Decorin inhibits macrophage proliferation through p27^Kip1 expression.

(A) Decorin induces the expression of both p21^Waf1 and p27^Kip1. The expression of p21^Waf1 and p27^Kip1 after treatment with decorin was analyzed by Western blotting. Macrophages were cultured in 100 μg/mL decorin precoated surface for the indicated times or for 24 hours at the indicated concentrations of decorin. Then 100 μg total protein was loaded per lane. Expression of p21^Waf1 and p27^Kip1 was analyzed using monoclonal antibodies as described in “Materials and methods.” The expression of β-actin was used as a control of sample loading and transfer efficiency. This is representative of 4 independent experiments. (B) Decorin inhibits cdk-2 activity. The cdk-2 activity induced by M-CSF in macrophages cultured on plates precoated with BSA or decorin was measured as histone H1 phosphorylation in vitro at the indicated times after M-CSF stimulation. (C) Decorin 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 in BSA or 10 μg/mL precoated plates in the presence of 1000 U/mL of 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.