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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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Inducing activity of PSMA cross-linking on IL-6 and CCL5 expression and role of PSMA-activated p38 and ERK1/2 .IL-6 (panel A) and CCL5 (panel B): production by LNCaP cells left untreated (open bars), stimulated by cross-linking via anti PSMA mAb (filled bars) or anti-VCAM-1 mAb (dashed bars) in the presence or in the absence of single or combined inhibitors (SB and PD) at the indicated doses (SB50 = 50 µg/ml, SB25 = 25 µg/ml; PD50 = 50 µg/ml, PD25 = 25 µg/ml; SB/PD25 = combinations of SB+PD at 25 µg/ml each). Control and treated cell cultures were washed, cultured for further 24 h before collecting cells and cell-free supernatants. IL-6 and CCL5 accumulation in each supernatant was measured by ELISA and expressed as pg/mg of cell lysates/24 h. Inhibitors were added to the cells 6 h prior to stimulation and maintained throughout the duration of the experiment. Values represent the mean±SEM of results of four experiments performed in duplicates. Stars on top of the bars indicate statistical significance as follows: p<0.001, ***; p<0.01, **; p<0.05, *.
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pone-0004608-g004: Inducing activity of PSMA cross-linking on IL-6 and CCL5 expression and role of PSMA-activated p38 and ERK1/2 .IL-6 (panel A) and CCL5 (panel B): production by LNCaP cells left untreated (open bars), stimulated by cross-linking via anti PSMA mAb (filled bars) or anti-VCAM-1 mAb (dashed bars) in the presence or in the absence of single or combined inhibitors (SB and PD) at the indicated doses (SB50 = 50 µg/ml, SB25 = 25 µg/ml; PD50 = 50 µg/ml, PD25 = 25 µg/ml; SB/PD25 = combinations of SB+PD at 25 µg/ml each). Control and treated cell cultures were washed, cultured for further 24 h before collecting cells and cell-free supernatants. IL-6 and CCL5 accumulation in each supernatant was measured by ELISA and expressed as pg/mg of cell lysates/24 h. Inhibitors were added to the cells 6 h prior to stimulation and maintained throughout the duration of the experiment. Values represent the mean±SEM of results of four experiments performed in duplicates. Stars on top of the bars indicate statistical significance as follows: p<0.001, ***; p<0.01, **; p<0.05, *.

Mentions: Results shown in Fig. 2 and 3 demonstrated that cross-linking of PSMA recruited a signalling pathway eventually leading to NF-κB activation and nuclear translocation. The contribution of PSMA to the regulation of the production of IL-6 and CCL5 in LNCaP cells was then investigated in cross-linking experiments carried out as in Fig. 2. Further, we analyzed the role of PSMA-activated p38 and ERK1/2 in a loss-of-function condition achieved by using two pharmacological inhibitors: SB 202190 (SB), a compound binding specifically p38 and blocking reversibly its enzymatic activity or PD 98059 (PD), an inhibitor specifically preventing the activation of the ERK1/2-activating kinase MEK-1 [23]. LNCaP cells were stimulated as confluent monolayer and harvested 24 h later. To determine the basal and inducible release of IL-6 and CCL5 in culture, LNCaP cells and the corresponding cell-free supernatants were separately recovered from each treated or untreated cell culture. Cell lysates and cell-free supernatants were used to determine respectively the total protein amount and IL-6 and CCL5 accumulation in the supernatant of each sample. The results of IL-6 and CCL5 measurements were then expressed as pg of IL-6 or CCL5/mg of cell protein lysates. As illustrated in Fig. 4 A, LNCaP cells produced low amounts of IL-6 and of CCL5 basally (11.3±2 and14±3.6 pg/mg total cell protein/24 h, respectively). The cross-linking of PSMA increased this production by three-fold in the case of IL-6 and by two-fold in the case of CCL5 (311%±48, p<0.001 and 214%±38 p<0.001, respectively), thus demonstrating that PSMA recruitment at the surface of LNCaP cells powerfully up-regulates the production of both IL-6 and CCL5 at the same time. The cross-linking of the isotype-matched anti VCAM-1 mAb lacked instead any significant activity (155%±52 and 133%±11, respectively).


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)

Inducing activity of PSMA cross-linking on IL-6 and CCL5 expression and role of PSMA-activated p38 and ERK1/2 .IL-6 (panel A) and CCL5 (panel B): production by LNCaP cells left untreated (open bars), stimulated by cross-linking via anti PSMA mAb (filled bars) or anti-VCAM-1 mAb (dashed bars) in the presence or in the absence of single or combined inhibitors (SB and PD) at the indicated doses (SB50 = 50 µg/ml, SB25 = 25 µg/ml; PD50 = 50 µg/ml, PD25 = 25 µg/ml; SB/PD25 = combinations of SB+PD at 25 µg/ml each). Control and treated cell cultures were washed, cultured for further 24 h before collecting cells and cell-free supernatants. IL-6 and CCL5 accumulation in each supernatant was measured by ELISA and expressed as pg/mg of cell lysates/24 h. Inhibitors were added to the cells 6 h prior to stimulation and maintained throughout the duration of the experiment. Values represent the mean±SEM of results of four experiments performed in duplicates. Stars on top of the bars indicate statistical significance as follows: p<0.001, ***; p<0.01, **; p<0.05, *.
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Related In: Results  -  Collection

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pone-0004608-g004: Inducing activity of PSMA cross-linking on IL-6 and CCL5 expression and role of PSMA-activated p38 and ERK1/2 .IL-6 (panel A) and CCL5 (panel B): production by LNCaP cells left untreated (open bars), stimulated by cross-linking via anti PSMA mAb (filled bars) or anti-VCAM-1 mAb (dashed bars) in the presence or in the absence of single or combined inhibitors (SB and PD) at the indicated doses (SB50 = 50 µg/ml, SB25 = 25 µg/ml; PD50 = 50 µg/ml, PD25 = 25 µg/ml; SB/PD25 = combinations of SB+PD at 25 µg/ml each). Control and treated cell cultures were washed, cultured for further 24 h before collecting cells and cell-free supernatants. IL-6 and CCL5 accumulation in each supernatant was measured by ELISA and expressed as pg/mg of cell lysates/24 h. Inhibitors were added to the cells 6 h prior to stimulation and maintained throughout the duration of the experiment. Values represent the mean±SEM of results of four experiments performed in duplicates. Stars on top of the bars indicate statistical significance as follows: p<0.001, ***; p<0.01, **; p<0.05, *.
Mentions: Results shown in Fig. 2 and 3 demonstrated that cross-linking of PSMA recruited a signalling pathway eventually leading to NF-κB activation and nuclear translocation. The contribution of PSMA to the regulation of the production of IL-6 and CCL5 in LNCaP cells was then investigated in cross-linking experiments carried out as in Fig. 2. Further, we analyzed the role of PSMA-activated p38 and ERK1/2 in a loss-of-function condition achieved by using two pharmacological inhibitors: SB 202190 (SB), a compound binding specifically p38 and blocking reversibly its enzymatic activity or PD 98059 (PD), an inhibitor specifically preventing the activation of the ERK1/2-activating kinase MEK-1 [23]. LNCaP cells were stimulated as confluent monolayer and harvested 24 h later. To determine the basal and inducible release of IL-6 and CCL5 in culture, LNCaP cells and the corresponding cell-free supernatants were separately recovered from each treated or untreated cell culture. Cell lysates and cell-free supernatants were used to determine respectively the total protein amount and IL-6 and CCL5 accumulation in the supernatant of each sample. The results of IL-6 and CCL5 measurements were then expressed as pg of IL-6 or CCL5/mg of cell protein lysates. As illustrated in Fig. 4 A, LNCaP cells produced low amounts of IL-6 and of CCL5 basally (11.3±2 and14±3.6 pg/mg total cell protein/24 h, respectively). The cross-linking of PSMA increased this production by three-fold in the case of IL-6 and by two-fold in the case of CCL5 (311%±48, p<0.001 and 214%±38 p<0.001, respectively), thus demonstrating that PSMA recruitment at the surface of LNCaP cells powerfully up-regulates the production of both IL-6 and CCL5 at the same time. The cross-linking of the isotype-matched anti VCAM-1 mAb lacked instead any significant activity (155%±52 and 133%±11, respectively).

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