CXCL12 up-regulates MMP-9 in B-CLL cells via the ERK1/2 signaling pathway. (A) B-CLL cells (2 × 106) from 4 different patients were suspended in RPMI, 0.1% FCS and incubated on wells coated with 0.5% BSA and with or without 150 ng/mL CXCL12. Pertussis toxin (PT, 200 ng/mL) was added to some cells. After 24 hours, the conditioned media were concentrated and analyzed by gelatin zymography. Values represent the average of the 6 samples studied, and basal levels of MMP-9 without CXCL12 were normalized to 100. (B) B-CLL cells from 4 patients were incubated for 1 hour with or without (None) the indicated inhibitors (same concentrations as in Figure 3), and added to BSA-coated wells in the presence or absence of 150 ng/mL CXCL12. After 24 hours, the conditioned media were concentrated and MMP-9 was analyzed and quantitated. (C) Cells from 2 patients were incubated on 0.5% BSA in the presence of 150 ng/mL CXCL12, and in the presence or absence of pertussis toxin (PT) or UO126. Control cells were incubated in the absence of CXCL12. Phospho- and total Akt and ERK were analyzed by Western blotting. (D) B-CLL cells from the same patients used in panel C were incubated with or without 150 ng/mL CXCL12; at the indicated times, RNA was extracted and expression of c-fos mRNA was analyzed by RT-PCR using specific primers. GAPDH was also amplified as an internal control for sample loading. Values represent the average of the 2 samples studied. (E) Cells (2 × 106) from the same patient used in Figure 3E were incubated with or without 150 ng/mL CXCL12; at the indicated times, cells were lysed and lysates analyzed by Western blotting using specific Abs to phospho-Akt and phospho-ERK. Relative P-Akt and P-ERK levels were quantitated and normalized with respect to constitutive levels, which were considered 1. ** P < .01.
