α4β1 integrin–induced MMP-9 up-regulation involves the PI3-K/Akt signaling pathway. (A) B-CLL cells from 3 different patients were incubated or not with either 30 nM wortmannin (Wmn) or 5 μM of the following: Bis I, SB203580 (SB), PP2, and UO126 for 1 hour at 37°C, and added to FN-H89–coated wells. Cells untreated or treated with 30 nM Wmn were also added to BSA-coated wells. After 24 hours, the conditioned media were concentrated and analyzed by gelatin zymography, and MMP-9 secretion was quantitated. Basal levels of MMP-9 on BSA were normalized to 100, and values represent arbitrary units. (B) B-CLL cells from one patient were preincubated for 1 hour with the indicated concentrations of wortmannin, LY294002 (LY), or API-2, and added to FN-H89–coated wells. After 24 hours, the conditioned media were analyzed by gelatin zymography. Values represent the average of duplicate determinations. (C) B-CLL cells (2 × 10⁶) from 2 patients were incubated on 0.5% BSA or 10 μg/mL FN-H89 in the absence or presence of wortmannin or API-2. Cells were lysed after 24 hours and phosphorylated, and total Akt and ERK were analyzed by Western blotting using specific Abs. (D) The same lysates used in panel C were also analyzed by Western blotting using specific Abs to IκB-α. Relative IκB-α levels were quantitated and values represent the average of the 2 samples studied. IκB-α levels of cells on BSA were normalized to 1. (E) B-CLL cells (2 × 10⁶) from one patient were incubated on 0.5% BSA or 10 μg/mL FN-H89, and at the indicated times phospho-Akt and phospho-ERK were analyzed as explained. Relative P-Akt and P-ERK levels were quantitated and constitutive levels were normalized to 1. Error bars indicate standard deviation. * P < .05; ** P < .01.