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Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.

Xu J, Rodriguez D, Petitclerc E, Kim JJ, Hangai M, Moon YS, Davis GE, Brooks PC, Yuen SM - J. Cell Biol. (2001)

Bottom Line: Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo.A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth.Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

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Integrin binding to triple helical and denatured human collagen IV. Microtiter wells were coated with either triple helical or denatured human collagen IV (25 μg/ml). Purified human integrins α1β1, α2β1, α5β1, or αvβ3 (0.5–4 μg/ml) were allowed to bind to triple helical collagen IV (A) or denatured collagen IV (B) for 1 h at 37°C. Integrin binding was detected with antiintegrin antibodies. (C) Purified human integrins α2β1 and αvβ3 (1.0 μg/ml) were allowed to bind to denatured collagen IV–coated plates for 1 h at 37°C in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. Integrin binding was detected by incubation with either polyclonal antibody directed to α2 or α3 integrins. Data bars represent the mean OD ± standard deviations from triplicate wells.
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fig7: Integrin binding to triple helical and denatured human collagen IV. Microtiter wells were coated with either triple helical or denatured human collagen IV (25 μg/ml). Purified human integrins α1β1, α2β1, α5β1, or αvβ3 (0.5–4 μg/ml) were allowed to bind to triple helical collagen IV (A) or denatured collagen IV (B) for 1 h at 37°C. Integrin binding was detected with antiintegrin antibodies. (C) Purified human integrins α2β1 and αvβ3 (1.0 μg/ml) were allowed to bind to denatured collagen IV–coated plates for 1 h at 37°C in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. Integrin binding was detected by incubation with either polyclonal antibody directed to α2 or α3 integrins. Data bars represent the mean OD ± standard deviations from triplicate wells.

Mentions: We sought to determine whether an integrin receptor was involved in mediating cellular interactions with the HUIV26 cryptic epitope. To facilitate these studies, microtiter wells were coated with either triple helical or denatured collagen IV. The wells were incubated with purified integrin receptors, including α1β1, α2β1, αvβ3, and α5β1. After incubation, bound integrins were detected with antiintegrin-specific antibodies. As shown in Fig. 7 A, the collagen-binding integrins α1β1 and α2β1 bound to triple helical collagen in a dose-responsive manner, whereas integrins αvβ3 and α5β1 showed little if any interaction. After denaturation, α1β1 binding was lost; however, denatured collagen IV acquired the capacity to bind to integrin αvβ3 (Fig. 7 B). Moreover, denatured collagen IV retained its ability to bind to α2β1, whereas the control fibronectin receptor integrin α5β1 failed to interact (Fig. 7 B). These findings suggest that denaturation of the triple helical structure of collagen IV can shift integrin binding specificity from that of a β1 dependency to both β1 and αvβ3. To determine whether integrin α2β1 or integrin αvβ3 interacts with the HUIV26 cryptic epitope, similar receptor binding assays were performed in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. As shown in Fig. 7 C, Mab HUIV26 failed to block the ability of purified integrin α2β1 to bind to denatured collagen IV, while inhibiting integrin αvβ3 binding by >70%. Together, these findings suggest that integrin αvβ3 may function as a receptor for the HUIV26 cryptic epitope.


Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.

Xu J, Rodriguez D, Petitclerc E, Kim JJ, Hangai M, Moon YS, Davis GE, Brooks PC, Yuen SM - J. Cell Biol. (2001)

Integrin binding to triple helical and denatured human collagen IV. Microtiter wells were coated with either triple helical or denatured human collagen IV (25 μg/ml). Purified human integrins α1β1, α2β1, α5β1, or αvβ3 (0.5–4 μg/ml) were allowed to bind to triple helical collagen IV (A) or denatured collagen IV (B) for 1 h at 37°C. Integrin binding was detected with antiintegrin antibodies. (C) Purified human integrins α2β1 and αvβ3 (1.0 μg/ml) were allowed to bind to denatured collagen IV–coated plates for 1 h at 37°C in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. Integrin binding was detected by incubation with either polyclonal antibody directed to α2 or α3 integrins. Data bars represent the mean OD ± standard deviations from triplicate wells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196184&req=5

fig7: Integrin binding to triple helical and denatured human collagen IV. Microtiter wells were coated with either triple helical or denatured human collagen IV (25 μg/ml). Purified human integrins α1β1, α2β1, α5β1, or αvβ3 (0.5–4 μg/ml) were allowed to bind to triple helical collagen IV (A) or denatured collagen IV (B) for 1 h at 37°C. Integrin binding was detected with antiintegrin antibodies. (C) Purified human integrins α2β1 and αvβ3 (1.0 μg/ml) were allowed to bind to denatured collagen IV–coated plates for 1 h at 37°C in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. Integrin binding was detected by incubation with either polyclonal antibody directed to α2 or α3 integrins. Data bars represent the mean OD ± standard deviations from triplicate wells.
Mentions: We sought to determine whether an integrin receptor was involved in mediating cellular interactions with the HUIV26 cryptic epitope. To facilitate these studies, microtiter wells were coated with either triple helical or denatured collagen IV. The wells were incubated with purified integrin receptors, including α1β1, α2β1, αvβ3, and α5β1. After incubation, bound integrins were detected with antiintegrin-specific antibodies. As shown in Fig. 7 A, the collagen-binding integrins α1β1 and α2β1 bound to triple helical collagen in a dose-responsive manner, whereas integrins αvβ3 and α5β1 showed little if any interaction. After denaturation, α1β1 binding was lost; however, denatured collagen IV acquired the capacity to bind to integrin αvβ3 (Fig. 7 B). Moreover, denatured collagen IV retained its ability to bind to α2β1, whereas the control fibronectin receptor integrin α5β1 failed to interact (Fig. 7 B). These findings suggest that denaturation of the triple helical structure of collagen IV can shift integrin binding specificity from that of a β1 dependency to both β1 and αvβ3. To determine whether integrin α2β1 or integrin αvβ3 interacts with the HUIV26 cryptic epitope, similar receptor binding assays were performed in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. As shown in Fig. 7 C, Mab HUIV26 failed to block the ability of purified integrin α2β1 to bind to denatured collagen IV, while inhibiting integrin αvβ3 binding by >70%. Together, these findings suggest that integrin αvβ3 may function as a receptor for the HUIV26 cryptic epitope.

Bottom Line: Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo.A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth.Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

Show MeSH
Related in: MedlinePlus