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Anthrax toxin receptors and infantile systemic hyalinosis
Matrix Biology 26 (2007) 73 –74 www.elsevier.com/locate/matbio
From the Editor's Desk
Anthrax toxin receptors and infantile systemic hyalinosis Bjorn R. Olsen A few editorials back (Olsen, 2004), I discussed how Bacillus anthracis uses two integrin-like proteins, TEM8 and CMG2 (Scobie et al., 2003), to get its toxin into cells. At the time, little was known about the physiological functions of TEM8 and CMG2, but I cited evidence that their expression in vascular endothelial cells is coupled to capillary morphogenesis and that TEM8 can bind the cleaved C5 domain of α3(VI) collagen chains (Nanda et al., 2004). I also mentioned that loss-offunction mutations in CMG2 cause the autosomal recessive conditions juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) (Dowling et al., 2003; Hanks et al., 2003). Stimulated by a recent paper in CELL (Wei et al., 2006), I now like to continue this discussion. So, here it is, the second installment in my anthrax receptor musings. In a screen for genes that affect the anthrax toxicity pathway, Wei et al. (2006) made the exciting discovery that decreased expression of low-density lipoprotein receptor-related protein 6 (LRP6) results in increased resistance to the toxin. The reader may recall (Olsen, 2004) that the toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). Upon binding to the cell surface receptor, the PA protein is cleaved by furin to a smaller form and assembles into a heptameric ring, a “prepore”, that is endocytosed with the clustered receptor molecules and bound LF or EF. In the acidic endosomal environment, the prepore forms a transmembrane channel through which LF and EF get into the cytoplasm. So where does LRP6 enter the picture? To address this question, Wei et al. (2006) used siRNA to reduce LRP6 expression in macrophages which are natural targets for LF. The results demonstrated that reduced LRP6 transcript levels are associated with a decrease in the level of intact PA bound to the cell surface and a reduced amount of an oligomeric internalized form of PA. In further experiments, the authors showed that the cytoplasmic portion of the LRP6 transmembrane receptor is essential for internalization of PA and for toxin-mediated cell killing. Given the roles of LRP6 (and its homologue, LRP5) as Wntbinding receptors (Pinson et al., 2000; Tamai et al., 2000; Wehrli et al., 2000), Wei et al. (2006) naturally asked whether the role of LRP6 in endocytosis of anthrax toxin requires LRP6dependent Wnt signaling. Surprisingly, the answer was negative, as impaired Wnt signaling did not affect toxin internalization and lethality. The effect of LRP6 on toxin
endocytosis appeared to be quite specific, since LRP6 deficiency did not result in a general defect in receptor internalization; also, induction of broad defects in endocytic receptor trafficking did not result in toxin resistance. In further experiments, Wei et al. (2006) provided evidence for a direct association between LRP6 and TEM8 and CMG2 and they could demonstrate that the extracellular domain of LRP6 is sufficient for this association. In contrast, the cytoplasmic domain of LRP6 did not show binding to the two receptors. Immunofluorescence experiments indicated that LRP6 colocalizes inside cells with PA and TEM8 or CMG2. Treatment with siRNA against LRP6 or expression of a dominant negative form (lacking the cytoplasmic domain) of LRP6 prevented the intracellular appearance of TEM8 and CMG2 after incubation of cells with PA. Finally, incubation of cells with antibodies against LRP6 protected macrophages from being killed by PA and LF. These experiments clearly demonstrate that LRP6 is crucial for endocytosis of anthrax toxin into target cells expressing the receptors TEM8 or CMG2 and the subsequent deadly action of the lethal and edema factors. However, what do these experiments tell us about the role of LRP6 for the physiological functions of TEM8 and CMG2? Importantly, Wei et al. (2006) find that LRP6 and TEM8/CMG2 form a complex even in the absence of the toxin and that binding of toxin to the complex triggers endocytosis by a mechanism that requires the LRP6 cytoplasmic domain. As pointed out by Wei et al. (2006), this endocytic enabling function of LRP6 in the presence of anthrax toxin may parallel its physiological role in Wnt-signaling in cells. In the conventional view of the canonical Wnt-signaling pathway, Wnt glycoproteins bind to Frizzled (Frz) receptors and their LRP5 or LRP6 coreceptors. This results in clathrinmediated endocytosis of the complex (and recent evidence suggests that endocytosis is critical for Wnt-signaling (Blitzer and Nusse, 2006)), and accumulation of β-catenin in the cytoplasm. Based on recent studies, it is now clear that this conventional view is too simple and that understanding how Wnt–Frz–LRP5/6 signaling works to control intracellular levels of β-catenin is best viewed in the context of many ligands and receptors that utilize LRP5/6 coreceptors. These receptors include transmembrane proteins called Kremen1 and 2; to these one can now also add the integrin-like proteins TEM8 and CMG2. Ligands other than Wnts include proteins
0945-053X/$ - see front matter © 2007 Published by Elsevier B.V./International Society of Matrix Biology. doi:10.1016/j.matbio.2007.01.007
74
B.R. Olsen / Matrix Biology 26 (2007) 73–74
such as Mesd (Li et al., 2005), Sclerostin (Ellies et al., 2006), Norrin (Xu et al., 2004), R-spondin (Nam et al., 2006) and Dickkopf1 (Mao et al., 2001). In some situations, as in the case of Norrin, these alternative ligands have high affinity for a specific Frz–LRP6 receptor complex and induce β-catenindependent signaling; in other situations, as in the case of Mesd or Sclerostin, they act as inhibitors of Wnt-signaling. For receptors such as TEM8 and CMG2, their association with LRP5 or LRP6 on the cell surface may likewise serve to modulate Wnt signaling. Since collagens I and VI are potential ligands for TEM8 and CMG2 appears to bind to collagen IV and laminin, one wonders whether their interaction with LRP5 and LRP6 may even represent a matrix-modulated mechanism for local control of Wnt-signaling. Recently, Werner et al. (2006) demonstrated that TEM8 can serve as an adhesion receptor and mediate spreading of cells on collagen I by an actin-dependent mechanism. Fibroblasts from patients with JHF or ISH are unable to adhere to laminin matrices (Dowling et al., 2003), suggesting that CMG2 also is involved in cell–matrix interactions. However, the phenotype of patients with these syndromes is difficult to explain by a simple loss of cell adhesion to a select group of matrix molecules. Given what Bacillus anthracis has known for a long time, and we now know as a result of the studies of Wei et al. (2006), it would be interesting to find out whether abnormal Wntsignaling contributes to the phenotypic aspects of the hyalinosis syndromes. References Blitzer, J.T., Nusse, R., 2006. A critical role for endocytosis in Wnt signaling. BMC Cell Biol. 7, 28. Dowling, O., Difeo, A., Ramirez, M.C., Tukel, T., Narla, G., Bonafe, L., Kayserili, H., Yuksel-Apak, M., Paller, A.S., Norton, K., et al., 2003. Mutations in capillary morphogenesis gene-2 result in the allelic disorders juvenile hyaline fibromatosis and infantile systemic hyalinosis. Am. J. Hum. Genet. 73, 957–966. Ellies, D.L., Viviano, B., McCarthy, J., Rey, J.P., Itasaki, N., Saunders, S., Krumlauf, R., 2006. Bone density ligand, Sclerostin, directly interacts with
LRP5 but not LRP5G171V to modulate Wnt activity. J. Bone Miner. Res. 21, 1738–1749. Hanks, S., Adams, S., Douglas, J., Arbour, L., Atherton, D.J., Balci, S., Bode, H., Campbell, M.E., Feingold, M., Keser, G., et al., 2003. Mutations in the gene encoding capillary morphogenesis protein 2 cause juvenile hyaline fibromatosis and infantile systemic hyalinosis. Am. J. Hum. Genet. 73, 791–800. Li, Y., Chen, J., Lu, W., McCormick, L.M., Wang, J., Bu, G., 2005. Mesd binds to mature LDL-receptor-related protein-6 and antagonizes ligand binding. J. Cell Sci. 118, 5305–5314. Mao, B., Wu, W., Li, Y., Hoppe, D., Stannek, P., Glinka, A., Niehrs, C., 2001. LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 411, 321–325. Nam, J.S., Turcotte, T.J., Smith, P.F., Choi, S., Yoon, J.K., 2006. Mouse cristin/ R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate beta-catenin-dependent gene expression. J. Biol. Chem. 281, 13247–13257. Nanda, A., Carson-Walter, E.B., Seaman, S., Barber, T.D., Stampfl, J., Singh, S., Vogelstein, B., Kinzler, K.W., St Croix, B., 2004. TEM8 interacts with the cleaved C5 domain of collagen alpha 3(VI). Cancer Res. 64, 817–820. Olsen, B., 2004. From the Editor's desk. Matrix Biol. 23, 205–206. Pinson, K.I., Brennan, J., Monkley, S., Avery, B.J., Skarnes, W.C., 2000. An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 407, 535–538. Scobie, H.M., Rainey, G.J., Bradley, K.A., Young, J.A., 2003. Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc. Natl. Acad. Sci. U. S. A. 100, 5170–5174. Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., Hess, F., Saint-Jeannet, J.P., He, X., 2000. LDL-receptor-related proteins in Wnt signal transduction. Nature 407, 530–535. Wehrli, M., Dougan, S.T., Caldwell, K., O'Keefe, L., Schwartz, S., VaizelOhayon, D., Schejter, E., Tomlinson, A., DiNardo, S., 2000. Arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 407, 527–530. Wei, W., Lu, Q., Chaudry, G.J., Leppla, S.H., Cohen, S.N., 2006. The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell 124, 1141–1154. Werner, E., Kowalczyk, A.P., Faundez, V., 2006. Anthrax toxin receptor 1/tumor endothelium marker 8 mediates cell spreading by coupling extracellular ligands to the actin cytoskeleton. J. Biol. Chem. 281, 23227–23236. Xu, Q., Wang, Y., Dabdoub, A., Smallwood, P.M., Williams, J., Woods, C., Kelley, M.W., Jiang, L., Tasman, W., Zhang, K., Nathans, J., 2004. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand–receptor pair. Cell 116, 883–895.
From the Editor's Desk
Anthrax toxin receptors and infantile systemic hyalinosis Bjorn R. Olsen A few editorials back (Olsen, 2004), I discussed how Bacillus anthracis uses two integrin-like proteins, TEM8 and CMG2 (Scobie et al., 2003), to get its toxin into cells. At the time, little was known about the physiological functions of TEM8 and CMG2, but I cited evidence that their expression in vascular endothelial cells is coupled to capillary morphogenesis and that TEM8 can bind the cleaved C5 domain of α3(VI) collagen chains (Nanda et al., 2004). I also mentioned that loss-offunction mutations in CMG2 cause the autosomal recessive conditions juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) (Dowling et al., 2003; Hanks et al., 2003). Stimulated by a recent paper in CELL (Wei et al., 2006), I now like to continue this discussion. So, here it is, the second installment in my anthrax receptor musings. In a screen for genes that affect the anthrax toxicity pathway, Wei et al. (2006) made the exciting discovery that decreased expression of low-density lipoprotein receptor-related protein 6 (LRP6) results in increased resistance to the toxin. The reader may recall (Olsen, 2004) that the toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). Upon binding to the cell surface receptor, the PA protein is cleaved by furin to a smaller form and assembles into a heptameric ring, a “prepore”, that is endocytosed with the clustered receptor molecules and bound LF or EF. In the acidic endosomal environment, the prepore forms a transmembrane channel through which LF and EF get into the cytoplasm. So where does LRP6 enter the picture? To address this question, Wei et al. (2006) used siRNA to reduce LRP6 expression in macrophages which are natural targets for LF. The results demonstrated that reduced LRP6 transcript levels are associated with a decrease in the level of intact PA bound to the cell surface and a reduced amount of an oligomeric internalized form of PA. In further experiments, the authors showed that the cytoplasmic portion of the LRP6 transmembrane receptor is essential for internalization of PA and for toxin-mediated cell killing. Given the roles of LRP6 (and its homologue, LRP5) as Wntbinding receptors (Pinson et al., 2000; Tamai et al., 2000; Wehrli et al., 2000), Wei et al. (2006) naturally asked whether the role of LRP6 in endocytosis of anthrax toxin requires LRP6dependent Wnt signaling. Surprisingly, the answer was negative, as impaired Wnt signaling did not affect toxin internalization and lethality. The effect of LRP6 on toxin
endocytosis appeared to be quite specific, since LRP6 deficiency did not result in a general defect in receptor internalization; also, induction of broad defects in endocytic receptor trafficking did not result in toxin resistance. In further experiments, Wei et al. (2006) provided evidence for a direct association between LRP6 and TEM8 and CMG2 and they could demonstrate that the extracellular domain of LRP6 is sufficient for this association. In contrast, the cytoplasmic domain of LRP6 did not show binding to the two receptors. Immunofluorescence experiments indicated that LRP6 colocalizes inside cells with PA and TEM8 or CMG2. Treatment with siRNA against LRP6 or expression of a dominant negative form (lacking the cytoplasmic domain) of LRP6 prevented the intracellular appearance of TEM8 and CMG2 after incubation of cells with PA. Finally, incubation of cells with antibodies against LRP6 protected macrophages from being killed by PA and LF. These experiments clearly demonstrate that LRP6 is crucial for endocytosis of anthrax toxin into target cells expressing the receptors TEM8 or CMG2 and the subsequent deadly action of the lethal and edema factors. However, what do these experiments tell us about the role of LRP6 for the physiological functions of TEM8 and CMG2? Importantly, Wei et al. (2006) find that LRP6 and TEM8/CMG2 form a complex even in the absence of the toxin and that binding of toxin to the complex triggers endocytosis by a mechanism that requires the LRP6 cytoplasmic domain. As pointed out by Wei et al. (2006), this endocytic enabling function of LRP6 in the presence of anthrax toxin may parallel its physiological role in Wnt-signaling in cells. In the conventional view of the canonical Wnt-signaling pathway, Wnt glycoproteins bind to Frizzled (Frz) receptors and their LRP5 or LRP6 coreceptors. This results in clathrinmediated endocytosis of the complex (and recent evidence suggests that endocytosis is critical for Wnt-signaling (Blitzer and Nusse, 2006)), and accumulation of β-catenin in the cytoplasm. Based on recent studies, it is now clear that this conventional view is too simple and that understanding how Wnt–Frz–LRP5/6 signaling works to control intracellular levels of β-catenin is best viewed in the context of many ligands and receptors that utilize LRP5/6 coreceptors. These receptors include transmembrane proteins called Kremen1 and 2; to these one can now also add the integrin-like proteins TEM8 and CMG2. Ligands other than Wnts include proteins
0945-053X/$ - see front matter © 2007 Published by Elsevier B.V./International Society of Matrix Biology. doi:10.1016/j.matbio.2007.01.007
74
B.R. Olsen / Matrix Biology 26 (2007) 73–74
such as Mesd (Li et al., 2005), Sclerostin (Ellies et al., 2006), Norrin (Xu et al., 2004), R-spondin (Nam et al., 2006) and Dickkopf1 (Mao et al., 2001). In some situations, as in the case of Norrin, these alternative ligands have high affinity for a specific Frz–LRP6 receptor complex and induce β-catenindependent signaling; in other situations, as in the case of Mesd or Sclerostin, they act as inhibitors of Wnt-signaling. For receptors such as TEM8 and CMG2, their association with LRP5 or LRP6 on the cell surface may likewise serve to modulate Wnt signaling. Since collagens I and VI are potential ligands for TEM8 and CMG2 appears to bind to collagen IV and laminin, one wonders whether their interaction with LRP5 and LRP6 may even represent a matrix-modulated mechanism for local control of Wnt-signaling. Recently, Werner et al. (2006) demonstrated that TEM8 can serve as an adhesion receptor and mediate spreading of cells on collagen I by an actin-dependent mechanism. Fibroblasts from patients with JHF or ISH are unable to adhere to laminin matrices (Dowling et al., 2003), suggesting that CMG2 also is involved in cell–matrix interactions. However, the phenotype of patients with these syndromes is difficult to explain by a simple loss of cell adhesion to a select group of matrix molecules. Given what Bacillus anthracis has known for a long time, and we now know as a result of the studies of Wei et al. (2006), it would be interesting to find out whether abnormal Wntsignaling contributes to the phenotypic aspects of the hyalinosis syndromes. References Blitzer, J.T., Nusse, R., 2006. A critical role for endocytosis in Wnt signaling. BMC Cell Biol. 7, 28. Dowling, O., Difeo, A., Ramirez, M.C., Tukel, T., Narla, G., Bonafe, L., Kayserili, H., Yuksel-Apak, M., Paller, A.S., Norton, K., et al., 2003. Mutations in capillary morphogenesis gene-2 result in the allelic disorders juvenile hyaline fibromatosis and infantile systemic hyalinosis. Am. J. Hum. Genet. 73, 957–966. Ellies, D.L., Viviano, B., McCarthy, J., Rey, J.P., Itasaki, N., Saunders, S., Krumlauf, R., 2006. Bone density ligand, Sclerostin, directly interacts with
LRP5 but not LRP5G171V to modulate Wnt activity. J. Bone Miner. Res. 21, 1738–1749. Hanks, S., Adams, S., Douglas, J., Arbour, L., Atherton, D.J., Balci, S., Bode, H., Campbell, M.E., Feingold, M., Keser, G., et al., 2003. Mutations in the gene encoding capillary morphogenesis protein 2 cause juvenile hyaline fibromatosis and infantile systemic hyalinosis. Am. J. Hum. Genet. 73, 791–800. Li, Y., Chen, J., Lu, W., McCormick, L.M., Wang, J., Bu, G., 2005. Mesd binds to mature LDL-receptor-related protein-6 and antagonizes ligand binding. J. Cell Sci. 118, 5305–5314. Mao, B., Wu, W., Li, Y., Hoppe, D., Stannek, P., Glinka, A., Niehrs, C., 2001. LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 411, 321–325. Nam, J.S., Turcotte, T.J., Smith, P.F., Choi, S., Yoon, J.K., 2006. Mouse cristin/ R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate beta-catenin-dependent gene expression. J. Biol. Chem. 281, 13247–13257. Nanda, A., Carson-Walter, E.B., Seaman, S., Barber, T.D., Stampfl, J., Singh, S., Vogelstein, B., Kinzler, K.W., St Croix, B., 2004. TEM8 interacts with the cleaved C5 domain of collagen alpha 3(VI). Cancer Res. 64, 817–820. Olsen, B., 2004. From the Editor's desk. Matrix Biol. 23, 205–206. Pinson, K.I., Brennan, J., Monkley, S., Avery, B.J., Skarnes, W.C., 2000. An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 407, 535–538. Scobie, H.M., Rainey, G.J., Bradley, K.A., Young, J.A., 2003. Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc. Natl. Acad. Sci. U. S. A. 100, 5170–5174. Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., Hess, F., Saint-Jeannet, J.P., He, X., 2000. LDL-receptor-related proteins in Wnt signal transduction. Nature 407, 530–535. Wehrli, M., Dougan, S.T., Caldwell, K., O'Keefe, L., Schwartz, S., VaizelOhayon, D., Schejter, E., Tomlinson, A., DiNardo, S., 2000. Arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 407, 527–530. Wei, W., Lu, Q., Chaudry, G.J., Leppla, S.H., Cohen, S.N., 2006. The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell 124, 1141–1154. Werner, E., Kowalczyk, A.P., Faundez, V., 2006. Anthrax toxin receptor 1/tumor endothelium marker 8 mediates cell spreading by coupling extracellular ligands to the actin cytoskeleton. J. Biol. Chem. 281, 23227–23236. Xu, Q., Wang, Y., Dabdoub, A., Smallwood, P.M., Williams, J., Woods, C., Kelley, M.W., Jiang, L., Tasman, W., Zhang, K., Nathans, J., 2004. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand–receptor pair. Cell 116, 883–895.