Limits...
Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells.

Otake S, Yoshida K, Seira N, Sanchez CM, Regan JW, Fujino H, Murayama T - Pharmacol Res Perspect (2014)

Bottom Line: The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA.The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density.This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University Chuo-ku, Chiba, 260-8675, Japan.

ABSTRACT
Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.

No MeSH data available.


Related in: MedlinePlus

Effects of cellular density on PGE2-induced COX-2 expression (B) or phosphorylation of ERKs (C) in HCA-7 cells. Cells were cultured at low (3 × 104 cells/each 6 well), middle (1 × 105 cells/each 6 well), and high (3 × 105 cells/each 6 well) densities (A). Each cellular density was treated with either vehicle (v) or 1 μmol/L PGE2 (P) for 6 h (B) or 15 min (C) at 37°C and then subjected to immunoblot analysis with an antibody against COX-2 (B) or phospho-ERKs 1 and 2 (pERK1/2) (C), as described in the Materials and Methods. The blots were stripped and re-probed with an antibody against β-tubulin (B) or ERKs 1 and 2 (ERK1/2) (C). The bar graphs represent the ratio of COX-2 to β-tubulin (B) or pERK1/2 to total ERK1/2 (C) as assessed with pooled densitometric data (mean ± SD) from more than three independent experiments. Data are normalized to the ratio of COX-2 to β-tubulin (B) or pERK1/2 to total ERK1/2 (C) of vehicle-treated controls at the low cellular density as 100%. *P < 0.05, ANOVA, significantly different from the vehicle treatment. †P < 0.05, ANOVA, significantly different from PGE2-stimulated HCA-7 cells at the low cellular density.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4317221&req=5

fig01: Effects of cellular density on PGE2-induced COX-2 expression (B) or phosphorylation of ERKs (C) in HCA-7 cells. Cells were cultured at low (3 × 104 cells/each 6 well), middle (1 × 105 cells/each 6 well), and high (3 × 105 cells/each 6 well) densities (A). Each cellular density was treated with either vehicle (v) or 1 μmol/L PGE2 (P) for 6 h (B) or 15 min (C) at 37°C and then subjected to immunoblot analysis with an antibody against COX-2 (B) or phospho-ERKs 1 and 2 (pERK1/2) (C), as described in the Materials and Methods. The blots were stripped and re-probed with an antibody against β-tubulin (B) or ERKs 1 and 2 (ERK1/2) (C). The bar graphs represent the ratio of COX-2 to β-tubulin (B) or pERK1/2 to total ERK1/2 (C) as assessed with pooled densitometric data (mean ± SD) from more than three independent experiments. Data are normalized to the ratio of COX-2 to β-tubulin (B) or pERK1/2 to total ERK1/2 (C) of vehicle-treated controls at the low cellular density as 100%. *P < 0.05, ANOVA, significantly different from the vehicle treatment. †P < 0.05, ANOVA, significantly different from PGE2-stimulated HCA-7 cells at the low cellular density.

Mentions: HCA-7 human colon cancer cells were cultured at low, middle, and high densities, as described in the Materials and Methods (Fig.1A). To examine the effects of cellular density on PGE2-induced COX-2 expression, HCA-7 cells cultured of different densities were treated with 1 μmol/L PGE2 for 6 h. As shown in Figure1B, stimulating cells with PGE2 significantly induced the expression of COX-2 at the low cellular density. COX-2 was also significantly induced by the treatment with PGE2 at the middle cellular density, whereas the production of COX-2 was significantly lower at the high cellular density than at the low cellular density. The induction of COX-2 was previously reported via the activation of ERKs in HCA-7 cells (Yoshida et al. 2013); therefore, the PGE2-stimulated phosphorylation of ERKs was examined at different cellular densities. As with the induction of COX-2, the treatment with PGE2 significantly induced the phosphorylation of ERKs at the low cellular density (Fig.1C). However, PGE2 only slightly induced the phosphorylation of ERKs at the middle cellular density. In contrast, the phosphorylation of ERKs was significantly reduced by the PGE2 treatment at the high cellular density. To ensure the equal loading of proteins, blots were stripped and re-probed with antibodies to β-tubulin or ERKs in Figure1B or C, respectively. As shown in the lower panels of Figure1B and C, nearly identical amounts of proteins were present under both experimental conditions.


Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells.

Otake S, Yoshida K, Seira N, Sanchez CM, Regan JW, Fujino H, Murayama T - Pharmacol Res Perspect (2014)

Effects of cellular density on PGE2-induced COX-2 expression (B) or phosphorylation of ERKs (C) in HCA-7 cells. Cells were cultured at low (3 × 104 cells/each 6 well), middle (1 × 105 cells/each 6 well), and high (3 × 105 cells/each 6 well) densities (A). Each cellular density was treated with either vehicle (v) or 1 μmol/L PGE2 (P) for 6 h (B) or 15 min (C) at 37°C and then subjected to immunoblot analysis with an antibody against COX-2 (B) or phospho-ERKs 1 and 2 (pERK1/2) (C), as described in the Materials and Methods. The blots were stripped and re-probed with an antibody against β-tubulin (B) or ERKs 1 and 2 (ERK1/2) (C). The bar graphs represent the ratio of COX-2 to β-tubulin (B) or pERK1/2 to total ERK1/2 (C) as assessed with pooled densitometric data (mean ± SD) from more than three independent experiments. Data are normalized to the ratio of COX-2 to β-tubulin (B) or pERK1/2 to total ERK1/2 (C) of vehicle-treated controls at the low cellular density as 100%. *P < 0.05, ANOVA, significantly different from the vehicle treatment. †P < 0.05, ANOVA, significantly different from PGE2-stimulated HCA-7 cells at the low cellular density.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317221&req=5

fig01: Effects of cellular density on PGE2-induced COX-2 expression (B) or phosphorylation of ERKs (C) in HCA-7 cells. Cells were cultured at low (3 × 104 cells/each 6 well), middle (1 × 105 cells/each 6 well), and high (3 × 105 cells/each 6 well) densities (A). Each cellular density was treated with either vehicle (v) or 1 μmol/L PGE2 (P) for 6 h (B) or 15 min (C) at 37°C and then subjected to immunoblot analysis with an antibody against COX-2 (B) or phospho-ERKs 1 and 2 (pERK1/2) (C), as described in the Materials and Methods. The blots were stripped and re-probed with an antibody against β-tubulin (B) or ERKs 1 and 2 (ERK1/2) (C). The bar graphs represent the ratio of COX-2 to β-tubulin (B) or pERK1/2 to total ERK1/2 (C) as assessed with pooled densitometric data (mean ± SD) from more than three independent experiments. Data are normalized to the ratio of COX-2 to β-tubulin (B) or pERK1/2 to total ERK1/2 (C) of vehicle-treated controls at the low cellular density as 100%. *P < 0.05, ANOVA, significantly different from the vehicle treatment. †P < 0.05, ANOVA, significantly different from PGE2-stimulated HCA-7 cells at the low cellular density.
Mentions: HCA-7 human colon cancer cells were cultured at low, middle, and high densities, as described in the Materials and Methods (Fig.1A). To examine the effects of cellular density on PGE2-induced COX-2 expression, HCA-7 cells cultured of different densities were treated with 1 μmol/L PGE2 for 6 h. As shown in Figure1B, stimulating cells with PGE2 significantly induced the expression of COX-2 at the low cellular density. COX-2 was also significantly induced by the treatment with PGE2 at the middle cellular density, whereas the production of COX-2 was significantly lower at the high cellular density than at the low cellular density. The induction of COX-2 was previously reported via the activation of ERKs in HCA-7 cells (Yoshida et al. 2013); therefore, the PGE2-stimulated phosphorylation of ERKs was examined at different cellular densities. As with the induction of COX-2, the treatment with PGE2 significantly induced the phosphorylation of ERKs at the low cellular density (Fig.1C). However, PGE2 only slightly induced the phosphorylation of ERKs at the middle cellular density. In contrast, the phosphorylation of ERKs was significantly reduced by the PGE2 treatment at the high cellular density. To ensure the equal loading of proteins, blots were stripped and re-probed with antibodies to β-tubulin or ERKs in Figure1B or C, respectively. As shown in the lower panels of Figure1B and C, nearly identical amounts of proteins were present under both experimental conditions.

Bottom Line: The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA.The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density.This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University Chuo-ku, Chiba, 260-8675, Japan.

ABSTRACT
Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.

No MeSH data available.


Related in: MedlinePlus