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The prostate specific membrane antigen regulates the expression of IL-6 and CCL5 in prostate tumour cells by activating the MAPK pathways.

Colombatti M, Grasso S, Porzia A, Fracasso G, Scupoli MT, Cingarlini S, Poffe O, Naim HY, Heine M, Tridente G, Mainiero F, Ramarli D - PLoS ONE (2009)

Bottom Line: As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-kappaB activation associated with an increased expression of IL-6 and CCL5 genes.Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident.Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Verona, Verona, Italy.

ABSTRACT
The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-kappaB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis.

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Reactivity of anti-PSMA mAb.Panel A: representative flow cytometry of LNCaP cells stained with anti-PSMA or with J591 mAb. Controls are represented by cells goat anti-mouse-FITC IgG only. Panel B: flow cytometry of LNCaP cells incubated with biotinylated J591 mAb (B-J591) with or without pretreatment (45 min on ice) with anti-PSMA at the indicated concentrations or with non-biotinylated J591 mAb as control. Antibody binding was revealed by FITC-Avidin (F-Avidin). The percentage of positive cells and the Mean Fluorescence Intensity observed under the different conditions are representative of two independent experiments performed with independent cultures. Panel C: Western blot analysis of LNCaP cell lysates immuneprecipitated with the indicated mAbs, and then probed with the anti-PSMA mAb followed by a goat anti-mouse antiserum labelled with horseradish peroxidase. The apparent molecular weight of PSMA band is indicated by the arrow.
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pone-0004608-g001: Reactivity of anti-PSMA mAb.Panel A: representative flow cytometry of LNCaP cells stained with anti-PSMA or with J591 mAb. Controls are represented by cells goat anti-mouse-FITC IgG only. Panel B: flow cytometry of LNCaP cells incubated with biotinylated J591 mAb (B-J591) with or without pretreatment (45 min on ice) with anti-PSMA at the indicated concentrations or with non-biotinylated J591 mAb as control. Antibody binding was revealed by FITC-Avidin (F-Avidin). The percentage of positive cells and the Mean Fluorescence Intensity observed under the different conditions are representative of two independent experiments performed with independent cultures. Panel C: Western blot analysis of LNCaP cell lysates immuneprecipitated with the indicated mAbs, and then probed with the anti-PSMA mAb followed by a goat anti-mouse antiserum labelled with horseradish peroxidase. The apparent molecular weight of PSMA band is indicated by the arrow.

Mentions: Biochemical and functional studies shown in this paper were performed with an anti PSMA mAb (henceforth designated “anti-PSMA mAb”) produced by our group. Specificity for PSMA is shown in Fig. 1. Staining, competition and immuneprecipitation experiments were performed with LNCaP cells and the anti-PSMA mAb was compared with the J591 mAb, a reference mAb recognizing an extra-cellular epitope of the PSMA molecule [20]. Flow cytometry of cells stained with anti-PSMA or J591 mAbs demonstrated that both mAbs recognized a surface molecule expressed at comparable levels on LNCaP cells (Fig. 1A). No staining was instead observed with the PSMA negative DU145 or PC3 cell lines (not shown). Competition experiments, performed with biotinilated J591 mAb revealed by FITC-streptavidin, demonstrated that the number of positive cells and the mean fluorescence observed following J591 staining was progressively decreased when cells were pre-treated with increasing amounts of non-conjugated anti-PSMA mAb (Fig. 1, B), thus demonstrating that our anti-PSMA mAb specifically recognizes an epitope of the extra-cellular domain of the molecule identical or spatially close to that identified by the J591 mAb. Immunoprecipitations of crude cell lysates revealed the ability of our anti-PSMA mAb to immunoprecipitate a protein of the same apparent molecular weight (110 kD) of that immunoprecipitated using J591 mAb or 7E11c mAb, this last recognizing an intracellular epitope of PSMA (Fig. 1C). Collectively, these results demonstrated that our anti-PSMA mAb specifically recognizes an extra cellular epitope of PSMA. Results obtained at a single cell level were extended by immunohistochemistry to human tissues, demonstrating that the reactivity of anti-PSMA mAb overlapped that of J591 (not shown)


The prostate specific membrane antigen regulates the expression of IL-6 and CCL5 in prostate tumour cells by activating the MAPK pathways.

Colombatti M, Grasso S, Porzia A, Fracasso G, Scupoli MT, Cingarlini S, Poffe O, Naim HY, Heine M, Tridente G, Mainiero F, Ramarli D - PLoS ONE (2009)

Reactivity of anti-PSMA mAb.Panel A: representative flow cytometry of LNCaP cells stained with anti-PSMA or with J591 mAb. Controls are represented by cells goat anti-mouse-FITC IgG only. Panel B: flow cytometry of LNCaP cells incubated with biotinylated J591 mAb (B-J591) with or without pretreatment (45 min on ice) with anti-PSMA at the indicated concentrations or with non-biotinylated J591 mAb as control. Antibody binding was revealed by FITC-Avidin (F-Avidin). The percentage of positive cells and the Mean Fluorescence Intensity observed under the different conditions are representative of two independent experiments performed with independent cultures. Panel C: Western blot analysis of LNCaP cell lysates immuneprecipitated with the indicated mAbs, and then probed with the anti-PSMA mAb followed by a goat anti-mouse antiserum labelled with horseradish peroxidase. The apparent molecular weight of PSMA band is indicated by the arrow.
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Related In: Results  -  Collection

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pone-0004608-g001: Reactivity of anti-PSMA mAb.Panel A: representative flow cytometry of LNCaP cells stained with anti-PSMA or with J591 mAb. Controls are represented by cells goat anti-mouse-FITC IgG only. Panel B: flow cytometry of LNCaP cells incubated with biotinylated J591 mAb (B-J591) with or without pretreatment (45 min on ice) with anti-PSMA at the indicated concentrations or with non-biotinylated J591 mAb as control. Antibody binding was revealed by FITC-Avidin (F-Avidin). The percentage of positive cells and the Mean Fluorescence Intensity observed under the different conditions are representative of two independent experiments performed with independent cultures. Panel C: Western blot analysis of LNCaP cell lysates immuneprecipitated with the indicated mAbs, and then probed with the anti-PSMA mAb followed by a goat anti-mouse antiserum labelled with horseradish peroxidase. The apparent molecular weight of PSMA band is indicated by the arrow.
Mentions: Biochemical and functional studies shown in this paper were performed with an anti PSMA mAb (henceforth designated “anti-PSMA mAb”) produced by our group. Specificity for PSMA is shown in Fig. 1. Staining, competition and immuneprecipitation experiments were performed with LNCaP cells and the anti-PSMA mAb was compared with the J591 mAb, a reference mAb recognizing an extra-cellular epitope of the PSMA molecule [20]. Flow cytometry of cells stained with anti-PSMA or J591 mAbs demonstrated that both mAbs recognized a surface molecule expressed at comparable levels on LNCaP cells (Fig. 1A). No staining was instead observed with the PSMA negative DU145 or PC3 cell lines (not shown). Competition experiments, performed with biotinilated J591 mAb revealed by FITC-streptavidin, demonstrated that the number of positive cells and the mean fluorescence observed following J591 staining was progressively decreased when cells were pre-treated with increasing amounts of non-conjugated anti-PSMA mAb (Fig. 1, B), thus demonstrating that our anti-PSMA mAb specifically recognizes an epitope of the extra-cellular domain of the molecule identical or spatially close to that identified by the J591 mAb. Immunoprecipitations of crude cell lysates revealed the ability of our anti-PSMA mAb to immunoprecipitate a protein of the same apparent molecular weight (110 kD) of that immunoprecipitated using J591 mAb or 7E11c mAb, this last recognizing an intracellular epitope of PSMA (Fig. 1C). Collectively, these results demonstrated that our anti-PSMA mAb specifically recognizes an extra cellular epitope of PSMA. Results obtained at a single cell level were extended by immunohistochemistry to human tissues, demonstrating that the reactivity of anti-PSMA mAb overlapped that of J591 (not shown)

Bottom Line: As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-kappaB activation associated with an increased expression of IL-6 and CCL5 genes.Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident.Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Verona, Verona, Italy.

ABSTRACT
The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-kappaB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis.

Show MeSH
Related in: MedlinePlus