Analysis of connexin expression in human conjunctival epithelial cells

Suzanne Hagan, Sapthagiri Ravikanti, Navin D. Rathna Kumar, Patricia Martin, Michael Doughty

Research output: Contribution to journalMeeting abstract

Abstract

Purpose: Dry eye disease (DED) is a common and distressing ocular condition in the over-55s, and a significant cause of visual morbidity. In DED, the conjunctival cells are physically altered, a process known as "squamous metaplasia". The cells become enlarged, flattened-out (lose cell-cell contacts) and adopt characteristics more typical of corneal epithelial cells. The mechanism by which this change occurs is poorly understood, but logically must affect epithelial transport and cell junctions as the cells react to hyperosmotic changes in the overlying tear fluid. Connexins (Cx) are the constituent proteins of Gap Junctions and play a central role in the coordination of cellular activities. These specialised connections are considered to be essential in maintaining the integrity and function of mucous membranes, permitting the exchange of small molecules and ions between neighbouring cells. Cx31.1 and Cx43 are known to have roles in corneal wound healing and apoptosis, however, little is known of Cx expresson in human conjunctiva. The aim of this study is to analyse Cx expression in a human conjunctival cell line to further our understanding of cell-cell communication in this tissue and in DED.

Methods: All experiments were performed on the immortalised Chang human conjunctival epithelial cell line. Morphometric analysis was performed to determine cell size. Reverse-Transcription-PCR (RT-PCR), western blotting (WB) and confocal immunofluorescence (IF) were used to assess Cx gene and protein expression.

Results: RT-PCR of Chang cells showed Cx43 and Cx45 expression at the mRNA level. Cx26 and Cx37 mRNA expression was absent.WB of Chang cell lysates indicated a band at 43kd, suggesting Cx43 protein expression. IF studies demonstrated punctate Cx43 and Cx45 staining, specific to intercellular junctions of confluent Chang cells.

Conclusions: This study shows for the first time that Chang conjunctival epithelial cells express Cx43 and Cx45 at both the mRNA and protein level, especially at cell confluence. This indicates a potential role for these specific Cxs in conjunctival epithelial cell communication at the ocular surface.
Original languageEnglish
Pages (from-to)6699
Number of pages1
JournalInvestigative Ophthalmology & Visual Science
Volume52
Issue number14
Publication statusPublished - Apr 2011

Fingerprint

Connexins
Epithelial Cells
Connexin 43
Eye Diseases
Intercellular Junctions
Cell Communication
Messenger RNA
Reverse Transcription
Fluorescent Antibody Technique
Western Blotting
Cell Line
Polymerase Chain Reaction
Proteins
Conjunctiva
Metaplasia
Tears
Cell Size
Wound Healing
Mucous Membrane
Ions

Keywords

  • conjunctiva
  • cell junction
  • gene expression
  • cell adhesions

Cite this

Hagan, S., Ravikanti, S., Rathna Kumar, N. D., Martin, P., & Doughty, M. (2011). Analysis of connexin expression in human conjunctival epithelial cells. Investigative Ophthalmology & Visual Science, 52(14), 6699.
Hagan, Suzanne ; Ravikanti, Sapthagiri ; Rathna Kumar, Navin D. ; Martin, Patricia ; Doughty, Michael. / Analysis of connexin expression in human conjunctival epithelial cells. In: Investigative Ophthalmology & Visual Science. 2011 ; Vol. 52, No. 14. pp. 6699.
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Hagan, S, Ravikanti, S, Rathna Kumar, ND, Martin, P & Doughty, M 2011, 'Analysis of connexin expression in human conjunctival epithelial cells', Investigative Ophthalmology & Visual Science, vol. 52, no. 14, pp. 6699.

Analysis of connexin expression in human conjunctival epithelial cells. / Hagan, Suzanne; Ravikanti, Sapthagiri; Rathna Kumar, Navin D.; Martin, Patricia; Doughty, Michael.

In: Investigative Ophthalmology & Visual Science, Vol. 52, No. 14, 04.2011, p. 6699.

Research output: Contribution to journalMeeting abstract

TY - JOUR

T1 - Analysis of connexin expression in human conjunctival epithelial cells

AU - Hagan, Suzanne

AU - Ravikanti, Sapthagiri

AU - Rathna Kumar, Navin D.

AU - Martin, Patricia

AU - Doughty, Michael

PY - 2011/4

Y1 - 2011/4

N2 - Purpose: Dry eye disease (DED) is a common and distressing ocular condition in the over-55s, and a significant cause of visual morbidity. In DED, the conjunctival cells are physically altered, a process known as "squamous metaplasia". The cells become enlarged, flattened-out (lose cell-cell contacts) and adopt characteristics more typical of corneal epithelial cells. The mechanism by which this change occurs is poorly understood, but logically must affect epithelial transport and cell junctions as the cells react to hyperosmotic changes in the overlying tear fluid. Connexins (Cx) are the constituent proteins of Gap Junctions and play a central role in the coordination of cellular activities. These specialised connections are considered to be essential in maintaining the integrity and function of mucous membranes, permitting the exchange of small molecules and ions between neighbouring cells. Cx31.1 and Cx43 are known to have roles in corneal wound healing and apoptosis, however, little is known of Cx expresson in human conjunctiva. The aim of this study is to analyse Cx expression in a human conjunctival cell line to further our understanding of cell-cell communication in this tissue and in DED.Methods: All experiments were performed on the immortalised Chang human conjunctival epithelial cell line. Morphometric analysis was performed to determine cell size. Reverse-Transcription-PCR (RT-PCR), western blotting (WB) and confocal immunofluorescence (IF) were used to assess Cx gene and protein expression.Results: RT-PCR of Chang cells showed Cx43 and Cx45 expression at the mRNA level. Cx26 and Cx37 mRNA expression was absent.WB of Chang cell lysates indicated a band at 43kd, suggesting Cx43 protein expression. IF studies demonstrated punctate Cx43 and Cx45 staining, specific to intercellular junctions of confluent Chang cells.Conclusions: This study shows for the first time that Chang conjunctival epithelial cells express Cx43 and Cx45 at both the mRNA and protein level, especially at cell confluence. This indicates a potential role for these specific Cxs in conjunctival epithelial cell communication at the ocular surface.

AB - Purpose: Dry eye disease (DED) is a common and distressing ocular condition in the over-55s, and a significant cause of visual morbidity. In DED, the conjunctival cells are physically altered, a process known as "squamous metaplasia". The cells become enlarged, flattened-out (lose cell-cell contacts) and adopt characteristics more typical of corneal epithelial cells. The mechanism by which this change occurs is poorly understood, but logically must affect epithelial transport and cell junctions as the cells react to hyperosmotic changes in the overlying tear fluid. Connexins (Cx) are the constituent proteins of Gap Junctions and play a central role in the coordination of cellular activities. These specialised connections are considered to be essential in maintaining the integrity and function of mucous membranes, permitting the exchange of small molecules and ions between neighbouring cells. Cx31.1 and Cx43 are known to have roles in corneal wound healing and apoptosis, however, little is known of Cx expresson in human conjunctiva. The aim of this study is to analyse Cx expression in a human conjunctival cell line to further our understanding of cell-cell communication in this tissue and in DED.Methods: All experiments were performed on the immortalised Chang human conjunctival epithelial cell line. Morphometric analysis was performed to determine cell size. Reverse-Transcription-PCR (RT-PCR), western blotting (WB) and confocal immunofluorescence (IF) were used to assess Cx gene and protein expression.Results: RT-PCR of Chang cells showed Cx43 and Cx45 expression at the mRNA level. Cx26 and Cx37 mRNA expression was absent.WB of Chang cell lysates indicated a band at 43kd, suggesting Cx43 protein expression. IF studies demonstrated punctate Cx43 and Cx45 staining, specific to intercellular junctions of confluent Chang cells.Conclusions: This study shows for the first time that Chang conjunctival epithelial cells express Cx43 and Cx45 at both the mRNA and protein level, especially at cell confluence. This indicates a potential role for these specific Cxs in conjunctival epithelial cell communication at the ocular surface.

KW - conjunctiva

KW - cell junction

KW - gene expression

KW - cell adhesions

M3 - Meeting abstract

VL - 52

SP - 6699

JO - Investigative Ophthalmology & Visual Science

JF - Investigative Ophthalmology & Visual Science

SN - 0146-0404

IS - 14

ER -