Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of tumour cell contact on endothelial cell OPG production. HuDMECs were co-cultured with the T47D cell line at HuDMEC: T47D ratios of 2:1, 4:1 and 10:1 for 72 hours as described in materials and methods. HuDMECs were separated from the T47D cell line and gene expression measured in the separate cell populations using real-time quantitative PCR (a). OPG secretion was assessed using ELISA (b). For real-time quantitative PCR, values were normalised to GAPDH and are given as fold expression compared to untreated HuDMECs. Data are represented as mean ± S.E.M. from three independent experiments. ***, p < 0.001 compared to control cells (HuDMEC only); **, p < 0.01 compared to control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2719583&req=5

Figure 4: Effect of tumour cell contact on endothelial cell OPG production. HuDMECs were co-cultured with the T47D cell line at HuDMEC: T47D ratios of 2:1, 4:1 and 10:1 for 72 hours as described in materials and methods. HuDMECs were separated from the T47D cell line and gene expression measured in the separate cell populations using real-time quantitative PCR (a). OPG secretion was assessed using ELISA (b). For real-time quantitative PCR, values were normalised to GAPDH and are given as fold expression compared to untreated HuDMECs. Data are represented as mean ± S.E.M. from three independent experiments. ***, p < 0.001 compared to control cells (HuDMEC only); **, p < 0.01 compared to control.

Mentions: An alternative mechanism by which OPG production by HuDMECs might be enhanced is through direct contact with tumour cells. Since clinical studies have demonstrated increased OPG expression in breast tumour endothelial cells, HuDMECs were co-cultured with the T47D breast cancer cell line, which did not produce detectable levels of OPG (data not shown). These co-cultures were established at HuDMEC: T47D ratios of 2:1, 4:1 and 10:1. Following a 72-hour incubation period, HuDMECs were separated from the T47D cells using CD31 Dynabeads and OPG gene expression assessed in each cell type. As demonstrated in figure 4(a), tumour cell contact significantly increased OPG gene expression levels compared to HuDMECs cultured alone. This was particularly noticeable at a 2:1 HuDMEC: T47D ratio, where OPG levels were increased 3-fold (p < 0.001). This was specific to HuDMECs as OPG gene expression was not detectable in the T47D cells following co-culture. The elevated levels of OPG expression were accompanied by increased HuDMEC OPG secretion. This increase was significant in co-cultures with a 2:1 HuDMEC: T47D ratio (100 pg/1000 cells) compared to HuDMECs cultured alone (42 pg/1000 cells) (p < 0.01) (Figure 4(b)). The increase in OPG expression seen in the co-cultures was dependent on direct cell-cell contact, as addition of conditioned medium from T47D monolayers had no effect on HuDMEC OPG levels (data not shown).


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 tumour cell contact on endothelial cell OPG production. HuDMECs were co-cultured with the T47D cell line at HuDMEC: T47D ratios of 2:1, 4:1 and 10:1 for 72 hours as described in materials and methods. HuDMECs were separated from the T47D cell line and gene expression measured in the separate cell populations using real-time quantitative PCR (a). OPG secretion was assessed using ELISA (b). For real-time quantitative PCR, values were normalised to GAPDH and are given as fold expression compared to untreated HuDMECs. Data are represented as mean ± S.E.M. from three independent experiments. ***, p < 0.001 compared to control cells (HuDMEC only); **, p < 0.01 compared to control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effect of tumour cell contact on endothelial cell OPG production. HuDMECs were co-cultured with the T47D cell line at HuDMEC: T47D ratios of 2:1, 4:1 and 10:1 for 72 hours as described in materials and methods. HuDMECs were separated from the T47D cell line and gene expression measured in the separate cell populations using real-time quantitative PCR (a). OPG secretion was assessed using ELISA (b). For real-time quantitative PCR, values were normalised to GAPDH and are given as fold expression compared to untreated HuDMECs. Data are represented as mean ± S.E.M. from three independent experiments. ***, p < 0.001 compared to control cells (HuDMEC only); **, p < 0.01 compared to control.
Mentions: An alternative mechanism by which OPG production by HuDMECs might be enhanced is through direct contact with tumour cells. Since clinical studies have demonstrated increased OPG expression in breast tumour endothelial cells, HuDMECs were co-cultured with the T47D breast cancer cell line, which did not produce detectable levels of OPG (data not shown). These co-cultures were established at HuDMEC: T47D ratios of 2:1, 4:1 and 10:1. Following a 72-hour incubation period, HuDMECs were separated from the T47D cells using CD31 Dynabeads and OPG gene expression assessed in each cell type. As demonstrated in figure 4(a), tumour cell contact significantly increased OPG gene expression levels compared to HuDMECs cultured alone. This was particularly noticeable at a 2:1 HuDMEC: T47D ratio, where OPG levels were increased 3-fold (p < 0.001). This was specific to HuDMECs as OPG gene expression was not detectable in the T47D cells following co-culture. The elevated levels of OPG expression were accompanied by increased HuDMEC OPG secretion. This increase was significant in co-cultures with a 2:1 HuDMEC: T47D ratio (100 pg/1000 cells) compared to HuDMECs cultured alone (42 pg/1000 cells) (p < 0.01) (Figure 4(b)). The increase in OPG expression seen in the co-cultures was dependent on direct cell-cell contact, as addition of conditioned medium from T47D monolayers had no effect on HuDMEC OPG levels (data not shown).

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