The collagen fibers that span the periodontal ligament (PDL) connect teeth to the bone socket by weaving through the cementum of each tooth and into the alveolar bone. PDL has high rates of extracellular matrix turnover, as compared to other collagen rich tissues, characterized by procollagen synthesis, processing, and deposition in addition to uptake of insoluble collagen. Thus PDL provides an excellent tissue milieu for investigating mechanisms of procollagen processing. SPARC, a collagen-binding protein, has been identified as a key factor in collagen ECM deposition. Accordingly, we reported that SPARC-null mice had significantly less collagen in PDL as compared to age-matched wild type (WT) PDL. Interestingly, the PDL collagen fibers in SPARC-null mice were also significantly thinner than WT PDL fibers. When challenged with lipopolysaccharide to induce periodontal disease, SPARC-null mice had more severe disease as measured by loss of alveolar bone and PDL collagen. To address cellular mechanisms of decreased homeostatic collagen content and response to disease in the absence of SPARC, we cultured PDL fibroblasts and used a novel organ culture model. Our preliminary results demonstrated, both in vitro and ex vivo, that SPARC-null PDL fibroblasts had a deficiency in procollagen processing, as indicated by an accumulation of procollagen in null cells and tissue that was not evident in WT conditions. Furthermore, increases in procollagen were found associated with cell surfaces in the absence of SPARC. These results suggested that SPARC acts to decrease collagen interaction with cell surface receptors and that collagen engagement by cell surface receptors hampered efficient procollagen processing.
These Specific Aims will test the hypothesis that SPARC mediates collagen homeostasis in PDL fibroblasts by competing with collagen receptor engagement, an activity required for efficient procollagen processing and subsequent collagen fibril incorporation. Mutant SPARC lacking collagen binding will be transfected into SPARC-null PDL fibroblasts to determine whether the collagen binding capacity of SPARC is required to modulate procollagen processing. In addition, adenovirus constructs will be used to knockdown two collagen cellular receptors known to share collagen binding sites with SPARC, integrin 2 and Discoidin Domain Receptor (DDR) 2, in both WT and SPARC-null PDL. We predict that knockdown of integrin 2 and DDR2 will promote procollagen processing in WT and SPARC-null PDL, thereby decreasing levels of procollagen and increasing levels of fully mature collagen 1. Furthermore, we will investigate the outcome of DDR2 and integrin 2 receptors knockdown on collagen fibril assembly using our ex vivo model. Thus, this project will determine the role of SPARC, DDR2, and integrin 2 on procollagen processing and collagen fibril formation. The training plan proposed here will test the above hypothesis and prepare me for a career as an academic scientist.

Public Health Relevance

This project focuses on the mechanism of SPARC in procollagen processing and fibril assembly in the periodontal ligament. As SPARC is highly expressed in fibrotic tissues, understanding the mechanism(s) of SPARC action could identify potential targets for therapeutic treatment of fibrosis as well as in periodontal ligament repair, and ideally, regeneration of this important tissue.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30DE023009-02
Application #
8598808
Study Section
NIDCR Special Grants Review Committee (DSR)
Program Officer
Frieden, Leslie A
Project Start
2012-07-05
Project End
2014-07-04
Budget Start
2013-07-05
Budget End
2014-07-04
Support Year
2
Fiscal Year
2013
Total Cost
$48,032
Indirect Cost
Name
Medical University of South Carolina
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
Trombetta-eSilva, Jessica; Rosset, Emilie A; Hepfer, R Glenn et al. (2015) Decreased Mechanical Strength and Collagen Content in SPARC-Null Periodontal Ligament Is Reversed by Inhibition of Transglutaminase Activity. J Bone Miner Res 30:1914-24
Trombetta-eSilva, Jessica; Eadie, Erik P; Zhang, Yuhua et al. (2013) The effects of age and the expression of SPARC on extracellular matrix production by cardiac fibroblasts in 3-D cultures. PLoS One 8:e79715
Trombetta-Esilva, Jessica; Bradshaw, Amy D (2012) The Function of SPARC as a Mediator of Fibrosis. Open Rheumatol J 6:146-55