title: Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching.
abstract: Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures.
authors.affiliation: University of Oxford, Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Oxford OX3 7LD, United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Kuzma-Kuzniarska
authors.affiliation: University of Oxford, Structural Genomics Consortium, Oxford OX3 7DQ, United Kingdom.
authors.forename: Clarence
authors.initials: C
authors.lastname: Yapp
authors.affiliation: University of Oxford, Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Oxford OX3 7LD, United Kingdom.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Pearson-Jones
authors.affiliation: Oxford Brookes University, Faculty of Health and Life Sciences, Department of Biological and Medical Sciences Oxford OX3 0BP, United Kingdom.
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Jones
authors.affiliation: University of Oxford, Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Oxford OX3 7LD, United Kingdom.
authors.forename: Philippa A
authors.initials: PA
authors.lastname: Hulley
mesh_terms: D000328:Adult; D002229:Carbenoxolone / Q000737:chemistry; D002450:Cell Communication; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D004058:Diffusion; D005260:Female; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D017629:Gap Junctions / Q000473:pathology*; D006034:Glycyrrhetinic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D013710:Tendons / Q000166:cytology* / Q000473:pathology*
chemical_list: C119129:18alpha-glycyrrhetinic acid; D003094:Collagen; D002229:Carbenoxolone; D006034:Glycyrrhetinic Acid