A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration

Sayan Ghosh, Peng Shang, Hiroto Terasaki, Nadezda Stepicheva, Stacey Hose, Meysam Yazdankhah, Joseph Weiss, Taiji Sakamoto, Imran A. Bhutto, Shuli Xia, J. Samuel Zigler, Ram Kannan, Jiang Qian, James T. Handa, Debasish Sinha

Research output: Contribution to journalArticle

Abstract

Purpose: The RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that βA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells.

Methods: RPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used. Spatial localization of proteins was demonstrated with immunofluorescence, gene expression levels were determined by quantitative PCR (qPCR), and protein levels by Western blotting. Cell movement was evaluated using wound healing and cell migration assays. Co-immunoprecipitation was used to identify binding partners of βA3/A1-crystallin.

Results: βA3/A1-crystallin is upregulated in polarized RPE cells compared to undifferentiated cells. Loss of βA3/A1-crystallin in murine and human RPE cells resulted in upregulation of Snail and vimentin, downregulation of E-cadherin, and increased cell migration. βA3/A1-crystallin binds to cortactin, and loss of βA3/A1-crystallin resulted in increased P-cortactinY421. The RPE from AMD samples had increased Snail and vimentin, and decreased E-cadherin, compared to age-matched controls.

Conclusions: We introduced a novel concept of dry AMD initiation induced by lysosomal clearance defects in the RPE and subsequent attempts by RPE cells to avoid the resulting stress by undergoing EMT. We demonstrate that βA3/A1-crystallin is a potential therapeutic target for AMD through rejuvenation of lysosomal dysfunction and potentially, reversal of EMT.

Original languageEnglish (US)
Pages (from-to)AMD104-AMD113
JournalInvestigative ophthalmology & visual science
Volume59
Issue number4
DOIs
StatePublished - Mar 20 2018

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Crystallins
Epithelial-Mesenchymal Transition
Macular Degeneration
Vimentin
Cadherins
Cell Movement
Cortactin
Cell Migration Assays
Rejuvenation
Proteins
Immunoprecipitation
Wound Healing
Fluorescent Antibody Technique
Melanoma
Up-Regulation
Down-Regulation
Western Blotting
Gene Expression
Cell Line
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Ghosh, S., Shang, P., Terasaki, H., Stepicheva, N., Hose, S., Yazdankhah, M., ... Sinha, D. (2018). A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration. Investigative ophthalmology & visual science, 59(4), AMD104-AMD113. https://doi.org/10.1167/iovs.18-24132

A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration. / Ghosh, Sayan; Shang, Peng; Terasaki, Hiroto; Stepicheva, Nadezda; Hose, Stacey; Yazdankhah, Meysam; Weiss, Joseph; Sakamoto, Taiji; Bhutto, Imran A.; Xia, Shuli; Zigler, J. Samuel; Kannan, Ram; Qian, Jiang; Handa, James T.; Sinha, Debasish.

In: Investigative ophthalmology & visual science, Vol. 59, No. 4, 20.03.2018, p. AMD104-AMD113.

Research output: Contribution to journalArticle

Ghosh, S, Shang, P, Terasaki, H, Stepicheva, N, Hose, S, Yazdankhah, M, Weiss, J, Sakamoto, T, Bhutto, IA, Xia, S, Zigler, JS, Kannan, R, Qian, J, Handa, JT & Sinha, D 2018, 'A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration', Investigative ophthalmology & visual science, vol. 59, no. 4, pp. AMD104-AMD113. https://doi.org/10.1167/iovs.18-24132
Ghosh, Sayan ; Shang, Peng ; Terasaki, Hiroto ; Stepicheva, Nadezda ; Hose, Stacey ; Yazdankhah, Meysam ; Weiss, Joseph ; Sakamoto, Taiji ; Bhutto, Imran A. ; Xia, Shuli ; Zigler, J. Samuel ; Kannan, Ram ; Qian, Jiang ; Handa, James T. ; Sinha, Debasish. / A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration. In: Investigative ophthalmology & visual science. 2018 ; Vol. 59, No. 4. pp. AMD104-AMD113.
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abstract = "Purpose: The RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that βA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells.Methods: RPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used. Spatial localization of proteins was demonstrated with immunofluorescence, gene expression levels were determined by quantitative PCR (qPCR), and protein levels by Western blotting. Cell movement was evaluated using wound healing and cell migration assays. Co-immunoprecipitation was used to identify binding partners of βA3/A1-crystallin.Results: βA3/A1-crystallin is upregulated in polarized RPE cells compared to undifferentiated cells. Loss of βA3/A1-crystallin in murine and human RPE cells resulted in upregulation of Snail and vimentin, downregulation of E-cadherin, and increased cell migration. βA3/A1-crystallin binds to cortactin, and loss of βA3/A1-crystallin resulted in increased P-cortactinY421. The RPE from AMD samples had increased Snail and vimentin, and decreased E-cadherin, compared to age-matched controls.Conclusions: We introduced a novel concept of dry AMD initiation induced by lysosomal clearance defects in the RPE and subsequent attempts by RPE cells to avoid the resulting stress by undergoing EMT. We demonstrate that βA3/A1-crystallin is a potential therapeutic target for AMD through rejuvenation of lysosomal dysfunction and potentially, reversal of EMT.",
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T1 - A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration

AU - Ghosh, Sayan

AU - Shang, Peng

AU - Terasaki, Hiroto

AU - Stepicheva, Nadezda

AU - Hose, Stacey

AU - Yazdankhah, Meysam

AU - Weiss, Joseph

AU - Sakamoto, Taiji

AU - Bhutto, Imran A.

AU - Xia, Shuli

AU - Zigler, J. Samuel

AU - Kannan, Ram

AU - Qian, Jiang

AU - Handa, James T.

AU - Sinha, Debasish

PY - 2018/3/20

Y1 - 2018/3/20

N2 - Purpose: The RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that βA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells.Methods: RPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used. Spatial localization of proteins was demonstrated with immunofluorescence, gene expression levels were determined by quantitative PCR (qPCR), and protein levels by Western blotting. Cell movement was evaluated using wound healing and cell migration assays. Co-immunoprecipitation was used to identify binding partners of βA3/A1-crystallin.Results: βA3/A1-crystallin is upregulated in polarized RPE cells compared to undifferentiated cells. Loss of βA3/A1-crystallin in murine and human RPE cells resulted in upregulation of Snail and vimentin, downregulation of E-cadherin, and increased cell migration. βA3/A1-crystallin binds to cortactin, and loss of βA3/A1-crystallin resulted in increased P-cortactinY421. The RPE from AMD samples had increased Snail and vimentin, and decreased E-cadherin, compared to age-matched controls.Conclusions: We introduced a novel concept of dry AMD initiation induced by lysosomal clearance defects in the RPE and subsequent attempts by RPE cells to avoid the resulting stress by undergoing EMT. We demonstrate that βA3/A1-crystallin is a potential therapeutic target for AMD through rejuvenation of lysosomal dysfunction and potentially, reversal of EMT.

AB - Purpose: The RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that βA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells.Methods: RPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used. Spatial localization of proteins was demonstrated with immunofluorescence, gene expression levels were determined by quantitative PCR (qPCR), and protein levels by Western blotting. Cell movement was evaluated using wound healing and cell migration assays. Co-immunoprecipitation was used to identify binding partners of βA3/A1-crystallin.Results: βA3/A1-crystallin is upregulated in polarized RPE cells compared to undifferentiated cells. Loss of βA3/A1-crystallin in murine and human RPE cells resulted in upregulation of Snail and vimentin, downregulation of E-cadherin, and increased cell migration. βA3/A1-crystallin binds to cortactin, and loss of βA3/A1-crystallin resulted in increased P-cortactinY421. The RPE from AMD samples had increased Snail and vimentin, and decreased E-cadherin, compared to age-matched controls.Conclusions: We introduced a novel concept of dry AMD initiation induced by lysosomal clearance defects in the RPE and subsequent attempts by RPE cells to avoid the resulting stress by undergoing EMT. We demonstrate that βA3/A1-crystallin is a potential therapeutic target for AMD through rejuvenation of lysosomal dysfunction and potentially, reversal of EMT.

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