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. 2004 Apr 1;294(2):550-8.
doi: 10.1016/j.yexcr.2003.11.023.

Extracellular alpha 6 integrin cleavage by urokinase-type plasminogen activator in human prostate cancer

Manolis C Demetriou  1 , Michael E Pennington, Raymond B Nagle, Anne E Cress
Affiliations

Extracellular alpha 6 integrin cleavage by urokinase-type plasminogen activator in human prostate cancer

Manolis C Demetriou et al. Exp Cell Res. .

Abstract

During human prostate cancer progression, the integrin alpha6beta1 (laminin receptor) is expressed on the cancer cell surface during invasion and in lymph node metastases. We previously identified a novel structural variant of the alpha6 integrin called alpha6p. This variant was produced on the cell surface and was missing the beta-barrel extracellular domain. Using several different concentrations of amiloride, aminobenzamidine and PAI-1 and the urokinase-type plasminogen activator (uPA) function-blocking antibody (3689), we showed that uPA, acting as a protease, is responsible for production of alpha6p. We also showed that addition of uPA in the culture media of cells that do not produce alpha6p, resulted in a dose-dependent alpha6p production. In contrast, the addition of uPA did not result in the cleavage of other integrins. Using alpha2-antiplasmin and plasmin depleted media, we observed that uPA cleaves the alpha6 integrin directly. Further, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) induced the production of alpha6p, and this induction was abolished by PAI-1 but not alpha2-antiplasmin. Finally, the alpha6p integrin variant was detected in invasive human prostate carcinoma tissue indicating that this is not a tissue culture phenomenon. These data, taken together, suggest that this is a novel function of uPA, that is, to remove the beta-barrel ligand-binding domain of the integrin while preserving its heterodimer association.

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Figures

Fig. 1
Fig. 1
Inhibitors and a blocking antibody of uPA reduce α6p levels in DU145H cells (A) DU145H cells were surface biotinylated and α6 and α6p were immunoprecipitated using the AA6A antibody. The samples were analyzed using streptavidin-HRP and the bands were quantified using the Scion Image software and the graph was plotted using Sigma Plot. (B) DU145H cells were treated with different concentrations of Amiloride or aminobenzamidine. The samples were analyzed for α6 and α6p integrin levels using the AA6A rabbit polyclonal antibody and α3 integrin using a rabbit polyclonal antibody (Chemicon, Temecula, CA, USA). (C) DU145H cells were treated with a uPA blocking antibody or control IgG for 3 days. The samples were analyzed for α6 and α6p integrin levels using the AA6A rabbit polyclonal antibody.
Fig. 2
Fig. 2
The production of α6p by addition of uPA in MCF10A cells is plasmin independent (A) MCF10A cells were cultured for 3 days and then treated for 90 min with different concentrations of purified uPA or DU145H conditioned media overnight. The samples were analyzed for α6 and α6p integrin levels using the AA6A rabbit polyclonal antibody. (B) DU145H cells were either untreated or treated with plasmin depleted media for 3 days. The samples were analyzed for α6 and α6p integrin levels using the AA6A rabbit polyclonal antibody. (C) DU145H cells were treated with α2-antiplasmin or PAI-1 for 3 days. The samples were analyzed for α6 and α6p integrin levels using the AA6A rabbit polyclonal antibody.
Fig. 3
Fig. 3
uPAR associates with the α6 integrin but not α6p, and uPAR signaling is not sufficient to produce α6p (A) DU145H and MCF10A cells were cultured for 3 days and immunoprecipitated with anti-uPAR antibody or with anti-α6 antibody J1B5. Samples were blotted for the α6 integrin using the AA6A-biotinylated antibody. (B) MCF10A and PC3N cells were cultured for 3 days and were then treated with 20 µg/ml uPA or 400 nM ATF for 1.5 h. The samples were analyzed for α6 and α6p integrin levels using the AA6A rabbit polyclonal antibody.
Fig. 4
Fig. 4
uPA can cleave the α6 integrin directly in vitro (A and B) Surface biotinylated proteins from MCF10A cells were retrieved by immunoprecipitation using the AA6A antibody and the washed immunoprecipitates were either untreated or treated with the different compounds indicated. Samples were analyzed by SDS-PAGE, and blotted for HRP-streptavidin. (C and D) Whole cell lysates from different cell lines were immunoprecipitated with different antibodies and the immunocomplexes were run on gelatin gels (C) or casein gels (D) to detect proteolytic activity. The proteolytic activity of the controls is indicated by arrows.
Fig. 5
Fig. 5
TPA induces α6p in MCF10A cells (A) MCF10A cells were cultured for four days and were then treated with different concentrations of TPA for 18 h. The samples were analyzed by SDS-PAGE and a Western blot was performed for the α6 integrin using the AA6A rabbit polyclonal antibody. (B) MCF10A cells were cultured for four days and were then treated with different concentrations of TPA or with TPA and different inhibitors for 18 h. The samples were analyzed by SDS-PAGE and a Western blot was performed for the α6 integrin using the AA6A rabbit polyclonal antibody. (C) Media from the samples from panel B was analyzed for uPA activity using the uPA activity assay kit (Chemicon International).
Fig. 6
Fig. 6
α6p is present in human prostate tissues. Normal and cancer human prostate tissues were analyzed for α6 and α6p levels by immunoprecipitation with the J1B5 rat monoclonal antibody and Western blotting using the AA6A rabbit polyclonal antibody.
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