Identification and functional characterization of two type VI collagen receptors, alpha 3 beta 1 integrin and NG2, during avian corneal stromal development.

Identification and functional characterization of two type VI collagen receptors, alpha 3 beta 1 integrin and NG2, during avian corneal stromal development.

Doane K J; Howell S J; Birk D E

Investigative ophthalmology & visual science

1998

1998-02

PURPOSE: The development and maintenance of extracellular matrix architecture in the corneal stroma is associated with abundant type VI collagen deposition. This collagen has been implicated in mediating both cell-matrix and matrix-matrix interactions. Although corneal fibroblasts spread extensively on this collagen, its role in corneal development has not been elucidated. METHODS: To clarify the role of this collagen, two type VI collagen receptors were studied during corneal development using immunochemical techniques: alpha 3 beta 1 integrin and an integral membrane proteoglycan, NG2. RESULTS: At embryonic day 6, these receptors were present in a diffuse pattern on cells within the cornea and juxtacorneal regions, indicating a migratory phenotype. At embryonic day 14, when the stroma is fully differentiated, alpha 3 and NG2 were localized in a punctate pattern on a subset of corneal fibroblasts, whereas beta 1 was more ubiquitously expressed. Colocalization of NG2 and type VI collagen indicated that this collagen was present and punctate in its organization was associated with NG2-positive cells. Immunochemical analyses at embryonic days 5 and 14 revealed alpha 3 and beta 1 at 155 kDa and 120 kDa, respectively, and demonstrated that these subunits were interacting to form a heterodimer. NG2 was present with a core protein of 330 kDa and an intact proteoglycan of approximately 600 kDa, and analysis of stromal lysates indicated a chondroitin sulfate-containing proteoglycan. Matrix-receptor cross-linking demonstrated the interaction of beta 1 and NG2 in periocular mesenchyme cells and corneal fibroblasts with type VI collagen, whereas only a subset of cells expressed alpha 3, indicating the presence of another beta 1 integrin. No variations between in vivo and in vitro expression of either alpha 3 beta 1 or NG2 were observed. CONCLUSIONS: These data indicate that two receptors for type VI collagen, alpha 3 beta 1 and NG2, are present during corneal stromal development, with a functional interaction of these receptors with type VI collagen. These interactions may play a role in corneal cell migration, development, and maintenance of corneal architecture.

Animals; Chick Embryo; Fibroblasts/metabolism; Cell Movement/physiology; Immunoblotting; Integrins/*metabolism; Fluorescent Antibody Technique; Cell Culture Techniques; Extracellular Matrix/metabolism; Antigens/*metabolism; Cell Polarity/physiology; Cell Size/physiology; Collagen/*metabolism; Corneal Stroma/*embryology/*metabolism; Integrin alpha3beta1; Proteoglycans/*metabolism; Receptors; Indirect; Laminin/*metabolism

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