Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions

Laura Soriano-Romaní, Laura García-Posadas, Antonio López-García, Luminita Paraoan, Yolanda Diebold

Research output: Contribution to journalArticle (journal)peer-review

Abstract

Recently, thrombospondin-1 (TSP-1) has been reported to be critical for maintaining a healthy ocular surface. The purpose of the study was to characterize the expression of TSP-1 and of its receptors CD36 and CD47 in corneal and conjunctival epithelial cells and determine the effect of exogenous TSP-1 treatment on these cells, following the induction of inflammation- and apoptosis-related changes. The expression of TSP-1, CD36 and CD47 by corneal and conjunctival cell lines was firstly characterized by ELISA, immunofluorescence analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Benzalkonium chloride (BAC) exposure for 5 or 15 min was used as pro-inflammatory and pro-apoptotic stimulus for corneal or conjunctival epithelial cells, respectively. To analyze inflammation and apoptosis-related changes, IL-6 and TGF-β2 secretion determined by ELISA was used as inflammatory markers, while activated caspase-3/7 levels and cell viability, determined by CellEvent™ Caspase-3/7 Green Detection Reagent and XTT cytotoxicity assay, respectively, were used as apoptotic markers. Changes in CD36 and CD47 mRNA expression were quantified by real time RT-PCR. Corneal epithelial cells secreted and expressed higher protein levels of TSP-1 than conjunctival epithelial cells, although TSP-1 mRNA expression levels were similar and had lower CD36 and CD47, both at protein and mRNA levels. Both cell lines responded to exogenous TSP-1 treatment increasing CD36 at protein and mRNA levels. Blocking experiments revealed a predominance of TSP-1/CD47 rather than TSP-1/CD36 interactions to up-regulate CD36 levels in conjunctival epithelial cells, but not in corneal epithelial cells. BAC exposure increased IL-6 secretion and caspase-3/7 levels and decreased cell viability in both, corneal and conjunctival epithelial cells. Moreover, BAC exposure increased latent TGF-β2 levels in conjunctival epithelial cells. Interestingly, CD36 mRNA expression was down-regulated after BAC exposure in both cell lines. Exogenous TSP-1 treatment reduced TGF-β2 up-regulated levels by BAC exposure in conjunctival epithelial cells and less pronounced reduced IL-6 in BAC-exposed corneal epithelial cells. The effect on CD36 and CD47 regulation was less pronounced or even opposite depending on the inflammation- and apoptosis-related markers tested. Our results show evidence of the capacity of corneal and conjunctival epithelial cells to respond to TSP-1 via CD36 or CD47. Experimental simulation of inflammation- and apoptosis-related conditions changed the effects differentially elicited by TSP-1 on corneal and conjunctival epithelial cells, suggesting an unexpected and relevant contribution of TSP-1 on ocular surface homeostasis regulation.

Original languageEnglish
Pages (from-to)1-14
Number of pages14
JournalExperimental Eye Research
Volume134
DOIs
Publication statusPublished - 1 May 2015
Externally publishedYes

Keywords

  • Apoptosis/physiology
  • Blotting, Western
  • CD36 Antigens/genetics
  • CD47 Antigen/genetics
  • Caspase 3/metabolism
  • Caspase 7/metabolism
  • Cell Line
  • Conjunctiva/cytology
  • Cornea/cytology
  • Enzyme-Linked Immunosorbent Assay
  • Epithelial Cells/drug effects
  • Fluorescent Antibody Technique, Indirect
  • Gene Expression Regulation/physiology
  • Homeostasis
  • Humans
  • Inflammation/metabolism
  • Interleukin-6/metabolism
  • Quinolines/toxicity
  • RNA, Messenger/genetics
  • Real-Time Polymerase Chain Reaction
  • Thrombospondin 1/genetics
  • Transforming Growth Factor beta2/metabolism

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