Establishment of a human conjunctival epithelial cell line lacking the functional TACSTD2 gene (an American Ophthalmological Society thesis)

Abstract

Purpose: To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene.

Methods: A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD.

Results: The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction-related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line.

Conclusions: Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD.

FIGURE 1

Corneal clinical phenotypes for gelatinous drop-like corneal dystrophy: (GDLD) mulberry type (left), band keratopathy type (middle), and kumquat-like type (right). Reprinted with permission from Developments in Ophthalmology. All rights reserved.

FIGURE 2

Epithelial barrier function is significantly decreased in cornea with gelatinous drop-like corneal dystrophy (GDLD). Top, Slit-lamp microscopy photograph of the hyperfluorescence of the cornea observed in a GDLD patient. Middle, Slit-lamp microscopy photograph demonstrating a GDLD cornea that underwent keratoplasty. Triangles indicate the boundary between the host corneal epithelium and the donor corneal epithelium. Note that the host corneal epithelium demonstrates hyperfluorescence, but that of the donor cornea does not. Bottom, Bar graph demonstrating fluorescein uptake among several corneal dystrophies. GCD, granular corneal dystrophy; LCD, lattice corneal dystrophy; MCD, macular corneal dystrophy. Reprinted with permission from Developments in Ophthalmology. All rights reserved.

FIGURE 3

Electron microscopy image showing decreased epithelial barrier function in a cornea with GDLD. Permeated horseradish peroxidase is demonstrated through the loosened epithelial barrier. Horseradish peroxidase is visible as an electron-dense tracer in a degenerated superficial epithelial cell (asterisk), and is seen penetrating beneath the epithelial surface (arrows). Reprinted with permission from Cornea. All rights reserved.

FIGURE 4

Electron micrographs showing excessive desquamation of superficial corneal epithelial cells of cornea (left) and enlarged intercellular space of corneal epithelium (right) in a patient with GDLD. Reprinted with permission from Cornea. All rights reserved.

FIGURE 5

Results of immunostaining analysis show that the expression of the tight junction–related proteins, zonula occludens-1 (ZO-1) (top), occuludin (OCLN) (middle upper), claudin 1 (CLDN1) (middle lower), as well as desmosome protein desmoplakin (bottom), was significantly attenuated in cornea with gelatinous drop-like corneal dystrophy (GDLD) (right) compared to normal cornea (left). Arrowheads indicate the apical expression of these tight junction–related proteins in normal epithelium. Arrows indicate the basement of corneal epithelium. Bar = 50 μm. Reprinted with permission from Investigative Ophthalmology & Visual Science. All rights reserved.

FIGURE 6

Data indicating that the tumor-associated calcium signal transducer 2 (TACSTD2) protein may protect CLDN1 and 7 proteins from protein degradation by the ubiquitin-proteasome system. Top, HeLa cells were introduced with CLDN1, 4, or 7 genes with or without the TACSTD2 gene. Introduction of the TACSTD2 gene significantly enhanced the expression of CLDN1 and 7, but not of CLDN4. Bottom left, HeLa cells introduced with CLDN1, 4, or 7 genes were treated with the proteasome inhibitor MG-132. The MG-132 treatment significantly enhanced the expression of CLDN1 and 7, but not of CLDN4. Bottom right, Immunostaining analysis data was confirmed by Western blotting analysis. Reprinted with permission from American Journal of Pathology. All rights reserved.

FIGURE 7

Clinical and genetic information for the gelatinous drop-like corneal dystrophy (GDLD) patient whose conjunctival tissue was obtained for the establishment of our immortalized conjunctival epithelial cell line. Top, Slit-lamp microscopy image demonstrating the clinical phenotype of the GDLD patient. Middle, Electropherogram data of the TACSTD2 gene from a normal volunteer (left) and the GDLD patient (right). Triangle denotes the c.352C>T mutation, which may produce a p.Gln118x mutation within the TACSTD2 gene. Bottom, Domain structure of the TACSTD2 protein with mutations thus far reported for the TACSTD2 gene. SS, signal sequence; EGF, epidermal growth factor–like repeat; TY, thyloglobulin repeat; TM, transmembrane domain; PIP2, PIP2-binding sequence. Note that the TM domain is located near the C-terminus of this protein; thus the p.Gln118x nonsense mutation (indicated by a triangle) may produce a truncated form of this protein.

FIGURE 8

Lentiviral introduction of SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes significantly elongated the life span of the conjunctival epithelial cells from a gelatinous drop-like corneal dystrophy (GDLD) patient. Top, Plasmid map of the lentiviral vector harboring the expression cassette for the SV40 large T antigen (left) and hTERT (right) genes. Middle left, Scatter diagram showing the population doubling analysis data of transfected or nontransfected GDLD conjunctival epithelial cells fitted by quadratic curve. Note that the fitted curve of the transduced cells curves downward, whereas that of the nontransduced cells curves upward. Middle right, Results of colony-forming assay. Bottom, Cell shape of conjunctival epithelial cells of the GDLD patient with or without the transduction.

FIGURE 9

Expression of SV40 large T antigen and telomerase in the immortalized conjunctival epithelial cells from a gelatinous drop-like corneal dystrophy (GDLD) patient. Results of immunostaining (top) and Western blotting (bottom left) analyses for the expression of the SV40 large T antigen and hTERT genes in the immortalized conjunctival (Cj) epithelial cells from a GDLD patient and immortalized human corneal epithelial cells (HCE-T). Lysate of HCE-T cells was used in the Western blotting analysis (bottom left) as a positive control. Bottom right, Results of telomeric repeat amplification protocol (TRAP) assay show the expression of functional telomerase. The ladder pattern seen at lanes 2, 3, 4, and 5 indicates the existence of telomerase activity. 1: size marker, 2: HCE-T cells, 3: HeLa cells, 4: immortalized human normal conjunctival epithelial cells, 5: immortalized human GDLD conjunctival epithelial cells, 6: heat inactivated HCE-T cells, 7: heat-inactivated HeLa cells, 8: heat-inactivated immortalized human normal conjunctival epithelial cells, 9: heat-inactivated immortalized human GDLD conjunctival epithelial cells and 10: buffer only.

FIGURE 10

Decreased epithelial barrier function in the immortalized conjunctival epithelial cells from a gelatinous drop-like corneal dystrophy (GDLD) patient. Epithelial barrier function was investigated using a commercial voltmeter (top). Epithelial barrier function was found to be significantly low in the immortalized conjunctival epithelial cells from the GDLD patient both in high (1 mM, bottom left) and middle (0.54 mM, bottom right) calcium media.

FIGURE 11

Expression of the TACSTD2 and tight junction–related proteins in the immortalized conjunctival epithelial cells from a gelatinous drop-like corneal dystrophy (GDLD) patient. Top, Results of immunostaining analysis for the expression of the TACSTD2 protein in the immortalized normal and GDLD conjunctival epithelial cells. Bottom, Protein expression of tight junction–related proteins CLDN1, CLDN4, CLDN7, OCLN, and tight junction protein-1 (TJP1) in the immortalized normal and GDLD conjunctival epithelial cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) was investigated as a loading control. HCE-T cells were also used as a positive control.