lrp5+/+ parental, Lrp5+/− heterozygous, and Lrp5−/− izygous MEFs demonstrated equal cleavage of MEK1 following treatment with PA plus LF (Figure 2C). These observations demonstrate that the loss of expression of LRP5 is not sufficient to prevent PA-mediated uptake of FP59 or LF. Similar tests were performed with MEFs from Lrp6+/+ parental, Lrp6+/− heterozygous, and Lrp6−/− izygous mice. Each of these MEFs demonstrated equal sensitivity to treatment with combinations of PA plus FP59 (Figure 2A and 2B). Again, as an independent measure of PA-mediated entry, MEFs were treated with PA plus LF, and lysates were immunoblotted for N-terminal proteolysis of MEK1. Lrp6+/+ parental, Lrp6+/− heterozygous, and Lrp6−/− izygous MEFs demonstrated equal MEK1 cleavage following treatment with PA plus LF (Figure 2C). These observations indicate that the loss of expression of Lrp6 is not sufficient to prevent PA-mediated uptake of FP59 or LF. Collectively, these results demonstrate that neither Lrp5 nor Lrp6 is essential for PA-mediated uptake of FP59 or LF.


LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin.

Young JJ, Bromberg-White JL, Zylstra C, Church JT, Boguslawski E, Resau JH, Williams BO, Duesbery NS - PLoS Pathog. (2007)

In Vitro Treatment of Embryonic Fibroblasts from LRP5- and LRP6-Deficient Mice with PA Plus FP59 or LF(A) To assess the effects of LRP5 or LRP6 deficiency upon LeTx-sensitivity in vitro, embryonic fibroblasts from LRP5 and LRP6 parental (+/+), heterozygous (ko/+), and izygous (ko/ko) mice were treated with PA (1 ug/ml) and FP59 (20 pg/ml–2 ug/ml). Cell viability (oordinate) at the end of 24 h of incubation is plotted versus the concentration of FP59 (abcissa). Error bars indicate standard deviation between three replicates in a single experiment.(B) Alternatively, embryonic fibroblasts from the same mice were treated with FP59 (1 ng/ml) and PA (1 pg/ml–10 ug/ml). Cell viability (oordinate) at the end of 24 h of incubation is plotted versus the concentration of PA (abcissa). Error bars indicate standard deviation between three replicates in a single experiment. This plot is representative of three independent experiments.(C) As an independent indicator of PA entry into embryonic fibroblasts derived from mice with targeted deletions of LRP5 (upper panel) and LRP6 (lower panel), cleavage of MEK1 was assessed by immunoblotting with antibodies that are specific for the NH2-terminus of MEK1. UT, untreated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1808072&req=5

ppat-0030027-g002: In Vitro Treatment of Embryonic Fibroblasts from LRP5- and LRP6-Deficient Mice with PA Plus FP59 or LF(A) To assess the effects of LRP5 or LRP6 deficiency upon LeTx-sensitivity in vitro, embryonic fibroblasts from LRP5 and LRP6 parental (+/+), heterozygous (ko/+), and izygous (ko/ko) mice were treated with PA (1 ug/ml) and FP59 (20 pg/ml–2 ug/ml). Cell viability (oordinate) at the end of 24 h of incubation is plotted versus the concentration of FP59 (abcissa). Error bars indicate standard deviation between three replicates in a single experiment.(B) Alternatively, embryonic fibroblasts from the same mice were treated with FP59 (1 ng/ml) and PA (1 pg/ml–10 ug/ml). Cell viability (oordinate) at the end of 24 h of incubation is plotted versus the concentration of PA (abcissa). Error bars indicate standard deviation between three replicates in a single experiment. This plot is representative of three independent experiments.(C) As an independent indicator of PA entry into embryonic fibroblasts derived from mice with targeted deletions of LRP5 (upper panel) and LRP6 (lower panel), cleavage of MEK1 was assessed by immunoblotting with antibodies that are specific for the NH2-terminus of MEK1. UT, untreated.
Mentions: Wei et al. [22] observed that antisense expression of an EST corresponding to an intronic sequence between exons 21 and 22 of the LRP6 gene could 1) reduce expression of LRP6, and 2) protect M2182 prostate carcinoma cells from PA-mediated uptake of FP59, a cytotoxic fusion protein consisting of the N-terminus of LF genetically fused with the ADP-ribosylating domain of Pseudomonas exotoxin A [43]. These observations formed the basis for their conclusion that LRP6 was essential for PA-dependent uptake into cells. To test whether Lrp5 was essential for PA-mediated internalization, we isolated murine embryonic fibroblasts (MEFs) from Lrp5+/+ parental, Lrp5+/− heterozygous, and Lrp5−/− izygous mice, and treated them with PA plus FP59. Lrp5+/+ parental, Lrp5+/− heterozygous, and Lrp5−/− izygous MEFs demonstrated equal sensitivity to treatment with a constant amount of PA plus varying concentrations of FP59 (Figure 2A), or with a varying amount of PA plus constant concentrations of FP59 (Figure 2B). These results indicate that loss of Lrp5 expression alters neither MEF sensitivity to FP59 nor the ability of PA to translocate FP59 into cells. As an independent measure of PA-mediated entry, MEFs were treated with PA plus LF, and lysates were immunoblotted for N-terminal proteolysis of mitogen activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1. Lrp5+/+ parental, Lrp5+/− heterozygous, and Lrp5−/− izygous MEFs demonstrated equal cleavage of MEK1 following treatment with PA plus LF (Figure 2C). These observations demonstrate that the loss of expression of LRP5 is not sufficient to prevent PA-mediated uptake of FP59 or LF. Similar tests were performed with MEFs from Lrp6+/+ parental, Lrp6+/− heterozygous, and Lrp6−/− izygous mice. Each of these MEFs demonstrated equal sensitivity to treatment with combinations of PA plus FP59 (Figure 2A and 2B). Again, as an independent measure of PA-mediated entry, MEFs were treated with PA plus LF, and lysates were immunoblotted for N-terminal proteolysis of MEK1. Lrp6+/+ parental, Lrp6+/− heterozygous, and Lrp6−/− izygous MEFs demonstrated equal MEK1 cleavage following treatment with PA plus LF (Figure 2C). These observations indicate that the loss of expression of Lrp6 is not sufficient to prevent PA-mediated uptake of FP59 or LF. Collectively, these results demonstrate that neither Lrp5 nor Lrp6 is essential for PA-mediated uptake of FP59 or LF.

Bottom Line: Recently, the low-density lipoprotein receptor-related protein LRP6 has been reported to mediate internalization and lethality of AnTx.Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression.Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer and Developmental Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus