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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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Generation of uPARAP/Endo180-targeted mice. (A) Diagram of the targeting strategy showing the structure of the uPARAP/Endo180-targeting vector (top), wild-type uPARAP/Endo180 allele (middle), and targeted uPARAP/Endo180 allele (bottom). Exons 2–6 of the uPARAP/Endo180 gene (encoding the cysteine-rich, the FN-II, and the first carbohydrate recognition domains) were replaced by the HPRT selection cassette. Exons are indicated with boxes and introns with a solid line. B, BamHI; E, EcoRI; H, HindIII; X, XhoI. (B) Southern blot of BamHI-digested mouse tail DNA from mice genotyped by PCR as uPARAP/Endo180+/+ (left lane), uPARAP/Endo180+/− (middle lane), and uPARAP/Endo180−/− (right lane). The hybridization probe (solid line below the wild-type allele in A) was located upstream of the targeted area. The expected DNA fragments of the wild-type (4.1 kb) and targeted (2.4 kb) alleles that hybridize to the probe are indicated with solid lines in A. (C) Northern blot of total RNA isolated from cultured fibroblasts. RNA from fibroblasts of mice genotyped as uPARAP/Endo180+/+ (left lane) and uPARAP/Endo180−/− (right lane) was hybridized with a cDNA probe complementary to the 3′ end of the uPARAP/Endo180 mRNA. (D) Western blot of lysates of cultured dermal fibroblasts from neonates genotyped as uPARAP/Endo180+/+ (left lane), uPARAP/Endo180+/− (middle lane), and uPARAP/Endo180−/− (right lane). The blot was probed with a murine anti–uPARAP/Endo180 peptide antibody prepared as described in Materials and methods. The positions of molecular mass markers (kD) are indicated left.
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fig1: Generation of uPARAP/Endo180-targeted mice. (A) Diagram of the targeting strategy showing the structure of the uPARAP/Endo180-targeting vector (top), wild-type uPARAP/Endo180 allele (middle), and targeted uPARAP/Endo180 allele (bottom). Exons 2–6 of the uPARAP/Endo180 gene (encoding the cysteine-rich, the FN-II, and the first carbohydrate recognition domains) were replaced by the HPRT selection cassette. Exons are indicated with boxes and introns with a solid line. B, BamHI; E, EcoRI; H, HindIII; X, XhoI. (B) Southern blot of BamHI-digested mouse tail DNA from mice genotyped by PCR as uPARAP/Endo180+/+ (left lane), uPARAP/Endo180+/− (middle lane), and uPARAP/Endo180−/− (right lane). The hybridization probe (solid line below the wild-type allele in A) was located upstream of the targeted area. The expected DNA fragments of the wild-type (4.1 kb) and targeted (2.4 kb) alleles that hybridize to the probe are indicated with solid lines in A. (C) Northern blot of total RNA isolated from cultured fibroblasts. RNA from fibroblasts of mice genotyped as uPARAP/Endo180+/+ (left lane) and uPARAP/Endo180−/− (right lane) was hybridized with a cDNA probe complementary to the 3′ end of the uPARAP/Endo180 mRNA. (D) Western blot of lysates of cultured dermal fibroblasts from neonates genotyped as uPARAP/Endo180+/+ (left lane), uPARAP/Endo180+/− (middle lane), and uPARAP/Endo180−/− (right lane). The blot was probed with a murine anti–uPARAP/Endo180 peptide antibody prepared as described in Materials and methods. The positions of molecular mass markers (kD) are indicated left.

Mentions: We targeted the uPARAP/Endo180 gene in mice by replacing exons 2–6 with an HPRT expression cassette (Fig. 1 A). The correct targeting of the uPARAP/Endo180 gene was confirmed by Southern hybridization (Fig. 1 B) and by the presence of a truncated uPARAP/Endo180 transcript in mice homozygous for the insertion (uPARAP/Endo180−/− mice), as determined by Northern blot hybridization (Fig. 1 C). Western blot analysis of extracts of cultured fibroblasts from uPARAP/Endo180−/− mice, using an antibody directed against an epitope (residue 809–829) located outside of the introduced deletion, confirmed the absence of intact uPARAP/Endo180 (Fig. 1 D). A weak band with an apparent Mr of 70 kD was observed in Western blots of homozygously targeted as well as wild-type and heterozygous mice (unpublished data). This product was seen in all genotypes, suggesting that it was due to a weak cross-reactivity of the antibody rather than a putative truncated uPARAP/Endo180 gene product. However, it should be noted that even though formation of truncated uPARAP/Endo180 in the targeted mice could not be formally excluded, any such product would be devoid of the FN-II domain due to the targeting strategy (Fig. 1 A, legend). uPARAP/Endo180−/− mice were born in a Mendelian ratio and were outwardly normal, viable, and able to reproduce. The observations documenting these conclusions are summarized in Fig. S1 and legend (available at http://www.jcb.org/cgi/content/full/jcb.200211091/DC1).


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)

Generation of uPARAP/Endo180-targeted mice. (A) Diagram of the targeting strategy showing the structure of the uPARAP/Endo180-targeting vector (top), wild-type uPARAP/Endo180 allele (middle), and targeted uPARAP/Endo180 allele (bottom). Exons 2–6 of the uPARAP/Endo180 gene (encoding the cysteine-rich, the FN-II, and the first carbohydrate recognition domains) were replaced by the HPRT selection cassette. Exons are indicated with boxes and introns with a solid line. B, BamHI; E, EcoRI; H, HindIII; X, XhoI. (B) Southern blot of BamHI-digested mouse tail DNA from mice genotyped by PCR as uPARAP/Endo180+/+ (left lane), uPARAP/Endo180+/− (middle lane), and uPARAP/Endo180−/− (right lane). The hybridization probe (solid line below the wild-type allele in A) was located upstream of the targeted area. The expected DNA fragments of the wild-type (4.1 kb) and targeted (2.4 kb) alleles that hybridize to the probe are indicated with solid lines in A. (C) Northern blot of total RNA isolated from cultured fibroblasts. RNA from fibroblasts of mice genotyped as uPARAP/Endo180+/+ (left lane) and uPARAP/Endo180−/− (right lane) was hybridized with a cDNA probe complementary to the 3′ end of the uPARAP/Endo180 mRNA. (D) Western blot of lysates of cultured dermal fibroblasts from neonates genotyped as uPARAP/Endo180+/+ (left lane), uPARAP/Endo180+/− (middle lane), and uPARAP/Endo180−/− (right lane). The blot was probed with a murine anti–uPARAP/Endo180 peptide antibody prepared as described in Materials and methods. The positions of molecular mass markers (kD) are indicated left.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172772&req=5

fig1: Generation of uPARAP/Endo180-targeted mice. (A) Diagram of the targeting strategy showing the structure of the uPARAP/Endo180-targeting vector (top), wild-type uPARAP/Endo180 allele (middle), and targeted uPARAP/Endo180 allele (bottom). Exons 2–6 of the uPARAP/Endo180 gene (encoding the cysteine-rich, the FN-II, and the first carbohydrate recognition domains) were replaced by the HPRT selection cassette. Exons are indicated with boxes and introns with a solid line. B, BamHI; E, EcoRI; H, HindIII; X, XhoI. (B) Southern blot of BamHI-digested mouse tail DNA from mice genotyped by PCR as uPARAP/Endo180+/+ (left lane), uPARAP/Endo180+/− (middle lane), and uPARAP/Endo180−/− (right lane). The hybridization probe (solid line below the wild-type allele in A) was located upstream of the targeted area. The expected DNA fragments of the wild-type (4.1 kb) and targeted (2.4 kb) alleles that hybridize to the probe are indicated with solid lines in A. (C) Northern blot of total RNA isolated from cultured fibroblasts. RNA from fibroblasts of mice genotyped as uPARAP/Endo180+/+ (left lane) and uPARAP/Endo180−/− (right lane) was hybridized with a cDNA probe complementary to the 3′ end of the uPARAP/Endo180 mRNA. (D) Western blot of lysates of cultured dermal fibroblasts from neonates genotyped as uPARAP/Endo180+/+ (left lane), uPARAP/Endo180+/− (middle lane), and uPARAP/Endo180−/− (right lane). The blot was probed with a murine anti–uPARAP/Endo180 peptide antibody prepared as described in Materials and methods. The positions of molecular mass markers (kD) are indicated left.
Mentions: We targeted the uPARAP/Endo180 gene in mice by replacing exons 2–6 with an HPRT expression cassette (Fig. 1 A). The correct targeting of the uPARAP/Endo180 gene was confirmed by Southern hybridization (Fig. 1 B) and by the presence of a truncated uPARAP/Endo180 transcript in mice homozygous for the insertion (uPARAP/Endo180−/− mice), as determined by Northern blot hybridization (Fig. 1 C). Western blot analysis of extracts of cultured fibroblasts from uPARAP/Endo180−/− mice, using an antibody directed against an epitope (residue 809–829) located outside of the introduced deletion, confirmed the absence of intact uPARAP/Endo180 (Fig. 1 D). A weak band with an apparent Mr of 70 kD was observed in Western blots of homozygously targeted as well as wild-type and heterozygous mice (unpublished data). This product was seen in all genotypes, suggesting that it was due to a weak cross-reactivity of the antibody rather than a putative truncated uPARAP/Endo180 gene product. However, it should be noted that even though formation of truncated uPARAP/Endo180 in the targeted mice could not be formally excluded, any such product would be devoid of the FN-II domain due to the targeting strategy (Fig. 1 A, legend). uPARAP/Endo180−/− mice were born in a Mendelian ratio and were outwardly normal, viable, and able to reproduce. The observations documenting these conclusions are summarized in Fig. S1 and legend (available at http://www.jcb.org/cgi/content/full/jcb.200211091/DC1).

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