Abstract
An intact Smad3 gene product is critical for a functioning signal transduction pathway following TGF binding to the TGF-β receptor. We have previously established Smad3−/− and Smad3+/+ long term bone marrow cultures (LTBMCs) and isolated clonal bone marrow stromal cell lines from each. The Smad3−/− cells were smaller in size but had a faster cell doubling time (24 hours compared to 48 hours) and increased saturation density compared to +/+ cells (15.3 ± 1.0 x 105 cells/25 mm2 flask compared to 3.8 ± 0.1 x 105, p = 0.003). The plating efficiency of the lines was similar (18.3 ± 2.7 compared to 15.5 ± 1.7, p = 0.417). We transfected the Smad3−/− cell line with a retrovirus containing the Smad3 transgene, and selected a subclone expressing the transgene mRNA, designated Smad3−/−(3). Smad3−/−(3) cells were increased in size to that of Smad3+/+ cells, and showed decreased cell saturation density. Using the Cytoworks computer controlled cell tracking Bioreactor, we measured the migration of each clonal line. Tissue culture wells of 100 cells per well were followed for 5 days tracking each cell in quadruplicate wells per cell line. Smad3+/+ cells migrated significantly faster over 5 days in culture compared to Smad3−/− cells. (The average velocities were 0.62 μm/min for Smad3+/+ and 0.36 μm/min for Smad3−/−, p<0.0001). Over 5 days, the average velocities for Smad3+/+ cells were 0.51, 0.51, 0.52, 0.72, 0.91 μm/min, and for Smad3−/− cells were 0.28, 0.38, 0.41, 0.37, 0.35 μm/min. The 5 p-values comparing these cell lines were all <0.0001. The 7 day clonagenic irradiation survival curve showed that Smad3+/+ and Smad3−/−(3) cells were significantly more sensitive (D0 = 1.75 ± 0.03 and 1.51 ± 0.07 Gy, respectively) compared to the Smad3−/− cell line (D0 = 2.43 ± 0.06 Gy, p=0.0016 and 0.0103). These results demonstrate a concordance of radioresistance and decreased migratory capacity in bone marrow stromal cells devoid of a functioning Smad3 gene product, and restoration of both properties following overexpression of the transgene product. These data may help explain the decreased radiation fibrosis observed in Smad3−/− mice.
Expression of the c-kitligand and interleukin 6 genes in mouse bone marrow stromal cell lines
