Fibronectin also inhibits macrophage proliferation through p27^Kip1 expression.

(A) Fibronectin induces p27^Kip1 but not p21^Waf1expression. The expression of these components of the cell cycle machinery was analyzed by Western blotting. Macrophages were cultured on plates precoated with 10 μg/mL decorin or fibronectin for 6 or 24 hours. Then, 100 μg total protein was loaded per lane. The expression of β-actin was used as a sample loading and transfer efficiency control. This is the result of 3 independent experiments. (B) Decorin and fibronectin elongate the activation of ERK induced by M-CSF. Quiescent macrophages were grown on plates precoated with 10 μg/mL fibronectin or 10 μg/mL decorin and were then treated with or without 1000 U/mL M-CSF for the indicated times. ERK activity was determined by in-gel kinase assay. (C) Fibronectin also inhibits MKP-1 expression induced by M-CSF. Quiescent macrophages cultured on plates precoated with BSA or 10 μg/mL fibronectin were stimulated with M-CSF for 30 minutes. The expression of MKP-1 was analyzed by Northern blotting. The levels of the 18S rRNA transcript were used as a loading and transfer control. (D) Fibronectin did not inhibit M-CSF–dependent proliferation of BMDMs from p27^Kip1 knock-out mice. After 7 days of culture, a total of 10⁵ macrophages from wild-type, p27^Kip1, or p21^Waf1 knock-out mice were cultured for 24 hours on plates precoated with BSA or 10 μg/mL fibronectin in the presence of 1000 U/mL M-CSF. Proliferation was determined by [³H]-thymidine incorporation. Each determination was made in triplicate, and the values represented correspond to the mean ± SD of 2 independent experiments.