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Breast cancer cells stimulate osteoprotegerin (OPG) production by endothelial cells through direct cell contact.

Reid PE, Brown NJ, Holen I - Mol. Cancer (2009)

Bottom Line: In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor.In contrast, the pro-angiogenic factors VEGF, bFGF and TGFbeta had no effect on HuDMEC OPG levels.These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Academic Units of Clinical Oncology and Surgical Oncology, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK. p.reid@sheffield.ac.uk

ABSTRACT

Background: Angiogenesis, the sprouting of capillaries from existing blood vessels, is central to tumour growth and progression, however the molecular regulation of this process remains to be fully elucidated. The secreted glycoprotein osteoprotegerin (OPG) is one potential pro-angiogenic factor, and clinical studies have demonstrated endothelial cells within a number of tumour types to express high levels of OPG compared to those in normal tissue. Additionally, OPG can increase endothelial cell survival, proliferation and migration, as well as induce endothelial cell tube formation in vitro. This study aims to elucidate the processes involved in the pro-angiogenic effects of OPG in vitro, and also how OPG levels may be regulated within the tumour microenvironment.

Results: It has previously been demonstrated that OPG can induce tube formation on growth factor reduced matrigel. In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor. We also show that cell contact between HuDMECs and the T47D breast cancer cell line increases endothelial cell OPG mRNA and protein secretion levels in in vitro co-cultures. These increases in endothelial cell OPG secretion were dependent on alphanubeta3 ligation and NFkappaB activation. In contrast, the pro-angiogenic factors VEGF, bFGF and TGFbeta had no effect on HuDMEC OPG levels.

Conclusion: These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro. Additionally, we show that breast cancer cells cause increased levels of OPG expression by endothelial cells, and that direct contact between endothelial cells and tumour cells is required in order to increase endothelial OPG expression and secretion. Stimulation of OPG secretion was shown to involve alphanubeta3 ligation and NFkappaB activation.

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Effect of NFκB inhibition on TNFα mediated endothelial cell OPG production. HuDMECs were treated with the NFκB inhibitor PDTC (50 μM), TNFα (10 ng/ml) or TNFα (10 ng/ml) in conjunction with PDTC (50 μM) for 24 hours. RNA was extracted and gene expression quantified using real-time PCR (a). For real-time quantitative PCR, values were normalised to GAPDH and are given as fold expression compared to untreated HuDMECs. OPG secretion was measured in the conditioned medium using ELISA as described in materials and methods (b). Data are represented as mean ± S.E.M. from three independent experiments performed in triplicate. ***, p < 0.001.
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Figure 6: Effect of NFκB inhibition on TNFα mediated endothelial cell OPG production. HuDMECs were treated with the NFκB inhibitor PDTC (50 μM), TNFα (10 ng/ml) or TNFα (10 ng/ml) in conjunction with PDTC (50 μM) for 24 hours. RNA was extracted and gene expression quantified using real-time PCR (a). For real-time quantitative PCR, values were normalised to GAPDH and are given as fold expression compared to untreated HuDMECs. OPG secretion was measured in the conditioned medium using ELISA as described in materials and methods (b). Data are represented as mean ± S.E.M. from three independent experiments performed in triplicate. ***, p < 0.001.

Mentions: Previous studies have demonstrated both the bacterial pathogen Porphyromonas gingivalis and the secreted glycoprotein osteopontin to increase endothelial cell OPG production in an NFκB-dependent manner [4,21]. To determine the involvement of NFkB in tumour cell contact-mediated HuDMEC production, co-cultures were established as previously and treated with the NFkB inhibitor PDTC (50 μM). As shown in figure 5(a), the tumour cell contact-mediated increase in HuDMEC OPG secretion was attenuated following NFκB inhibition with PDTC. Whilst untreated co-cultures at a 2:1 HuDMEC: T47D ratio secreted 30 pg/1000 cells of OPG, this was decreased by 53% to 14 pg/1000 cells in those treated with the PDTC (p < 0.001). In contrast, NFkB inhibition did not affect the tumour cell contact-mediated increase in HuDMEC OPG gene expression, suggesting NFκB involvement at the post-transcriptional level (Figure 5(b)). As a control, the effect of NFκB inhibition on TNFα induced HuDMEC OPG production was assessed. As demonstrated in figure 6, in contrast to the co-cultures, the TNFα mediated increase in both HuDMEC OPG gene expression and secretion was attenuated following treatment with PDTC. In terms of gene expression, PDTC significantly inhibited the TNFα mediated increase in OPG levels (p < 0.001). Similarly, whilst treatment of HuDMECs with TNFα significantly increased OPG secretion from 74.53 pg/1000 cells to 1065 pg/1000 cells (p < 0.001), in the presence of PDTC this was significantly decreased to 5.76 pg/1000 cells (p < 0.001).


Breast cancer cells stimulate osteoprotegerin (OPG) production by endothelial cells through direct cell contact.

Reid PE, Brown NJ, Holen I - Mol. Cancer (2009)

Effect of NFκB inhibition on TNFα mediated endothelial cell OPG production. HuDMECs were treated with the NFκB inhibitor PDTC (50 μM), TNFα (10 ng/ml) or TNFα (10 ng/ml) in conjunction with PDTC (50 μM) for 24 hours. RNA was extracted and gene expression quantified using real-time PCR (a). For real-time quantitative PCR, values were normalised to GAPDH and are given as fold expression compared to untreated HuDMECs. OPG secretion was measured in the conditioned medium using ELISA as described in materials and methods (b). Data are represented as mean ± S.E.M. from three independent experiments performed in triplicate. ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719583&req=5

Figure 6: Effect of NFκB inhibition on TNFα mediated endothelial cell OPG production. HuDMECs were treated with the NFκB inhibitor PDTC (50 μM), TNFα (10 ng/ml) or TNFα (10 ng/ml) in conjunction with PDTC (50 μM) for 24 hours. RNA was extracted and gene expression quantified using real-time PCR (a). For real-time quantitative PCR, values were normalised to GAPDH and are given as fold expression compared to untreated HuDMECs. OPG secretion was measured in the conditioned medium using ELISA as described in materials and methods (b). Data are represented as mean ± S.E.M. from three independent experiments performed in triplicate. ***, p < 0.001.
Mentions: Previous studies have demonstrated both the bacterial pathogen Porphyromonas gingivalis and the secreted glycoprotein osteopontin to increase endothelial cell OPG production in an NFκB-dependent manner [4,21]. To determine the involvement of NFkB in tumour cell contact-mediated HuDMEC production, co-cultures were established as previously and treated with the NFkB inhibitor PDTC (50 μM). As shown in figure 5(a), the tumour cell contact-mediated increase in HuDMEC OPG secretion was attenuated following NFκB inhibition with PDTC. Whilst untreated co-cultures at a 2:1 HuDMEC: T47D ratio secreted 30 pg/1000 cells of OPG, this was decreased by 53% to 14 pg/1000 cells in those treated with the PDTC (p < 0.001). In contrast, NFkB inhibition did not affect the tumour cell contact-mediated increase in HuDMEC OPG gene expression, suggesting NFκB involvement at the post-transcriptional level (Figure 5(b)). As a control, the effect of NFκB inhibition on TNFα induced HuDMEC OPG production was assessed. As demonstrated in figure 6, in contrast to the co-cultures, the TNFα mediated increase in both HuDMEC OPG gene expression and secretion was attenuated following treatment with PDTC. In terms of gene expression, PDTC significantly inhibited the TNFα mediated increase in OPG levels (p < 0.001). Similarly, whilst treatment of HuDMECs with TNFα significantly increased OPG secretion from 74.53 pg/1000 cells to 1065 pg/1000 cells (p < 0.001), in the presence of PDTC this was significantly decreased to 5.76 pg/1000 cells (p < 0.001).

Bottom Line: In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor.In contrast, the pro-angiogenic factors VEGF, bFGF and TGFbeta had no effect on HuDMEC OPG levels.These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Academic Units of Clinical Oncology and Surgical Oncology, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK. p.reid@sheffield.ac.uk

ABSTRACT

Background: Angiogenesis, the sprouting of capillaries from existing blood vessels, is central to tumour growth and progression, however the molecular regulation of this process remains to be fully elucidated. The secreted glycoprotein osteoprotegerin (OPG) is one potential pro-angiogenic factor, and clinical studies have demonstrated endothelial cells within a number of tumour types to express high levels of OPG compared to those in normal tissue. Additionally, OPG can increase endothelial cell survival, proliferation and migration, as well as induce endothelial cell tube formation in vitro. This study aims to elucidate the processes involved in the pro-angiogenic effects of OPG in vitro, and also how OPG levels may be regulated within the tumour microenvironment.

Results: It has previously been demonstrated that OPG can induce tube formation on growth factor reduced matrigel. In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor. We also show that cell contact between HuDMECs and the T47D breast cancer cell line increases endothelial cell OPG mRNA and protein secretion levels in in vitro co-cultures. These increases in endothelial cell OPG secretion were dependent on alphanubeta3 ligation and NFkappaB activation. In contrast, the pro-angiogenic factors VEGF, bFGF and TGFbeta had no effect on HuDMEC OPG levels.

Conclusion: These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro. Additionally, we show that breast cancer cells cause increased levels of OPG expression by endothelial cells, and that direct contact between endothelial cells and tumour cells is required in order to increase endothelial OPG expression and secretion. Stimulation of OPG secretion was shown to involve alphanubeta3 ligation and NFkappaB activation.

Show MeSH
Related in: MedlinePlus