FIGURE 5.

Reinforcement of αvβ3 integrin survival and proliferation signaling by TIMP-1. A, TIMP-1 was co-immunoprecipitated (IP) with αvβ3 integrin in a TIMP-1 level-dependent manner. The heavily glycosylated TIMP-1 interacted more readily with the integrin, and the aglycosylated form of TIMP-1 did not bind to the integrin dimer. WB, Western blot. B, cells were treated with TNF-α and IL-2 as for Fig. 4, and the activation of caspase-8 and ERK was monitored. C, cells were treated with TNF-α and IL-2, and the proliferation signaling that is mediated by αvβ3 integrin and the downstream ERK activation was investigated, including cyclin D1 and cyclin A expression. D, incubated with 50 ng/ml TNF-α and 200 ng/ml IL-2 for 48 h, cells were then pulsed with BrdU for 1 h prior to analysis by FACS. Due to the aneuploidy of the cells, a gate in the region below 500 on the x axis was chosen, and thus the percentages do not total to 100%. E, cells were incubated in the presence or absence of the neutralizing TIMP-1 antibody at a concentration of 10 μg/ml. Effects of the TIMP-1 antibody on G₁/S transition were tested by a BrdU incorporation assay. Change in cell proliferation was also investigated following treatment with the cytokines. Results were obtained from three independent experiments. * and #, p < 0.05. Error bars, S.E.