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Pertubation of beta1 integrin function using anti-sense or function-blocking antibodies on corneal cells grown on
Kathleen J Doane1, Raka Bhattacharya, Jeff Marchant
1Department of Anatomy, Northeastern Ohio Universities College of Medicine, 4209 State Route 44, Rootstown, OH 44272-0095, USA. kd@neoucom.edu
Abstract:
During corneal development, neural crest derivatives from the periocular mesenchyme migrate into the cornea and differentiate into corneal fibroblasts. During this time, these cells interact with a variety of extracellular matrices for proper orientation and development. In the present studies, we have examined the interaction of beta(1) integrins on periocular mesenchyme cells (POM) and corneal fibroblasts (CF) with fibronectin and tenascin by perturbing the function of this integrin. POM and CF attached and spread to a much greater extent on fibronectin than on tenascin. An antibody against beta(1) integrin, CSAT, decreased spreading and attachment, and resulted in a lack of immuno-detectable beta(1) integrin in focal adhesions on fibronectin; few beta(1) positive focal adhesions were observed in cells grown on tenascin. An anti-sense retroviral construct decreased endogenous levels of beta(1) integrin protein, and caused decreased attachment and spreading as well as sparse, disorganized focal adhesions. These data indicate that in vitro, both POM and CF have beta(1) integrins that interact with fibronectin and allow them to attach and spread, while tenascin is anti-adhesive. Further studies using both of these experimental paradigms will clarify whether these interactions also occur in vivo.
Insights
Periocular mesenchyme cells and corneal fibroblasts use beta(1) integrins to attach and spread on fibronectin. Tenascin acts as an anti-adhesive substrate for these developing corneal cells.
Area of Science:
- Developmental Biology
- Cell Biology
- Ophthalmology
Background:
- Corneal development involves neural crest cell migration and differentiation.
- Extracellular matrix interactions are crucial for cell orientation and development.
- Beta(1) integrins mediate cell adhesion and signaling.
Purpose of the Study:
- To investigate the role of beta(1) integrins in periocular mesenchyme cell (POM) and corneal fibroblast (CF) interactions with fibronectin and tenascin.
- To understand how perturbing beta(1) integrin function affects cell attachment and spreading on different extracellular matrices.
Main Methods:
- Utilized antibody perturbation (CSAT) to inhibit beta(1) integrin function.
- Employed anti-sense retroviral constructs to reduce endogenous beta(1) integrin levels.
- Assessed cell attachment and spreading on fibronectin and tenascin substrates.
- Examined beta(1) integrin localization in focal adhesions via immunodetection.
Main Results:
- POM and CF exhibited significantly greater attachment and spreading on fibronectin compared to tenascin.
- CSAT antibody treatment reduced cell attachment and spreading, and diminished beta(1) integrin in focal adhesions on fibronectin.
- Reduced beta(1) integrin levels led to decreased attachment, spreading, and disorganized focal adhesions.
- Few beta(1) positive focal adhesions were observed on tenascin.
Conclusions:
- In vitro, beta(1) integrins on POM and CF interact with fibronectin, promoting cell attachment and spreading.
- Tenascin functions as an anti-adhesive matrix for these corneal cells.
- Further in vivo studies are needed to confirm these extracellular matrix interactions during corneal development.