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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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NF-κB activation induced by PSMA crosslinking.LNCaP cells were subjected to stimulation by cross-linking of PSMA or of VCAM-1 for 45 min at room temperature and then cultured for the indicated times at 37°C. The cells were then harvested, washed, permeabilized and stained with a PE-labelled anti-phosphorylated NF-κB p65 antibody. Cells were then analyzed by flow cytometry. The MFI was recorded. An isotype-matched IgG1 antibody was used to mock-treat LNCaP cells; the Figure illustrates a representative experiment out of three performed independently. MFI values of controls were: untreated cells 9.22±0.9, cells treated with an anti-VCAM-1 mAb 5.74+0.09, cells treated with a mouse IgG1 mAb 5.02±0.3. No substantial differences in the control samples were observed at the various time points of the experiments. MFI of Anti-PSMA cross-linked cells were: 22.03±2.2 and 15.6±1.3 at 15 min and 30 min, respectively. p values of samples treated with anti-PSMA mAb were <0.01 at 15 min and 30 min, respectively, with respect to the untreated cells. The first panel from top (mouse IgG1) is the plot corresponding to time 0.
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pone-0004608-g003: NF-κB activation induced by PSMA crosslinking.LNCaP cells were subjected to stimulation by cross-linking of PSMA or of VCAM-1 for 45 min at room temperature and then cultured for the indicated times at 37°C. The cells were then harvested, washed, permeabilized and stained with a PE-labelled anti-phosphorylated NF-κB p65 antibody. Cells were then analyzed by flow cytometry. The MFI was recorded. An isotype-matched IgG1 antibody was used to mock-treat LNCaP cells; the Figure illustrates a representative experiment out of three performed independently. MFI values of controls were: untreated cells 9.22±0.9, cells treated with an anti-VCAM-1 mAb 5.74+0.09, cells treated with a mouse IgG1 mAb 5.02±0.3. No substantial differences in the control samples were observed at the various time points of the experiments. MFI of Anti-PSMA cross-linked cells were: 22.03±2.2 and 15.6±1.3 at 15 min and 30 min, respectively. p values of samples treated with anti-PSMA mAb were <0.01 at 15 min and 30 min, respectively, with respect to the untreated cells. The first panel from top (mouse IgG1) is the plot corresponding to time 0.

Mentions: It is well established that p38 and ERK1/2 have a role in the process of activation of NF-κB transcription factor, and that NF-κB plays a major function in the transcriptional control of various cytokines and chemokines, including IL-6 and CCL5 [1]. To investigate whether NF-κB activation was induced in LNCaP cells by the cross-linking of PSMA, cells were treated as described in Fig. 2 and stained with a mAb recognizing the phosphorylated Serine 529 (pS529) located in the transactivation domain of the p65 subunit of the human NF-κB. As the pS529 containing domain is unmasked only after detachment of NF-κB inhibitors induced by stimulation, the exposure of pS529 is considered a marker of NF-κB nuclear translocation and activation in normal and neoplastic cells of different origin. Additionally, phosphorylated S529 further augments NF-κB transactivation potential [22]. Controls were represented by cells left untreated, treated with an isotype-matched IgG1 antibody or subjected to VCAM-1 cross-linking. The results of flow cytometry analysis (Fig. 3) demonstrated that the mean fluorescence intensity (MFI) of LNCaP cells (9.2±0.9 SEM) was greatly augmented following PSMA cross-linking, peaking at 15 min from stimulation and declining 30 min later (230%±30 SEM and 160%±20 SEM of control cells, respectively), thus implying that NF-κB activation had occurred. No increase of MFI was instead detected at the various points of the time course in the presence of the control antibody or VCAM-1 cross-linking.


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)

NF-κB activation induced by PSMA crosslinking.LNCaP cells were subjected to stimulation by cross-linking of PSMA or of VCAM-1 for 45 min at room temperature and then cultured for the indicated times at 37°C. The cells were then harvested, washed, permeabilized and stained with a PE-labelled anti-phosphorylated NF-κB p65 antibody. Cells were then analyzed by flow cytometry. The MFI was recorded. An isotype-matched IgG1 antibody was used to mock-treat LNCaP cells; the Figure illustrates a representative experiment out of three performed independently. MFI values of controls were: untreated cells 9.22±0.9, cells treated with an anti-VCAM-1 mAb 5.74+0.09, cells treated with a mouse IgG1 mAb 5.02±0.3. No substantial differences in the control samples were observed at the various time points of the experiments. MFI of Anti-PSMA cross-linked cells were: 22.03±2.2 and 15.6±1.3 at 15 min and 30 min, respectively. p values of samples treated with anti-PSMA mAb were <0.01 at 15 min and 30 min, respectively, with respect to the untreated cells. The first panel from top (mouse IgG1) is the plot corresponding to time 0.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2643478&req=5

pone-0004608-g003: NF-κB activation induced by PSMA crosslinking.LNCaP cells were subjected to stimulation by cross-linking of PSMA or of VCAM-1 for 45 min at room temperature and then cultured for the indicated times at 37°C. The cells were then harvested, washed, permeabilized and stained with a PE-labelled anti-phosphorylated NF-κB p65 antibody. Cells were then analyzed by flow cytometry. The MFI was recorded. An isotype-matched IgG1 antibody was used to mock-treat LNCaP cells; the Figure illustrates a representative experiment out of three performed independently. MFI values of controls were: untreated cells 9.22±0.9, cells treated with an anti-VCAM-1 mAb 5.74+0.09, cells treated with a mouse IgG1 mAb 5.02±0.3. No substantial differences in the control samples were observed at the various time points of the experiments. MFI of Anti-PSMA cross-linked cells were: 22.03±2.2 and 15.6±1.3 at 15 min and 30 min, respectively. p values of samples treated with anti-PSMA mAb were <0.01 at 15 min and 30 min, respectively, with respect to the untreated cells. The first panel from top (mouse IgG1) is the plot corresponding to time 0.
Mentions: It is well established that p38 and ERK1/2 have a role in the process of activation of NF-κB transcription factor, and that NF-κB plays a major function in the transcriptional control of various cytokines and chemokines, including IL-6 and CCL5 [1]. To investigate whether NF-κB activation was induced in LNCaP cells by the cross-linking of PSMA, cells were treated as described in Fig. 2 and stained with a mAb recognizing the phosphorylated Serine 529 (pS529) located in the transactivation domain of the p65 subunit of the human NF-κB. As the pS529 containing domain is unmasked only after detachment of NF-κB inhibitors induced by stimulation, the exposure of pS529 is considered a marker of NF-κB nuclear translocation and activation in normal and neoplastic cells of different origin. Additionally, phosphorylated S529 further augments NF-κB transactivation potential [22]. Controls were represented by cells left untreated, treated with an isotype-matched IgG1 antibody or subjected to VCAM-1 cross-linking. The results of flow cytometry analysis (Fig. 3) demonstrated that the mean fluorescence intensity (MFI) of LNCaP cells (9.2±0.9 SEM) was greatly augmented following PSMA cross-linking, peaking at 15 min from stimulation and declining 30 min later (230%±30 SEM and 160%±20 SEM of control cells, respectively), thus implying that NF-κB activation had occurred. No increase of MFI was instead detected at the various points of the time course in the presence of the control antibody or VCAM-1 cross-linking.

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