Abstract
The contraction of floating collagen gels is suggested to mimic the reorganization of collagenous matrix during development and tissue healing. Here, we have studied two osteogenic cell lines, namely MG-63 and HOS, and a chemically transformed subclone of HOS cells, HOS-MNNG. Transforming growth factor-beta (TGF-beta), a putative regulator of bone fracture healing, increased collagen gel contraction by MG-63 and HOS-MNNG, but not by HOS cells. Our data show that TGF-beta-induced fibronectin synthesis is not sufficient for the process. Instead, anti-beta 1 integrin antibodies could prevent the contraction. There are three different integrin heterodimers that are known to mediate the cell-collagen interaction, namely alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1. In MG-63 cells TGF-beta increased the expression of alpha 2 beta 1 integrin and decreased the expression of alpha 3 beta 1 integrin, whereas alpha 1 beta 1 integrin is not expressed. HOS cells had no alpha 2 beta 1 integrin, neither did TGF-beta induce its expression. However, HOS-MNNG cells expressed more alpha 2 beta 1 integrin when treated with TGF-beta. Thus, we suggest that the mechanism of the enhanced collagen gel contraction by TGF-beta is the increased expression of alpha 2 beta 1 integrin heterodimer. To further test this hypothesis, we expressed a full-length alpha 2 integrin cDNA in HOS cells and in MG-63 cells. We obtained HOS cell clones that expressed alpha 2 beta 1 heterodimer, and the ability of these cells to contract collagen gels was greatly enhanced. Furthermore, the contraction by MG-63 cells transfected with alpha 2 integrin cDNA was enhanced, and the contraction by cells transfected with antisense oriented alpha 2 integrin cDNA was decreased. Thus, both in MG-63 and HOS cells the increased alpha 2 integrin expression alone was sufficient for the enhanced contraction of collagen gels. Furthermore, the amount of alpha 2 integrin is critical for the process, and its decrease leads to diminished ability to contract gels.
Highlights
From the Department of Medical Biochemistry and the MediCity Research Laboratory, University of Turhu, FIN-20520 Turku, Finland
The contraction by MG-63 cells transfected with 0'2 integrin eDNA was enhanced, and the contraction by cells transfected with antisense oriented 0'2 integrin eDNA was decreased
Type I collagen is the major component of bone matrix, proposing its importance for osteogenic cells
Summary
Cell Cultures-Human osteosarcoma cells used were MG-63, HOS, and HOS-MNNG (HaS cells transformed with N-methyl-N' -nitro-Nnitrosoguanidine, tumorigenic) all from American Type Culture Collection. Immunocomplexes were recovered by binding to protein A-sepharose and washing the beads four times with 25 mM Tris-buffered isotonic saline (pH 7.4) containing 0.5% Triton X-100 and 1 mg/ml bovine serum albumin and twice with 0.5 M NaCI and 25 mM Tris-HCI (pH 7.4). An equal volume of medium and phosphate-buffered isotonic saline containing 100 roM n-octyl-/3-D-glucopyranoside were precleaned with packed protein A-sepharose and incubated with polyclonal antiserum against human plasma fibronectin (Chen et at., 1986) at 4°C for 12 h. Statistical Analysis-The statistical significance of differences in cell adhesion and in collagen gel contraction assays was assessed with general mixed model analysis of variance (BMDPlDynamic, 7.0, BMDP Statistical Software Inc., Cork, Ireland), when multiple independent experiments were analyzed (random factor being the experiment and fixed factor being the treatment). Experiments with two groups were analyzed with Student's t test for independent samples
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