MDA-MB231 breast cancer cells were cultured in duplicate 6-well plates in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum to 80% confluence. To test the effect of an acidic pH on lysosome distribution and cath D routing, cells were transferred on glass coverslips and medium was changed to a 50 mM HEPES-buffered Ringer’s solution with 70 mM Na acetate as described (11). To prepare conditioned media during longer time of treatment, the cells were transferred in duplicate in 6-well plates in DMEM solution as described (12). Briefly, the culture medium was DMEM without FCS, with Hank’s balanced salt solution. It was adjusted to pH 6.6 or 7.4 using HCO₃/HCl according to the equation [HCO₃] = (1.52 mM of CO₂) × [10 (pH 6.24)]. In experiments with pepstatin A (50 or 100 μM), this aspartyl protease inhibitor was dissolved in DMSO and its effect was compared to control media containing the same amount of DMSO alone. The pH of conditioned media measured before addition to the cells and at the end of incubation were controlled to be constant. Under these conditions, MDA-MB231 cells were viable for at least 4 days. Cell number was evaluated by DNA assay using the DABA colorimetric method (13).

Media conditioned by these cells during increasing periods of time were normalised to the number of cells (determined by DNA assay), and centrifuged to remove cell debris. The supernatant was then layered on a PAGE for zymography or western blot analysis or were assayed for tPA activity and for tPA and PAI-1 antigen concentrations.

PA activity was analysed after separation by electrophoresis in 10% polyacrylamide SDS gel copolymerized with 1 mg/ml of casein (Sigma, St. Louis, MO, USA) and 30 μg/ml human plasminogen (Sigma) under non-reducing conditions (without mercaptoethanol) and in the absence of serine protease inhibitors, as described (14,15). The caseinolytic bands were revealed and quantified by scanning. Molecular weight markers (Amersham, Piscataway, NJ, USA) were run in parallel.

PA activity in conditioned media was determined in triplicate using the human PA chromogenic enzymatic kit (Assay pro ref CT1001, AssaySense, St. Charles, MO, USA) according to instructions of this laboratory. This assay was not specific for tPA or uPA. Briefly, plasmin produced from plasminogen activation was quantified using a specific plasmin substrate releasing para-nitroaniline, a yellow chromophore. Amiloride (1 mM), which inhibits uPA activity, was used to discriminate tPA activities (16). The plate was incubated at 37°C in a humid incubator for increasing periods of time. The optical density at 405 nm determined using a microplate spectrophotometer MRX (Dynatech Laboratories, Chantilly, VA, USA) and checked to be proportional to PA activities.

The 3-day conditioned media were prepared from MDA-MB231 cells as described above, supplemented with protease inhibitors (Complete, Roche Diagnostics, Meylan, France), and 5 mM mercaptoethanol and concentrated 20-fold using 10K Amicon Ultra-0.5 ml (Millipore, Molsheim, France). A total of 10 μl of concentrated media were analysed on 7.5% SDS-PAGE and transferred to nitrocellulose as described (17). Membranes were probed with a rabbit polyclonal antibody to human tPA (ab28219 from Abcam, Paris, France) with 1:1,000 dilution. After washing, membranes were incubated with peroxidaseconjugated secondary antibody (dilution 1:5,000; Amersham) and revealed by bioluminescence with the ECL detection system (Amersham). The apparent molecular weights were estimated with pre-stained standard proteins (Bio-Rad, Marnes-la-Coquette, France) run in parallel. Intracellular protein amounts were quantified by the Bradford method.

The Imubind tPA and PAI-1 ELISA kits (American Diagnostica GmbH, Pfungstadt, Germany) were used according to manufacturer’s instructions. Briefly, tPA or PAI-1 antigens were retained on a goat polyclonal antibody anti-tPA or a mouse monoclonal anti-PAI-1 adsorbed on the microtiter plate and quantified by a second peroxidase-conjugated polyclonal anti-tPA or anti-PAI-1 antibody. The concentrations of tPA and PAI-1 in the samples were determined by plotting the values of peroxidase activities to a standard curve obtained from purified antigens.

Recombinant active PAI-1 (∼43 kDa) and cath D from human liver (∼34 kDa) were purchased from Sigma-Aldrich (Lyon, France). The ability of cath D to cleave PAI-1 was investigated with an enzyme:substrate ratio of 1:5 in a 100 μM sodium acetate buffer at 37°C at pH 6.0, 5.2 or 4.0 as previously described (23). Inhibition of this digestion was performed with pepstatin at 0.1 μM. After the indicated time of incubation, the reaction was stopped by freezing. Samples were then boiled in electrophoresis buffer for 3 min, and fragments of PAI-1 were separated on a 12% polyacrylamide SDS-PAGE. The SDS-PAGE was stained with Brilliant Blue.

MDA-MB231 cells were cultured on glass coverslips at pH 7.4 or 6.6 for 3 h. They were then fixed with paraformaldehyde and glutaraldehyde and permeabilized by Triton X-100. Lysosomes vesicles were stained using the anti-cath D M1G8 mAb (18), the cath D swine polyclonal antibodies (kindly provided by Dr M. Fusek) or the anti-LAMP1 antibody (Abcam). The M1G8 mAb used (Figs. 1 and 2) recognises all forms of cath D, while the M2E8 mAb is specific of the pro-enzyme. These antibodies have been characterized previously (18,19 and the references within).

MDA-MB231 breast cancer cells were plated in 12-well plates to reach 30% confluence and then incubated in the FCS free media at pH 7.4 or 6.6 for up to 3 days. Their growth was decreased by 35% at pH 6.6 compared to pH 7.4, but not altered by pepstatin A, at either pH value (results not shown). We have also verified that cells plated at 80% confluence, remained quiescent without cell lysis at pH 6.6 for at least 36 h allowing the preparation of conditioned media for secreted proteins analysis (data not shown).

As shown on Fig. 1, the lysosomes stained by different cath D antibodies or Lamp1 antibodies, were delocalised at the cell periphery by medium acidification from pH 7.4 (30% at the cell periphery) to pH 6.6 (70% at periphery). The effect was progressive from pH 7.4 to 6.3 and was optimal at pH 6.6 which was chosen for further experiments. This peripheral location of lysosomes at pH 6.6 was rapid, within 7 min and stable for 3 days (Fig. 2). It was rapidly reversible at pH 7.4 indicating that the cells were still viable (data not shown).

The conditioned media of MDA-MB231 cells, collected after 1 to 3 days of culture at pH 7.4 or 6.6, were analysed for PA activities on a non-reducing SDS-PAGE containing plasminogen and casein as described in Materials and methods. In acidic and neutral media the same caseinolytic large bands were observed migrating with apparent molecular weight of 110–130, 63–75 and 45 kDa. However, when cells had been treated with pepstatin, the caseinolytic bands of higher molecular weight (63–75 kDa) were decreased in acidic conditions but 45 kDa bands were unaffected (Fig. 3). The molecular weights of the cath D stimulated bands were in agreement with those of the unbound tPA (63–75 kDa) and of a PA/PAI-1 complex (110–130 kDa) (16,20,21). The absence of caseinolytic bands in PAGE analysis performed in the same experiments without plasminogen (Fig. 3A, lanes 9 and 10) or without conditioned media showed an effect due to the activation of plasminogen rather than the activation of other proteases secreted or present in the gel. The 45 kDa band (Fig. 3) corresponded to uPA on the basis of its molecular weight and specific sensitivity to amiloride (16,22) as shown in Fig. 4. This band was not modified by pepstatin treatment suggesting that uPA activity was not stimulated by cath D activity (Figs. 3 and 4). Quantification by scanning of lanes 1 to 4 of the zymograph (Fig. 3A) showed that after 3 days of conditioning, the activity of the different molecular weight PA forms, was decreased at the acidic pH (Fig. 3B). This decreased PA-induced caseinolytic activity at pH 6.6 compared to pH 7.4 was reproducibly observed in independent experiments (Fig. 5) and was associated with a decrease of the secreted cath D (see previous section). Fig. 3B also shows that pepstatin decreased specifically the 75 to 63 kDa activities at pH 6.6 but not at pH 7.4 while the uPA activities were not altered. The effect of pepstatin on the 63–75 kDa lytic bands was slow and optimal after 3 days of treatment (Fig. 5).

The effect of pepstatin on the PA proteolytic activity of conditioned media was also evaluated by a PA colorimetric assay. After 3 days of culture at pH 6.6, the total PA activity and the amiloride resistant tPA activity were partly inhibited by pepstatin (Table I, compare lanes a and b).

In order to confirm that the effect of cath D was not due to an increase of the secreted tPA, we estimated its concentration using western immunoblot analysis. As shown in Fig. 6A, the human tPA present as a large 75 kDa band appeared unaffected in the presence of pepstatin at pH 6.6. Excluding a non-specific 55 kDa band also revealed with the secondary antibodies alone (lanes 4–6), the tPA antibodies recognised at pH 7.4 a 75 kDa band corresponding to tPA and a predominant 110 kDa band corresponding to tPA/PAI-1 inhibitor complexes (lane 1) as previously described (20,21). At pH 6.6 the amount of the secreted tPA was decreased compared to pH 7.4, but pepstatin had no effect on the level of the 75 kDa band (Fig. 6). While the 110 kDa entity was predominant at pH 7.4, (lane 1) it was not seen at pH 6.6 (lanes 2 and 3) which is consistent with a dissociation of the tPA/PAI-1 inhibitor complex facilitated at an acidic pH as shown previously (14,15,25). The contrast between the larger lytic bands (75 to 63 kDa) of the zymography experiments, compared to the distinct 75 kDa bands of the western blot analysis, might be due to the absence of added serine protease inhibitors and mercaptoethanol in the zymograpy experiments.

These data suggest that the mechanism of the cath D activation of tPA was probably indirect and due to the degradation of a tPA inhibitor by cath D. We demonstrate that pepstatin markedly increased PAI-1 concentration in the same conditioned media (Table I, line 4). PAI-1 degradation by cath D has been previously described in monocytes at a more acidic condition (23). We complete these data in Fig. 7 by finding that cath D slowly degraded pure PAI-1 even at pH 6.0 in a cell free system. Collectively, these data indicate that cath D increased the activity of secreted plasminogen activators (PA) at acidic pH and decreased in the same conditions the level of PAI-1.