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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.

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The Effect of Lrp5 Knockdown upon PA-Dependent Uptake of FP59 and Anthrax LF in Lrp6−/− MEFs(A) The efficacy with which LRP5-specific siRNA knocked down target gene expression was measured by qPCR. Results are presented as an average of two independent samples, each of which was run in duplicate. The error bars indicate the standard deviation.(B) The effect of Lrp5 knockdown on cell sensitivity to PA plus FP59 (1 ng/ml) was assessed in Lrp6−/− MEFs using toxicity assays as described in Materials and Methods. 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 two independent experiments, each of which was run in quadruplicate.(C) As an independent indicator of PA-mediated entry into the siRNA-treated CHO cells, cleavage of MEK1 was assessed by immunoblotting with antibodies that are specific for the NH2-terminus of MEK1. Only representative data for negative control siRNA (med GC) and LRP5 siRNA2 are shown, though identical results were obtained for LRP5 siRNA 1 and 3. An immunoblot with an antibody against the carboxy-terminus of MEK1 (MEK1-COOH) is shown as a control for loading and protein degradation.
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ppat-0030027-g005: The Effect of Lrp5 Knockdown upon PA-Dependent Uptake of FP59 and Anthrax LF in Lrp6−/− MEFs(A) The efficacy with which LRP5-specific siRNA knocked down target gene expression was measured by qPCR. Results are presented as an average of two independent samples, each of which was run in duplicate. The error bars indicate the standard deviation.(B) The effect of Lrp5 knockdown on cell sensitivity to PA plus FP59 (1 ng/ml) was assessed in Lrp6−/− MEFs using toxicity assays as described in Materials and Methods. 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 two independent experiments, each of which was run in quadruplicate.(C) As an independent indicator of PA-mediated entry into the siRNA-treated CHO cells, cleavage of MEK1 was assessed by immunoblotting with antibodies that are specific for the NH2-terminus of MEK1. Only representative data for negative control siRNA (med GC) and LRP5 siRNA2 are shown, though identical results were obtained for LRP5 siRNA 1 and 3. An immunoblot with an antibody against the carboxy-terminus of MEK1 (MEK1-COOH) is shown as a control for loading and protein degradation.

Mentions: Finally, we examined the possibility that Lrp5 or Lrp6 are functionally redundant and that PA-mediated internalization requires expression of either Lrp5 or Lrp6. MEFs isolated from Lrp6 knockout mice were treated with Lrp5-specific siRNA and assayed for sensitivity to PA plus FP59 or LF. Using real–time PCR, we established that Lrp5-siRNA1, Lrp5-siRNA2, and Lrp5-siRNA3 were similarly effective in reducing Lrp5 expression by approximately 66%–80% (Figure 5A). Despite this, reduced expression of Lrp5 in this Lrp6 background had no effect upon sensitivity of MEFs to PA plus FP59 (Figure 5B) or upon LF cleavage of MEK1 (Figure 5C). Similar results were obtained when we treated Lrp5 MEFs with siRNA specific for Lrp6, or when we treated T-CHO cells with a combination of Lrp5-siRNA2 and Lrp6-siRNA1 (unpublished data). These results indicate that PA-mediated internalization by MEFs or CHO cells proceeds independently of the expression of Lrp5 and Lrp6.


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)

The Effect of Lrp5 Knockdown upon PA-Dependent Uptake of FP59 and Anthrax LF in Lrp6−/− MEFs(A) The efficacy with which LRP5-specific siRNA knocked down target gene expression was measured by qPCR. Results are presented as an average of two independent samples, each of which was run in duplicate. The error bars indicate the standard deviation.(B) The effect of Lrp5 knockdown on cell sensitivity to PA plus FP59 (1 ng/ml) was assessed in Lrp6−/− MEFs using toxicity assays as described in Materials and Methods. 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 two independent experiments, each of which was run in quadruplicate.(C) As an independent indicator of PA-mediated entry into the siRNA-treated CHO cells, cleavage of MEK1 was assessed by immunoblotting with antibodies that are specific for the NH2-terminus of MEK1. Only representative data for negative control siRNA (med GC) and LRP5 siRNA2 are shown, though identical results were obtained for LRP5 siRNA 1 and 3. An immunoblot with an antibody against the carboxy-terminus of MEK1 (MEK1-COOH) is shown as a control for loading and protein degradation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1808072&req=5

ppat-0030027-g005: The Effect of Lrp5 Knockdown upon PA-Dependent Uptake of FP59 and Anthrax LF in Lrp6−/− MEFs(A) The efficacy with which LRP5-specific siRNA knocked down target gene expression was measured by qPCR. Results are presented as an average of two independent samples, each of which was run in duplicate. The error bars indicate the standard deviation.(B) The effect of Lrp5 knockdown on cell sensitivity to PA plus FP59 (1 ng/ml) was assessed in Lrp6−/− MEFs using toxicity assays as described in Materials and Methods. 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 two independent experiments, each of which was run in quadruplicate.(C) As an independent indicator of PA-mediated entry into the siRNA-treated CHO cells, cleavage of MEK1 was assessed by immunoblotting with antibodies that are specific for the NH2-terminus of MEK1. Only representative data for negative control siRNA (med GC) and LRP5 siRNA2 are shown, though identical results were obtained for LRP5 siRNA 1 and 3. An immunoblot with an antibody against the carboxy-terminus of MEK1 (MEK1-COOH) is shown as a control for loading and protein degradation.
Mentions: Finally, we examined the possibility that Lrp5 or Lrp6 are functionally redundant and that PA-mediated internalization requires expression of either Lrp5 or Lrp6. MEFs isolated from Lrp6 knockout mice were treated with Lrp5-specific siRNA and assayed for sensitivity to PA plus FP59 or LF. Using real–time PCR, we established that Lrp5-siRNA1, Lrp5-siRNA2, and Lrp5-siRNA3 were similarly effective in reducing Lrp5 expression by approximately 66%–80% (Figure 5A). Despite this, reduced expression of Lrp5 in this Lrp6 background had no effect upon sensitivity of MEFs to PA plus FP59 (Figure 5B) or upon LF cleavage of MEK1 (Figure 5C). Similar results were obtained when we treated Lrp5 MEFs with siRNA specific for Lrp6, or when we treated T-CHO cells with a combination of Lrp5-siRNA2 and Lrp6-siRNA1 (unpublished data). These results indicate that PA-mediated internalization by MEFs or CHO cells proceeds independently of the expression of Lrp5 and Lrp6.

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