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Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye.

Bauskar A, Mack WJ, Mauris J, Argüeso P, Heur M, Nagel BA, Kolar GR, Gleave ME, Nakamura T, Kinoshita S, Moradian-Oldak J, Panjwani N, Pflugfelder SC, Wilson MR, Fini ME, Jeong S - PLoS ONE (2015)

Bottom Line: When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress.CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component.Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure.

View Article: PubMed Central - PubMed

Affiliation: USC Institute for Genetic Medicine and Graduate Program in Medical Biology, Keck School of Medicine of USC, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye.

No MeSH data available.


Related in: MedlinePlus

Topical CLU binds selectively to the ocular surface subjected to desiccating stress, and to LGALS3 in vitro.(A) The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions were included for comparison. Eyes were treated with CF-594-anti-His antibody that binds to the His tag of recombinant human CLU (rhCLU), or with a complex of the antibody-rhCLU for 15 min, followed by confocal imaging of central cornea. Images were taken at 10X magnification. Scale bar = 100 um. (B) A DS eye was treated with a complex of the antibody-rhCLU (red) as in (A), as well as a fluorescent membrane tracer DiO (green). Images were taken at 20X magnification. In the left panel only CLU was projected. The right three panels show one Z-section plane with cross-sections oriented to the XY, YZ, and XZ axes, generated using Image J software. Yellow indicates regions of co-localization of the red and green signal. Scale bar = 100 um. (C) LGALS3-Sepharose affinity column chromatography. 1.5 ug rhCLU was applied to a 300 uL LGALS3 affinity column equilibrated in PBS containing 0.1% Triton X-100 (PBST) and the column was washed with PBST. To test sugar-binding specificity, the column was then treated sequentially with a non-competing disaccharide, sucrose (0.1 M), and then a competing disaccharide, 0.1 M lactose, dissolved in PBST. Western blotting was used to quantify CLU in the resulting fractions. Loading of the “Lac” lane represents a 1:10 dilution of the input and the “Beads” lane is a 1:4 dilution of the input, thus ~2.5X more CLU was Lac-eluted than retained on the beads. FT = flow-through; Suc = sucrose; Lac = lactose
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pone.0138958.g005: Topical CLU binds selectively to the ocular surface subjected to desiccating stress, and to LGALS3 in vitro.(A) The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions were included for comparison. Eyes were treated with CF-594-anti-His antibody that binds to the His tag of recombinant human CLU (rhCLU), or with a complex of the antibody-rhCLU for 15 min, followed by confocal imaging of central cornea. Images were taken at 10X magnification. Scale bar = 100 um. (B) A DS eye was treated with a complex of the antibody-rhCLU (red) as in (A), as well as a fluorescent membrane tracer DiO (green). Images were taken at 20X magnification. In the left panel only CLU was projected. The right three panels show one Z-section plane with cross-sections oriented to the XY, YZ, and XZ axes, generated using Image J software. Yellow indicates regions of co-localization of the red and green signal. Scale bar = 100 um. (C) LGALS3-Sepharose affinity column chromatography. 1.5 ug rhCLU was applied to a 300 uL LGALS3 affinity column equilibrated in PBS containing 0.1% Triton X-100 (PBST) and the column was washed with PBST. To test sugar-binding specificity, the column was then treated sequentially with a non-competing disaccharide, sucrose (0.1 M), and then a competing disaccharide, 0.1 M lactose, dissolved in PBST. Western blotting was used to quantify CLU in the resulting fractions. Loading of the “Lac” lane represents a 1:10 dilution of the input and the “Beads” lane is a 1:4 dilution of the input, thus ~2.5X more CLU was Lac-eluted than retained on the beads. FT = flow-through; Suc = sucrose; Lac = lactose

Mentions: To visualize CLU binding to the ocular surface, we used the technique of direct immunostaining with an antibody conjugated to CF-594 dye. To differentiate topically applied rhCLU from endogenous CLU, we took advantage of the C-terminal His tag incorporated into the rhCLU molecule. Representative results are shown in Fig 5A. Eyes subjected to desiccating stress, then treated with CF-594 dye conjugated anti-His antibody alone, showed some diffuse fluorescence over the ocular surface subjected to desiccating stress. However, when the ocular surface of these mice was treated with rhCLU, substantial punctate binding of dye-conjugated antibody to the ocular surface subjected to desiccating stress was observed, indicating the location of direct CLU binding. In contrast, the NS eye showed far less binding. In a second set of experiments, the fluorescent lipophilic membrane tracer DiO was used to delineate individual cells. Representative results are shown in Fig 5B. This showed that the CLU “spots” were approximately the size of cells. In some cases, the CLU spots (red) filled the entire area of individual cells marked by the dyed membrane (green), overlapping completely (yellow color). In other cases, CLU spots were clearly separate.


Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye.

Bauskar A, Mack WJ, Mauris J, Argüeso P, Heur M, Nagel BA, Kolar GR, Gleave ME, Nakamura T, Kinoshita S, Moradian-Oldak J, Panjwani N, Pflugfelder SC, Wilson MR, Fini ME, Jeong S - PLoS ONE (2015)

Topical CLU binds selectively to the ocular surface subjected to desiccating stress, and to LGALS3 in vitro.(A) The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions were included for comparison. Eyes were treated with CF-594-anti-His antibody that binds to the His tag of recombinant human CLU (rhCLU), or with a complex of the antibody-rhCLU for 15 min, followed by confocal imaging of central cornea. Images were taken at 10X magnification. Scale bar = 100 um. (B) A DS eye was treated with a complex of the antibody-rhCLU (red) as in (A), as well as a fluorescent membrane tracer DiO (green). Images were taken at 20X magnification. In the left panel only CLU was projected. The right three panels show one Z-section plane with cross-sections oriented to the XY, YZ, and XZ axes, generated using Image J software. Yellow indicates regions of co-localization of the red and green signal. Scale bar = 100 um. (C) LGALS3-Sepharose affinity column chromatography. 1.5 ug rhCLU was applied to a 300 uL LGALS3 affinity column equilibrated in PBS containing 0.1% Triton X-100 (PBST) and the column was washed with PBST. To test sugar-binding specificity, the column was then treated sequentially with a non-competing disaccharide, sucrose (0.1 M), and then a competing disaccharide, 0.1 M lactose, dissolved in PBST. Western blotting was used to quantify CLU in the resulting fractions. Loading of the “Lac” lane represents a 1:10 dilution of the input and the “Beads” lane is a 1:4 dilution of the input, thus ~2.5X more CLU was Lac-eluted than retained on the beads. FT = flow-through; Suc = sucrose; Lac = lactose
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581869&req=5

pone.0138958.g005: Topical CLU binds selectively to the ocular surface subjected to desiccating stress, and to LGALS3 in vitro.(A) The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions were included for comparison. Eyes were treated with CF-594-anti-His antibody that binds to the His tag of recombinant human CLU (rhCLU), or with a complex of the antibody-rhCLU for 15 min, followed by confocal imaging of central cornea. Images were taken at 10X magnification. Scale bar = 100 um. (B) A DS eye was treated with a complex of the antibody-rhCLU (red) as in (A), as well as a fluorescent membrane tracer DiO (green). Images were taken at 20X magnification. In the left panel only CLU was projected. The right three panels show one Z-section plane with cross-sections oriented to the XY, YZ, and XZ axes, generated using Image J software. Yellow indicates regions of co-localization of the red and green signal. Scale bar = 100 um. (C) LGALS3-Sepharose affinity column chromatography. 1.5 ug rhCLU was applied to a 300 uL LGALS3 affinity column equilibrated in PBS containing 0.1% Triton X-100 (PBST) and the column was washed with PBST. To test sugar-binding specificity, the column was then treated sequentially with a non-competing disaccharide, sucrose (0.1 M), and then a competing disaccharide, 0.1 M lactose, dissolved in PBST. Western blotting was used to quantify CLU in the resulting fractions. Loading of the “Lac” lane represents a 1:10 dilution of the input and the “Beads” lane is a 1:4 dilution of the input, thus ~2.5X more CLU was Lac-eluted than retained on the beads. FT = flow-through; Suc = sucrose; Lac = lactose
Mentions: To visualize CLU binding to the ocular surface, we used the technique of direct immunostaining with an antibody conjugated to CF-594 dye. To differentiate topically applied rhCLU from endogenous CLU, we took advantage of the C-terminal His tag incorporated into the rhCLU molecule. Representative results are shown in Fig 5A. Eyes subjected to desiccating stress, then treated with CF-594 dye conjugated anti-His antibody alone, showed some diffuse fluorescence over the ocular surface subjected to desiccating stress. However, when the ocular surface of these mice was treated with rhCLU, substantial punctate binding of dye-conjugated antibody to the ocular surface subjected to desiccating stress was observed, indicating the location of direct CLU binding. In contrast, the NS eye showed far less binding. In a second set of experiments, the fluorescent lipophilic membrane tracer DiO was used to delineate individual cells. Representative results are shown in Fig 5B. This showed that the CLU “spots” were approximately the size of cells. In some cases, the CLU spots (red) filled the entire area of individual cells marked by the dyed membrane (green), overlapping completely (yellow color). In other cases, CLU spots were clearly separate.

Bottom Line: When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress.CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component.Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure.

View Article: PubMed Central - PubMed

Affiliation: USC Institute for Genetic Medicine and Graduate Program in Medical Biology, Keck School of Medicine of USC, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye.

No MeSH data available.


Related in: MedlinePlus