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BMP9-Induced Survival Effect in Liver Tumor Cells Requires p38MAPK Activation.

García-Álvaro M, Addante A, Roncero C, Fernández M, Fabregat I, Sánchez A, Herrera B - Int J Mol Sci (2015)

Bottom Line: However, the precise mechanisms driving these effects have not yet been established.In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK.However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, Complutense University of Madrid. San Carlos Clinical Hospital Health Research Institute (IdISSC), Plaza Ramón y Cajal S/N, Madrid 28040, Spain. mariagarciaalvaro@gmail.com.

ABSTRACT
The study of bone morphogenetic proteins (BMPs) role in tumorigenic processes, and specifically in the liver, has gathered importance in the last few years. Previous studies have shown that BMP9 is overexpressed in about 40% of hepatocellular carcinoma (HCC) patients. In vitro data have also shown evidence that BMP9 has a pro-tumorigenic action, not only by inducing epithelial to mesenchymal transition (EMT) and migration, but also by promoting proliferation and survival in liver cancer cells. However, the precise mechanisms driving these effects have not yet been established. In the present work, we deepened our studies into the intracellular mechanisms implicated in the BMP9 proliferative and pro-survival effect on liver tumor cells. In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK. However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells. Using genetic and pharmacological approaches, we demonstrate that p38MAPK activation, although dispensable for the BMP9 proliferative activity, is required for the BMP9 protective effect on serum withdrawal-induced apoptosis. These findings contribute to a better understanding of the signaling pathways involved in the BMP9 pro-tumorigenic role in liver tumor cells.

No MeSH data available.


Related in: MedlinePlus

BMP9 increases HepG2 cell number: effects on cell cycle and survival. (A) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for four days, and then, the cell number was analyzed by crystal violet staining. Data from two independent experiments with n = 6 displayed as the percentage of 0% FBS-treated cells (mean ± S.E.M.). Statistical analysis was carried out using the paired t-test, and data were compared to untreated cells (0% FBS samples), ***p < 0.001 or compared as indicated. n.s., not significant; (B) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for three days. Cells were visualized, and pictures were taken by phase contrast microscopy. Scale bars represent 50 μm; (C) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for two days, and proliferation by BrdU incorporation was analyzed. Data from one representative experiment out of four, with n = 6, displayed as the percentage of untreated cells (0% FBS samples) (mean ± S.D.). Statistical analysis was carried out as in (A); (D) HepG2 cells were incubated for different periods of time −/+ BMP9 (5 ng/mL) in 0% FBS media. Western blots were performed with antibodies that recognize cyclin D1, p27 and Smad2,3 analyzed as the loading control. Optical density values relative to the loading control were calculated. A representative experiment of two is shown; (E) HepG2 cells were incubated in 10% FBS media or in 0% FBS media −/+ BMP9 (5 ng/mL) for four days, and then, nuclear DNA content was analyzed by flow cytometry. SubG1 cells are hypodiploid cells, cells with a DNA content lower than 2n. Data from one representative out of four experiments, with n = 2, are shown; (F) HepG2 cells were incubated in 10% FBS media or in 0% FBS media −/+ BMP9 (5 ng/mL) for 24 h and Bim, Puma, Bax and Bcl-xL mRNA levels were analyzed by RT-qPCR and normalized to Gusb. Fold changes relative to 10% FBS media samples were determined (mean ± S.E.M., n = 4). Statistical analysis was carried out using the paired t-test, and data were compared to 10% FBS samples, **p < 0.01, ***p < 0.001 or compared to 0% FBS samples, &p < 0.05, &&&p < 0.001; (G) HepG2 cells were incubated −/+ BMP9 (5 ng/mL) in 0% FBS media or in the presence of 10% FBS media for different periods of time. Western blots were performed with antibodies that recognize Bim, Puma and GAPDH (loading control). A representative experiment of three is shown.
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ijms-16-20431-f001: BMP9 increases HepG2 cell number: effects on cell cycle and survival. (A) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for four days, and then, the cell number was analyzed by crystal violet staining. Data from two independent experiments with n = 6 displayed as the percentage of 0% FBS-treated cells (mean ± S.E.M.). Statistical analysis was carried out using the paired t-test, and data were compared to untreated cells (0% FBS samples), ***p < 0.001 or compared as indicated. n.s., not significant; (B) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for three days. Cells were visualized, and pictures were taken by phase contrast microscopy. Scale bars represent 50 μm; (C) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for two days, and proliferation by BrdU incorporation was analyzed. Data from one representative experiment out of four, with n = 6, displayed as the percentage of untreated cells (0% FBS samples) (mean ± S.D.). Statistical analysis was carried out as in (A); (D) HepG2 cells were incubated for different periods of time −/+ BMP9 (5 ng/mL) in 0% FBS media. Western blots were performed with antibodies that recognize cyclin D1, p27 and Smad2,3 analyzed as the loading control. Optical density values relative to the loading control were calculated. A representative experiment of two is shown; (E) HepG2 cells were incubated in 10% FBS media or in 0% FBS media −/+ BMP9 (5 ng/mL) for four days, and then, nuclear DNA content was analyzed by flow cytometry. SubG1 cells are hypodiploid cells, cells with a DNA content lower than 2n. Data from one representative out of four experiments, with n = 2, are shown; (F) HepG2 cells were incubated in 10% FBS media or in 0% FBS media −/+ BMP9 (5 ng/mL) for 24 h and Bim, Puma, Bax and Bcl-xL mRNA levels were analyzed by RT-qPCR and normalized to Gusb. Fold changes relative to 10% FBS media samples were determined (mean ± S.E.M., n = 4). Statistical analysis was carried out using the paired t-test, and data were compared to 10% FBS samples, **p < 0.01, ***p < 0.001 or compared to 0% FBS samples, &p < 0.05, &&&p < 0.001; (G) HepG2 cells were incubated −/+ BMP9 (5 ng/mL) in 0% FBS media or in the presence of 10% FBS media for different periods of time. Western blots were performed with antibodies that recognize Bim, Puma and GAPDH (loading control). A representative experiment of three is shown.

Mentions: We have previously described that BMP9 is a strong mitogen for liver tumor cells in the presence of 0.1% FBS [15]. Our current study also shows this effect in the absence of serum. In fact, when HepG2 cells were incubated with BMP9 for four days in 0% FBS, we found that the number of viable adherent cells doubled in comparison to untreated cells. Indeed, BMP9 treatment in the absence of serum resulted in cell growth rates similar to those observed in the presence of 10% FBS (normal growing conditions). Furthermore, the BMP9 cell growth effect was readily visible by phase contrast microscopy (Figure 1A,B). Consistently, BMP9 induces an increase in BrdU incorporation to nearly the same extent as that obtained when cells were incubated in 10% FBS (Figure 1C). Increased cell proliferation induced by BMP9 was accompanied by changes in the expression of cell cycle regulators: BMP9 enhanced cyclinD1 expression and decreased CDK interacting protein/kinase inhibitory protein p27 expression (Figure 1D), both events involved in the progression from the G0/G1 phases towards the S phase of the cell cycle [18]. We had documented before that incubation of HepG2 cells in low serum, 0.1% FBS, resulted in an apoptotic cell death that was rescued by BMP9 [15]. Data presented here indicate that the BMP9 pro-survival effect is also observed when cells are incubated in the complete absence of serum (Figure 1E). It is well established that serum deprivation in HCC cells results in a mitochondrial apoptosis characterized by mitochondrial membrane potential depletion, cytochrome c release and Bcl-2 family member modulation [19,20,21]. Our results are in agreement with these previous results, as we observed that serum starvation resulted in the upregulation of several pro-apoptotic members of the Bcl-2 family, in particular Bim, Puma and Bax, both at the mRNA and the protein level. Importantly, BMP9 treatment blocked these effects (Figure 1F,G). In parallel, we analyzed the antiapoptotic protein Bcl-xL, but no modulation was observed in any of the conditions tested (Figure 1G).


BMP9-Induced Survival Effect in Liver Tumor Cells Requires p38MAPK Activation.

García-Álvaro M, Addante A, Roncero C, Fernández M, Fabregat I, Sánchez A, Herrera B - Int J Mol Sci (2015)

BMP9 increases HepG2 cell number: effects on cell cycle and survival. (A) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for four days, and then, the cell number was analyzed by crystal violet staining. Data from two independent experiments with n = 6 displayed as the percentage of 0% FBS-treated cells (mean ± S.E.M.). Statistical analysis was carried out using the paired t-test, and data were compared to untreated cells (0% FBS samples), ***p < 0.001 or compared as indicated. n.s., not significant; (B) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for three days. Cells were visualized, and pictures were taken by phase contrast microscopy. Scale bars represent 50 μm; (C) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for two days, and proliferation by BrdU incorporation was analyzed. Data from one representative experiment out of four, with n = 6, displayed as the percentage of untreated cells (0% FBS samples) (mean ± S.D.). Statistical analysis was carried out as in (A); (D) HepG2 cells were incubated for different periods of time −/+ BMP9 (5 ng/mL) in 0% FBS media. Western blots were performed with antibodies that recognize cyclin D1, p27 and Smad2,3 analyzed as the loading control. Optical density values relative to the loading control were calculated. A representative experiment of two is shown; (E) HepG2 cells were incubated in 10% FBS media or in 0% FBS media −/+ BMP9 (5 ng/mL) for four days, and then, nuclear DNA content was analyzed by flow cytometry. SubG1 cells are hypodiploid cells, cells with a DNA content lower than 2n. Data from one representative out of four experiments, with n = 2, are shown; (F) HepG2 cells were incubated in 10% FBS media or in 0% FBS media −/+ BMP9 (5 ng/mL) for 24 h and Bim, Puma, Bax and Bcl-xL mRNA levels were analyzed by RT-qPCR and normalized to Gusb. Fold changes relative to 10% FBS media samples were determined (mean ± S.E.M., n = 4). Statistical analysis was carried out using the paired t-test, and data were compared to 10% FBS samples, **p < 0.01, ***p < 0.001 or compared to 0% FBS samples, &p < 0.05, &&&p < 0.001; (G) HepG2 cells were incubated −/+ BMP9 (5 ng/mL) in 0% FBS media or in the presence of 10% FBS media for different periods of time. Western blots were performed with antibodies that recognize Bim, Puma and GAPDH (loading control). A representative experiment of three is shown.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4613212&req=5

ijms-16-20431-f001: BMP9 increases HepG2 cell number: effects on cell cycle and survival. (A) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for four days, and then, the cell number was analyzed by crystal violet staining. Data from two independent experiments with n = 6 displayed as the percentage of 0% FBS-treated cells (mean ± S.E.M.). Statistical analysis was carried out using the paired t-test, and data were compared to untreated cells (0% FBS samples), ***p < 0.001 or compared as indicated. n.s., not significant; (B) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for three days. Cells were visualized, and pictures were taken by phase contrast microscopy. Scale bars represent 50 μm; (C) HepG2 cells were incubated in 10% FBS media or in 0% FBS media with or without BMP9 (5 ng/mL) for two days, and proliferation by BrdU incorporation was analyzed. Data from one representative experiment out of four, with n = 6, displayed as the percentage of untreated cells (0% FBS samples) (mean ± S.D.). Statistical analysis was carried out as in (A); (D) HepG2 cells were incubated for different periods of time −/+ BMP9 (5 ng/mL) in 0% FBS media. Western blots were performed with antibodies that recognize cyclin D1, p27 and Smad2,3 analyzed as the loading control. Optical density values relative to the loading control were calculated. A representative experiment of two is shown; (E) HepG2 cells were incubated in 10% FBS media or in 0% FBS media −/+ BMP9 (5 ng/mL) for four days, and then, nuclear DNA content was analyzed by flow cytometry. SubG1 cells are hypodiploid cells, cells with a DNA content lower than 2n. Data from one representative out of four experiments, with n = 2, are shown; (F) HepG2 cells were incubated in 10% FBS media or in 0% FBS media −/+ BMP9 (5 ng/mL) for 24 h and Bim, Puma, Bax and Bcl-xL mRNA levels were analyzed by RT-qPCR and normalized to Gusb. Fold changes relative to 10% FBS media samples were determined (mean ± S.E.M., n = 4). Statistical analysis was carried out using the paired t-test, and data were compared to 10% FBS samples, **p < 0.01, ***p < 0.001 or compared to 0% FBS samples, &p < 0.05, &&&p < 0.001; (G) HepG2 cells were incubated −/+ BMP9 (5 ng/mL) in 0% FBS media or in the presence of 10% FBS media for different periods of time. Western blots were performed with antibodies that recognize Bim, Puma and GAPDH (loading control). A representative experiment of three is shown.
Mentions: We have previously described that BMP9 is a strong mitogen for liver tumor cells in the presence of 0.1% FBS [15]. Our current study also shows this effect in the absence of serum. In fact, when HepG2 cells were incubated with BMP9 for four days in 0% FBS, we found that the number of viable adherent cells doubled in comparison to untreated cells. Indeed, BMP9 treatment in the absence of serum resulted in cell growth rates similar to those observed in the presence of 10% FBS (normal growing conditions). Furthermore, the BMP9 cell growth effect was readily visible by phase contrast microscopy (Figure 1A,B). Consistently, BMP9 induces an increase in BrdU incorporation to nearly the same extent as that obtained when cells were incubated in 10% FBS (Figure 1C). Increased cell proliferation induced by BMP9 was accompanied by changes in the expression of cell cycle regulators: BMP9 enhanced cyclinD1 expression and decreased CDK interacting protein/kinase inhibitory protein p27 expression (Figure 1D), both events involved in the progression from the G0/G1 phases towards the S phase of the cell cycle [18]. We had documented before that incubation of HepG2 cells in low serum, 0.1% FBS, resulted in an apoptotic cell death that was rescued by BMP9 [15]. Data presented here indicate that the BMP9 pro-survival effect is also observed when cells are incubated in the complete absence of serum (Figure 1E). It is well established that serum deprivation in HCC cells results in a mitochondrial apoptosis characterized by mitochondrial membrane potential depletion, cytochrome c release and Bcl-2 family member modulation [19,20,21]. Our results are in agreement with these previous results, as we observed that serum starvation resulted in the upregulation of several pro-apoptotic members of the Bcl-2 family, in particular Bim, Puma and Bax, both at the mRNA and the protein level. Importantly, BMP9 treatment blocked these effects (Figure 1F,G). In parallel, we analyzed the antiapoptotic protein Bcl-xL, but no modulation was observed in any of the conditions tested (Figure 1G).

Bottom Line: However, the precise mechanisms driving these effects have not yet been established.In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK.However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, Complutense University of Madrid. San Carlos Clinical Hospital Health Research Institute (IdISSC), Plaza Ramón y Cajal S/N, Madrid 28040, Spain. mariagarciaalvaro@gmail.com.

ABSTRACT
The study of bone morphogenetic proteins (BMPs) role in tumorigenic processes, and specifically in the liver, has gathered importance in the last few years. Previous studies have shown that BMP9 is overexpressed in about 40% of hepatocellular carcinoma (HCC) patients. In vitro data have also shown evidence that BMP9 has a pro-tumorigenic action, not only by inducing epithelial to mesenchymal transition (EMT) and migration, but also by promoting proliferation and survival in liver cancer cells. However, the precise mechanisms driving these effects have not yet been established. In the present work, we deepened our studies into the intracellular mechanisms implicated in the BMP9 proliferative and pro-survival effect on liver tumor cells. In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK. However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells. Using genetic and pharmacological approaches, we demonstrate that p38MAPK activation, although dispensable for the BMP9 proliferative activity, is required for the BMP9 protective effect on serum withdrawal-induced apoptosis. These findings contribute to a better understanding of the signaling pathways involved in the BMP9 pro-tumorigenic role in liver tumor cells.

No MeSH data available.


Related in: MedlinePlus