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M6P/IGF2R modulates the invasiveness of liver cells via its capacity to bind mannose 6-phosphate residues.

Puxbaum V, Nimmerfall E, Bäuerl C, Taub N, Blaas PM, Wieser J, Mikula M, Mikulits W, Ng KM, Yeoh GC, Mach L - J. Hepatol. (2012)

Bottom Line: Forced expression of M6P/IGF2R restores intracellular transport of cathepsins to lysosomes and concomitantly reduces the tumorigenicity and invasive potential of these cells.We also demonstrate that functional M6P-binding sites are important for the anti-invasive properties of M6P/IGF2R, whereas the capacity to bind IGF-II is dispensable for the anti-invasive activity of the receptor in liver cells.M6P/IGF2R restricts liver cell invasion by preventing the pericellular action of M6P-modified proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.

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Effects of stable M6P/IGF2R knock-down on MIM-1–4 cells. (A) Immunoblot of membrane extracts with antibodies against M6P/IGF2R. GM130 was used as loading control. (B) Cell lysates (C; 20 μg protein) and conditioned media (M) corresponding to 100 μg (CD) or 4 μg (CL) total cellular protein were subjected to immunoblotting with antibodies to cathepsin D (CD) or cathepsin L (CL). proCD/proCL, procathepsin D/L; scCD/scCL, mature single-chain cathepsin D/L; tcCD/tcCL, mature two-chain cathepsin D/L. (C) Cells were incubated for 24 h with or without 10 mM NH4Cl prior to determination of β-N-acetylhexosaminidase (HEX) activity in cell lysates and media. The secretion levels are presented as means ± SEM of three independent experiments. ∗p < 0.05 compared to cells transfected with control shRNA. (D) Analysis of cell migration using the monolayer wound healing assay. Data are presented as means of 3–4 wounds. Error bars have been omitted to avoid obstruction of the individual data points. IGF2R shRNA-1: p = 0.001 compared to parental cells; p = 0.044 compared to cells transfected with control shRNA. (E) Phase contrast microscopy images of representative wounds at the time of wounding (0 h) and after incubation for 8.5 h. Scale bar, 100 μm. (F) Invasion assays using 10% FBS ± 10 ng/ml mouse HGF as chemoattractant. Data are presented as means ± SEM of five independent experiments.
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f0020: Effects of stable M6P/IGF2R knock-down on MIM-1–4 cells. (A) Immunoblot of membrane extracts with antibodies against M6P/IGF2R. GM130 was used as loading control. (B) Cell lysates (C; 20 μg protein) and conditioned media (M) corresponding to 100 μg (CD) or 4 μg (CL) total cellular protein were subjected to immunoblotting with antibodies to cathepsin D (CD) or cathepsin L (CL). proCD/proCL, procathepsin D/L; scCD/scCL, mature single-chain cathepsin D/L; tcCD/tcCL, mature two-chain cathepsin D/L. (C) Cells were incubated for 24 h with or without 10 mM NH4Cl prior to determination of β-N-acetylhexosaminidase (HEX) activity in cell lysates and media. The secretion levels are presented as means ± SEM of three independent experiments. ∗p < 0.05 compared to cells transfected with control shRNA. (D) Analysis of cell migration using the monolayer wound healing assay. Data are presented as means of 3–4 wounds. Error bars have been omitted to avoid obstruction of the individual data points. IGF2R shRNA-1: p = 0.001 compared to parental cells; p = 0.044 compared to cells transfected with control shRNA. (E) Phase contrast microscopy images of representative wounds at the time of wounding (0 h) and after incubation for 8.5 h. Scale bar, 100 μm. (F) Invasion assays using 10% FBS ± 10 ng/ml mouse HGF as chemoattractant. Data are presented as means ± SEM of five independent experiments.

Mentions: For permanent receptor knock-down, MIM-1–4 cells were stably transfected with an M6P/IGF2R-specific shRNA construct. Densitometric analysis of immunoblots revealed that the residual receptor content of the two MIM-1–4/IGF2R shRNA clones chosen for further studies was <1% as compared with cells transfected with a control shRNA sequence (Fig. 4A). This goes in hand with enhanced β-N-acetylhexosaminidase secretion, with MIM-1–4/IGF2R shRNA cells (line 1: 55%; line 2: 47%) secreting far more of this lysosomal marker enzyme than parental (7%) and control cells (16%). Similar observations were made for cathepsin D. MIM-1–4/IGF2R shRNA cells secreted substantially more of this lysosomal proteinase (11%) than parental MIM-1–4 cells (1%). The difference in cathepsin L secretion between MIM-1–4/IGF2R shRNA and parental cells was less pronounced (87% and 81%, respectively). NH4Cl treatment resulted in strongly enhanced β-N-acetylhexosaminidase secretion by parental and control MIM-1–4 cells. Conversely, MIM-1–4/IGF2R shRNA cells displayed no further increase in β-N-acetylhexosaminidase secretion upon addition of NH4Cl (Fig. 4B and C).


M6P/IGF2R modulates the invasiveness of liver cells via its capacity to bind mannose 6-phosphate residues.

Puxbaum V, Nimmerfall E, Bäuerl C, Taub N, Blaas PM, Wieser J, Mikula M, Mikulits W, Ng KM, Yeoh GC, Mach L - J. Hepatol. (2012)

Effects of stable M6P/IGF2R knock-down on MIM-1–4 cells. (A) Immunoblot of membrane extracts with antibodies against M6P/IGF2R. GM130 was used as loading control. (B) Cell lysates (C; 20 μg protein) and conditioned media (M) corresponding to 100 μg (CD) or 4 μg (CL) total cellular protein were subjected to immunoblotting with antibodies to cathepsin D (CD) or cathepsin L (CL). proCD/proCL, procathepsin D/L; scCD/scCL, mature single-chain cathepsin D/L; tcCD/tcCL, mature two-chain cathepsin D/L. (C) Cells were incubated for 24 h with or without 10 mM NH4Cl prior to determination of β-N-acetylhexosaminidase (HEX) activity in cell lysates and media. The secretion levels are presented as means ± SEM of three independent experiments. ∗p < 0.05 compared to cells transfected with control shRNA. (D) Analysis of cell migration using the monolayer wound healing assay. Data are presented as means of 3–4 wounds. Error bars have been omitted to avoid obstruction of the individual data points. IGF2R shRNA-1: p = 0.001 compared to parental cells; p = 0.044 compared to cells transfected with control shRNA. (E) Phase contrast microscopy images of representative wounds at the time of wounding (0 h) and after incubation for 8.5 h. Scale bar, 100 μm. (F) Invasion assays using 10% FBS ± 10 ng/ml mouse HGF as chemoattractant. Data are presented as means ± SEM of five independent experiments.
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f0020: Effects of stable M6P/IGF2R knock-down on MIM-1–4 cells. (A) Immunoblot of membrane extracts with antibodies against M6P/IGF2R. GM130 was used as loading control. (B) Cell lysates (C; 20 μg protein) and conditioned media (M) corresponding to 100 μg (CD) or 4 μg (CL) total cellular protein were subjected to immunoblotting with antibodies to cathepsin D (CD) or cathepsin L (CL). proCD/proCL, procathepsin D/L; scCD/scCL, mature single-chain cathepsin D/L; tcCD/tcCL, mature two-chain cathepsin D/L. (C) Cells were incubated for 24 h with or without 10 mM NH4Cl prior to determination of β-N-acetylhexosaminidase (HEX) activity in cell lysates and media. The secretion levels are presented as means ± SEM of three independent experiments. ∗p < 0.05 compared to cells transfected with control shRNA. (D) Analysis of cell migration using the monolayer wound healing assay. Data are presented as means of 3–4 wounds. Error bars have been omitted to avoid obstruction of the individual data points. IGF2R shRNA-1: p = 0.001 compared to parental cells; p = 0.044 compared to cells transfected with control shRNA. (E) Phase contrast microscopy images of representative wounds at the time of wounding (0 h) and after incubation for 8.5 h. Scale bar, 100 μm. (F) Invasion assays using 10% FBS ± 10 ng/ml mouse HGF as chemoattractant. Data are presented as means ± SEM of five independent experiments.
Mentions: For permanent receptor knock-down, MIM-1–4 cells were stably transfected with an M6P/IGF2R-specific shRNA construct. Densitometric analysis of immunoblots revealed that the residual receptor content of the two MIM-1–4/IGF2R shRNA clones chosen for further studies was <1% as compared with cells transfected with a control shRNA sequence (Fig. 4A). This goes in hand with enhanced β-N-acetylhexosaminidase secretion, with MIM-1–4/IGF2R shRNA cells (line 1: 55%; line 2: 47%) secreting far more of this lysosomal marker enzyme than parental (7%) and control cells (16%). Similar observations were made for cathepsin D. MIM-1–4/IGF2R shRNA cells secreted substantially more of this lysosomal proteinase (11%) than parental MIM-1–4 cells (1%). The difference in cathepsin L secretion between MIM-1–4/IGF2R shRNA and parental cells was less pronounced (87% and 81%, respectively). NH4Cl treatment resulted in strongly enhanced β-N-acetylhexosaminidase secretion by parental and control MIM-1–4 cells. Conversely, MIM-1–4/IGF2R shRNA cells displayed no further increase in β-N-acetylhexosaminidase secretion upon addition of NH4Cl (Fig. 4B and C).

Bottom Line: Forced expression of M6P/IGF2R restores intracellular transport of cathepsins to lysosomes and concomitantly reduces the tumorigenicity and invasive potential of these cells.We also demonstrate that functional M6P-binding sites are important for the anti-invasive properties of M6P/IGF2R, whereas the capacity to bind IGF-II is dispensable for the anti-invasive activity of the receptor in liver cells.M6P/IGF2R restricts liver cell invasion by preventing the pericellular action of M6P-modified proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.

Show MeSH
Related in: MedlinePlus