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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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Related in: MedlinePlus

Effects of pro-proliferative stimuli on LNCaP cells.A) LNCaP proliferation induced by treatment with CCL5 and/or IL-6. 3H-TdR uptake of cells after 24 h of culture with CCL5 (grey bars), 72 h of culture with IL-6 (hatched bars) or 24 h of culture with combinations of CCL5 and IL-6 (crossed bars) at the indicated concentrations. B) 3H-TdR uptake of cells after 24 h culture with 5 ng/ml of CCL5 in the presence or in the absence of anti-CCR5 or 7E11c antibodies at the indicated concentrations; C) Western blot analysis of cell extracts (20 µg/lane) of LNCaP cells treated with CCL5 (5 ng/ml) and probed with anti-STAT 5 or with anti-cyclin D1 antibodies. Equal protein loading in all the lanes is confirmed by not specific bands shown in the middle gel. Arrows indicate phosphorylated STAT5 proteins. Western blotting results are representative of two independent experiments. D) 3H-TdR uptake of cells 72 h after binding or cross-linking of 7E11c or anti-PSMA antibodies. Values represent the mean±SEM of three independent experiments performed in triplicates. Stars on the bars indicate statistical significance as in Fig. 4.
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pone-0004608-g005: Effects of pro-proliferative stimuli on LNCaP cells.A) LNCaP proliferation induced by treatment with CCL5 and/or IL-6. 3H-TdR uptake of cells after 24 h of culture with CCL5 (grey bars), 72 h of culture with IL-6 (hatched bars) or 24 h of culture with combinations of CCL5 and IL-6 (crossed bars) at the indicated concentrations. B) 3H-TdR uptake of cells after 24 h culture with 5 ng/ml of CCL5 in the presence or in the absence of anti-CCR5 or 7E11c antibodies at the indicated concentrations; C) Western blot analysis of cell extracts (20 µg/lane) of LNCaP cells treated with CCL5 (5 ng/ml) and probed with anti-STAT 5 or with anti-cyclin D1 antibodies. Equal protein loading in all the lanes is confirmed by not specific bands shown in the middle gel. Arrows indicate phosphorylated STAT5 proteins. Western blotting results are representative of two independent experiments. D) 3H-TdR uptake of cells 72 h after binding or cross-linking of 7E11c or anti-PSMA antibodies. Values represent the mean±SEM of three independent experiments performed in triplicates. Stars on the bars indicate statistical significance as in Fig. 4.

Mentions: As illustrated in Fig. 5 A, the rate of unlimited proliferation of LNCaP cells was increased by the addition of recombinant IL-6 or CCL5 in a dose-dependent manner. IL-6 exerted the maximal activity at 100 ng/ml (175±16, p<0.01) whereas CCL5 promoted a similar enhancement already at 5 ng/ml (146%±2 of increase, p<0.05), although only a subset (8–10%) of strongly positive CCR5 cells could be detected in our LNCaP cells population when FBS was lowered in culture to 1% (S. Grasso, personal observation). IL-6 and CCL5 appeared to act synergistically in inducing LNCaP cells proliferation, as demonstrated by the finding that the addition of as little as 1 ng/ml of IL-6 to the most effective dose of CCL5 raised the LNCaP cells proliferation up to values which were significantly higher than those of controls (168%±2.9, p<0.001), and, perhaps more importantly, greater than those observed in the presence of 5 ng/ml of CCL5 (p<001). As shown in Fig. 5 B the induction of proliferation induced by CCL5 was almost abrogated by blocking CCR5 with specific antibodies. No interference was instead observed in the presence of 7E11c antibody, used as isotype-matched control, thus putting into evidence a direct correlation between the burst of LNCaP cells proliferation and the CCL5 interaction with its receptor CCR5. It has been reported that the triggering of CCR5 by CCL5 is rapidly followed by JAK-STAT phosphorylation, more specifically by STAT1 and/or STAT3 and/or STAT5, depending on the cellular system [26]–[28]. It has been also reported that STAT5 phosphorylation regulates the expression of cyclin D1 in tumor cells including those of prostate [29], [30]. To investigate the downstream effects of CCR5 engagement in LNCaP cells, the cells were cultured in the presence of 5 ng/ml of CCL5 for the indicated times and then analysed as for the expression of STAT 5 and Cyclin D1 by immunoblotting with specific antibodies. As illustrated in Fig. 5 C phosphorylated STAT5 appeared as a doublet at 30 min. The doublet lowered its intensity at 1 h to return to the almost undetectable level observed in untreated cell at 3 h. Consistently, the amount of cyclin D1 was found more than doubled at 1 h and at 3 h, thus indicating that CCL5 can regulate LNCaP cells proliferation in a STAT 5-Cyclin D1 dependent way. Taken toghether, these results demonstrate that the antibody-mediated clustering of PSMA at the LNCaP cells surface activates signalling pathways able to directly or indirectly control the LNCaP cells growth. To confirm that the complex series of phenomena reported in the previous paragraphs led to LNCaP cells proliferation we decided to test if the cross-linking of PSMA would indeed induce the LNCaP cells proliferation. As shown in Fig. 5D LNCaP cells proliferation was found to be increased following PSMA cross-linking (175%±11, p<0.05), whereas the bindings of the sole anti-PSMA, of 7E11c or the cross-linking of 7E11c were all devoid of significant activity.


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)

Effects of pro-proliferative stimuli on LNCaP cells.A) LNCaP proliferation induced by treatment with CCL5 and/or IL-6. 3H-TdR uptake of cells after 24 h of culture with CCL5 (grey bars), 72 h of culture with IL-6 (hatched bars) or 24 h of culture with combinations of CCL5 and IL-6 (crossed bars) at the indicated concentrations. B) 3H-TdR uptake of cells after 24 h culture with 5 ng/ml of CCL5 in the presence or in the absence of anti-CCR5 or 7E11c antibodies at the indicated concentrations; C) Western blot analysis of cell extracts (20 µg/lane) of LNCaP cells treated with CCL5 (5 ng/ml) and probed with anti-STAT 5 or with anti-cyclin D1 antibodies. Equal protein loading in all the lanes is confirmed by not specific bands shown in the middle gel. Arrows indicate phosphorylated STAT5 proteins. Western blotting results are representative of two independent experiments. D) 3H-TdR uptake of cells 72 h after binding or cross-linking of 7E11c or anti-PSMA antibodies. Values represent the mean±SEM of three independent experiments performed in triplicates. Stars on the bars indicate statistical significance as in Fig. 4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2643478&req=5

pone-0004608-g005: Effects of pro-proliferative stimuli on LNCaP cells.A) LNCaP proliferation induced by treatment with CCL5 and/or IL-6. 3H-TdR uptake of cells after 24 h of culture with CCL5 (grey bars), 72 h of culture with IL-6 (hatched bars) or 24 h of culture with combinations of CCL5 and IL-6 (crossed bars) at the indicated concentrations. B) 3H-TdR uptake of cells after 24 h culture with 5 ng/ml of CCL5 in the presence or in the absence of anti-CCR5 or 7E11c antibodies at the indicated concentrations; C) Western blot analysis of cell extracts (20 µg/lane) of LNCaP cells treated with CCL5 (5 ng/ml) and probed with anti-STAT 5 or with anti-cyclin D1 antibodies. Equal protein loading in all the lanes is confirmed by not specific bands shown in the middle gel. Arrows indicate phosphorylated STAT5 proteins. Western blotting results are representative of two independent experiments. D) 3H-TdR uptake of cells 72 h after binding or cross-linking of 7E11c or anti-PSMA antibodies. Values represent the mean±SEM of three independent experiments performed in triplicates. Stars on the bars indicate statistical significance as in Fig. 4.
Mentions: As illustrated in Fig. 5 A, the rate of unlimited proliferation of LNCaP cells was increased by the addition of recombinant IL-6 or CCL5 in a dose-dependent manner. IL-6 exerted the maximal activity at 100 ng/ml (175±16, p<0.01) whereas CCL5 promoted a similar enhancement already at 5 ng/ml (146%±2 of increase, p<0.05), although only a subset (8–10%) of strongly positive CCR5 cells could be detected in our LNCaP cells population when FBS was lowered in culture to 1% (S. Grasso, personal observation). IL-6 and CCL5 appeared to act synergistically in inducing LNCaP cells proliferation, as demonstrated by the finding that the addition of as little as 1 ng/ml of IL-6 to the most effective dose of CCL5 raised the LNCaP cells proliferation up to values which were significantly higher than those of controls (168%±2.9, p<0.001), and, perhaps more importantly, greater than those observed in the presence of 5 ng/ml of CCL5 (p<001). As shown in Fig. 5 B the induction of proliferation induced by CCL5 was almost abrogated by blocking CCR5 with specific antibodies. No interference was instead observed in the presence of 7E11c antibody, used as isotype-matched control, thus putting into evidence a direct correlation between the burst of LNCaP cells proliferation and the CCL5 interaction with its receptor CCR5. It has been reported that the triggering of CCR5 by CCL5 is rapidly followed by JAK-STAT phosphorylation, more specifically by STAT1 and/or STAT3 and/or STAT5, depending on the cellular system [26]–[28]. It has been also reported that STAT5 phosphorylation regulates the expression of cyclin D1 in tumor cells including those of prostate [29], [30]. To investigate the downstream effects of CCR5 engagement in LNCaP cells, the cells were cultured in the presence of 5 ng/ml of CCL5 for the indicated times and then analysed as for the expression of STAT 5 and Cyclin D1 by immunoblotting with specific antibodies. As illustrated in Fig. 5 C phosphorylated STAT5 appeared as a doublet at 30 min. The doublet lowered its intensity at 1 h to return to the almost undetectable level observed in untreated cell at 3 h. Consistently, the amount of cyclin D1 was found more than doubled at 1 h and at 3 h, thus indicating that CCL5 can regulate LNCaP cells proliferation in a STAT 5-Cyclin D1 dependent way. Taken toghether, these results demonstrate that the antibody-mediated clustering of PSMA at the LNCaP cells surface activates signalling pathways able to directly or indirectly control the LNCaP cells growth. To confirm that the complex series of phenomena reported in the previous paragraphs led to LNCaP cells proliferation we decided to test if the cross-linking of PSMA would indeed induce the LNCaP cells proliferation. As shown in Fig. 5D LNCaP cells proliferation was found to be increased following PSMA cross-linking (175%±11, p<0.05), whereas the bindings of the sole anti-PSMA, of 7E11c or the cross-linking of 7E11c were all devoid of significant activity.

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