Thrombospondin-1 promotes fibroblast-mediated collagen gel contraction caused by activation of latent transforming growth factor beta-1.
BACKGROUND
Grafting of cultured epithelium has become a useful technique for the treatment of epithelial defects, since grafted epithelial cells secrete factors promoting wound healing. We identified one such factor produced by cultured oral epithelial cells as thrombospondin-1 (TSP-1). Recently, TSP-1 was reported to act as an activator of transforming growth factor-beta1 (TGF-beta1).


OBJECTIVE
The role of TSP-1 in wound healing and its mechanism were investigated in vitro and in vivo.


METHODS
The cultured oral epithelial cell-conditioned medium was harvested and applied to Heparin-Sepharose affinity chromatography. Proteins were analyzed by N-Terminal sequencer. TSP-1 and the other factors were applied to fibroblasts-mediated collagen gel contraction assay. The amount of TGF-beta1 (latent TGF-beta1 (LTGF) and active TGF-beta1) in collagen gels was quantified by ELISA and Western blotting analysis. Collagen sponges were soaked with TSP-1 and implanted subcutaneously into rats.


RESULTS
A 38 kDa protein secreted from cultured oral epithelial cells was found to be human TSP-1. TSP-1 promoted collagen gel contraction activity, and anti-human TSP-1 and TGF-beta1 antibody inhibited the activity. The diameters of the gels treated with LTGF and TSP-1 were reduced to a greater extent than those of gels treated with either factor alone. Although there were no significant differences in the amounts of total TGF-beta1, which include LTGF, the amount of 25 kDa TGF-beta1 was 3.30-fold greater in TSP-1-treated samples than controls. In vivo, 7 days after implantation, increased numbers of fibroblasts were observed in the sponges treated with TSP-1.


CONCLUSIONS
These findings suggested that TSP-1 causes collagen gel contraction by activation of LTGF. TSP-1 is expected to be especially suitable for regulating wound healing.
Sakai
Kensuke
K
Department of Oral and Maxillofacial Surgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, 466-8550, Nagoya, Japan.
Sumi
Yukio
Y
Muramatsu
Hisako
H
Hata
Ken-ichiro
K
Muramatsu
Takashi
T
Ueda
Minoru
M
D000293:Adolescent; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D005782:Gels; D006801:Humans; D051381:Rats; D012867:Skin / Q000166:cytology; D015503:Surgical Sponges; D019700:Thrombospondin 1 / Q000302:isolation & purification / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D014945:Wound Healing / Q000187:drug effects*
D005782:Gels; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D019700:Thrombospondin 1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D003094:Collagen