Regeneration of the periodontal tissues lost due to periodontal disease involves complex interactions of cells and extracellular matrix (ECM) molecules. Tenascin and fibronectin are two examples of the ECM molecules involved in cell-matrix interactions during biologic events such as embryonic development and of wound healing. The proposed studies are aimed at analyzing the interactions cells derived from periodontium with one extracellular matrix molecule -Tenascin- which is known to be prominently expressed during tooth development, in the adult periodontal ligament and, appears to play important roles in wound healing/tissue regeneration. The proposed studies will enable us to analyze the biologic functions of tenascin and map the location of active sites within the various domains of this large molecule.
In specific aim 1, we propose to prepare recombinant proteins expressed in E.coli, corresponding to specific fragments of the tenascin subunit. The recombinant proteins, carefully designed to begin and end at the junctions between independently folding domains, will span the entire length of the tenascin subunit. Three well known activities will be tested initially: a weak cell adhesion to tenascin-coated substrates, demonstrated by a centrifugal assay; an """"""""anti-adhesion"""""""" effect, in which soluble tenascin induces cells to shed their focal adhesions and; a down-regulating effect on total protein synthesis, which we have recently demonstrated for soluble tenascin. In addition, effects on cell proliferation and migration will be evaluated. Normal adult periodontal cells and embryonic and transformed cell lines will be used. This will provide us a low resolution map of the functional activities to different regions of the tenascin subunit.
In specific aim 2, we will obtain a high resolution map of the functional domains of the tenascin subunit eventually identifying small peptide sequences that mimic or inhibit interaction of tenascin with specific cell receptors. The paradigm of these studies is the mapping of the major fibronectin cell attachment to a single. FN-Type III domain and eventually to the sequence RGD. Here, in addition to high resolution mapping, the contributions of neighboring-domains to all active domains of tenascin will be studied. PCR techniques, site-directed mutations and synthetic peptides will be used to accomplish the studies in specific aim 2. Data from these studies will help us understand better the roles and mechanisms of the matrix glycoprotein tenascin in biologic events such as tooth development, wound healing and tissue regeneration.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE007801-05
Application #
3221571
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Project Start
1988-07-01
Project End
1995-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
5
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Dentistry
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Sahlberg, C; Aukhil, I; Thesleff, I (2001) Tenascin-C in developing mouse teeth: expression of splice variants and stimulation by TGFbeta and FGF. Eur J Oral Sci 109:114-24
Aukhil, I (2000) Biology of wound healing. Periodontol 2000 22:44-50
Hashimoto-Uoshima, M; Yan, Y Z; Schneider, G et al. (1997) The alternatively spliced domains EIIIB and EIIIA of human fibronectin affect cell adhesion and spreading. J Cell Sci 110 ( Pt 18):2271-80
Leahy, D J; Aukhil, I; Erickson, H P (1996) 2.0 A crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region. Cell 84:155-64
Aukhil, I; Sahlberg, C; Thesleff, I (1996) Basal layer of epithelium expresses tenascin mRNA during healing of incisional skin wounds. J Periodontal Res 31:105-12
Leahy, D J; Erickson, H P; Aukhil, I et al. (1994) Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine. Proteins 19:48-54
Joshi, P; Chung, C Y; Aukhil, I et al. (1993) Endothelial cells adhere to the RGD domain and the fibrinogen-like terminal knob of tenascin. J Cell Sci 106 ( Pt 1):389-400
Aukhil, I; Joshi, P; Yan, Y et al. (1993) Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression proteins. J Biol Chem 268:2542-53
Leahy, D J; Hendrickson, W A; Aukhil, I et al. (1992) Structure of a fibronectin type III domain from tenascin phased by MAD analysis of the selenomethionyl protein. Science 258:987-91
Aukhil, I (1991) Biology of tooth-cell adhesion. Dent Clin North Am 35:459-67

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