All ETDs from UAB

Advisory Committee Chair

Joanne E Murphy-Ullrich

Advisory Committee Members

Anne Woods

Janusz Kabarowski

Elizabeth Sztul

Dennis Kucik

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Thrombospondin-1 (TSP1) is a multifunctional matricellular protein released by platelets in response to injury and secreted by cells under stress. TSP1 is cleaved into functional N- and C-terminal domain fragments, which have paradoxical actions with respect to angiogenesis, cell survival, and cell adhesion. Wound healing studies by others demonstrated that TSP1 knockout mice have delayed rates of wound closure, which was attributed to actions of the C-terminal domain. We wished to investigate the specific role of the N-terminal domain in tissue remodeling. Previously, we established that a sequence (aa 17-35) in the N-terminal domain of TSP1 induces focal adhesion disassembly, stimulates cell migration, and prevents anoikis. These actions occur when TSP1 binds to cell surface calreticulin (CRT) associated with LDL receptor-related protein 1 (LRP1). To determine the role of TSP1 signaling through the CRT/LRP1 co-complex in tissue remodeling, we utilized a model of the foreign body response. Surgical sponges filled with collagen and a plasmid encoding the TSP1 signal peptide, followed by the CRT-binding sequence tagged with enhanced green fluorescent protein [NTD (1-35)-EGFP] were implanted subcutaneously in mice. Cells responding to the insult of implantation ingest the collagen-plasmid mixture, initiating localized transfection. A plasmid encoding two amino acid substitutions within the CRT-binding sequence was used as an inactive control [NTD mod (1-35)-EGFP]. EGFP expression in the sponge implants from the transfection of invading cells was confirmed over days 5-21. Unexpectedly, mice with induced expression of NTD (1-35)-EFGP showed an accelerated and highly organized collagen encapsulation of the implant. Therefore, human dermal fibroblasts were treated with the TSP1, a recombinant NTD, or the TSP1-CRT binding sequence to determine whether TSP1 directly regulates collagen. TSP1 and the TSP1-CRT binding sequence stimulated increased expression of fibrillar collagens, collagen deposition into the ECM, and levels of collagen I and III mRNA. TSP1 stimulation of collagen was blocked by a peptide (CRT19-36) that inhibits TSP binding to CRT. Furthermore, the increase in collagen signaled by the TSP1-CRT binding sequence was independent of TGFß-1 activation and Smad-2 phosphorylation. These results demonstrate a novel role for the N-terminal domain of TSP1 in tissue remodeling and collagen regulation.

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