Cell Attachment Activity of Cementum Proteins and Mechanism of Endotoxin Inhibition

Abstract

Cementum occupies a unique anatomical location where soft connective tissues of the periodontium are attached to root surfaces. Cell attachment properties of proteins present in cementum were studied. Human and bovine cementum were extracted with 0.5 mol/L CH3COOH followed by 4 mol/L guanidine, and proteins were separated by ion-exchange chromatography and SDS-polyacrylamide gel electrophoresis. Cells were labeled with radioactive amino acids and added to tissue-culture plastic plates incubated with cementum proteins, and attachment was measured. Results showed that cementum proteins promoted the attachment of smooth muscle cells, endothelial cells, and fibroblasts, but not epithelial cells. Fibroblasts attached more efficiently than other cell types, and they manifested spreading with re-organization of actin filaments. No attachment occurred to plates incubated with endotoxin from A. actinomycetemcomitans. Fewer fibroblasts attached to plates treated with cementum proteins in the presence of endotoxin, but cells pre-treated with endotoxin attached normally. Attachment was not inhibited when plates were incubated first with attachment proteins and then with endotoxin; however, it was decreased when endotoxin or bovine serum albumin preceded cementum proteins. Cementum proteins with Mr 68,000, 61,000, 55,000, and 36,000 (p68, p61, p55, and p36, respectively) manifested attachment activity, while protein(s) with Mr 23,000-24,000 did not. Western blots revealed that guanidine extracts contained three bands cross-reacting with anti-bovine sialoprotein-II antibody, but the p61, p55, and p36 were negative. We conclude that cementum contains bovine sialoprotein-II and at least four other fibroblast attachment proteins, and that they do not support epithelial cell attachment.(ABSTRACT TRUNCATED AT 250 WORDS)

Signaling Reactions Induced in Human Fibroblasts During Adhesion to Cementum-Derived Attachment Protein

“…(24,25) The following antibodies were used: monoclonal antibodies against phosphotyrosine (PY-20; Transduction Laboratory, Lexington, KY, U.S.A.) and FAK (2 A7, Upstate Biotechnology Inc., Lake Placid, NY, U.S.A.), polyclonal antibodies against FAK (C-903), Cas (C-20; Santa Cruz Biochemistry, Santa Cruz, CA, U.S.A.), and …”

“…(26) The CAP promotes cell adhesion at least as well as FN, and among the periodontal cells, alveolar bone cells and fibroblast subpopulations bind and adhere to CAP-coated surfaces with different affinities. (24,27) In contrast, epithelial cells do not adhere to CAP. (24) The CAP binding to periodontal fibroblast populations appears to be related to their ability to differentiate into osteoblastic phenotype.…”

“…(24,27) In contrast, epithelial cells do not adhere to CAP. (24) The CAP binding to periodontal fibroblast populations appears to be related to their ability to differentiate into osteoblastic phenotype. (28) To understand the molecular basis for the biological functions mediated by the CAP, we have studied the signaling events transduced by this protein during cell adhesion.…”

“…(22,23) We recently reported that the cementum, which is the calcified outer layer of teeth through which collagen fibers of connective tissues are anchored onto tooth root surfaces, contains a M r 56,000 collagenous protein, which promotes the adhesion and spreading of mesenchymal cell types. (24,25) The distribution of this protein, cementumderived attachment protein (CAP), appears to be limited to cementum. (26) The CAP promotes cell adhesion at least as well as FN, and among the periodontal cells, alveolar bone cells and fibroblast subpopulations bind and adhere to CAP-coated surfaces with different affinities.…”

Cementum and Periodontal Wound Healing and Regeneration

“…In bovine tooth germs, the CAP is expressed by cementoblasts, and in cementum its expression pattern is different from that of type I collagen (Saito et al, 2001). The CAP promotes the adhesion and spreading of mesenchymal cells; however, it promotes the adhesion of mineralized-tissue-forming cells preferentially (Olson et al, 1991;Pitaru et al, 1995).…”