Limits...
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.

Show MeSH

Related in: MedlinePlus

Kinetics of ERK1/21/2, p38, RAC 1 and RAS phosphorylation induced by PSMA cross-linking.LNCaP cells were left untreated or subjected to PSMA cross-linking for the indicated times at 37°C. An anti-VCAM-1 mAb was used as the isotype matched control. Panels A and B: p38 and ERK1/2 activation was assessed in crude lysates. Equal amounts (20 µg) of total proteins were boiled in sample buffer and separated by SDS-PAGE. Following immunoblotting with an anti-phospho-p38 or anti-phospho-ERK1/2 mAb, immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and the ECL system. Panels C and D: RAC1 and RAS activation was evaluated by a specific assay as described in the Material and Methods section. Bound active GTP-RAC and GTP-RAS molecules were analyzed by Western blotting using an anti-RAC1 or anti-Ras mAb and visualized as above. Total amount of ERK1/2, p38, RAC1 and RAS in crude lysates are shown as loading control at the bottom of each gel. Results are representative of one of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2643478&req=5

pone-0004608-g002: Kinetics of ERK1/21/2, p38, RAC 1 and RAS phosphorylation induced by PSMA cross-linking.LNCaP cells were left untreated or subjected to PSMA cross-linking for the indicated times at 37°C. An anti-VCAM-1 mAb was used as the isotype matched control. Panels A and B: p38 and ERK1/2 activation was assessed in crude lysates. Equal amounts (20 µg) of total proteins were boiled in sample buffer and separated by SDS-PAGE. Following immunoblotting with an anti-phospho-p38 or anti-phospho-ERK1/2 mAb, immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and the ECL system. Panels C and D: RAC1 and RAS activation was evaluated by a specific assay as described in the Material and Methods section. Bound active GTP-RAC and GTP-RAS molecules were analyzed by Western blotting using an anti-RAC1 or anti-Ras mAb and visualized as above. Total amount of ERK1/2, p38, RAC1 and RAS in crude lysates are shown as loading control at the bottom of each gel. Results are representative of one of three independent experiments.

Mentions: To test whether PSMA was able to transduce intracellular signals leading to p38 and/or ERK1/2 phosphorylation, LNCaP cells were subjected to PSMA cross-linking by anti-PSMA-specific antibodies as adherent, confluent monolayers. PSMA molecules, recognized by the anti-PSMA mAb were induced to cluster at the cell membrane due to the binding of a goat anti-mouse IgG antiserum (Gam) added to cell cultures at 37°C for the indicated times. Controls included treatment with anti VCAM-1, with 7E11c or with Gam alone and the use of untreated cells. Cells were then washed and used as a source of cell lysates. We first assayed the phosphorylation of p38 and ERK1/2 in crude lysates obtained from the same cell cultures immunoblotted with antibodies specifically detecting the active, phosphorylated form of the two kinases. As shown in Fig. 2A and B, ERK1/2 and p38 were found activated at low levels in untreated LNCaP cells. Upon PSMA cross-linking, both ERK1/2 and p38 became considerably phosphorylated at 10 min. Sustained levels of phosphorylation lasted up to 20 min, and then returned to basal levels. It is known that the small GTPases RAS and RAC1 lie upstream on the MAPKs activation pathways [21]. Thus, to examine if PSMA cross-linking could activate also RAC1 in LNCaP cells, we performed an activation assay with a GST-Pak fusion protein which is able to bind and precipitate the active form of RAC1. As shown in Fig. 2C, PSMA cross-linking determined endogenous RAC1 activation in the same lysates. Similar results were obtained in the case of RAS, whose constitutive activation level was greatly augmented upon PSMA cross-linking (Fig. 2D). Cross-linking performed with an isotype matched mAb recognizing VCAM-1 failed to induce activation in all assays and at all points of the time-course (Fig. 2, all panels). Gel densitometry of independent experiments performed with different cell lysates confirmed the significance of the increased activation of ERK1/2, p38, RAC1 and RAS observed following PSMA, but not VCAM-1 cross-linking (Table 1). Because the time-course of RAS and RAC1 activation paralleled that of p38 and ERK1/2, these results overall indicate that PSMA is able to induce a sustained activation of the RAS- and RAC-MAPK pathways.


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)

Kinetics of ERK1/21/2, p38, RAC 1 and RAS phosphorylation induced by PSMA cross-linking.LNCaP cells were left untreated or subjected to PSMA cross-linking for the indicated times at 37°C. An anti-VCAM-1 mAb was used as the isotype matched control. Panels A and B: p38 and ERK1/2 activation was assessed in crude lysates. Equal amounts (20 µg) of total proteins were boiled in sample buffer and separated by SDS-PAGE. Following immunoblotting with an anti-phospho-p38 or anti-phospho-ERK1/2 mAb, immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and the ECL system. Panels C and D: RAC1 and RAS activation was evaluated by a specific assay as described in the Material and Methods section. Bound active GTP-RAC and GTP-RAS molecules were analyzed by Western blotting using an anti-RAC1 or anti-Ras mAb and visualized as above. Total amount of ERK1/2, p38, RAC1 and RAS in crude lysates are shown as loading control at the bottom of each gel. Results are representative of one of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004608-g002: Kinetics of ERK1/21/2, p38, RAC 1 and RAS phosphorylation induced by PSMA cross-linking.LNCaP cells were left untreated or subjected to PSMA cross-linking for the indicated times at 37°C. An anti-VCAM-1 mAb was used as the isotype matched control. Panels A and B: p38 and ERK1/2 activation was assessed in crude lysates. Equal amounts (20 µg) of total proteins were boiled in sample buffer and separated by SDS-PAGE. Following immunoblotting with an anti-phospho-p38 or anti-phospho-ERK1/2 mAb, immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and the ECL system. Panels C and D: RAC1 and RAS activation was evaluated by a specific assay as described in the Material and Methods section. Bound active GTP-RAC and GTP-RAS molecules were analyzed by Western blotting using an anti-RAC1 or anti-Ras mAb and visualized as above. Total amount of ERK1/2, p38, RAC1 and RAS in crude lysates are shown as loading control at the bottom of each gel. Results are representative of one of three independent experiments.
Mentions: To test whether PSMA was able to transduce intracellular signals leading to p38 and/or ERK1/2 phosphorylation, LNCaP cells were subjected to PSMA cross-linking by anti-PSMA-specific antibodies as adherent, confluent monolayers. PSMA molecules, recognized by the anti-PSMA mAb were induced to cluster at the cell membrane due to the binding of a goat anti-mouse IgG antiserum (Gam) added to cell cultures at 37°C for the indicated times. Controls included treatment with anti VCAM-1, with 7E11c or with Gam alone and the use of untreated cells. Cells were then washed and used as a source of cell lysates. We first assayed the phosphorylation of p38 and ERK1/2 in crude lysates obtained from the same cell cultures immunoblotted with antibodies specifically detecting the active, phosphorylated form of the two kinases. As shown in Fig. 2A and B, ERK1/2 and p38 were found activated at low levels in untreated LNCaP cells. Upon PSMA cross-linking, both ERK1/2 and p38 became considerably phosphorylated at 10 min. Sustained levels of phosphorylation lasted up to 20 min, and then returned to basal levels. It is known that the small GTPases RAS and RAC1 lie upstream on the MAPKs activation pathways [21]. Thus, to examine if PSMA cross-linking could activate also RAC1 in LNCaP cells, we performed an activation assay with a GST-Pak fusion protein which is able to bind and precipitate the active form of RAC1. As shown in Fig. 2C, PSMA cross-linking determined endogenous RAC1 activation in the same lysates. Similar results were obtained in the case of RAS, whose constitutive activation level was greatly augmented upon PSMA cross-linking (Fig. 2D). Cross-linking performed with an isotype matched mAb recognizing VCAM-1 failed to induce activation in all assays and at all points of the time-course (Fig. 2, all panels). Gel densitometry of independent experiments performed with different cell lysates confirmed the significance of the increased activation of ERK1/2, p38, RAC1 and RAS observed following PSMA, but not VCAM-1 cross-linking (Table 1). Because the time-course of RAS and RAC1 activation paralleled that of p38 and ERK1/2, these results overall indicate that PSMA is able to induce a sustained activation of the RAS- and RAC-MAPK pathways.

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