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uPARAP/Endo180 is essential for cellular uptake of collagen and promotes fibroblast collagen adhesion.

Engelholm LH, List K, Netzel-Arnett S, Cukierman E, Mitola DJ, Aaronson H, Kjøller L, Larsen JK, Yamada KM, Strickland DK, Holmbeck K, Danø K, Birkedal-Hansen H, Behrendt N, Bugge TH - J. Cell Biol. (2003)

Bottom Line: However, the molecular mechanisms governing this pathway are poorly understood.Here, we show that the urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process.These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions.

View Article: PubMed Central - PubMed

Affiliation: Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process. Fibroblasts from mice with a targeted deletion in the uPARAP/Endo180 gene displayed a near to complete abrogation of collagen endocytosis. Furthermore, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions.

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uPARAP/Endo180 is required for collagen internalization. (A) Representative examples of the ability of dermal fibroblasts from uPARAP/Endo180−/− (black bars) or littermate uPARAP/Endo180+/+ (gray bars) neonates to internalize the 125I-labeled ligands indicated above each panel. Col I, IV, and V designate collagen subtypes. In panel 2, uPARAP/Endo180−/− fibroblasts were transiently transfected with a uPARAP/Endo180 expression plasmid, or with a neomycin resistance gene control expression plasmid, as indicated. The transfection efficiency was ∼10%. The data are expressed as the total amount of 125I-labeled ligand internalized per cell within a 3-h period at 37°C. Error bars indicate SDs. Asterisk in Col V, P < 0.000001; Col V in transfected cells, P < 0.000003; Col I, P < 0.00000001; and Col IV, P < 0.00005. (B) Cells with the genotype indicated were preincubated in the presence (hatched columns) or absence (unhatched columns) of a blocking antibody against β1 integrins, and the internalization of collagen V and I was measured as above.
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fig2: uPARAP/Endo180 is required for collagen internalization. (A) Representative examples of the ability of dermal fibroblasts from uPARAP/Endo180−/− (black bars) or littermate uPARAP/Endo180+/+ (gray bars) neonates to internalize the 125I-labeled ligands indicated above each panel. Col I, IV, and V designate collagen subtypes. In panel 2, uPARAP/Endo180−/− fibroblasts were transiently transfected with a uPARAP/Endo180 expression plasmid, or with a neomycin resistance gene control expression plasmid, as indicated. The transfection efficiency was ∼10%. The data are expressed as the total amount of 125I-labeled ligand internalized per cell within a 3-h period at 37°C. Error bars indicate SDs. Asterisk in Col V, P < 0.000001; Col V in transfected cells, P < 0.000003; Col I, P < 0.00000001; and Col IV, P < 0.00005. (B) Cells with the genotype indicated were preincubated in the presence (hatched columns) or absence (unhatched columns) of a blocking antibody against β1 integrins, and the internalization of collagen V and I was measured as above.

Mentions: Strikingly, the uPARAP/Endo180-deficient fibroblasts displayed a virtually complete abrogation of the cellular uptake of collagen types I, IV, and V (Fig. 2 A, panels 3, 4, and 1, respectively). This deficiency in collagen internalization was observed with several independent isolates of uPARAP/Endo180−/− fibroblasts and could be alleviated by the reintroduction of uPARAP/Endo180 by transient transfection of the cells with a uPARAP/Endo180 cDNA expression vector (Fig. 2 A, panel 2; unpublished data). In contrast, the cellular uptake of holotransferrin, MMP-13, and fibronectin was unaffected (Fig. 2 A, panels 5–7). The internalization of MMP-13 has been shown previously to be uPARAP/Endo180-independent (Bailey et al., 2002), but depends on the low density lipoprotein receptor–related protein (LRP; Barmina et al., 1999). Therefore, as an additional test of our assay system, we included samples with LRP-deficient cells (LRP−/− and matched controls) in the same manner to study the internalization of 125I-labeled MMP-13. This experiment confirmed the essential role of LRP in this process (P < 0.0007), whereas LRP deficiency had no effect on the cellular uptake of 125I-labeled collagen, examined as described for uPARAP/Endo180−/− cells (unpublished data). These data show that uPARAP/Endo180 has a critical and specific role in the uptake of collagen by fibroblasts.


uPARAP/Endo180 is essential for cellular uptake of collagen and promotes fibroblast collagen adhesion.

Engelholm LH, List K, Netzel-Arnett S, Cukierman E, Mitola DJ, Aaronson H, Kjøller L, Larsen JK, Yamada KM, Strickland DK, Holmbeck K, Danø K, Birkedal-Hansen H, Behrendt N, Bugge TH - J. Cell Biol. (2003)

uPARAP/Endo180 is required for collagen internalization. (A) Representative examples of the ability of dermal fibroblasts from uPARAP/Endo180−/− (black bars) or littermate uPARAP/Endo180+/+ (gray bars) neonates to internalize the 125I-labeled ligands indicated above each panel. Col I, IV, and V designate collagen subtypes. In panel 2, uPARAP/Endo180−/− fibroblasts were transiently transfected with a uPARAP/Endo180 expression plasmid, or with a neomycin resistance gene control expression plasmid, as indicated. The transfection efficiency was ∼10%. The data are expressed as the total amount of 125I-labeled ligand internalized per cell within a 3-h period at 37°C. Error bars indicate SDs. Asterisk in Col V, P < 0.000001; Col V in transfected cells, P < 0.000003; Col I, P < 0.00000001; and Col IV, P < 0.00005. (B) Cells with the genotype indicated were preincubated in the presence (hatched columns) or absence (unhatched columns) of a blocking antibody against β1 integrins, and the internalization of collagen V and I was measured as above.
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Related In: Results  -  Collection

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fig2: uPARAP/Endo180 is required for collagen internalization. (A) Representative examples of the ability of dermal fibroblasts from uPARAP/Endo180−/− (black bars) or littermate uPARAP/Endo180+/+ (gray bars) neonates to internalize the 125I-labeled ligands indicated above each panel. Col I, IV, and V designate collagen subtypes. In panel 2, uPARAP/Endo180−/− fibroblasts were transiently transfected with a uPARAP/Endo180 expression plasmid, or with a neomycin resistance gene control expression plasmid, as indicated. The transfection efficiency was ∼10%. The data are expressed as the total amount of 125I-labeled ligand internalized per cell within a 3-h period at 37°C. Error bars indicate SDs. Asterisk in Col V, P < 0.000001; Col V in transfected cells, P < 0.000003; Col I, P < 0.00000001; and Col IV, P < 0.00005. (B) Cells with the genotype indicated were preincubated in the presence (hatched columns) or absence (unhatched columns) of a blocking antibody against β1 integrins, and the internalization of collagen V and I was measured as above.
Mentions: Strikingly, the uPARAP/Endo180-deficient fibroblasts displayed a virtually complete abrogation of the cellular uptake of collagen types I, IV, and V (Fig. 2 A, panels 3, 4, and 1, respectively). This deficiency in collagen internalization was observed with several independent isolates of uPARAP/Endo180−/− fibroblasts and could be alleviated by the reintroduction of uPARAP/Endo180 by transient transfection of the cells with a uPARAP/Endo180 cDNA expression vector (Fig. 2 A, panel 2; unpublished data). In contrast, the cellular uptake of holotransferrin, MMP-13, and fibronectin was unaffected (Fig. 2 A, panels 5–7). The internalization of MMP-13 has been shown previously to be uPARAP/Endo180-independent (Bailey et al., 2002), but depends on the low density lipoprotein receptor–related protein (LRP; Barmina et al., 1999). Therefore, as an additional test of our assay system, we included samples with LRP-deficient cells (LRP−/− and matched controls) in the same manner to study the internalization of 125I-labeled MMP-13. This experiment confirmed the essential role of LRP in this process (P < 0.0007), whereas LRP deficiency had no effect on the cellular uptake of 125I-labeled collagen, examined as described for uPARAP/Endo180−/− cells (unpublished data). These data show that uPARAP/Endo180 has a critical and specific role in the uptake of collagen by fibroblasts.

Bottom Line: However, the molecular mechanisms governing this pathway are poorly understood.Here, we show that the urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process.These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions.

View Article: PubMed Central - PubMed

Affiliation: Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process. Fibroblasts from mice with a targeted deletion in the uPARAP/Endo180 gene displayed a near to complete abrogation of collagen endocytosis. Furthermore, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions.

Show MeSH
Related in: MedlinePlus