Periodontal-derived cells attach to cementum attachment protein via alpha 5 beta 1 integrin.

Abstract

A specific collagenous cementum attachment protein (CAP) has been identified in human cementum which promotes selective cell migration towards and attachment of various periodontal derived cell populations to root surfaces in vitro. The CAP is known to support attachment of periodontal-derived cell via an RGD motif, which suggests an integrin-mediated mode of attachment. The purpose of the present study was to ascertain which integrin(s) are involved in the attachment of periodontal-derived cells to CAP. The integrins examined comprised subunits of the major receptors for fibronectin (alpha 5) and collagen (alpha 2, alpha 3), as well as the common beta 1 subunit which is present in many extracellular matrix receptors. The wells of 48-well non-tissue culture treated plates were coated with CAP (2 micrograms/ml). For negative and positive controls the wells were coated with bovine serum albumin and fibronectin (5 micrograms/ml), respectively. Human gingival fibroblasts and periodontal ligament fibroblasts were labeled with [3H]-proline, incubated with anti-integrin antibodies and added to the precoated wells. Attachment was assessed after incubating the cells for 1 h at 37 degrees C in the presence of the antibodies. Antibodies to alpha 5 and beta 1 inhibited the attachment of both human gingival fibroblasts and human periodontal ligament fibroblasts to CAP, while anti alpha 2 and alpha 3 antibodies did not affect the attachment. The binding of the fibroblasts to fibronectin was also inhibited by anti-alpha 5 and beta 1 antibodies, both of which are components of the "classical" fibronectin receptor and remained unaffected by the addition of anti-alpha 2 and alpha 3 antibodies. Proteins migrating in SDS-polyacrylamide gels in positions similar to the alpha 5 and beta 1 integrin subunits were present in fractions bound to a column of CAP coupled to Sepharose CL-4B. These results indicate that the attachment to CAP of the periodontal-derived cells, human gingival fibroblasts and human periodontal ligament fibroblasts, is mediated primarily via the integrin alpha 5 beta 1.